POSITRON EMISSION TOMOGRAPHY IMAGING
OF HEPATOCELLULAR CARCINOMA WITH
RADIOLABELED CHOLINE
By
YU KUANG
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Adviser: Zhenghong Lee, Ph.D.
Department of Biomedical Engineering
CASE WESTERN RESERVE UNIVERSITY
May, 2009
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
______
candidate for the ______degree *.
(signed)______(chair of the committee)
______
______
______
______
______
(date) ______
*We also certify that written approval has been obtained for any proprietary material contained therein.
To my parents
献给我亲爱的父母
ii
Table of Contents
Table of Contents ...... i
List of Tables ...... xi
List of Figures ...... xii
Acknowledgments...... xv
List of Abbreviations ...... xvi
Abstract ...... xviii
Chapter 1 Introduction ...... 1
1.1 Molecular Imaging of Cancer ...... 1
1.1.1 Positron emission tomography for cancer imaging ...... 1
1.1.2 Molecular imaging modalities ...... 3
1.1.3 Positron emission tomography imaging for hepatocellular carcinoma ...... 7
1.2 Basic Principle of Positron Emission Tomography ...... 8
1.2.1 Positron emission and annihilation ...... 8
1.2.2 511 keV photon interactions in matter ...... 10
1.2.2.1 Compton scattering ...... 10
1.2.2.2 Rayleigh scattering...... 12
1.2.2.3 Photoelectric effect ...... 12
1.2.3 Data collection ...... 13
i
1.2.3.1 Coincidence detection ...... 13
1.2.3.2 Electronic timing window ...... 14
1.2.3.3 Time-of-flight ...... 14
1.2.4 Detectors and scanner design ...... 15
1.2.4.1 Scintillation crystals ...... 15
1.2.4.2 Dead time ...... 17
1.2.4.3 PET/CT ...... 18
1.2.4.4 Collimation ...... 18
1.2.5 LORs and projection data ...... 19
1.2.5.1 Lines of response (LORs) ...... 19
1.2.5.2 Organization of data ...... 20
1.2.5.3 Projections and sinograms ...... 21
1.2.6 Image reconstruction ...... 22
1.2.6.1 Forward problem ...... 22
1.2.6.2 Radon transform...... 22
1.2.6.3 Analytical reconstruction ...... 23
1.2.6.3.1 Simple backprojection ...... 23
1.2.6.3.2 Filtered backprojection (FBP) ...... 24
1.2.6.4 Iterative reconstruction ...... 24
1.2.6.4.1 Five basic steps...... 24 ii
1.2.6.4.2 Maximum-likelihood expectation-maximization ...... 25
1.2.7 Attenuation correction ...... 27
1.2.8 Scatter and randoms ...... 28
1.2.8.1 Coincidence events ...... 28
1.2.8.2 Scatter Correction ...... 30
1.2.8.3 Random Correction ...... 30
1.3 Review of the literatures ...... 31
1.3.1 Hepatitis B viral infection induced hepatocellular carcinoma ...... 31
1.3.1.1 Chronic hepatitis B viral Infection and hepatocellular carcinoma ...... 31
1.3.1.2 Woodchuck hepatitis virus infection induced woodchuck Model of
hepatocellular carcinoma ...... 34
1.3.2 Medical imaging of hepatocellular carcinoma ...... 36
1.3.2.1 Diagnosis techniques for hepatocellular carcinoma ...... 36
1.3.2.2 Tumor glucose metabolism and FDG-PET imaging on cancer ...... 38
1.3.2.3 Tumor-associated de novo fatty acid synthesis ...... 39
1.3.3 Imaging lipid synthesis in Cancer with PET ...... 43
1.4 Organization of the thesis ...... 45
Chapter 2 2-Deoxy-2-[18F]-fluoro-D-glucose Positron Emission Tomography Imaging of
Hepatocellular Carcinoma ...... 46
2.1 Introduction ...... 46
iii
2.2 Materials and methods ...... 48
2.2.1 Materials ...... 48
2.2.2 Animals ...... 48
2.2.3 Radiopharmaceuticals ...... 49
2.2.4 Imaging protocol ...... 49
2.2.5 Image analysis ...... 50
2.2.6 Histology ...... 51
2.2.7 Tissue excision ...... 51
2.2.8 Hexokinase activity assay ...... 52
2.2.9 Glucose-6-phosphatase activity assay...... 53
2.2.10 Statistical Analysis ...... 54
2.3 Results ...... 54
2.3.1 PET imaging ...... 54
2.3.2 Enzyme activity ...... 58
2.4 Discussion ...... 59
Chapter 3 Transport Mechanism and Metabolic Fate of Radiolabeled Choline in
Hepatocellular Carcinoma: an in vitro Comparative Study ...... 61
3.1 Introduction ...... 61
3.2 Materials and Methods ...... 65
3.2.1 Materials ...... 65
iv
3.2.2 Primary rat hepatocytes preparation ...... 65
3.2.3 WCH17 Cell culture and media ...... 67
3.2.4 [Methyl-14C]-Choline uptake and metabolism ...... 67
3.2.5 Pulse and chase study and efflux ...... 68
3.2.6 Extraction of radiolabeled metabolites and lipids using Bligh and Dyer method . 69
3.2.7 Analysis of radiolabeled metabolites ...... 70
3.2.7.1 HPLC system...... 70
3.2.7.2 Water soluble phase metabolites analysis ...... 70
3.2.7.3 Lipid soluble phase metabolites analysis ...... 71
3.2.8 [methyl-14C]-betaine uptake in WCH17 cells ...... 72
3.2.8.1 Preparation of 14C-betaine...... 72
3.2.8.2 [methyl-14C]-betaine uptake in WCH17 cells ...... 72
3.2.9 Kinetics of choline transport in WCH17 cells in vitro ...... 73
3.2.9.1 Kinetics of choline transport in WCH17 cells and effects of lithium-for-sodium
replacement ...... 73
3.2.9.2 Inhibition of choline transporter in WCH17 cells ...... 73
3.2.9.3 Parameter Estimation for choline transporter in WCH17 cells ...... 74
3.2.10 Assay of choline kinase in WCH17 cells ...... 75
3.2.11 Phosphocholine production (whole-cell choline kinase assay) in WCH17 cells . 75
3.2.12 Protein assay ...... 76
v
3.2.13 Liquid scintillation counting ...... 76
3.2.14 Statistical analysis ...... 76
3.3 Results ...... 77
3.3.1 Separation of the radioactive metabolites derived from [methyl-14C]-choline
(authentic 14C standards) ...... 77
3.3.2 [methyl-14C]-Choline uptake patterns and metabolism in WCH17 cells and
primary rat hepatocytes ...... 81
3.3.2.1 Time course of [methyl-14C]choline uptake ...... 81
3.3.2.2 Metabolism of [methyl-14C]choline in WCH17 cells and primary rat
hepatocytes ...... 82
3.3.3 Pulse and chase study and efflux in WCH17 cells and primary rat hepatocytes ... 85
3.3.4 The effect of oleic acid on the metabolism of [14C]choline in WCH17 cells and
primary rat hepatocytes ...... 93
3.3.5 Kinetics of choline transport system in WCH17 Cells ...... 97
3.3.5.1 Kinetics parameters of the choline transport system in WCH17 cells ...... 97
3.3.5.2 Effect of hemicholinium-3 inhibitor on the choline transporter system in
cultured WCH17 cells ...... 100
3.3.5.3 Effect of other inhibitors on the choline transporter system in cultured WCH17
cells ...... 101
3.3.6 Choline kinase activity in WCH17 cells ...... 104
3.4 Discussion ...... 105 vi
3.4.1 Metabolic fate of radiolabeled choline ...... 106
3.4.2 Choline incorporation to lipids ...... 107
3.4.3 Choline transport ...... 108
Chapter 4 Imaging Lipid Synthesis in Hepatocellular Carcinoma Correlated with Metabolites
Study in vivo ...... 115
4.1 Introduction ...... 115
4.2 Materials and Methods ...... 119
4.2.1 Materials ...... 119
4.2.2 Animals ...... 119
4.2.3 Radiopharmaceuticals ...... 120
4.2.3.1 [Methyl-11C]-Choline ...... 120
18 4.2.3.2 2-Deoxy-2[ F]Fluoro-D-Glucose (FDG) ...... 120
4.2.3.3 1-11C-acetate (Act) ...... 121
4.2.4 Imaging Protocol ...... 121
4.2.5 Image Analysis...... 122
4.2.6 Histology ...... 123
4.2.7 Metabolites study in woodchuck model of hepatocellular carcinoma ...... 124
4.2.7.1 [Methyl-14C]-CHOL metabolism in woodchuck model of hepatocellular
carcinoma ...... 124
4.2.7.2 Extraction of radiolabeled metabolites and lipids using Folch method ...... 124
vii
4.2.7.3 Analysis of radiolabeled metabolites ...... 126
4.2.7.3.1 HPLC system...... 126
4.2.7.3.2 Analysis of water soluble phase metabolites ...... 126
4.2.7.3.3 Lipid soluble phase metabolites analysis ...... 127
4.2.8 Contribution of CDP-choline pathway and PE methylation pathway to phosphoatidylcholine synthesis in woodchuck model of hepatocellular carcinoma .... 128
4.2.8.1 Argentation TLC to separate different molecular species of PC synthesized
from CDP-choline pathway or PE methylation pathway in woodchuck model of
hepatocellular carcinoma ...... 128
4.2.8.2 Verify the contribution of PE methylation pathway to phosphoatidylcholine
synthesis in hepatocellular carcinoma ...... 129
4.2.9 Assay of choline kinase activity ...... 130
4.2.10 Assay of choline-phosphate cytidylyltransferase activity ...... 132
4.2.10.1 Preparation of cytosolic and microsomal pellet fraction of liver tissue...... 132
4.2.10.2 Assay of choline-phosphate cytidylyltransferase activity ...... 132
4.2.11 Effect of oleic acid on phosphatidylcholine synthesis derived from radiolabeled choline tracer in cultured WCH17 well-differentiated hepatoma cells ...... 133
4.2.11.1 Preparation of 1mM oleic acid ...... 133
4.2.11.2 Pulse and chase study of phosphatidycholine synthesis from [methyl-14C]-
choline in cultured WCH17 woodchuck hepatoma cells with 1mM oleic acid treatment
...... 133
viii
4.2.12 Protein assay ...... 134
4.2.13 Liquid scintillation counting ...... 134
4.2.14 Statistical analysis ...... 134
4.3 Results ...... 135
4.3.1 PET/CT imaging and histology ...... 135
4.3.2 Metabolites study in woodchuck model of hepatocellular carcinoma ...... 139
4.3.3 Contribution of CDP-choline pathway and PE methylation pathway to
phosphatidylcholine synthesis ...... 144
4.3.4 Choline kinase activity ...... 148
4.3.5 Choline-phosphate cytidydyltransfearse ...... 149
4.3.6 Effect of oleic acid on phosphatidylcholine synthesis ...... 151
4.4 Discussion ...... 152
4.4.1 PET/CT imaging ...... 152
4.4.2 Metabolites study in woodchuck model of hepatocellular carcinoma ...... 155
4.4.3 Contribution of CDP-choline pathway and PE methylation pathway to
phosphatidylcholine synthesis ...... 158
4.4.4 Choline kinase ...... 161
4.4.5 Choline-phosphate cytidydyltransfearse ...... 162
4.4.6 Effect of oleic acid on phosphatidylcholine synthesis ...... 164
4.4.7 Summary ...... 165
ix
Chapter 5 Conclusions and Future Perspectives ...... 167
5.1 Conclusions ...... 167
5.2 Future Prospectives ...... 169
5.2.1 Effect of hypoxia on the tracer metabolism in hepatocellular carcinoma ...... 169
5.2.2 Ethanolamine and N, N'-dimethyl ethanolamine as probes for early detection of
hepatocellular carcinoma ...... 170
Bibliography ...... 173
x
List of Tables
Table 1.1 Characteristics of molecular imaging modalities...... 5
Table 1.2 Common PET Radionuclides ...... 11
Table 3.1 Parameter estimation by nonlinear least-squares fit to [methyl-14C]-choline uptake in cultured WCH17 cells ...... 100
Table 3.2 Effect of other inhibitors on the uptake of [methyl-14C]-choline in cultured WCH17
Cells ...... 103
Table 3.3 Characteristics of carrier-mediated choline transport system in mammalian cells
...... 113
Table 4.1 Tumor/Liver ratio from the PET imaging with FDG, 11C-Act, 11C-CHOL ...... 135
Table 4.2 Molecular species of 14C-phosphatidylholine formed in woodchuck model of hepatocellular carcinoma receiving [methyl-14C]-choline IV injection ...... 147
xi
List of Figures
Figure 1.1 A positron-emitting radionuclide decays by transforming a proton into a neutron, neutrino (v), and positron (e+)...... 10
Figure 1.2 Annihilation photons may interact with matter by (A) Compton scattering, (B) photoelectric effect scattering...... 12
Figure 1.3 Time-of flight PET...... 15
Figure 1.4 Typical energy distribution measured by a scintillation detector system exposed to gamma rays ...... 17
Figure 1.5 Three types of coincidences in a PET detector: (A) True, (B) Scatter, and (C)
Random...... 29
Figure 2.1 FDG-PET imaging on the woodchuck model of HCC...... 55
Figure 2.2 PET imaging on well-differentiated HCC ...... 57
Figure 2.3 dynamic PET scan with FDG on well-differentiated HCC and normal liver...... 57
Figure 2.4 PET imaging on poorly-differentiated HCC ...... 58
Figure 2.5 Hexokinase and Glucose-6-phosphatase activity in woodchuck model of HCC. . 59
Figure 3.1 Metabolic fate of radiolabeled choline...... 63
Figure 3.2 Representative HPLC radiochromatograms of water soluble metabolites derived from [methyl-14C]-choline...... 78
Figure 3.3 Representative HPLC radiochromatograms of lipid soluble metabolites derived from [methyl-14C]-choline...... 79
Figure 3.4 Autoradiogram of labeled authentic 14C standards ...... 80
Figure 3.5 Time course of incorporation of [14C]choline into lipid-, water-soluble and insoluble phases ...... 81
xii
Figure 3.6 Comparison of uptake of [14C]betaine and [14C]CHOL in WCH17 cells...... 82
Figure 3.7 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites during the incubation period in WCH17 cells and primary rat hepatocytes...... 83
Figure 3.8 Pattern of [14C]choline metabolites in the water- and lipid-soluble fraction during the 60 min incubation...... 84
Figure 3.9 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites in pulse and chase study in WCH17 cells and primary rat hepatocytes...... 87
Figure 3.10 Pulse-chase study on the metabolism of [methyl-14C]-CHOL by WCH17 cells and primary rat hepatocytes...... 89
Figure 3.11 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites in pulse and chase study in oleic acid treated WCH17 cells and primary rat hepatocytes. .... 94
Figure 3.12 Effect of oleic acid on the phosphatidylcholine synthesis...... 96
Figure 3.13 Kinetics of [methyl-14C]-choline uptake in WCH17 cells and effects of lithium- for-sodium replacement...... 99
Figure 3.14 Inhibition of [methyl-14C]-choline uptake in WCH17 cells by hemicholinium 3.
Lines show the fit of the modified Michaelis-Menten equation (Eq 4.1) to the data...... 101
Figure 3.15 Choline kinase activity in WCH17 cells...... 104
Figure 3.16 Whole cell choline kinase activity in WCH17 cells...... 105
Figure 4.1 Metabolic fate of [methyl-11C]-choline...... 118
Figure 4.2 Coronal PET/CT imaging of well-differentiated HCC with CHOL, Act and FDG
...... 136
Figure 4.3 PET imaging of a woodchuck model of HCC with [11C]-CHOL and [11C]-Act . 138
Figure 4.4 Histology of well-differentiated HCC ...... 138
xiii
Figure 4.5 A woodchuck model of HCC that used for 11C-Choline metabolites study ...... 139
Figure 4.6 The distribution of C-14 activity between HCCs and surrounding hepatic tissues.
...... 141
Figure 4.7 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites in woodchuck model of HCC...... 142
Figure 4.8 Pattern of 14C-choline metabolites in the water- and lipid-soluble fraction from woodchuck model of HCC...... 143
Figure 4.9 Pulse and chase study in WCH17 cells using L-[methyl-14C]Methionine...... 148
Figure 4.10 Choline kinase activity in woodchuck model of hepatocellular carcinoma...... 149
Figure 4.11 Activation of CTP:phosphocholine cytidylyltransferase by 1mM oleic acid. .. 150
Figure 4.12 Effect of oleic acid on the phosphatidylcholine synthesis...... 152
Figure 4.13 Hypothetical mechanism for the down-regulation of the gene for CCT as a result of over expression of PEMT2 [221]...... 161
Figure 5.14 Comparison of metabolic pathway of choline and ethanolamine...... 172
xiv
Acknowledgments
I would like to express my most sincere thanks and gratitude to my advisor, Dr. Zhenghong
Lee, for his excellent guidance and generous support throughout my Ph.D. study. I would also like to thank Dr. David Wilson, Dr. Xin Yu and Dr. Thomas Kelley for their knowledgeable advice and kindness to serve on my Ph.D. thesis guidance committee. My
Ph.D. study would have never been completed without their supervision. It is a rare privilege and a great honor for me to work with them. Both their professional advice and plentiful experience help me to further improve myself.
I also acknowledge my labmates for their advice, suggestion, support and friendship, in particular, Nick Salem, Dr. Haibin Tian, Fangjing Wang, Jeff Kolthammer, David Corn, Joe
Molter. I benefited greatly from the detailed discussions with them. In addition, I wish to thank Dr. Chunying Wu for helping the tracer synthesis.
I am also grateful to Dr. Bernard Landau, Dr. Visvanathan Chandramouli, Dr. Bill Schumann,
Dr. Ann-Marie Broome and Ms. Irene Panagopoulos. They have provided very useful discussions and laboratory techniques. Thanks also go to Dr. Jim Basilion, for allowing us to use his excellent lab resources. I would also like to thank Mr. Steve Schomisch, Dr. Judy Zu
Jin, Ms. Bernadette Erowku, Ms. Rachel Moore, Ms. Megan Pope, Ms. Angie Estok for their help on the animal surgery.
Last but not least, I would like to especially thank my parents. Without their continuing support and encouragement, this study would never have been possible. xv
List of Abbreviations
1,2-DAG 1,2-diacylglycerol
2D two-dimensional
3D three-dimensional
Act Acetate
AFP α-fetoprotein
APF acid precipitable fraction
ASF acid soluble fraction
BLI bioluminescence imaging
CCT CTP:phosphocholine cytidylyltransferase
CDP-Cho CDP-choline
ChoK Choline kinase
CHOL choline
CT computed tomography
DMEM Dulbecco's Modified Eagle's Medium
DOPC dioleoyl phosphatidylcholine
DOPE dioleoyl phosphatidylethanolamin
FBP filtered backprojection
FBS Fetal bovine serum
FCH [18F]-fluorocholine
FDG 2-Deoxy-2-[18F]-fluoro-D-glucose
FI fluorescence Imaging
GLUT glucose transporter
xvi
HBV Hepatitis B virus
HCC hepatocellular carcinoma
HCV Hepatitis C virus
Met Methionine miR-21 microRNA 21 miRNAs micro RNA
ML-EM Maximum-likelihood expectation-maximization
MRI magnetic resonance imaging
MRS Magnetic Resonance Spectroscopy
MRS Magnetic resonance spectroscopy
P.I. post injection
PC Phosphoatidylcholine
Pcho phosphocholine
PE phosphoethanolamine
PEMT phosphatidylethanolamine-N-methyltransferase
PET positron emission tomography
PSRF point source response function
SAM S-adenosylmethionine
SPECT Single photon emission computed tomography
SREBP sterol regulatory element binding protein
SUV standard uptake value
US ultrasound
WHV woodchuck hepatitis virus
xvii
Positron Emission Tomography Imaging of
Hepatocellular Carcinoma with Radiolabeled Choline
Abstract
by
YU KUANG
Hepatocellular Carcinoma (HCC) is one of the most common malignancies throughout the world and its five-year survival rate has been dismal (5%). The carcinogenesis is frequently associated with the metabolic changes that precede the morphological changes. Therefore, a non-invasive, fast, quantitative technique for detection of HCC is much needed. Positron emission tomography (PET), a molecular imaging technique, holds particular promise for diagnostic imaging of neoplasms. The focus of this thesis is on the diagnostic utility of PET metabolic imaging on HCC and the mechanisms underlying the imaging using radiolabeled choline (CHOL) as the tracer. (1) Many cancers display a high rate of aerobic glycolysis, a phenomenon that is exploited by 2-Deoxy-2-[18F]-fluoro-D-glucose (FDG) PET imaging for the detection of tumors. Up-regulation of glycolytic metabolism plays a role in tumor progression by contributing to tumor growth or survival. In this study, the usefulness of
FDG-PET for HCC was investigated. The study addressed the correlation between FDG-PET images with pathologic types of HCC. The overall sensitivity of FDG-PET in the detection of
HCC is low (50-55%). This can be explained by the wide variability in enzyme activity in the individual HCC. In well-differentiated HCC, FDG metabolism may be similar to that of the surrounding liver, leading to a false negative result, while higher sensitivity was reported in poorly differentiated HCC. (2) Increased lipid synthesis is required by a growing tumor cell
xviii
to synthesize membranes and lipid-modified signaling molecules. The radiolabeled choline
(CHOL) was used to probe lipid synthesis in HCC. In this study, PET/CT imaging was correlated with metabolites analysis in vivo and in vitro, which helps to explain the heterogenous uptake of radiolabeled CHOL in HCC. Transport and phosphorylation of
CHOL are responsible for the tracer accumulation during [11C]-CHOL PET imaging in well- differentiated HCC. Moreover, Basal oxidation and phosphorylation activities in surrounding hepatic tissue contribute to the background signal seen in [11C]-CHOL PET images.
Furthermore, PET imaging of lipid synthesis with radiolabeled CHOL is useful in well- differentiated HCC that is not FDG avid. PET/CT imaging with radiolabeled CHOL could thus be a very promising diagnostic tool in patients with suspicious liver masses.
xix
Chapter 1 Introduction
1.1 Molecular Imaging of Cancer
1.1.1 Positron emission tomography for cancer imaging
The last two decades have witnessed tremendous advances and the extraordinary innovation in medical imaging and its clinical significance in diagnosis and treatment of diseases. The medical imaging is often thought of visualization of tissue characteristics, which traditionally focused on the acquisition of structural (anatomic) and functional (physiologic) information about patients at the organ and tissue levels [1]. Indeed, X-ray computed tomography (CT) and magnetic resonance imaging (MRI) can yield exquisitely detailed images of morphological changes which generally occur at the advanced stages of malignant tumors.
Thus, the radical intervention is required and the effectiveness of treatment is compromised.
In many of these cases, detection and diagnosis at an earlier stage in the progression of the cancer would improve the effectiveness of treatment and enhance the well-being of patients.
On the other hand, the molecular and metabolic changes precede structural and morphological changes in all forms of human diseases. Therefore, the objective of early detection of cancer demands that medical imaging expand its focus from the organ and tissue levels to the cellular and molecular levels.
Now things are changing, as we become increasingly able to characterize lesions by their molecular and metabolic characteristics by the use of molecular imaging technique. The term molecular imaging can be broadly defined as the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms [2, 3]. In contrast to “classical” diagnostic imaging, it sets forth to probe the
1
molecular abnormalities that are the basis of disease rather than to image the end effects of these molecular alterations [2].
Molecular imaging has its roots in nuclear medicine and in many ways is a direct extension of this existing discipline [2]. The main developing area of Nuclear Medicine with many applications and great potential is especially positron emission tomography (PET), which is the leading modality for imaging physiological, metabolic and molecular processes in vivo.
PET has a marked impact on cancer patient management by providing prognostic indications, improving tumor staging, detecting recurrences, identifying the primary tumor when secondary cancers are present, radiation treatment planning, and monitoring of tumor response to therapy [4]. Furthermore, PET is beginning to have an expanded role in therapy. The same radiopharmaceuticals for diagnostic imaging which target neoplasia can also carry high amounts of radioactivity to cancer cells and thus selectively deliver a lethal irradiation dose to the tumor [5].
Through quantitatively extracting pathophysiological information from the complex dynamic processes such as carcinogenesis, PET imaging is helping to establish the diagnosis at a much earlier stage of cancer for the clinical setting. Today, by combining new PET imaging probes with PET/CT scanner, PET imaging makes it possible to perform molecular imaging assays simultaneously with anatomic analyses for cancers. Information derived from structural studies and from noninvasive, dynamic monitoring of radiotracer distribution and concentration in the tumor can then be correlated with abnormal biological effects of signal transduction pathways, target enzyme activities, antigen levels, receptor activation, cell
2
proliferation, proteasome activity, etc [6]. These noninvasive molecular imaging using PET will permit real-time monitoring and modification of targeted interventions and therapeutic strategies of cancers.
1.1.2 Molecular imaging modalities
Molecular imaging encompasses a new imaging paradigm that includes multiple image modalities, cell and molecular biology, chemistry, pharmacology, medical physics, biomathematics, and bioinformatics. As conventional imaging techniques, X-ray CT, MRI and ultrasound (US) primarily rely on physical parameters such as absorption, scattering, proton density, and relaxation rates as the primary source of contrast for imaging [7]. These conventional imaging techniques focus mostly on nonspecific macroscopic physical, physiological changes that differentiate pathological from normal tissue rather than identifying specific molecular or metabolic events responsible for disease. In contrast, the major molecular imaging modalities comprise optical imaging, MRI and spectroscopy, PET and Single photon emission computed tomography (SPECT) etc. Molecular imaging techniques exploit specific molecular probes as the source of image contrast to identify the up-regulated specific molecular or metabolic event in the diseases. This change in emphasis from a nonspecific to a specific approach represents a significant paradigm shift, the impact of which is that imaging can now provide the potential for understanding of integrative biology, earlier detection and characterization of disease, and evaluation of treatment [2].
By choosing the suitable modalities, we can obtain temporal and spatial information of normal as well as abnormal cellular, genetic, molecular and biochemical events in vivo. The
3
prevailing modalities for molecular imaging were summarized in Table 1.1. [2, 7-9]. As one of the forerunners of molecular imaging techniques, PET has the advantages of high intrinsic sensitivity, unlimited depth penetration, and a broad range of clinically available molecular imaging probes. However, a limitation of PET imaging is its requirement for a cyclotron to generate imaging tracers. PET also has a lower resolution (>1 mm for microPET; ~4-5 mm for clinical PET) compared with other molecular imaging modalities. This constraint has been addressed by the introduction of PET/CT and PET/MR combined scanners. These fusion systems integrate the lower resolution molecular information from PET with higher resolution anatomical detail from CT or MR. They will provide a significant improvement in the diagnosis and staging of malignant tumors and for identifying and localizing metastases.
One of the potential benefits of molecular imaging is to earlier and specific detection of cancers [7]. Traditional imaging methods often lack the necessary sensitivity and specificity for early diagnoses of many cancers and for the detection of subcentimeter neoplasms and preneoplastic disease. Thus, a compelling need exists for more sensitive and specific detection of early malignancies. Many of the molecular imaging probes possess high avidity and specificity for targeted biomarkers of cancer [7]. This allows the possibility of earlier disease detection than is possible by anatomical or conventional contrast-enhanced imaging techniques.
4
Table 1.1 Characteristics of molecular imaging modalities.
(Modified from [2, 7-9]) Portion of EM radiation Temporal Imaging Spatial spectrum used resolution Sensitivity‡ Depth Advantage Disadvantage Modality Resolution* in image † generation Positron High energy ≥ 1mm (micro 10 second Picomolar range No High Low spatial emission rays PET); ~4-5mm to minutes limit sensitivity, resolution, tomography (clinical PET) short-lived cyclotron (PET) isotopes required for generating some isotopes Single Low energy ≥1mm Minutes Picomolar range No High Low spatial photon rays (microSPECT); limit sensitivity resolution,
5 emission ≥3 mm long-lived computed (clinical isotopes tomography SPECT) (SPECT)
Optical Visible light 3-5mm Seconds to ≥1000 cells 1-2cm High Restricted bioluminesce minutes sensitivity, depth detection nce quick, relative imaging high-through (BLI) put
Optical Visible light or 2-3mm Seconds to Nanomolar range: <1cm High Restricted fluorescence near-infrared minutes ≥50 cells sensitivity, depth detection Imaging (FI) detects fluorochrome in live and
5
dead cells Magnetic Radiowaves 4 µm Minutes to T2-contrast, iron No High spatial Particle size of resonance (experimental hours oxide nano-particles: limit resolution contrast agent, imaging MRI), nanomolar– reporter is (MRI) 250 µm in micromolar range; relative large, plane (clinical T1-contrast, which restricts MRI) multilabeled targeted in vivo Gd-DTPA delivery macromolecules: >10 µM Magnetic Radiowaves ≥0.5 cm (3 Minutes to Millimolar range (1H No Detection of Low sensitivity resonance Tesla), 0.7 cm hours at 4.7–11 Tesla) limit endogenous results in low Spectroscopy (1.5 Tesla) metabolites spatial (MRS) resolution
Computed X-rays >10 µm Minutes 500µmolar (Gd- No High spatial Patients are tomography DTPA) - low mmolar limit resolution exposed to 6 (CT) (Iodine) range radiation
Ultrasound( High- >40 µm Seconds to >106 microbubbles mm to High spatial Few probes US) frequency minutes per ml blood cm resolution, cost available sound effective
*Spatial resolution is a measure of the accuracy or detail of graphic display in the images expressed in millimeters. It is the minimum distance between two independently measured objects that can be distinguished separately [2]. It is a measure of how fine the image is. †Temporal resolution is the frequency at which the final interpretable version of images can be recorded/captured from the subject once the imaging process is initiated [2]. This relates to the time required to collect enough events to form an image, and to the responsiveness of the imaging system to rates of any change induced by the operator or in the biological system at hand. ‡Sensitivity, the ability to detect a molecular probe when it is present, relative to the background, measured in moles per liter [2].
6
1.1.3 Positron emission tomography imaging for hepatocellular carcinoma
As a molecular imaging technique, PET provides unique possibilities for the direct quantification of regional blood perfusion of organs and metabolism of substances in the tumor in vivo. However, PET imaging on the early detection of hepatocellular carcinoma
(HCC) is still evolving in spite of the fact that HCC is a major clinical problem that carries a considerable risk of death. The liver is the largest organ of the human body, which is normally metabolically active. Liver performs over 500 metabolic functions, resulting in synthesis of products that are released into the blood stream (e.g. glucose derived from glycogenesis, plasma proteins, clotting factors and urea), or that are excreted to the intestinal tract (bile); Also, several products are stored in liver parenchyma (e.g. glycogen, fat and fat soluble vitamins) [10].
As the predominant type of primary liver cancer, HCC has seen rapid increase in its incidence over the last 10 years in U.S.A. This increase is caused, in part, by the epidemic of hepatitis viral infections, which can lead to HCC. The incidence and death rates are similar indicating the overall poor survival rate of HCC. Therefore, strategies to improve the accuracy in early detection of HCC, by way of molecular imaging, are of paramount importance.
Imaging has had a central role in the diagnosis of HCC. A major challenge is to differentiate
HCC from coexisting regenerative and dysplastic nodules and detect HCC at an early stage.
To add to the challenge is the stepwise development of HCC in persons with cirrhosis or hepatic viral infection. This development starts with regenerative nodules and progresses to
7
low grade dysplastic nodules, high grade dysplastic nodules, well-differentiated HCC and poorly-differentiated HCC [11]. The use of PET for the studying of liver diseases remains on cancer detection. My thesis described the development of state-of-the-art PET imaging technique to delineate HCC from surrounding hepatic tissue through targeting the characteristic changes in tumor metabolism. Toward this goal I have the following specific aims:
(1) To investigate the usefulness of PET imaging with 2-Deoxy-2-[18F]-fluoro-D-glucose
(FDG) for detection of HCC.
(2) To characterize the transport mechanism and metabolic fate of radiolabeled choline
(CHOL) in HCC in vitro.
(3) To investigate the utility of PET imaging with [methyl-11C]-CHOL for detection of
HCC and correlate with the metabolites study in vivo.
1.2 Basic Principle of Positron Emission Tomography
1.2.1 Positron emission and annihilation
PET is a nuclear imaging technique, in which tracer compounds labeled with positron- emitting radionuclides are injected into the subject of the study. These tracers can then be used to track biochemical and physiological processes in vivo. One of the prime reasons for the importance of PET in medical research and practice is the existence of positron-emitting isotopes of elements such as carbon, nitrogen, oxygen and fluorine which can be processed to create a range of radioactive compounds similar to naturally occurring substances in the body.
8
PET tracers decay by positron emission. They must be manufactured using a charged particle accelerator (cyclotron) or a generator system. A positron is an antielectron, which is a positively charged electron. Positron emitters (11C, 13N, I8F, 15O, etc.) are neutron-deficient atoms. Positron emission occurs when positron-emitters undergo nuclear transformation by converting a proton into a neutron (n), a positron (β+) and a neutrino (υ).
(Eq. 1.1)
The emitted positron (β+) travels through surrounding matter (tissues) in a range of a few millimeters depending on its energy (varies from 0-1.7 MeV) [12] and material of the surrounding matter until it has lost its energy in collisions with electrons of other atoms. The distance traveled by the positron, called positron range, depends on the initial kinetic energy as well as the density and atomic number of the surrounding matter.
After loss of the energy, the positron annihilates with an electron, yielding two photons with energy of 511 KeV each (annihilation photons) that leave the annihilation site in an antiparallel direction (Figure 1.1). Annihilation photon pairs have a chance of escaping the body and are targeted for detection in PET.
Because the positron and electron usually have some residual kinetic energy at the time of annihilation, the direction traveled by the two photons is not exactly 180° apart. The angular distribution can be described by a Gaussian distribution with a full width at half maximum (FWHM) of approximately 0.5° [13, 14]. In addition,
Positron range is one factor contributing to the limited spatial resolution in PET.
9
The half-life, average positron kinetic energy, spatial resolution effects, and method of production for several radionuclides used in PET are listed in Table 1.2 [15, 16].
Figure 1.1 A positron-emitting radionuclide decays by transforming a proton into a neutron, neutrino (v), and positron (e+). The positron travels a short distance in the surrounding matter and interacts with an electron, resulting in annihilation of both particles and formation of two anti-parallel 511 keV gamma () photons. These annihilation photons are targeted for detection in PET and provide information about the distribution of the radiolabeled tracer molecule administered to the patient.
1.2.2 511 keV photon interactions in matter
1.2.2.1 Compton scattering
Ideally, annihilation photons would travel uninterrupted from the source to the detector; however, their course is often impeded by photon interactions with matter.
The most important interaction in tissue is Compton scattering. The 511-KeV photons scatters off a free or loosely electron in the medium, transferring some of its energy to the electron and changing direction in the process (Figure 1.2 (A)). This results in a lower energy photon and the emission of a high energy electron.
10
Table 1.2 Common PET Radionuclides [15, 16]
Average positron Spatial Resolution* Method of Radionuclide Half-life energy (keV) FWHM (mm) Production 11C 20.38 min 386 1.88 Cyclotron 13N 9.965 min 492 2.06 Cyclotron 15O 123 sec 735 2.50 Cyclotron 18F 109.77 min 250 1.67 Cyclotron 61Cu 3.35 hr 500 2.08 Cyclotron 62Cu 9.74 min 1314 3.89 Generator 64Cu 12.701 hr 278 1.71 Cyclotron 68Ga 68.3 min 830 2.74 Generator 76Br 16.1 hr 1180 2.52 Cyclotron 82Rb 78 sec 1479 4.29 Generator 124I 4.18 days 820 2.70 Cyclotron *Gaussian full width half maximum (FWHM) for a hypothetical imaging system with intrinsic spatial resolution of 1.5 mm FWHM.
The incident photon (energy E0) transfers energy to the electron, which recoils, and a lower energy photon (Escat,) is emitted in a different direction [17]:
(E.q. 1.2) where mc is the rest energy of an electron, and θ is the scattering angle. The angular distribution of scattered photons depends on the energy of the incident photons and can be described by the Klein-Nishina formula [18, 19]. Compton scattering contributes to the process of attenuation in PET and may also result in detection of scattered photons that provide incorrect information about the location of the radioactive source (Figure 1.2).
11
1.2.2.2 Rayleigh scattering
Rayleigh scattering is an interaction between a photon and an atom as a whole.
Since the mass of the atom is relatively large it absorbs very little energy, and the photon is essentially deflected with no change in energy. Rayleigh scattering occurs primarily at relatively low energies in tissues and infrequently effects 511 keV photons [20].
1.2.2.3 Photoelectric effect
Photoelectric effect, or photoelectric absorption, is an interaction where the energy of an incident photon is absorbed by an inner shell electron [21]. This electron is given enough energy to escape the atom but is quickly absorbed in solids and liquids. An x- ray with an energy equal to the binding energy of the electron is also generated as the vacancy in the electron shell of the atom is filled (Figure 1.2(B)). Like Rayleigh scattering, photoelectric effect has a negligible effect on 511 keV photons in tissue.
A B
Figure 1.2 Annihilation photons may interact with matter by (A) Compton scattering, (B) photoelectric effect scattering.
12
Compton scattering is the main interaction occurring in tissues that results in a reduction in the number of coincidence events that are detected. Compton scattering results in a change in direction and loss of energy for the incoming photon, while the photoelectric effect results in photon absorption.
1.2.3 Data collection
1.2.3.1 Coincidence detection
Annihilation photons can be detected using scintillation crystals surrounding the patient coupled to light-sensitive photomultiplier tubes (PMT) and coincidence detection electronics.
The simultaneous pulses from the detectors indicate that an annihilation occurred somewhere along the path between the detectors. This is because the photons leave the annihilation point in opposite directions. The path between two detectors is referred to as a line of response
(LOR). The simultaneous detection of two photons is referred to as a coincidence. The number of coincidence events occurring between detectors indicates how much radioactivity there was on the LOR between the detectors.
Collimators are not required in PET; instead, near-simultaneous detection of the anti-parallel photon pairs provides information about the location of the annihilation event. A coincidence event is assigned to LOR joining the two relevant detectors. In this way, positional information is gained from the detected radiation without the need for a physical collimator. This is known as electronic collimation. Electronic collimation has two
13
major advantages over physical collimation. These are improved sensitivity and improved uniformity of the point source response function (PSRF).
1.2.3.2 Electronic timing window
PET requires detectors and coincidence detection electronics with timing resolution on the order of nanoseconds, allowing coincidence events to be selected from the broader background of all single photons hitting the detector. All PET scanners require an electronic timing window for coincidence detection, which is typically 6-
12 nanoseconds in duration [22, 23]. This allows enough time for the different distances traveled by the photons, signal generation through scintillators and PMT tubes, and signal transit through cables and electronics. Typically, the electronic timing window limits the precision of spatial positioning information for each coincidence event to any point in the sensitive volume of space located between the two detectors involved in the measurement.
1.2.3.3 Time-of-flight
An ideal time-of-flight PET scanner would have perfect timing resolution (Figure
1.3), allowing the exact position of the annihilation event along the line between the two detectors to be determined using the formula [24-27] :
∆ (E.q. 1.2) where s is the position relative to the midpoint between the detectors, c is the speed of light, and ∆t is the difference in detection times for each photon. Unless the event occurs in the exact center of the detection ring, one of the photons will arrive before the other.
14
The time difference will be proportional to the difference in distances traveled by the two photons and can be used to calculate the position of the event along the line connecting the detectors. Current detector technology can provide timing resolutions of around 300-
500 picoseconds, which gives the ability to incorporate some partial time-of-flight information to improve image reconstruction [27, 28].
Figure 1.3 Time-of flight PET.
1.2.4 Detectors and scanner design
1.2.4.1 Scintillation crystals
The scintillation process involves the conversion of high-energy photons (e.g. 511KeV) into visible light via interaction with scintillation materials (crystals) [23, 28]. A photon incident on the scintillator creates an energetic electron, either by Compton scatter or by photoelectric absorption. As the electron passes through the scintillator, it loses energy and excites other electrons in the process. These excited electrons decay back to their ground state and give off light.
15
Some examples of scintillation materials used in PET include sodium iodide (NaI), bismuth-germanate (BGO). gadolinium-oxyorthosilicate (GSO), and lutetium- oxyorthosilicate (LSO).
Crystals should have high stopping power for 511 keV photons, high light output, and fast temporal response [29]. Stopping power refers to the crystal’s ability to absorb the energy of incoming photons, as opposed to letting them to pass through undetected. Higher stopping power improves detection efficiency and spatial resolution due to reduced depth-of-interaction. Light output refers to the number, or flux, of light photons emitted by the crystal upon absorbing the energy of an incoming photon. Higher light output allows the position of photon interaction with the crystal and the energy of the incident photon to measure more accurately. Fast temporal response is important for several reasons. The rise time, or delay between photon interaction and light output, is essential for determining the timing of detected events. Crystals with faster rise times allow shorter coincidence timing windows to be used, which reduces random coincidences and may provide useful time-of-flight information [29].
When real scintillation detectors are exposed to mono-energetic photons, the energy measured is not that of the electron generated by the initial interaction, but rather the total energy deposited by the photon in the detector [30]. This distinction is important because photons initially interacting by Compton scatter may subsequently be involved in further interactions within the detector. In a sufficiently large detector, most Compton-scattered
16
photons will eventually deposit all their energy and most events will register in the photon energy peak [30]. Consequently, the energy spectrum will have a full energy-peak and a lower energy Compton scatter plateau as shown in Figure 1.4.
Figure 1.4 Typical energy distribution measured by a scintillation detector system exposed to gamma rays
The energy resolution of the detector is defined as the ratio of the full-width at half- maximum (FWHM) of the full energy peak to its value. In the case of thallium doped sodium iodide NaI(Tl) at 511 keV gamma ray energy, 18% of the events interact by photoelectric effect. The cross-section for photo-electric absorption is proportional to Z5 whereas for
Compton scattering it is proportional to Z. Scintillators with higher atomic numbers (Z) therefore have better energy resolution [30, 31].
1.2.4.2 Dead time
Interaction between the incident photon and the scintillation crystal generates light flashes, which are converted to electronic pulses that are recorded by the electronic device. During
17
the time from interaction until recording, detector(s) are paralyzed implying that during this short period of time the detector is not able to sense other photons, which is termed dead- time [32]. This results in a loss of valid events called dead time losses and must be corrected for to obtain an estimate of the true count rate. Dead-time limits the counting rate of the tomograph depending on the amount of activity in the Field of View (FOV) and applied acquisition mode.
1.2.4.3 PET/CT
PET scanners have been combined with X-ray CT scanners to create dual-modality systems which can provide co-registered images of physiology and anatomy in a short period of time [33]. This dual-modality approach offers potential advantages for applications such as lesion localization, radiotherapy treatment planning, and improved diagnostic performance. Measurements of PET attenuation factors can be made from lower energy X-ray photons, but these values need to be converted into the appropriate units for 511 keV photons.
1.2.4.4 Collimation
Physical collimation is not necessary in PET because positioning information is obtained by coincidence detection. However, collimators may be employed to enhance the performance of PET scanners. Collimators in PET are often called interslice septa, and usually consist of parallel slats of high-attenuation material, such as lead or tungsten, positioned perpendicular to the axis of the detector [29-31].
The use of parallel interslice septa essentially creates a two-dimensional (2D)
18
imaging geometry where the coincidence events between detectors in different rings can be ignored. Each separate ring or slice of the scanner can then be treated independently for the purposes of image reconstruction. Fully three-dimensional
(3D) PET allows coincidence events from all possible detector pairs in the scanner for acquisition and reconstruction. Fully 3D PET has much higher sensitivity than
2D since the interslice septa are removed and more coincidence events are detected.
However, fully 3D PET has much higher levels of randoms and scatter than does 2D
PET. And the reconstruction problem in 3D is much more computationally challenging.
1.2.5 LORs and projection data
1.2.5.1 Lines of response (LORs)
The two annihilated photons emitted in opposite directions can be detected together to form a coincidence. Then, one can estimate the position of the annihilation event by drawing a line between the two detectors of the coincidence. When incident photons are detected within a short and predefined timing window (~10-12 ns, called true coincidence timing window)
[12], by a pair of detectors positioned along a line called Line of Response (LOR), a true coincidence is registered.
The size and sensitivity of LORs is nonuniform in dedicated PET scanners, due to geometric factors and variations in detection efficiency for each pair of crystals.
Consider the parallel LORs for one angle in a full-ring detector system; the LORs near the edges of the field of view are narrower and closer together because of the
19
curvature of the ring. Also, the sensitivity within the volume of an LOR is nonuniform, and depends on the solid angle subtended by lines passing through the point of interest and intersecting both detectors. The detection efficiency is slightly higher for annihilation events occurring near the center of the LOR as compared to events close to either detector. This effect contributes to nonuniform spatial resolution in PET [29]. PET data are basically a record of the number of coincidence events detected along each LOR during the scan timeframe.
1.2.5.2 Organization of data
The coincidence is detected if each photon in the pair is detected within the coincidence timing window of the detector (usually a few nanoseconds). The information about the coincidences is processed electronically and is recorded in the form of a sinogram or list mode data.
A sinogram gives the signal intensity for position versus angle, whereas list mode data is simply a list indicating which detectors were involved in detecting each coincidence [31].
This data is then used to reconstruct the image. Reconstruction involves summing up all these lines of response (LOR) to produce the image.
All LORs, which are parallel at a definite angle through the object, form a single projection view that is stored as a single row in a so called sinogram. Projections obtained from all parallel LORs from different angles through the object are stored similarly but into different rows in the sinogram. Generated sinograms are referred to as raw data in PET, and are used
20
for further processing and reconstruction. Detector systems in modern scanners consist of thousands of detectors which allow the system to store millions of LORs in sequences of sinograms. On the contrary, in list mode, each coincidence event is recorded individually, with information of the LOR and the time of occurrence. List mode data occupy more memory but provide information of the time. They can be histogramed to form sinogram data
[29, 31].
1.2.5.3 Projections and sinograms
After all corrections (e.g. for scatter, randoms and the effects of attenuation) have been applied to data acquired in a PET camera, the number of counts assigned to an LOR joining a pair of detectors is proportional to a line integral of the activity along that LOR. Parallel sets of such line integrals are known as projections [34, 35]. PET data are usually grouped into parallel sets of LORs prior to reconstruction, although in some cases reconstruction is performed directly from LORs. PET data can be viewed prior to reconstruction by forming sinograms. In the sinogram, which is a matrix that can be displayed as an image, the first row of pixels represents the number of counts at a single angle. The first row typically represents the angle made from vertical LORs. The next row represents the next angle, which is only slightly different. The row halfway down the sinogram represents the horizontal LORs, and the last row represents the lines almost 180° from the starting lines. Unlike SPECT imaging, in which the LORs are different if measured with the camera below the patient than with the camera 180° around at the top of the patient
(because of collimator distance-dependence, attenuation, and scatter effects), all the information in a PET scan can be represented by a 180° angular range.
21
1.2.6 Image reconstruction
Reconstruction is the process of creating transaxial slices from projection views. There are two basic approaches to creating the transaxial slices, i.e., filtered Backprojection and iterative reconstruction.
1.2.6.1 Forward problem
Imaging can be considered as a forward problem [29]. The forward problem can be written as a linear system of equations
(E.q 1.3)
where x is a vector of N image voxel values, p is a vector of M projection bin values, and F is an M x V system matrix or transfer matrix. The transfer matrix F describes how the projection measurements p are related to the image x, i.e., Fij is the probability that a positron emission from voxel i will result in a prompt coincidence detection in projection bin j. Ideally, F completely describes all aspects of image acquisition including the effects of positron range, noncollinearity, detector response, dead time losses, scanner geometry, attenuation, scatter, randoms, etc. In other words, the objective of F is to simulate the PET measurement.
1.2.6.2 Radon transform
In its simplest form, F describes the Radon transform, which is a set of integrals along all lines through the source activity. The Radon Transform was first introduced by
22
J. Radon in 1917 [29]. For example in 2D reconstruction, if lines of response are represented using
(E.q 1.4) where s is the shortest distance from the LOR to the origin and is the angle the line makes with the y-axis, then the Radon transform can be written:
∞ ∞ (E.q 1.5) , ∞ ∞ , where f(x,y) is proportional to the source radioactivity concentration for PET.
1.2.6.3 Analytical reconstruction
1.2.6.3.1 Simple backprojection
Analytical reconstruction algorithms estimate the image from the measured data by inverting the transfer matrix using analytical mathematics [30]. First, an image matrix is defined (typically, 128 X 128 pixels for PET). For a valid line of response, a line is drawn between the detectors and through the image matrix. The value added to each pixel that is intersected by the line is given by N X w, where N is the number of counts detected by the detector pair (after all corrections described in Data Correction) and w is a weighting factor proportional to the path-length of the line through the pixel. The value is therefore larger if the line passes across the center of the pixel and smaller if the line passes through the corner of the pixel. In essence, the counts from a detector pair are being projected back along the line from which they originated. This process is repeated for all valid detector pairs in the
PET system, the counts from each subsequent detector pair being added to the counts that have been backprojected for all preceding detector pairs, hence the name linear superposition of backprojections. 23
1.2.6.3.2 Filtered backprojection (FBP)
Filtered backprojection reconstruction (FBP) methods were originally developed for solving gravitational equations by Radon in 1917 [29]. In FBP, projection data are filtered prior to backprojection in order to remove the radial blurring effect observed using simple backprojection. The filtering process amplifies high frequency components of the projection data, and can be implemented in the spatial domain by a convolution or in the Fourier domain by multiplying by a ramp filter. The filtered projection data are then backprojected to complete the reconstructed. The FBP algorithm is fast, easy to apply, well established, and works well with X-ray CT and transmission scan data. However, FBP amplifies the high frequency noise in PET images, commonly results in streak-like artifacts, and is not well suited for compensating for image degrading factors [29, 30].
1.2.6.4 Iterative reconstruction
1.2.6.4.1 Five basic steps
Iterative reconstruction is the other major class of reconstruction methods used in
PET [36]. Iterative reconstruction can be considered to include five basic steps: 1) estimate the image. An initial guess is made of the image distribution (often a blank or uniform grayscale image). 2) simulate a measurement, i.e. to calculate what projection data would be measured for the radioactivity distribution in the initial guess. 3) compare the simulated data to the actual measured data, 4) based on the differences between simulated and measured data (projections), the initial guess is then adjusted, and update the
24
image estimate, and 5) repeat steps 2-4, until the simulated data agree with the measured data within a desired accuracy [29]. If the method by which the image estimate is updated is properly formulated, then with each successive iteration through this process, the image estimate will start to converge towards the true image.
Many types of iterative reconstruction have been developed, which accomplish steps
2-4 in different ways. The most important difference between them is usually the criterion, or objective function, which is minimized or maximized during the reconstruction process. Examples of different criteria include maximum-likelihood
(ML), maximum entropy, and weighted least squares. Algorithm generally refers to the mathematical recipe used to move the image estimate in the direction specified by the criteria. The main advantages of iterative reconstruction are that it can accurately model the physics of the measurement process and the Poisson statistics of the measured data [29].
1.2.6.4.2 Maximum-likelihood expectation-maximization
Maximum-likelihood expectation-maximization (ML-EM) is one of the most widely accepted iterative reconstruction methods in PET [29, 37].
In ML-EM, the expectation-maximization (EM) algorithm is used to maximize the posterior probability of the reconstructed image given the measured data. According to Poisson (counting) statistics, where the variance of a distribution is equal to the
25
mean, the probability of obtaining a certain value, n(d), for a non-negative integer random variable sampled over D (d = 1 to D) independent trials can be written:
| ∏ (E.q 1.6) ! given that the mean (expected value) for each trial is λ(d). Using the notation introduced in E.q 1.3 to represent the forward imaging problem, the Poisson equation can be applied to compute the probability of obtaining the actual measured data in projection bin j, given the current image estimate, [29, 37]:
(E.q 1.7) ! probability of obtaining the entire set of projection data can be written:
| ∏ (E.q 1.8) !
By applying the expectation-maximization algorithm to this equation [29, 38] , the following ML-EM update equation can be derived:
∑ (E.q 1.9) ∑ ∑
26
where N is the number of voxels, M is the number of projection bins, is the
current estimate of the value of voxel i, and is the updated estimate. MLEM reconstruction is based on the Poisson statistics of the data, converges to the maximum-likelihood solution, provides non-negative image estimates, and maintains the same number of counts in the simulated data as in the measured data after each iteration. However, one limitation of using MLEM is that it takes a long time to converge. Different methods to speed up convergence of MLEM have been developed, such as ordered-subsets expectation-maximization (OSEM) [29, 39].
OSEM is similar to MLEM, but performs subiterations using subsets of the projection data.
1.2.7 Attenuation correction
Attenuation refers to a reduction in the number of true coincidences detected due to photon interactions with matter, primarily by Compton scattering. The probability of photon attenuation depends on the energy of the incident photons and the composition and thickness of the materials through which the photons pass [29].
Attenuation causes a major increase in statistical noise, and leads to incorrect quantification if not corrected for during image reconstruction. Even when attenuation correction is performed, small errors in the estimated attenuation factors can translate to large errors in quantitation. Attenuation can also lead to data inconsistencies and artifacts in reconstructed images [29].
27
Attenuation correction factors are usually measured by illuminating the FOV with circular or rotating rod sources with the subject in the field of view. Sources containing quite large amounts of activity can be used to speed up the process, and scatter can be minimised by a technique called rod windowing, whereby only LORs passing through the rod source are used for the transmission measurement [30]. In certain cases it may be possible dispense with the measurement by using a calculated attenuation correction [30], or to improve it by reconstructing the attenuation data and segmenting it into regions with similar linear attenuation factors. This segmented image may then be reprojected to obtain the attenuation correction factors for each LOR [30].
1.2.8 Scatter and randoms
1.2.8.1 Coincidence events
Coincidence events in PET include four categories: true, scattered, random and multiple
(Figure 1.5) [30].
True coincidences occur when both photons from an annihilation event are detected by detectors in coincidence, neither photon undergoes any form of interaction prior to detection, and no other event is detected within the coincidence time-window (Figure 1.5 (A)).
A scattered coincidence is one in which at least one of the detected photons has undergone at least one Compton scattering event prior to detection (Figure 1.5 (B)). Since the direction of the photon is changed during the Compton scattering process, it is highly likely that the resulting coincidence event will be assigned to the wrong LOR. Scattered coincidences add a background to the true coincidence distribution which changes slowly with position,
28
decreasing contrast and causing the isotope concentrations to be overestimated [30, 31]. They also add statistical noise to the signal. The number of scattered events detected depends on the volume and attenuation characteristics of the object being imaged, and on the geometry of the camera.
A B C
Figure 1.5 Three types of coincidences in a PET detector: (A) True, (B) Scatter, and (C)
Random.
Randoms occur whenever photons arising from different annihilation events are detected in the same timing window (Figure 1.5 (C)). For a given detector pair, the randoms counting rate is given by [40]
2 (E.q 1.10) where τ is the coincidence timing window, and st and sj are the single-photon event rates on the two detectors. The number of random coincidences in a given LOR is closely linked to the rate of single events measured by the detectors joined by that LOR and the rate of random coincidences increase roughly with the square of the activity in the FOV. As with scattered events, the number of random coincidences detected also depends on the volume and attenuation characteristics of the object being imaged, and on the geometry of the 29
camera. The distribution of random coincidences is fairly uniform across the FOV, and will cause isotope concentrations to be overestimated if not corrected for. Random coincidences also add statistical noise to the data.
1.2.8.2 Scatter Correction
Many schemes have been proposed for scatter correction in 3D mode [30]. These include convolution-subtraction techniques [41], Monte-Carlo modeling techniques [30], direct measurement techniques [42] and multiple energy window [43]. The methods in widest use to date are the Gaussian fit technique [30], and model-based scatter correction algorithms
[30].
1.2.8.3 Random Correction
Several methods have been developed to correct for randoms [29, 40], One common approach is to measure delayed coincidence events, or “delays”, which can be immediately subtracted from the measured prompts data or can be modeled as an additive effect during iterative reconstruction. Delays are counted when two single photons from different annihilation events are measured in two different timing windows spaced widely apart in time relative to the duration of the timing window.
The probability of detecting a delay is equal to that of detecting a random coincidence in the prompt timing window, so delays provide a direct estimate of the randoms. The randoms estimate can be subtracted from the prompts data prior to reconstruction, or modeled as an additive component during iterative reconstruction.
30
Another form of randoms, called multiples, occurs when three or more photons are detected in a timing window. These events are usually rejected.
1.3 Review of the literatures
1.3.1 Hepatitis B viral infection induced hepatocellular carcinoma
1.3.1.1 Chronic hepatitis B viral Infection and hepatocellular carcinoma
Worldwide, Hepatitis B virus (HBV) and hepatitis C virus (HCV) infect hundreds of millions of people and induce a spectrum of chronic liver diseases. HCC has become the most frequent cause of death in individuals persistently infected with HBV or HCV [44].
HBV is able to persist in the host and cause chronic hepatitis. Inflammation forms the pathogenetic basis of chronic hepatitis that can lead to nodular fibrosis, which can progress to cirrhosis and, eventually, HCC. It has been proposed that hepatitis viruses can cause HCC by a combination of two mechanisms: first by cell lysis and stimulation of mitosis, leading to an accumulation of events necessary for transformation, and second by an increase in chromosomal instability mediated by induced recombinogeneic protein(s) during chronic hepatitis [45]. HCC is a consequence of accumulative somatic mutations in the genome of virus-infected liver cells. It remains a challenge to provide a clear picture of the connection between HBV and HCC.
MicroRNAs (miRNAs) have recently been reported to be one kind of host genetic factors associated with the carcinogenic process of liver cancers. By analyzing the miRNA expression profiles of paired HCC and surrounding hepatic tissues, several miRNAs showed
31
abnormal expression patterns. The functional role of some miRNAs targeting specific oncogenes or tumour suppressor genes have been identified, such as let-7 family miRNAs targeting the Ras oncogene, microRNA 21 (miR-21) targeting the PTEN tumour suppressor gene and miR-122 targeting the cyclin G1 cell-cycle regulator [46]. Therefore, the involvement of cellular miRNAs in HBV-related hepatocarcinogenesis is highly implicated, either affecting the virus replication or the host carcinogenic process.
The replication strategy of HBV includes the reverse transcription of an RNA intermediate.
In a patient with chronic hepatitis, HBV DNA sequences have been shown to integrate into cellular DNA in HCC tissue and in non-tumorous tissue. In some cases, the HBV DNA can also be integrated during the early stages of infection. When HBV integrations occur, they produce a wide range of genetic changes within the host genome, including deletions, translocations, production of fusion transcripts and generalized genomic instability. Studies have shown that the integration of viral DNA is associated with deletions in portions of the host’s chromosomes. Many of these chromosomal segments contain known tumour suppressor genes such as p53, Rb, cyclin D1 and p16 [47]. It has been shown that among the different HBV genes, the HBx gene seems to play a more causal role in HBV-related HCC because it is the most commonly integrated viral gene [47]. Among the pathobiological effects of HBx are transcriptional coactivation of cellular and viral genes (by transcriptional alteration through modulation of RNA polymerases II and III); action as a cotranscription factor for the major histocompatibility complex, epidermal growth factor receptor and oncogenes like the c-myc, c-jun/fos or Ras-signalling pathway; decrease of nucleotide excision repair and interaction with the cellular DNA repair system; and deregulation of cell-
32
cycle checkpoint controls. These HBx-related effects provide many different ways as to how
HBV contributes to HCC development.
There are also several more direct interactions between HBx and p53 functions. HBx binds to p53 and suppresses a number of p53-dependent functions: p53 sequence-specific DNA- binding activity in vitro, p53-mediated transcriptional activation in vivo and p53 transcription. HBx is capable of blocking p53-mediated apoptosis. Additionally, by decreasing p53’s binding to XBP, HBx indirectly reduces nucleotide excision repair and
XBP functions as a basic transcription factor [48]. Therefore, it seems likely that the HBx gene plays a role in the initiation of liver tumour formation.
The truncated form of the pre-S2/S gene is another HBV gene product that has been found to have transactivational properties. Truncated pre-S2/S sequences are often found in HBV
DNA integration sites in HCC [49]. Specific activation of mitogen-activated protein kinases’ signalling by the truncated pre-S2/S protein has been shown to result in an activation of transcription factors such as AP-1 and nuclear factor kB. Furthermore, by activation of this signaling cascade, the pre-S2/S activators cause an increase in the proliferation rate of hepatocytes [50]. Cytologically, overproduction of HBV envelope proteins (pre-S2/S), particularly L and possibly M, results in their intracellular accumulation and may predispose the cell to stress, which in turn may lead to the development of HCC [50]. HBV envelope protein (pre-S2/S) mutants that overaccumulate envelope polypeptides within the cell have also been found to be associated with advancing liver disease and may in part be responsible for ground glass hepatocytes and perhaps even HCC lesions [51]. In addition, mutations of
33
the core promoter region of the virus have also been described in integrated HBV sequences
[51].
1.3.1.2 Woodchuck hepatitis virus infection induced woodchuck Model of hepatocellular carcinoma
My thesis used the eastern woodchuck (Marmota monax) as animal model for the study.
Woodchuck Model of HCC harbors a DNA virus - woodchuck hepatitis virus (WHV). WHV is a member of the family Hepadnaviridae, genus Orthohepadnavirus, of which human HBV is the prototype. Like HBV, WHV infects the liver and can cause acute and chronic hepatitis.
Furthermore, chronic WHV infection in woodchucks usually leads to development of HCC within the first 2–4 years of life.
The WHV viral infection-induced HCC in the woodchucks is regarded as a natural occurring animal model of human HCC with similar pathology and natural history [52]. Analogous to
HCC tumors from human HBV carriers, WHV sequences have been demonstrated to be integrated into the host genome of woodchucks with HCC [53]. It has been shown that the hepatocarcinogenesis associated with WHV infection is related with the myc family of transcription factors. While the functional significance of increased expression of myc gene products is still unclear, there exists much evidence that up-regulation of N-myc2, an intronless retroposon found in woodchucks, occurs frequently in the HCCs associated with
WHV infection [54].
The relationship between the integration of WHV DNA sequences and tumor size and grade was also evaluated. 43% of well-differentiated HCCs that were smaller in size had integrated
34
WHV DNA but no molecular evidence of N-myc rearrangements; whereas, 79% of the generally larger, poorly differentiated HCCs had integrated WHV DNA and 74% had rearrangements in N-myc [55].
In addition to the presence of integrated viral DNA in liver tumors, WHV DNA has also been identified in nontumorous hepatic tissue as well as in early neoplastic nodules [53]. These observations suggest that a spectrum of molecular alterations exists from initial WHV DNA integration and early HCC to more advanced tumors [52]. Histologically, this is manifested in woodchucks by the progression from early foci of altered hepatocytes (FAH), to preneoplastic nodules, and to the development of HCC. By analogy, in humans, FAH have been found in patients with chronic hepatitis undergoing liver transplantation, and likely represent early dysplastic changes that are destined to progress to HCC [56].
While insertions of WHV DNA have been frequently demonstrated in the N-myc2 oncogene among woodchucks, integration of HBV DNA sequences occurs in a greater number of genetic loci in humans. Prior studies have shown that certain genes involved in cell signaling and cell growth in humans are recurrently targeted by the viral genome, including genes for human telomerase reverse transcriptase and the platelet-derived growth factor receptor [57,
58]. However, integration of HBV DNA into known oncogenes or tumor suppressor genes among humans occurs much less often than in woodchucks and no favored locus of integration for HBV DNA has been discovered [59]. The integration of WHV DNA sequences in specific oncogenes among woodchucks suggests that the insertion may directly result in dysregulation of proteins important to the cell cycle. Conversely, among humans,
35
while insertions of HBV DNA at key sites have been shown to directly disrupt tumor suppressor genes or alter cellular proliferation pathways, there appears to be a greater role for integrated HBV DNA to indirectly affect carcinogenesis [59].
Interestingly, compared with experimentally infected woodchucks, woodchucks with naturally acquired WHV infection had fewer WHV insertions near the N-myc2 locus. It was suggested that in naturally acquired WHV infection, the indirect effects of viral integration may be more important in hepatocarcinogenesis, as is believed to be the case in human HBV infection [60].
Taken together, WHV infection induced woodchuck model of HCC is an interesting animal model of human HCC. It has been used for preclinical evaluation of efficacy and safety of antiviral drugs, chemoprevention research and assessment of diagnostic imaging technology.
For example, it has been effectively for the development of new imaging agents for enhancement of detection of hepatic neoplasms by ultrasound and MRI. In this project, it will be more valuable in helping improve the PET utility for early detection and treatment evaluation of HCC.
1.3.2 Medical imaging of hepatocellular carcinoma
1.3.2.1 Diagnosis techniques for hepatocellular carcinoma
In clinical practice, ultrasound combining with α-fetoprotein (AFP) is used as screening tool.
However, the increase in serum AFP is not specific to HCC because it can be seen in a variety of non malignant conditions such as chronic hepatitis, fulminant hepatic failure or
36
cirrhosis [61]. These conditions are all characterized by hepatic necrosis and/or inflammation of the hepatic parenchyma. At the same time, AFP levels in most well differentiated HCCs whose sizes are smaller than 2 cm are often normal and the sensitivity of the test is blunted
[61]. On the other hand, in addition to difficulties and pitfalls in interpreting histological findings, percutaneous biopsy for HCC is not always feasible (due to the position of the nodule in the liver), needs a skilled operator to correctly target the nodule in the case of small lesions, and involves procedure-related morbidity and mortality risks. Consequently, imaging modalities able to differentiate the various steps of HCC development have been researched. Significant advances were produced in recent years, largely based on the capacity to characterize intranodular vascularization.
A non-invasive diagnosis of HCC based on imaging techniques can be made when abnormal arterial vascularization is documented in a nodule on the liver. Doppler US, CT and
MRI can diagnose HCC through translating the vascular derangement into morphologic
(direct visualization of the nodule) and functional (vascular pattern) alternations. However, any single of these imaging techniques is not considered sufficiently specific and sensible in small nodules especially 1-2cm in diameter [62].
On the contrast, numerous studies have demonstrated that malignant tumors can be detected with high sensitivity and specificity by imaging their increased metabolic rates for glucose, amino acids, or lipids. The metabolic imaging strategy of PET is totally different with above imaging modalities. PET with the glucose analog FDG has become a routine clinical test for staging and restaging of malignant lymphoma [63] and most solid tumors [64], [11C]-CHOL
37
and [18F]-fluorocholine (FCH) are used for detection of recurrent prostate cancer [65], and various radiolabeled amino acids have been shown to be clinically useful for brain tumor imaging [66]. The success of metabolic imaging motivates us to study using metabolic probes of PET, such as glucose and lipid analogs, to image the potentially up-regulated metabolic process inside the HCC tumor cells. In primary liver cancer such as HCC, the metabolic fates of imaging probes of PET are not entirely understood. In the thesis, the mechanism(s) underlying the PET imaging of HCC tumor cell metabolism was investigated, which will help to improve the PET utility for early detection and treatment evaluation of
HCC. It also shed new lights on the optimal protocol design for PET imaging on HCC.
1.3.2.2 Tumor glucose metabolism and FDG-PET imaging on cancer
The first steps of glucose metabolism (cellular uptake and phosphorylation by hexokinase) can be traced using the glucose analog FDG. As a common phenotype of cancer cells, i.e.
Warburg effect, glucose is largely metabolized to lactate (aerobic glycolysis) despite the presence of oxygen. Aerobic glycolysis and an overall increase in glucose metabolism are characteristics of cancer cells as compared with normal tissue [4]. The increased glucose metabolism in cancer is mediated through increased expression and activity of glucose transporters (GLUT) in the cell surface membrane and through characteristic changes in glycolytic enzyme expression and activity. These alterations in glucose metabolism are an early event in cancer development [4].
FDG PET is by far the most commonly used imaging technique to study glucose metabolism of cancer cells in vivo. After intravenous injection, FDG is transported across the cell
38
membrane by sodium-independent, facilitative GLUT. These transporters allow energy- independent transport of glucose across the cell membrane down a concentration gradient.
Intracellularly, FDG and glucose are phosphorylated by hexokinase to glucose-6-phosphate and FDG-6 phosphate. Glucose-6-phosphate is then further metabolized to fructose-1,6- biphosphate and enters glycolysis. Alternatively, glucose-6-phosphate enters the pentose phosphate pathway and is eventually converted to ribose-5-phosphate, which can serve as a building block for DNA and RNA synthesis. In contrast to this metabolic fate of glucose-6- phosphate, FDG-6-phosphate cannot be further metabolized in the glycolytic pathway because the fluorine atom at the C2 position prevents 18F-FDG-6P from further degradation.
As the cell membrane is impermeable for phosphorylated FDG, FDG-6 phosphate becomes trapped and steadily accumulates in metabolically active cells. The often increased glucose metabolism of malignant tissue offers an approach to using FDG and PET for in vivo investigations on tumors.
1.3.2.3 Tumor-associated de novo fatty acid synthesis
Many malignancies (including prostate, breast, head and neck, esophageal, gastric, hepatocellular, and colorectal cancers) are characterized by alterations in fatty acid metabolism, which can be summarized as the “lipogenic phenotype” of cancer [67].
Although glucose and fatty acid metabolism are interrelated, increased de novo fatty acid synthesis is also an independent mechanism in cancer pathogenesis, in particular through upregulation of the critical enzyme fatty acid synthase (FAS) [68]. This upregulation of FAS occurs in response to growth factor receptor activation or direct (i.e., growth-factor independent) activation of receptor tyrosine kinases, which initiate or enhance signal
39
transduction cascades, such as the Akt/PI-3-kinase pathway [69]. The common element through which these pathways induce transcription (and thus increased synthesis) of FAS is the sterol regulatory element binding protein (SREBP). This protein binds to the sterol regulatory element in the promoter region of FAS on the DNA[70]. In addition to increased de novo synthesis, greater levels of FAS in cancers cells also can be the result of decreased enzyme degradation due to removing ubiquitin from FAS, thus preventing FAS from proteasomal degradation [70].
In highly lipogenic tissues such as liver and adipose tissue, a variety of oncogenic changes
(H-ras, erb B-2, etc.) may result in the constitutive activation of MAPK and PI-3’K/AKT signaling cascades, which, in turn, can activate SREBP-1c and, subsequently, tumor- associated FAS-catalyzed endogenous lipogenesis. Thereafter, high levels of FAS are maintained in coordination with increased demand for fatty acid metabolism and/or membrane synthesis in response to cancer-related overexpression of growth factors (e.g.,
EGF, heregulin) and/or growth factor receptors (e.g., EGFR, Her-2/neu). The aberrant
MAPK and PI-30K/AKT cascades driven by these oncogenic changes subvert the downregulatory effects of physiological concentrations of dietary fatty acids, resulting in a cancer-associated FAS insensitivity to nutritional signals [4].
On the other hand, according to the extremely attractive theory recently proposed by
Hochachka et al [71], it is also conceivable that enhanced lipogenesis in tumor cells represents an adaptive response to control redox imbalance caused by glycolytic metabolism.
It is well known that cancer cells have an unusual tolerance to limiting O2 availability with an
40
unusual carbohydrate metabolism exhibiting excessive lactate production by aerobic glycolysis. In both normal and malignant prostate cells, three major pathways—lactate dehydrogenase, the respiratory chain, and lipid synthesis—are involved in the regulation of
the redox balance. Interestingly, in prostate cancer cells, because intracellular O2 supplies decline (i.e., hypoxic condition), the respiratory chain cannot fully oxidize the reducing equivalents being formed and, in this metabolic context, increased lipogenesis has been proposed to be the main physiologic function to maintain redox balance through oxidation of
NADPH during fatty acid synthesis. This hypothesis envisaged fatty acid synthesis as a means to mop up excess reducing potential in cancer cells. Finally, the three speculations— roles of lipogenesis in proliferation, mitochondria function, and hypoxia defense—are not exclusive.
In the liver and adipose tissue, FAS serves to store energy derived from carbohydrate metabolism as triglycerides. In contrast, human cancer cells do not store significant amounts of triglycerides but esterify fatty acids to phospholipids, such as phophatidylcholine (PC) [4].
Increased fatty acid synthesis ultimately leads to increased membrane lipid biosynthesis, for which choline kinase (ChoK) is a critical enzyme in cancer development and progression.
ChoK enables the conversion of choline to phosphatidylcholine, which is a major component of all membranes. As a practical clinical consequence, the imaging of fatty acid synthesis through choline or acetate tracers should enable us to study cancer development, aggressiveness, and its response to therapies. Thus, imaging with agents tracing fatty acid synthesis may be of particular interest in malignancies that are not imaged well with the
41
standard clinical PET tracer FDG, such as the low uptake of FDG in well-differentiated
HCC.
The first step of CHOL accumulation by tumor cells is transport across the cell membrane by various transporters. Since CHOL is a polar molecule, uptake by passive diffusion is low.
There are three known CHOL transport systems in human cells. These systems have initially been classified functionally as low-affinity, high-affinity, and intermediateaffinity transport
[64].
ChoK phosphorylates free intracellular CHOL to phosphocholine (PCho). Catalyzed by choline-cytidyltransferase (CCT), PCho can then react with CTP (cytosine triphosphate) to form cytosine diphosphate-choline (CDP-choline). The phosphorylcholine unit of CDP-
CHOL is then transferred to a diacylglycerol (DAG) by cholinephosphotransferase (CPT) to form PC, a major constituent of the mammalian cell membrane. In addition, during evolution, the liver has retained a backup pathway for choline production from the second most abundant membrane phospholipids, phosphatidylethanolamine (PE), to provide this essential metabolite when dietary choline is limited, as, for example, during starvation, embryonic development, pregnancy, or lactation. PE is transformed to PC in a three-step methylation by S-adenosylmethionine (SAM), which is catalyzed by phosphatidylethanolamine-N-methyltransferase (PEMT). Meanwhile, only a small amount of choline can be acetylated to acetylcholine, an important neurotransmitter.
42
It is currently still unclear whether the degree of CHOL uptake in HCC correlates indeed with the expression levels of critical enzymes (ChoK, CCT, FAS, etc.) and the specific changing of lipid metabolism in the tumor cells. There was a cell line study to investigate the metabolic fate of radiolabeled choline in ten cancer cells as compared to the normal fibroblast cell [72]. The cancer cell lines included glioblastoma, ovary carcinoma, malignat melanoma, lung carcinoma, adenocarcinoma, epidermiod carcinoma, fibrosarcoma and colorectal adenocarcinoma etc. After incubated with 14C-CHOL for 40 mins, the intracellular components were separate into lipid-soluble and water-soluble fractions using Bligh and
Dyer extraction method. The water-soluble fraction was applied to Radio-thin layer chromatography (TLC) to further separate into different metabolite components. The study indicated that 14C-CHOL uptake was higher in tumor cells than in fibroblasts and was correlated with the proliferative activity, though the sensitivity of 14C-choline uptake to proliferative activity was less than that of 14C-acetate. 14C-PCho produced from 14C-PC by phosphorylation mainly contributed to this accumulation. 14C-CHOL can be used for the evaluation of tumor proliferation through estimating choline kinase activity.
1.3.3 Imaging lipid synthesis in Cancer with PET
Methyl-CHOL labelled with 11C or 18F has been introduced as a tumor-seeking agent for the detection of malignancies. When injected into the body 11C-CHOL is incorporated into the kidney and liver where it is converted into the metabolite betaine. This is then released into the circulation and used for transmethylation reactions in various organs. However, in tumors, the metabolic fate of CHOL tracer is still unclear. It is believed that the metabolic
43
pathway of 11C-CHOL in cancer might be its integration into phospholipids. The biosynthesis of cell membranes is rapid most likely as a result of the fast replication of tumor cells.
Several investigations have been done on the 11C-CHOL PET imaging in different tumors, with brain and prostate tumors being the most common. In the central neuron system, the circulation is a major source of CHOL. Its uptake by the brain is slow, probably because healthy brain cells are in a non-dividing postmitotic state and, therefore, do not need much
CHOL for the formation of cell membranes. However, the increased vascularity of cerebral neoplasms results in strong uptake of CHOL into the endothelium of the new vessels. Thus, there is a good contrast between tumor tissue and healthy tissue in the brain. FDG is unable to produce such a contrast because healthy brain cells metabolise glucose intensely and, therefore, high background cerebral uptake exists. 11C-CHOL can also aid the differentiation between postsurgical or postradiotherapy changes in the tumor and those related to tumour reoccurrence. However, it is not good at discriminating benign from malignant brain tumors as both often have equal uptake of this tracer [73].
When used for imaging prostate cancer, 11C-CHOL has shown several advantages over FDG.
Prostate carcinoma is often a low-grade tumor, and, therefore, tends to show poor FDG uptake. Moreover, the prostate anatomically abuts the bladder resulting in the excretion of
FDG in the urine, providing a further challenge for PET imaging. By contrast, 11C-CHOL is taken up by many different grades of prostate carcinoma and only a relatively small amount of this tracer is excreted in the urine. Additionally, this tracer has shown to be useful in delineating local, regional, and distant disease and recurrence [74].
44
1.4 Organization of the thesis
The work described in this thesis has made distinct contributions to PET imaging of HCC using radiotracers FDG and 11C-choline.
(1) In chapter 1, the physics of PET and anatomy of liver were introduced. Moreover, the
work from the literatures was also reviewed. Current diagnosis techniques for HCC
were compared and non-invasive imaging techniques were highlighted. Furthermore,
potential metabolic pathways for radiotracers FDG and [11C]-CHOL were
summarized. At the same time, current status of application of radiotracers FDG and
11C-choline in nuclear medicine, especially in cancer imaging, was reviewed.
(2) In chapter 2, the evaluation of utility of FDG for imaging well- and poorly-
differentiated HCC was described. Two key enzyme activity, hexokinase and
glucose-6-phosphotase, was assayed and correlated with imaging data.
(3) In chapter 3, the metabolic fate of radiolabeled CHOL tracer in a well-differentiated
woodchuck cell line WCH17 was investigated as compared to primary rat
hepatocytes. The role of CHOL transporter and ChoK for the retention of choline
tracer in HCC was discussed. The main objective of this chapter is to understand the
exact mechanism of CHOL tracer uptake in HCC for PET imaging.
(4) In chapter4, the utility of [11C]-CHOL for imaging HCC was investigated. The
metabolic fate of radiolabeled choline in HCC in vivo were described. PET/CT
findings correlated with metabolites analysis in vivo and in vitro helped to elucidate
the exact mechanism(s) responsible for the heterogeneous uptake of radiolabeled
CHOL in HCC.
(5) In Chapter 5, Conclusions and future directions were discussed in this chapter.
45
Chapter 2 2-Deoxy-2-[18F]-fluoro-D-glucose Positron Emission
Tomography Imaging of Hepatocellular Carcinoma
2.1 Introduction
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Chronic infection with hepatitis B or C virus are important risk factors for the development of liver cirrhosis and HCC [75]. A widely accepted strategy for the early detection of HCC is 6- month surveillance by careful ultrasound scanning of the liver combined with determination of the serum level of α-fetoprotein (AFP) [76, 77]. However, in HBV carriers with liver cirrhosis combined nodular regeneration or other benign focal lesions, it may be difficult to differentiate from HCC by ultrasound studies only [78, 79].
Most tumor cells have increased glucose metabolism because of increased levels of glucose transporter and increased levels of intracellular enzymes that promote glycolysis such as hexokinase and phosphofructokinase [80-82]. The increased uptake of glucose is a marker of tumor viability, based upon the enhanced glucose metabolism of cancer cells. Positron emission tomography (PET) is a noninvasive imaging technique allowing direct evaluation of cellular glucose metabolism using FDG as a tracer. PET imaging with FDG has proven useful in differentiating malignant tumors from benign lesions based on differences in their metabolic activity for tumors of the central nervous system[83, 84] and various body tumors including: colorectal cancer, lung cancer, melanoma, and lymphoma, among others [85].
Several studies have also demonstrated the usefulness of FDG-PET to detect malignancy
46
recurrence, evaluate tumor stage, and monitor therapy for various malignant neoplasms [86-
88].
Two steps are required to accumulate FDG in cancer cells: facilitated diffusion through a glucose transport protein; and subsequent phosphorylation to FDG-6-P by one of the hexokinase isoforms,
FDG-6-P is not transported out of cells nor undergoes glycolytic breakdown; it is metabolically trapped inside cells. Therefore, in this study, we evaluated the usefulness of PET imaging with FDG on early detection of HCC.
Early diagnosis of HCC at a potentially curable stage is of highest priority, because only patients with small tumors (<5 cm) without extrahepatic metastases benefit from partial liver resection or liver transplantation as potentially curative treatment [89, 90]. In this study, we were experimenting with PET imaging on the woodchuck model of HCC induced by woodchuck hepatitis virus (WHV) infection. The activities of the two key enzymes that control glucose 6-phosphate (G-6-P) and 2-fluoro-2-deoxyglucosc 6-phosphate (FDG-6-P) levels, hexokinase (HK) and glucose-6-phosphatase (G6Pase), were also determined in both
HCC and surrounding hepatic tissues. Thw WHV infection-induced hepatoma in the woodchucks is a natural occurring animal model of human HCC with similar pathology and natural history [91, 92]. The size of the animal and tumor is suitable, compared to the rodents that are too small, for the developed or developing image-guided interventional procedures that can be translated directly into human applications. Woodchucks are often naturally infected with WHV, a virus similar to human hepatitis B virus, and develop liver cancer with
47
a high prevalence [91]. It takes about two years to develop this HCC model, and it is thus authentic as compared to some of the xenograft models.
2.2 Materials and methods
2.2.1 Materials
All chemical reagents used were obtained from Sigma Chemicals (St. Louis, MO) unless otherwise stated.
2.2.2 Animals
All procedures in this study followed the guidelines and recommendations of the Institutional
Animal Care and Use Committee of Case Western Reserve University (Cleveland, OH).
Fourteen chronic WHV-carrier and one normal (control) woodchucks with HCC obtained from Cornell University (Ithaca, NY) and Northeastern Wildlife, Inc. (Harrison, ID) weighing between 2.3 and 5 kg were used in this study. The animals were fasted for twelve hours before imaging. Otherwise they had access to water and food (Teklad laboratory diets for rodents 8864; Madison, WI) per normal husbandry. The woodchucks were initially sedated with an intramuscular injection of 5 mg/kg of Xylazine and 50 mg/kg of Ketamine. A sublingual catheter was inserted for intravenous injection of the radiolabeled compounds.
When this injection route failed, an incision was made in the upper thigh and a catheter was directly inserted in the femoral vein. Hydration of the animal and patency of the catheter and were maintained with a 0.9% sodium chloride injection (Baxter Healthcare Corporation;
Deerfield, IL). Pentobarbital was administered as incremental doses to keep the woodchucks under anesthesia.
48
2.2.3 Radiopharmaceuticals
FDG was produced on site. Briefly, 18O water was recovered using ion exchange resin to extract the fluoride. Unreacted fluoride and aminopolyether were removed using a silica cartridge eluted with acetonitrile/ether, and hydrolysis was done using 1N HCl which was then neutralized with NaOH and NaHCO3 to produce a buffered, hypertonic, sterile and pyrogen-free product. Nine woodchucks with HCC and one normal healthy woodchuck were included in the experiments. Each animal was injected with a 37 MBq bolus of 2FDG and dynamic emission scans were acquired for 60 minutes. The time-frame between 50 and 60 minutes was used to quantify the SUV and the tumor-to-liver uptake ratio (T/L).
2.2.4 Imaging protocol
Before imaging, the animals were anesthetized with an intramuscular injection of 5 mg/kg of
Xylazine and 50 mg/kg of Ketamine. Thereafter, incremental injections of pentobarbital were used to maintain anesthesia. A small incision was made in the inner left thigh to allow catheterization of the femoral vein. A whole-body CT scan was performed on the MX 8000
IDT scanner (Philips Medical Systems, Inc.; Cleveland, OH) with and without contrast agent
(Optiray 300 ioversol injection 64%; Mallinckrodt; Hazelwood, MO) and was later used for tumor localization when aligned with PET images. For the PET scanning, each animal was injected with a 37 MBq bolus of FDG and scanned on an Allegro PET scanner (Philips
Medical Systems, Inc.; Cleveland, OH) for a total duration of 90 minutes. Dynamic data from list-mode acquisition were re-binned into a total of 48 frames (6 × 5, 6 × 10, 6 × 30, 6 × 60,
4 × 90, 4 × 120, 4 × 180, 6 × 300, and 6 × 500 seconds). The 3-D emission scans were
49
reconstructed using filtered backprojection with attenuation and scatter correction as implemented in version 8.1.4 of the reconstruction software of the Allegro.
2.2.5 Image analysis
PET images were first assessed visually, using transaxial, sagittal, and coronal displays. To allow an objective assessment of the amount of tracer uptake, we evaluated the FDG uptake by semi-quantitative analysis using the standardized uptake value (SUV) and the tumor-to- liver (T/L) ratio for each abnormal focus. Circular regions of interest with a diameter of 1.2 cm were drawn around tumors (identified on the CT scans) or around the regions of the highest uptake within tumors. Similar regions were drawn around the hepatic tissues surrounding the HCCs. A tumor was considered detected if T/L was greater than 1.2. Values are reported as mean ± standard deviation. The standardized uptake value (SUV) was calculated as the activity concentration within an ROI normalized by the ratio of the injected dose and the animal weight. SUV is defined as:
where the radioactivity concentration in a pixel (Bq/ml) was to be determined from an apparent pixel count (cps/pixel volume) and a predetermined cofactor.
The largest diameter of each tumor was measured on the contrast-enhanced CT images. The intensity within a ROI on the CT images was reported in Hounsfield units
1000 where STP stands for the standard temperature and pressure conditions (0° C and 100 kPa).
50
2.2.6 Histology
The diagnosis of HCC was also based on routine histological examination. After PET and CT imaging, the animals were euthanized using FatalPlus (Vortech Pharmaceuticals Inc,
Dearborn, MI). For some woodchucks, HCCs and surrounding hepatic tissues were obtained post-mortem and fixed in formalin. Some tissues were also chosen for enzyme assay. Thin sections of paraffin-embedded tissues were prepared and stained with hematoxylin-eosin
(H&E). The fat content, presence of necrosis, and the tumor differentiation were assessed from these sections. Cells in well-differentiated tumors have distinct cell membranes, minimal atypia, a moderate amount of finely granular eosinophilic cytoplasm and bile cannaliculi are present. In moderately-differentiated tumors the trabecular structures are three or more cells wide, the cytoplasm is abundant and eosinophilic and the nuclei are round with prominent nucleoli. Multinucleated and giant cells are present in poorly-differentiated tumors and the nuclear/cytoplasm ratio is high and can be noticed by the increased nuclear density.
Nuclear atypia is also pronounced [93]. When different levels of differentiation were observed within one tumor, the poorest was reported.
2.2.7 Tissue excision
A pathologist separated HCC portion from the surrounding hepatic liver. Specimens were chosen and snap frozen in liquid nitrogen and stored at -80oC for later enzyme activity assays. Both HCC specimens and surrounding hepatic liver specimens from infected woodchucks were processed for HK and G6Pase activity assays. The normal liver specimens from healthy woodchuck were used for the same assays as a control.
51
2.2.8 Hexokinase activity assay
The method used for extracting and assaying the HK from liver tissues was describe previously [81]. The liver samples were homogenized in pre-cooled homogenization buffer containing Tris-HCl, 0.05M; sucrose, 0.25M; EDTA, 0.005M (pH 7.4); 2-mercaptoethanol,
5mM; glucose, 0.01M (1g/3ml). The homogenate was centrifuged at 4,500 rpm for 15 min at
4C (Sorvall RT 6000D centrifuge, Fisher Scientific Inc., Pittsburgh, PA). The supernatant was centrifuged at 45,000 rpm for 70min at 4C (Beckman L7 ultracentrifuge, Ti90 rotor,
Beckman Coulter Inc., Fullerton, CA). The supernatant was used for determination of HK activity. Aliquots (1.0ml each) were stored at -80C until further use.
The HK activity was measured spectrophtometerically through NADP+ reduction in the glucose-6-phosphate dehydrogenase-coupled reaction. Incubation medium for HK activity measurements contained 1ml triethanolamine buffer, 0.05M (pH=7.6); 1ml D-Glucose solution, 0.555M; 0.2ml magnesium chloride, 0.1M; 0.2ml NADP+, 0.014M; 0.02ml glucose-6-phosphate dehydrogenase, 125U/ml. 0.2ml HK extract from woodchuck liver was mixed with incubation medium and equilibrated to 25C. The reaction was started with 0.1ml
0.019M ATP addition and absorbance was continuously recorded for 5 min at 340nm
(Beckman DCI 520 general purpose UV/VIS, Beckman Coulter Inc., Fullerton, CA).
Standard 1U/ml HK solution (Sigma Inc., St. Louis, MO) was used as a positive control. The total protein content in the HK extract from woodchuck liver sample was also determined by the method of Bradford (Bio-Rad labortaries, Inc., Hercules, CA) [94]. The activity was normalized to the total protein content. One unit of HK will phosphorylate 1.0 μmole of D- glucose per minute at pH 7.6 at 25°C.
52
2.2.9 Glucose-6-phosphatase activity assay
The method used for determining the G6Pase activity in liver tissues was described previously [95-97]. Liver samples were homogenized in ice-cold 0.25M sucrose solution
(1g/3ml). The homogenate was centrifuged at 4C 14,000 rpm for 30 min. The supernatant was centrifuged again at 4C 46,000 rpm for 60 min. The resultant microsomal pellets were suspended in 0.25M/0.001M sucrose/EDTA solution and stored at -80C for enzyme assay.
The determination of G6Pase activity in woodchuck liver tissues consists of incubating a specific substrate glucose-6-phosphate (37C, pH 6.5) with tissue microsomal pellets and determining the amount of orthophosphate liberated. To a mixture of 0.1 ml 0.25M/0.001M sucrose/EDTA solution, 0.1 ml 0.1M cacodylate buffer (pH 6.5) and 0.1 ml 0.1M glucose-6- phosphate, 0.1 ml of samples was added. The system was incubated at 37C for 10 min. At the end of the incubation period, 2.0 ml of 2%/10% (w/v) ascorbic acid/trichloracetic acid solution was added to stop the reaction. The samples were centrifuged (3 min, 3000 rpm). A
1.0-ml supernatant fluid was used for the determination of phosphate by adding dye reagent
(0.5ml, 1% ammonium molybdate solution). 1 ml 2% arsenie/citrate solution was finally added into the end solution.
The amount of phosphate liberated per unit time was determined as the blue phosphomolybdous complex at 700 nm wavelength using spectrophotometer. The enzyme activity of the liver tissue is calculated by subtracting the inorganic phosphorus content of tissue incubated without substrate (enzyme blank) from that of complete system incubated
53
with substrate. A blank containing all reagents except the tissue (substrate blank) revealed no free inorganic phosphorus. Standard 1 U/ml G6Pase solution (Sigma Inc.) was used as a positive control. Bradford (Bio-Rad labortaries, Inc., Hercules, CA) [94]. The activity was normalized to the total protein content. One unit of G6Pase will release 1.0 μmole of inorganic phosphate from glucose 6-phosphate per minute at pH 6.5 and 37°C
2.2.10 Statistical Analysis
Comparisons of HK and G6Pase activities in the HCC region, surrounding hepatic tissue and normal liver tissue were performed by one way analysis of variance (ANOVA) or ANOVA on ranks when appropriate. The level of significance was set at p < 0.05.
2.3 Results
2.3.1 PET imaging
As shown in Figure 2.1, visual inspection of the dynamic image sequences showed a difference between the woodchuck HCC. The HCCs were divided into two groups based on their FDG uptake one hour after the bolus injection. HCCs in group 1 (Figure 2.1 A, C and
E) had a FDG uptake similar to the surrounding hepatic tissues (tumor-to-liver ratio ≤ 1.15).
In contrast, HCCs in group 2 (Figure 2.1 B, D and F) had a higher FDG uptake than the surrounding hepatic tissues (tumor-to-liver ratio > 1.15). H & E staining confirmed that the groups 1 HCCs were mostly well-differentiated which has a similar histological finding with surrounding hepatic tissue and the group 2 HCCs were poorly-differentiated which showed a very good FDG avidness.
54
Figure 2.1 FDG-PET imaging on the woodchuck model of HCC.
(A) Contrast-enhanced transverse CT image of well-differentiated HCCs. (B) Contrast- enhanced transverse CT scan of poorly-differentiated HCCs. Note that grayscale intensities on both CT images were adjusted for enhanced visualization. Smaller tumors can be found in the quadrate (outlined by a red dashed line). (C) Well-differentiated HCCs 60 minutes after FDG injection (later phase image). The HCCs are indicated by the arrows. (D) Poorly- differentiated HCCs 60 minutes after FDG injection (later phase image). The HCCs are indicated by the arrows. Note the hypointense region in the center of the tumor. (E) H&E stain of a well-differentiated HCC. (F) H&E stain of pseudo glandular structures seen in some poorly-differentiated HCCs.
In figure 2.2, a well-differentiated woodchuck showed FDG uptake at the 15 to 20 sec images but no any clear contrast was observed in the region after one hour time point post
FDG injection. These early images also helped in localizing the tumors in animals for which the tumor-to-normal ratio on the one-hour images was not high enough for detection. Thus, 55
in well-differentiated HCC, FDG worked like a perfusion tracer at early time point, which showed contrast.
A comparison between normal liver and well-differentiated HCC was also shown in Figure
2.3. ROI were drawn on both the normal and the tumor portions of the liver to quantify the tumor-to-normal SUV ratio at early time points (15-20 seconds) and at later time points (66.5 to 75 minutes) for all woodchucks. At the 18 seconds time point, the tumor/normal activity ratio is 3.25. At one hour time point, the tumor/normal activity ratio is only 1.16.
For the SUV, at the early arterial phase, the tumor-to-hepatic tissues SUV ratio was
2.33 ± 1.21 in HCCs in group 1 and 2.45 ± 0.76 in HCCs in group 2. At the one-hour time point, this ratio was 0.97 ± 0.14 and 1.24 ± 0.04 in HCCs in groups 1 and 2, respectively.
For animals in group 1, HCCs were moderately- to well-differentiated. As shown in Figure
2.4, only moderately-to-poorly-differentiated cells effectively entraped glucose and the FDG analog and, therefore, appear more intense on FDG PET images. Higher FDG uptake during
PET imaging was associated with a less differentiated HCC phenotype and the presence of increased necrosis. HCCs of Group 1 woodchucks with less FDG uptake had significant amounts of fat but little necrosis.
In contrast, well-differentiated HCC showed show an increased FDG uptake only at very early time frames (15-20 seconds post-injection). At this time point, the bolus of FDG has yet to reach the portal vein, and remains in the arterial circulation. Thus, this might be due to
56
different blood supply between HCC and surrounding hepatic tissue [98, 99]. Since HCC derive their afferent blood supply from the arterial circulation (in contrast to normal liver tissue which draws its blood supply primarily from the portal vein), hepatic neoplasms are the only structures that appear on the early images of the liver.
Figure 2.2 PET imaging on well-differentiated HCC (A) PET-CT overlay of a well-differentiated woodchuck at 20 to 25 seconds after FDG injection (early phase image). (B) Same HCCs 110 minutes after FDG injection. (C) Picture of the excised liver where the tumors are pointed out by the arrows.
C D
Figure 2.3 dynamic PET scan with FDG on well-differentiated HCC and normal liver.
(A) PET-CT overlay of a normal woodchuck at 15 to 20 seconds after injection. (B) Same animal at 66.5 to 75 minutes after injection. (C) PET-CT overlay of well-differentiated woodchuck at 15 to 20 seconds after injection. Note the contrast between HCC (outlined in a green dashed line) and normal tissue. (D) Same animal at 66.5 to 75 minutes after injection.
57
A B
C
Figure 2.4 PET imaging on poorly-differentiated HCC (A) Coronal slice of a PET image taken between 73 and 83 minutes post-injection. (B) Normal portion of the hisological slide. Note the thinness of the trabeculae. (C) Tumor portion of the histological slide (poorly differentiated).
2.3.2 Enzyme activity
The activities of HK and G-6-Pase in HCC, the surrounding hepatic tissue and the normal liver were shown in Figure 2.5. HCC had a significantly higher HK activity than the surrounding hepatic tissue and the normal liver tissue. In contrast, HCC had a significantly lowest G6Pase activity than the surrounding hepatic tissue and the normal liver tissues. In addition, normal liver tissue also had a higher G6Pase activity than the surrounding hepatic tissue.
58
A B
Figure 2.5 Hexokinase and Glucose-6-phosphatase activity in woodchuck model of HCC.
(A) HK, (B) G6Pase
2.4 Discussion
The current diagnostic strategy for HCC consists of an ultrasound study of the liver combined with serum AFP measurement twice a year [100]. Although the intimate association of AFP to HCC has been well established, recent studies have demonstrated the presence of a minute quantity of AFP in the sera of patients with benign liver diseases and even in healthy adults. Therefore, AFP screening is usually combined with abdominal ultra- sound [101]. Up to 80% of small HCC can be detected by ultrasound, but tumors smaller than 2cm may often escape detection [102]. In addition, the major problem of ultrasound screening in HBV carriers is the occurrence of false-positive results, e.g., nodular regeneration or hemangiomas
The role of FDG-PET is based on fundamental aspects of tumor metabolism: rapidly- dividing tumor cells have accelerated glucose utilization under anaerobic conditions [103]. 59
Explanations for this phenomenon include increased tissue concentration of glucose transport proteins and/or increased HK activity in tumor cells. The use of FDG-PET for detecting HCC presents a particularly interesting imaging situation because the liver is one of the tissues with the high metabolic activity. The conversion of glucose-6-phosphate to glucose is due to a higher G6Pase in liver. In well-differentiated HCC, not only surrounding hepatic tissue but also HCC might have high levels of G6Pase, the accumulation of FDG is relatively low [77].
In woodchuck HCC, both well-differentiated and poorly-differentiated HCC showed higher
HK activity, which helped accumulate the FDG intracellularly. But well-differentiated HCC only showed uptake at a very early time point, suggesting FDG-6-P was de-phosphorylated and. leave the liver cells. However, our enzyme assay for G6Pase did not support this hypothesis. Therefore, in HCC, the principle of FDG as a trapped tracer might be increased or decreased as in the present study.
Our results indicate that FDG-PET is not sensitive to HCC. It can be very useful for staging poorly-differentiated HCC from well-differentiated HCC. Future studies with more HCC are necessary to further evaluate the practical implications of FDG-PET as a tool in detecting and differentiating HCCs.
60
Chapter 3 Transport Mechanism and Metabolic Fate of Radiolabeled
Choline in Hepatocellular Carcinoma: an in vitro Comparative Study
3.1 Introduction
The development of an effective noninvasive imaging technique for early detection, staging and monitoring of the therapeutic outcome of hepatocellular carcinoma (HCC) remains a topic of intense interests. Molecular imaging modalities, including positron emission tomography (PET), promise to differentiate cancer from normal tissues based on abnormal metabolic patterns within neoplastic cells. The most commonly used PET tracer is 2-[18F]- fluoro-deoxyglucose (FDG), which has proved effective in detecting many tumor types.
However, FDG has been shown to be ineffective for imaging HCC since up to 50% HCC did not shown FDG uptake comparing to the surrounding hepatic tissues, and most of uptake could be found only in the poorly-differentiated HCC, a late stage of HCC with poor prognosis.
Recently, up-regulated choline (CHOL) uptake and metabolism, relative to benign lesions and normal tissue, has been used as a diagnostic marker of malignancy [104, 105]. It has been suggested that malignant transformation of cells is associated with the induction of choline kinase (ChoK) activity resulting in increased levels of phosphocholine (PCho) [104,
106-110]. Furthermore, it is also known that rapidly proliferating tumors contain large amounts of phospholipids, particularly phosphatidylcholine (PC) [104, 105]. Thus, this provides the rationale for the use of [methyl-11C]-CHOL as probe for PET imaging of early detection of HCC.
61
The quantitative analysis of PET images requires our understanding of the transport and metabolism of the radiotracer administered. The extent of metabolism of the tracer in tissue depends on the chemical and biological properties of the tracer as well as the specific metabolic characteristics of cancer. After intravenous injection, [methyl-11C]-CHOL is rapidly cleared from the blood circulation. Blood sampling has shown that the predominant metabolite in blood is betaine [110]. However, the metabolic fate of the [methyl-11C]-CHOL within HCC during a PET scan is not fully understood yet. Therefore, it is important to clarify the metabolism of radiolabeled CHOL leading to the enhanced uptake in HCC as compared to primary hepatocytes during PET imaging.
CHOL is a quaternary ammonium base and crucial for mammalian cells. All cells utilize
CHOL as a precursor for the biosynthesis of phospholipids, e.g. PC (lecithin), which are essential components of all membranes [110, 111]. Magnetic Resonance Spectroscopy
(MRS) studies of biopsied cancerous tissues and in vivo tumors have generally shown elevated levels of choline-containing metabolites in cancer relative to normal tissue [112-
115]. Of these CHOL containing metabolites, PCho has been found to most closely correlate with malignant transformation [116, 117], tumor growth rate [115], metastatic potential
[115], and therapeutic response [118]. This is logical since highly proliferating tumor cells require more building blocks for membranes.
Routing CHOL to its different metabolic pathways is cell and tissue specific. CHOL is transported into the cell by both facilitative transport mechanisms and diffusion. A diagram
62
of the metabolic fate of radiolabeled CHOL in the cell is shown in Figure 3.1. After transported into the cells, CHOL is then phosphorylated by ChoK to PCho for the synthesis of PC (CDP-CHOL pathway). In neuron cells, CHOL can be also converted to acetylcholine, an important neuron transmitter. PCho is the first intermediate in the stepwise incorporation of CHOL into phospholipids by the CDP-CHOL pathway. In the next step, PCho is converted to cytidine diphosphate (CDP)-CHOL by catalysis of cytidine triphosphate
(CTP):phosphocholine cytidylyltransferase (CTT). CDP-CHOL can further react with 1,2- diacylglycerol (DAG) to produce PC.
Figure 3.1 Metabolic fate of radiolabeled choline. 1, 2-DAG: 1,2-diacylglycerol; CCT: CTP:phosphocholine cytidylyltransferase; CDP-Cho: CDP-choline; ChoK; choline kinase; HCC: hepatocellular carcinoma; PCho: phosphocholine; PC: phosphoatidylcholine; PE: phosphoethanolamine; P.I.: post injection; SAM: S-adenosylmethionine.
In the meantime, CHOL can be oxidized to betaine aldehyde, which is then converted into betaine by the enzyme system of CHOL oxidase (CHOL dehydrogenase and betaine 63
aldehyde dehydrogenase) exclusively in the liver and kidney [110]. Betaine serves as an organic osmolyte in the cell, i.e. compound which is accumulated or released by cells in order to maintain cell volume homeostasis [108]. Betaine, the end product of CHOL oxidation, cannot be reduced to form CHOL, but it can donate one of its methyl groups and thereby produce methionine (Met) [110, 111]. Met can further incorporate into PC via
Phosphoatidylethanolamine (PE) methylation pathway. But, the precise metabolic fate of
[methyl-11C-CHOL] in HCC is not entirely clear.
In this study, the metabolism of radiolabeled CHOL was evaluated in a well-differentiated woodchuck HCC cell line WCH17 (an early stage of HCC) against the primary rat hepatocytes. As a rate-liming step for CHOL uptake, the transport mechanism for CHOL is still unknown in HCC tumor cells. Thus, we also performed mechanistic studies on the transport of CHOL in the WCH17 cells to provide further clarification of the properties of the CHOL transporter in HCC.
WCH17 is a well differentiated HCC cell line derived from an adult woodchuck hepatitis virus (WHV) induced woodchuck hepatoma by Bruce Fernie (Georgetown University). It contains hepadnavirus. The woodchuck contains woodchuck hepatitis virus (WHV). WHV belongs to the family hepadnaviridae, of which human hepatitis B virus (HBV) is the prototype. This cell line has not been extensively characterized, but it resembles human hepatoma cell lines such as PLC/PRF/5 and Hep3B, which contain HBV, in appearance.
64
Due to the very short physical half life of 11C (20 minutes), we used 14C labeled CHOL at the imaging tracer dose for this study. In addition, using cultured WCH17 cells and primary rat hepatocytes allow us to control potentially confounding variables, such as blood flow and necrosis, which are present when studying these biochemical parameters in animal tumor models or in human tumors. It helps to map out a clear figure about the mechanism regarding
PET imaging with radiolabeled CHOL tracer before we move on to the animal model.
3.2 Materials and Methods
3.2.1 Materials
All chemical reagents used were obtained from Sigma Chemicals (St. Louis, MO) unless otherwise stated. [methyl-14C]-CHOL chloride (specific activity 1.85-2.22 GBq/mmol), were obtained from American radiochemical Inc. (St. Louis, MO). Liver Perfusion Medium, Liver
Digest Medium, L-15 Medium, Hepatocyte Wash Medium, Hepatocyte Wash Medium, 20%
NYCODENZ®, Williams' Medium E, Hepatocyte SFM, Hexobarbital, Dulbecco's Modified
Eagle's Medium, Penicillin Streptomycin were obtained from Invitrogen Co. (Carlsbad, CA).
WCH17 cell line was purchased from American Type Culture Collection (ATCC)
(Manassas, VA). Organic solvents were purchased from Fisher Scientific (Pittsburgh, PA).
3.2.2 Primary rat hepatocytes preparation
Primary rat hepatocytes were fresh prepared as a negative control. Hepatocytes were prepared by the method as described previously [119-121]. Briefly, the rat liver was isolated in situ and perfused with 350 ml of warm (37°C) Liver Perfusion Medium through the abdominal aorta at a rate of 35 ml/minute with the perfusate exiting through the severed vena cava. This was followed by a Collagenase-Dispase digestion with Liver Digest Medium at a 65
rate of 35 ml/minute. The complete digestion of the liver was 10-12 min. The digestion step resulted in blanching, softening and dissociation of liver tissue. Then the liver was aseptically removed to a sterile 50 ml conical tube containing 15ml cold L-15 Medium and transferred to the cell culture laboratory on ice for the following steps. In the 50 ml conical tube, the hepatocytes were released by gently using a cell scrapper and pipetting with a large bore pipette. The cell suspension was spun down at 50 ×g for 5 min, resuspended in wash media and filtered through a sterile 100 μm nylon mesh into a 50 ml conical tube placed on ice. The component in the conical tube was sedimented by centrifugation at 50 ×g for 5min. The resultant sediments were resuspended and washed 2-3 times in 50 ml cold Hepatocyte Wash
Medium. Hepatocytes were further purified by centrifuging sediments on either 20%
NYCODENZ® or alternatively by Percoll density gradient separation and washed twice more before being resuspended in the attachment medium. Either of these separation procedures will give a cell yield of about 2.5 x 108 cells with 90-95% viability, as determined by trypan blue exclusion. Approximately 10 x 106 cells in 25 ml of Williams' Medium E, supplemented with 5ml penicillin-streptomycin (10,000 unit/ml penicillin, 10µg/ml streptomycin) and 10 % fetal bovine serum (FBS), were plated in 150-cm2 tissue culture flasks precoated with a Collagen 1 matrix (12.5 μg/cm2) or with the EHS matrix (100 μg/cm2
Matrigel, Collaborative Research Inc., Bedford, MA) and incubated in a humidified atmosphere of 10% CO2 in air at 37°C. Unattached cells were poured off 2-3 hours after plating and replaced with 25 ml HepatocyteZYME-SFM supplemented with 1.25 μg/cm2 rat tail Collagen to provide a sandwich matrix. Cultures were re-fed with HepatocyteZYME-
SFM (without collagen) at 24 hours and every 48 hours thereafter.
66
3.2.3 WCH17 Cell culture and media
WCH17 cells were cultured in the 75 cm2 corning cell culture flasks with Dulbecco's
Modified Eagle's Medium (DMEM) (Contains 28.5µM CHOL choride, 4,500 mg/L D- glucose, L-glutamine, and 110 mg/L sodium pyruvate; GIBCO/Invitrogen, Grand Island,
NY) with 10% FBS and 1% Penicillin Streptomycin solution (10,000 unit/ml penicillin,
10µg/ml streptomycin) under a 10% CO2-humidified atmosphere at 37°C.
3.2.4 [Methyl-14C]-Choline uptake and metabolism
WCH17 cells in the exponential phase were trypsinized and 1 × 107 cells were plated in 75 cm2 corning cell culture flasks. 5 × 106 cells of primary rat hepatocytes were plated in 150- cm2 tissue culture dish precoated with Collagen 1 matrix (12.5 μg/cm2). DMEM (GIBCO,
Grand Island, NY) supplemented with 10% FBS was used as the assay medium. Cells were incubated with 10 ml of 74 KBq [methyl-14C]-CHOL chloride (American Radiolabeled
Chemicals, Inc., St. Louis, MO) at 37°C for 5, 15, 30, 45 and 60 min. After incubation, the medium was removed and the cells were washed twice with ice-cold phosphate buffer saline
(PBS), trypsinised with 3 ml trypsin and neutralized with 3 ml media. The resultant cell suspensions were centrifuged at 2500 rpm for 10 min at 4 °C. Cell pellets were resuspended in 1.2 ml water containing 100 mg/l antioxidant Butylated Hydroxytolune (BHT) and homogenized for the extraction of metabolites and lipids (Bligh and Dyer extraction method) and metabolites analysis of intracellular fate of [methyl-14C]-CHOL using high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) as described below.
67
3.2.5 Pulse and chase study and efflux
For the pulse and chase experiment, untreated and 1mM oleic acid treated cells were used for the study respectively. The 1mM oleic acid was prepared as described by Van Harken et al.
[122]. Sodium oleate (0.1 mmol) was dissolved in 5.0 ml of warm saline and then mixed this with 5.0 ml of 20% bovine serum albumin (fatty acid-free). The mixture (10.0 ml) was diluted to 100 ml with DMEM. The final concentration was 1% bovine serum albumin
(BSA) containing 1 mM sodium oleate.
1 × 107 WCH17 Cells in 75 cm2 corning cell culture flasks or 4 × 106 primary rat hepatocytes in 150-cm2 tissue culture plates precoated with a Collagen 1 matrix (12.5 μg/cm2) were incubated in triplicate for 5 min at 37°C with 370 KBq [methyl-14C]-CHOL (specific activity: 2.035 MBq/µmol) (pulse). After incubation, the radioactive media were removed and the cells were washed three times with ice-cold PBS. And the cells were then incubated with DMEM with 10% FBS containing 18 µM non-radioactive CHOL for 10, 25, 40 and 55 min (chase). The DMEM contains either 1% bovine BSA, 1 mM sodium oleate (treated) or none of oleate (untreated). After chase, the chase media were collect and separated into water-, lipid-soluble and insoluble phase as described below (Bligh and Dyer extraction method) to determine effluxed activity. The cell layers were rinsed with ice-cold PBS twice, trypsinised with 3 ml trypsin and neutralised with 3 ml medium. The resultant cell suspension centrifuged at 2500 rpm for 10 min at 4°C. The supernatants were discarded. Cell pellets were resuspended in 1.2 ml water containing antioxidant 100 mg/l Butylated
Hydroxytolune (BHT) and homogenized for the extraction of metabolites and lipids (Bligh and Dyer extraction method) and metabolites analysis of intracellular fate of methyl-14C-
68
CHOL using high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) as described below.
3.2.6 Extraction of radiolabeled metabolites and lipids using Bligh and Dyer method
1.2 ml homogenate of cell pellets from incubation and pulse and chase study were used to extract the cellular CHOL metabolites using Bligh and Dyer method [123]. 0.1 ml of homogenate of cell pellets was used to measure the total radioactivity of uptake in whole cells in a liquid scintillation counter. Another 0.1 ml of homogenate of cell pellets was used for protein measurement. The remaining 1ml homogenate of cell pellets were added 3.75ml mixture of chloroform/methanol (1/2. v/v) and vortex for 10-15 min, then 1.25 ml chloroform was added into mixture and mixing 1 minute. 1.25 ml 1M NaCl was then added and mix another 1 minute to block the binding of some acidic lipids to denatured lipids. The whole extract was centrifuged at 2500 rpm 10 min at 4°C. The water-soluble (upper layer), lipid-soluble metabolites (lower layer) and insoluble phase (including RNA, DNA and protein) were separated. After evaporation under the nitrogen gas, the lipid soluble phase was redissolved in 0.7ml of chloroform/methanol (2/1, v/v); the water soluble phase was redissolved in 0.7 ml of methanol/water (1/1, v/v). 0.1 ml from each phase was used for measurement of radioactivity. The radioactivity from each phase was normalized to total protein content. The metabolites in the water- and lipid-soluble phases were analyzed by radio-HPLC and radio-TLC as described below.
69
The chase media from pulse and chase study were also applied to Bligh and Dyer method to separate into water-, lipid-soluble and insoluble phases. The radioactivity in each phase was counted in a liquid scintillation counter and normalized to the total volume of media.
3.2.7 Analysis of radiolabeled metabolites
3.2.7.1 HPLC system
The HPLC system consisted of HP1050 Instruments (Agilent Technologies Inc., Santa Clara,
CA): 1050 Pumps (isocratic and quaternary), 1050 Diode Array and Multiple UV
Wavelength Detectors, temperature controller (Waters Co., Milford, MA), followed by a radioisotope (14C/3H) detector (Radiomatic 150TR, Flow Scintillation Analyzer;
PerkinElmer, Waltham, Massachusetts, USA) and with computer data acquisition. HPLC analysis was performed on an Adsorbosphere Silica normal phase HPLC column
(250×4.6mm, Prevail Silica 5µ; Grace Davison Discovery Sciences Inc., Deerfield, IL) with an guard column (Guard 7.5×4.6mm, Prevail Silica 5µ; Grace Davison Discovery Sciences
Inc., Deerfield, IL) used as the stationary phase. The TLC technique on the silica gel G plate
(Merck KGaA, Gibbstown, NJ) was used to confirm the HPLC result. The metabolites were identified by comparing the retention time (HPLC) or retention factor (Rf) (TLC) to those of the authentic 14C standard compounds (American radiochemical Inc., St. Louis, MO).
3.2.7.2 Water soluble phase metabolites analysis
For HPLC, the mobile phase was composed of two buffers: Buffer A, containing acetonitrile/water/ethyl alcohol/aceticacid/0.83 M Sodium acetate (800/127/68/2/3, v/v/v/v), pH 3.6, and buffer B (400/400/68/53/79, v/v/v/v) [124]. A linear gradient from 0 to 100%
70
buffer B, with a slope of 5%/min, was started 15 min after injection. Flow rate was 2.7 ml/min and column temperature was maintained at 45°C. The 14C labeled metabolites: betaine, betaine aldehyde, CHOL, CDP-CHOL and PCho were identified in the water-soluble phase via comparing the retention time with the authentic 14C-labeled standards.
For TLC, a solvent system [125] was composed of methanol/0.5% NaCl/aqueous ammonium
(100/100/2, v/v/v). In the solvent system, the water-soluble metabolites were separated and identified via comparing the retention factor (Rf) with the authentic 14C-labeled standards.
The plates were exposed on an imaging storage phosphor screen (Molecular Dynamics,
Sunnyvale, CA, USA) and the exposed screens were scanned with a bio-imaging analyzer
(Typhoon Variable Mode Imager, GE, Piscataway, NJ) to detect the radioactivity.
3.2.7.3 Lipid soluble phase metabolites analysis
For HPLC, phospholipids and neutral lipids used different mobile phases. Phospholipid metabolites were analyzed with the mobile phase: acetonitrile/hexane/methanol/phosphoric acid (918/30/30/7.5,v/v/v/v) [126]. The flow rate was 1.5 ml/min. Neutral lipids metabolites were analyzed with the mobile phase hexane/isopropanol/acetic acid (100/2/0.02, v/v/v)
[127]. The flow rate was 2 ml/min. The non-radioactive phospholipid metabolites were monitored at 206 nm. The 14C-phospholipid metabolites were detected by flow scintillation analyzer and identified via comparing the retention time with the authentic 14C-labeled standards.
For TLC, phospholipid and neutral lipid metabolites were separated with a single step. An aliquot of the fraction was spotted on a silica gel G plate (Whatman, Tokyo, Japa) and
71
developed the plate in mobile phase: chloroform/methanol water (60/30/5, v/v/v) up to 7cm to separate phospholipids and then change the mobile phase to hexane diethyl ether-acetic acid (80/20/1.5, v/v/v) to migrate another 10 cm [128]. The lipid-soluble metabolites were separated and identified via as comparing the retention factor (Rf) with the authentic 14C- labeled standards.
3.2.8 [methyl-14C]-betaine uptake in WCH17 cells
3.2.8.1 Preparation of 14C-betaine.
It was prepared from [methyl-14C]-CHOL chloride in a reaction catalyzed by choline oxidase
[124]. Sodium phosphate buffer (100 µL, 0.2 M, pH 7.8), containing 1 µmol of 14C-CHOL chloride and 0.08 U of CHOL oxidase (Sigma-Aldrich, St. Louis, MO) was incubated for 1 hr at 37°C. At the end of the incubation, 400 µL of methanol and 200 µL of chloroform were added to extract the product. 100 µl of CHC13 and 100 µL of water were added to separate the aqueous and organic phases. The aqueous phase (top) containing 14C-betaine was dried down and 14C-betaine was purified by HPLC.
3.2.8.2 [methyl-14C]-betaine uptake in WCH17 cells
The experiment was conducted in the similar way as described previously [72]. Briefly, the
14 DPBS containing CaCl2 and MgCl2 (10 ml) containing 74 KBq of [methyl- C]-betaine or
[methyl-14C]-CHOL (for comparison) was added to each well. After incubation, the medium was removed and the cells were washed twice with ice-cold phosphate buffer saline (PBS).
The cells were then detached by cell lysis buffer and used to measure total uptake radioactivity in whole cells with a liquid scintillation counter. 0.1 ml of whole cell lysate was used for protein measurement. The total uptake was normalized to the total protein content.
72
3.2.9 Kinetics of choline transport in WCH17 cells in vitro
3.2.9.1 Kinetics of choline transport in WCH17 cells and effects of lithium-for-sodium replacement
The CHOL transporter assay was followed as previously described [129]. Briefly, WCH17 cells (3×105 cells) were cultured in the 24-well plate with DMEM Medium (Contains
28.5µM CHOL choride, 4,500 mg/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate; GIBCO/Invitrogen, Carlsbad, California, USA) with 10% FBS and 1% Penicillin
Streptomycin solution under a 10% CO2-humidified atmosphere at 37°C. The assay was performed after replacing the medium with the uptake medium (20 mM HEPES/Tris buffer, pH 7.4, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgCl2, and 10 mM glucose), By incubating the cells with 5 to 500 µM choline (containing [methyl-14C]-CHOL) at 37C for 5 minutes. The uptake reaction was stopped by removing the uptake medium, followed by washing with ice-cold phosphate-buffered saline (PBS) three times. The intracellular 14C radioactivity was counted in a liquid scintillation counter. The sodium dependence of the transporter was examined by using the uptake medium containing 141 mM LiCl instead of
141 mM NaCl.
3.2.9.2 Inhibition of choline transporter in WCH17 cells
The effects of CHOL transport inhibitor: 20–200 mM hemicholinium-3 (HC-3) (Sigma-
Aldrich, St. Louis, MO), 0.2 and 2.0 mM ouabain (Sigma-Aldrich, St. Louis, MO) and 0.2 and 2.0 mM dinitrophenol (Sigma-Aldrich, St. Louis, MO), were tested by adding the inhibitors to the incubation medium during the 15 min preincubation and [methyl-14C]-
73
CHOL 5 min incubation periods to repeat the CHOL transporter assay. Ouabain and dinitrophenol are metabolic inhibitors. Ouabain was used to determine the sensitivity of choline transport rates to inhibition of Na/K-dependent adenosine triphosphatase (ATPase).
The oxidative phosphorylation uncoupler dinitrophenol was used to inhibit of cellular adenosine triphosphate (ATP).
3.2.9.3 Parameter Estimation for choline transporter in WCH17 cells
The uptake rate from transporter assay were then fit by the nonlinear least-squares curve fitting algorithm [112] to the following equation including a non facilitative diffusion term:
/ / (Eq. 3.1) where [S] (µM) is CHOL concentration, [I] (µM) is HC-3 concentration, Vmax (nmol/mg protein/min) and KM (µM) are the Michaelis-Menten constants for maximal transport velocity and concentration at half-maximal velocity, respectively, KI (µM) is the inhibition constant for WCH17, and D (mL/mg protein/min) is the diffusion coefficient for nonfacilitative diffusion. The model assumes Michealis-Menten kinetics, competitive inhibition by WCH17 at the CHOL transporter, and first-order diffusion kinetics for the nonfacilitative diffusion component. The r2 statistic was used as a test of goodness of fit
[112]:
∑ (Eq. 3.2) ∑
where yi is the measured uptake rate, is the mean uptake rate; and is the model-predicted uptake rate at the ith measurement.
74
3.2.10 Assay of choline kinase in WCH17 cells
The cells were scraped and homogenized in chilled extraction buffer pH 7.5 (30 mM HEPES pH 7.5, 3% Triton X-100, 2 mM EDTA, 2% bovine adult serum, 10 µM TPCK, 10 mg/ml trypsin inhibitor, 1 µM leupeptin, 0.75 mg/ml DTT, 0.4 mg/ml PMSF). The cell homogenate was centrifuged for 10,000 x g for 20 min at 4°C. The supernatant was used for the assay as described by Ishidate and Nakazawa [130]. The assay mixture (300 µL) contained 0.1 M Tris-
14 HCl, pH 7.5, 10 mM ATP, 12 mM MgCl2, 10 µM CHOL, [methyl- C]-CHOL (0.7 MBq per assay mixture)). Samples were incubated at 37°C for 30 min. The reaction was stopped by adding 0.5 ml chloroform/methonal (2/1, v/v) solution. The reaction mix was then separated into two phase by centrifugation. The upper (aqueous) phase from this step was added 0.65 ml of 12 mM sodium phosphate (pH 7.0) to decrease the methanol concentration. The ion-pairing reagent sodium tetraphenylboron (TPB) in heptan-4-one (1 ml of a 5 mg/ml solution of TPB) was then added into the resultant mix.
After being vigorously mixed for 5 min and phase separation by brief centrifugation, the lower phase containing produced 14C-PCho from 14C-CHOL during the enzyme reaction was use to determine the radioactivity using a liquid scintillation counter. The activity of Chok was normalized to total protein content.
3.2.11 Phosphocholine production (whole-cell choline kinase assay) in WCH17 cells
Following HC-3 incubation for 30 min, cells with HC-3 containing medium were added 74
KBq [methyl-14C]-CHOL chloride per 7 cm3 flask and incubated for 1 hour. After incubation, cells were washed twice with PBS, then detached by cell lysis buffer. Cells were then resuspended 0.5 ml chloroform/methanol (2/1, v/v) solution. The mix was then
75
separated into two phase by centrifugation. The lower (aqueous) phase from this step was added 0.65 ml of 12 mM sodium phosphate (pH 7.0) to decrease the methanol concentration. The the ion-pairing reagent sodium tetraphenylboron (TPB) in heptan-4-one (1 ml of a 5 mg/ml solution of TPB) was then added into the resultant mixture.
After being vigorously mixed for 5 min and phase separation by brief centrifugation, the lower phase containing produced 14C-PCho from 14C-CHOL during the enzyme reaction was use to determine the radioactivity using a liquid scintillation counter. The activity of whole cell Chok was normalized to total protein content. WCH17 cells with non HC-3 incubation was used as a control.
3.2.12 Protein assay
Protein content was determined using the by the method of Bradford (Bio-Rad labortaries,
Inc., Hercules, CA) [94].
3.2.13 Liquid scintillation counting
Radioactivity of 14C was determined with a Beckman LS-6500 Liquid Scintillation Counter
(Beckman Coulter Inc., Fullerton, CA) and Bio-safe II (Fisher Scientific Inc., Pittsburgh, PA) as scintillation fluid. Disintegrations per minute (dpm) were obtained by correcting for background activity and efficiency based on calibrated standards. 14C counts were also corrected for quenching effects.
3.2.14 Statistical analysis
All data, unless otherwise stated, are expressed as mean ± standard deviation of the mean
(SD). The data were compared using one way analysis of variance (ANOVA) or ANOVA on 76
ranks when appropriate. All pairwise multiple comparison procedures used Tukey test.
Differences were regarded as statistically significant for p<0.05.
3.3 Results
3.3.1 Separation of the radioactive metabolites derived from [methyl-14C]-choline
(authentic 14C standards)
Under the HPLC conditions described above, typical HPLC radiochromatograms of authentic
14C standards were shown in Figure 3.2 and Figure 3.3. Using TLC method, authentic 14C standards were separated as shown in Figure 3.4 and Figure 3.5
A
.
77
B
Figure 3.2 Representative HPLC radiochromatograms of water soluble metabolites derived from [methyl-14C]-choline. (A) Authentic 14C-labeled standards: 14C-betaine, 14C-choline, 14C-CDP-CHOL, 14C-PCho; (B) Preparation of betaine from [methyl-14C]-choline. Incubation of [methyl-14C]-CHOL with CHOL oxidase to form of betaine aldehyde and betaine. Upper panel: 1-min incubation; lower-left panel: 3-min incubation. Lower-right panel: 10-min incubation. The reaction mix (100 uL) contained Sodium phosphate buffer (100 uL, 0.2 M, pH 7.8), 1 µmol of 14C-CHOL chloride and 0.08 U of CHOL oxidase. Abbreviation: Bet: Betaine; Bet Ad: Betaine aldyhyde.
78
A
B
Figure 3.3 Representative HPLC radiochromatograms of lipid soluble metabolites derived from [methyl-14C]-choline. (A) Authentic 14C-labeled standards of phoshpholipids: 14C-PI, 14C-PS, 14C-PE, 14C-PC, 14C- LPC, 14C-SM. Two small panels are the chromatograms of LPC and SM under UV light (206 nm); (B) Authentic 14C-labeled standards of neutral lipids: 14C-fatty acid, 14C-1,3-DAG, 14C- 1,2-DAG, 14C-Cholesterol. Abbreviation: SF: solvent front; PI: phosphatidylinositol; PS: phosphatidylserine; PE: phosphoethanolamine; PC: phosphoatidylcholine; LPC: lysophosphoatidylcholine; SM: sphingomyeline; 1,2-DAG: 1,2-diacylglycerol; 1,3-DAG:1,3- diacylglycerol;
79
A B Solvent Solvent front front
1 2 3 4 5 6 7 8 j i h g ef d
c b
Bet. Ad. a Origin Origin
Figure 3.4 Autoradiogram of labeled authentic 14C standards (A) radio-TLC of authentic 14C-labeled standards of water soluble metabolites. Lane 1, 14C- betaine; Lane 2, 14C-choline; Lane 3, 14C-PCho; Lane 4, 14C-CDP-CHOL; Lane 5, mix of 14C-betaine, 14C-choline, 14C-PCho and 14C-CDP-CHOL; Lane 6, incubation of [methyl-14C]- CHOL with CHOL oxidase to form of betaine aldehyde and betaine (1-min incubation); Lane 7, incubation of [methyl-14C]-CHOL with CHOL oxidase to form of betaine aldehyde and betaine (3-min incubation); Lane 8, incubation of [methyl-14C]-CHOL with CHOL oxidase to form of betaine aldehyde and betaine (10-min incubation). (B) radio-TLC of authentic 14C- labeled standards of lipid soluble metabolites. a. lysoPC; b. SM; C: PC; d: PI + PS; e: PE; f: MAG; g: 1,3-DAG; h: 1,2-DAG + Cholesterol; i: free fatty acid; j: TAG.
80
3.3.2 [methyl-14C]-Choline uptake patterns and metabolism in WCH17 cells and primary rat hepatocytes
3.3.2.1 Time course of [methyl-14C]choline uptake
7 Total Cellular Radioactivity A 6 Water Soluble Phase 10000
Lipid Soluble Phase x 5 Insoluble Phase (DNA/RNA/Protein precipitates) protein) 4 3
(dpm/mg 2
1 uptake
0 C
14 5 15304560 Incubation Time (mins)
7 Total Cellular Radioactivity B 6 Water Soluble Phase 10000 Lipid Soluble Phase x 5 Insoluble Phase(DNA/RNA/Protein precipitates) protein)
4 3 (dpm/mg 2 1 uptake
C 0 14 5 15304560 Incubation time (mins)
Figure 3.5 Time course of incorporation of [14C]choline into lipid-, water-soluble and insoluble phases (A) WCH17 cells; (B) Primary rat hepatocyes. Cells were incubated with 74 kBq [methyl- 14C]CHOL in DMEM containing 10% FBS for 5, 15, 30, 45 and 60 min. After incubation, cells were extracted into water-, lipid-soluble and insoluble phase using the Bligh and Dyer extraction method.
14C-CHOL uptake in WCH17 cells and primary rat hepatocytes was showed in Fig. 3.1.
Both cells showed increasing cellular uptake of 14C activity over time. The total uptake values in WCH17 cells were significantly higher than those in primary rat hepatocytes during
81
all the time points within 60 min (P < 0.05). Most of the radioactivity was found in the water- soluble phase. Only a small amount of lipid soluble phase and insoluble phase was found in both cells. On the other hand, WCH17 showed significantly lower uptake of [14C]betaine, a major metabolite of choline found in blood, as compared to the uptake of [14C]CHOL (Fig.
3.2).
30 14C‐Betaine 14C‐Choline 25
10000
x 20 15 (dpm/mg
10 protein) uptake
5 C 14 0 5 15304560 Incubation time (mins)
Figure 3.6 Comparison of uptake of [14C]betaine and [14C]CHOL in WCH17 cells. Cells were incubated with 18.5 kBq of [14C]betaine or [methyl-14C]CHOL in DPBS containing CaCl2 and MgCl2 for 5, 15, 30, 45 and 60 min. After incubation, cells were homogenized for radioactive counting of total cellular uptake.
3.3.2.2 Metabolism of [methyl-14C]choline in WCH17 cells and primary rat hepatocytes
4.5 PCh 4
10000 A 3.5 x 3 2.5 (dpm/mg Bet CH 2 60 1.5 45
protein) 1 30 Incubation time 0.5 15 (min) Radioactivity 5
C 0 14 0 102030405060 Retention time (min)
82
0.8 PC 0.7 B
10000
x 0.6 0.5 60 (dpm/mg 0.4 45 0.3 30Incubation time protein) 0.2 15 (min)
Radioactivity 0.1 5
C
14 0 0 5 10 15 20 25 30 35 40 45 50 Retention time (min)
0.8 C 0.7 PCh
10000
x 0.6 Bet 0.5 (dpm/mg 0.4 CH 0.3 60 45 protein) 0.2 30 15 Incubation time 0.1 Radioactivity 5 (min) C
14 0 0 1020304050 Retention time (min)
0.45 0.4 PC D 10000
0.35x
0.3 0.25 60
(dpm/mg 0.2 45
0.15 30 Incubation time 0.1
protein) (min) 0.05 5 0 Radioactivity
C
14 0 5 10 15 20 25 30 35 40 45 Retention time (min) Figure 3.7 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites during the incubation period in WCH17 cells and primary rat hepatocytes. A. Water soluble metabolites in WCH17 cells. B. Phospholipid metabolites in WCH17 cells. C. Water soluble metabolites in primary rat hepatocytes. D. Phospholipid metabolites in primary rat hepatocytes. 83
7 Betaine Choline Phosphocholine Phosphatidylcholine 6 A
10000
x 5 4
protein) 3
metabolites
2 1 choline ‐ (dpm/mg 0 14C 5 15304560 Incubation Time (mins)
7 Betaine & Betaine aldehyde Choline
6 B
10000 Phosphocholine Phosphatidylcholine
x 5 4 protein)
metabolites 3
2 1 choline ‐ (dpm/mg 0 14C 5 15304560 Incubation time (mins) Figure 3.8 Pattern of [14C]choline metabolites in the water- and lipid-soluble fraction during the 60 min incubation (A) WCH17 cells. (B) primary rat hepatocytes. Cells were incubated with 74 kBq [methyl- 14C]CHOL in DMEM containing 10% FBS for 5, 15, 30, 45 and 60 min. After incubation, cells were extracted into water-, lipid-soluble and insoluble phases using the Bligh and Dyer extraction method. The water- and lipid-soluble phases were applied to radio-HPLC and radio-TLC techniques respectively to quantify the different metabolites of [14C]CHOL.
Through quantifying the peak area in the HPLC radiochromatograms in Figure 3.7, Figure
3.8 showed that there are different metabolic patterns of methyl-14C-CHOL between WCH17 cells and primary rat hepatocyes. In WCh17 cells, radioactivity derived from [methyl-14C]-
CHOL was mainly in the water-soluble phase (87.0-91.8% of total accumulation in cells)
(Figure 3.5(A)). The major components of water-soluble metabolites were 14C-PCho as identified by radio-HPLC and radio-TLC (Figure 3.8 (A)). 14C-PCho levels increased over
84
time in WCH17 and accounted for over 94% of radioactivity in the water-soluble phase, indicating rapid phosphorylation of transported 14C-CHOL. Very small amounts of 14C-PC were found in the lipid-soluble phase. The levels of the 14C-CHOL/betaine pool were much lower than 14C -PCho. 14C-CDP-CHOL was non-detectable level.
In contrast, although over 90% of radioactivity in the primary rat hepatocytes was also found in the water-soluble phase, the major components of water-soluble metabolites were 14C-
PCho and 14C-Betaine (Figure 3.8(B)). Before 45 min time point, 14C-Betaine accounted for around 55% of radioactivity in the water-soluble phase; the rest 45% of radioactivity in the water-soluble phase was 14C-PCho. At 60 min time point, the amount of 14C-PCho kept increasing but the amount of 14C-Betaine started to decrease. Thus, the amount of 14C-PCho was slight exceeded to that of 14C-Betaine.On the other hand, very small amounts of 14C-PC were found in the lipid-soluble phase of primary rat hepatocytes. The levels of the 14C-CDP-
CHOL were also non-detectable in primary rat hepatocytes.
The amount of 14C-PCho and 14C-PC were significantly higher in WCH17 than in primary rat hepatocytes respectively (P < 0.05). On the contrary, the amount of 14C-betaine was significantly lower in WCH17 than in primary rat hepatocytes (P < 0.05).
3.3.3 Pulse and chase study and efflux in WCH17 cells and primary rat hepatocytes
Radiolabel CHOL has a rapid circulation and clearance. In order to mimic this in vivo situation, we will use a pulse and chase experiment to study the transient change of the intracellular metabolites of 14C-CHOL and the efflux.
85
1.8
1.6 PCho A 10000
x 1.4 1.2 (dpm/mg 1 Bet CH 0.8 55 40
protein) 0.6 25 0.4 Chase time 10 Radioactivity 0.2 C 0 (min) 14 0 0 102030405060 Retention time (min)
1.2 PC
B
10000 1
x 55 0.8 (dpm/mg
40 0.6 25 Chase time
protein) 0.4 10 (min)
Radioactivity 0.2
C 0 14 0 0 10203040506070 Retention time (min)
1.2 C PCh 1 Bet 10000
CH x protein)
55 0.8 40 0.6 (dpm/mg 25Chase time 0.4 10 (min) 0.2 0
Radioactivity 0 Bet. Ad. 0 1020304050 Retention time (min)
86
0.7
D 0.6 PC 10000
x 0.5 55 (dpm/mg 0.4 40 0.3 25 Chase time protein) 0.2 10 (min) Radioactivity
C 0.1 0 14 0 0 102030405060 Retention time (min)
Figure 3.9 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites in pulse and chase study in WCH17 cells and primary rat hepatocytes. (A) Water soluble metabolites in WCH17 cells. (B) Phospholipid metabolites in WCH17 cells. (C) Water soluble metabolites in primary rat hepatocytes. (D) Phospholipid metabolites in primary rat hepatocytes.
6 Total Cellular Radioactivity Lipid Soluble Phase A
10000 5
Water Soluble Phase x Insoluble Phase (DNA/RNA/Protein precipitates) 4 (dpm/mg
3 uptake
protein) 2
choline 1 ‐ C 14 0 0 10254055 Chase time (min)
87
8 B 7 10000
x 6 ml) 5
4 Total Media Activity (dpm/10 Water soluble phase 3 Lipid soluble phase 2 Insoluble phase
1 Radioactivity
0 14C 0 102030405060 Chase time (min)
5 Betaine Choline Phosphocholine Phosphatidylcholine 4.5 C 10000
4 x
(dpm/mg 3.5
3 2.5 2 protein)
metabolites 1.5
1 0.5 choline
‐ 0 C
14 0 10254055 Chase time (min)
6 Total Cellular Activity D Lipid Soluble Phase
10000 5
x Water soluble Phase (dpm/mg
4 Insoluble Phase (DNA/RNA/Protein Precipitates
3 protein) 2 Radioactovivity
1 14C 0 0 10254055 Chase time (min)
88
8 Total Media Activity E ml)
7 10000 6 Water Soluble Phase x 5 Lipid Soluble Phase (dpm/10 4 Insoluble Phase 3 2 1 Radioacitivty 0 4C 1 0 102030405060 Chase time (min)
5.0 Betaine & Betaine aldehyde 4.5 F Choline
10000 4.0
3.5 Phosphocholine (dpm/mg
3.0 Phosphatidylcholine 2.5 2.0 1.5 protein) x Metabolites 1.0 0.5 0.0 Choline ‐
C 0 10254055 14 Chase time (min)
Figure 3.10 Pulse-chase study on the metabolism of [methyl-14C]-CHOL by WCH17 cells and primary rat hepatocytes. Cells were incubated with [methyl-14C]-CHOL in DMEM with 10% FBS for 5 min. The radioactive media was removed and replaced with DMEM with 10% FBS containing 18 µM non-radioactive CHOL. The cells were then incubated further (Chase). The chase time at 0 represent the radioactivity after the initial 5-min pulse (no chase). (A) Incorporation of [methyl-14C]-CHOL into different phases in WCH17 cells. (B) The efflux of 14C radioactivity into the culture media after 5-min pulse in WCH17 cells with [methyl-14C]- CHOL. (C) Incorporation of [methyl-14C]-CHOL into different phases in WCH17 cells. (D) Incorporation of [methyl-14C]-CHOL into different phases in primary rat hepatocytes. (E) The efflux of 14C radioactivity into the culture media after 5-min pulse in primary rat hepatocytes with [methyl-14C]-CHOL. (F) Incorporation of [methyl-14C]-CHOL into different phases in primary rat hepatocytes.
89
Figure 3.9 showed the representative HPLC radiochromatograms of metabolites derived from
[methyl-14C]-CHOL in WCH17 cells and primary rat hepatocytes. In order to simulate the rapid blood circulation and clearance of [methyl-14C]-CHOL, WCH17 cells and primary rat hepatocytes were pulsed with [methyl-14C]-CHOL for 5 min followed by incubation in non- radioactive medium containing cold CHOL for 0, 10, 25, 40 and 55 min to simulate the time period of a dynamic PET scan.
There were different uptake patterns between WCH17 and primary rat hepatocytes (Figure
3.10). In WCH17 cells, the total radioactivity was decreased at first 10 min chase time and then maintained at fairly stable afterward within 50 min chase duration (Fig. 3.10 (A)). Most of radioactivity was found in water soluble phase. On the contrary, in primary rat hepatocytes, the total radioactivity remained stable at first 10 min chase time and then continuously decreased within 50 min chase duration (Fig. 3.10 (B)). This suggested that there was radioactivity released from intracellular content into culture media. However, the major radioactivity of 14C distribution in primary rat hepatocytes was still in the water soluble phase. Only small amount of radioactivity was in the lipid soluble phase and insoluble phase for both cells. Furthermore, WCH17 cells showed a significantly higher uptake than primary rat hepatocytes within 55 min chase duration (P < 0.05).
For the metabolite analysis in the pulse and chase study (Figure 3.10(C), (F)), the major metabolites in WCH17 was 14C-Pcho in WCH17 cells. The conversion from 14C-Pcho to 14C-
PC occurred slowly within 55 min chase duration (Figure 3.10(C)). There were only small amount of 14C-CHOL and 14C-betaine found in WCH17 cells. However, different
90
metabolism was observed in primary rat hepatocytes as compared to WCH17 cells (Figure
3.10(F)). The cultured hepato¢ytes efficiently incorporated and metabolized the labeled choline from the medium during the 5 min pulse period. Choline transported into the cells was rapidly oxidized to betaine or phosphorylated to PCho. Only small amount of the incorporated label was associated with free cellular choline at the end of the pulse period.
Betaine aldehyde was also found in some cases at the time of initial 5-min pulse.
Therefore, the major metabolites were 14C-PCho and 14C-betaine in primary rat hepatocytes.
14C-betaine started to continuously decrease after 10 min chase duration (Figure 3.10(F)).
14C-betaine can be released into the chase media or transferred as methyl groups to homocysteine for the synthesis of methionine, which caused the decrease of intracellular 14C- betaine. 14C-PCho was also decrease between 10 min and 25 min chase duration and then remained stable afterward. 14C-PC was slightly increased within 55 min chase duration.
In addition, 14C-PCho and 14C-PC were significantly higher in WCH17 cells than in primary rat hepatocytes. On the contrary, 14C-betaine was significantly lower in WCH17 cells than in primary rat hepatocytes.
Different metabolic pathways between WCH17 cells and primary rat hepatocytes were also confirmed in figure 3.10 (B), (E). Figure 3.10 (B) and (E) showed the activity in the media during the chase period that had effluxed. In the chase media of WCH17 (Fig. 3.10(B)), the total activity remained fairly stable from 10 min to 55 min chase duration. The radioactivity was located in water soluble phase. The components of water soluble phase of chase media
91
were found by TLC to consist of 14C-CHOL and a very small amount of 14C-betaine. In contrast, in primary rat hepatocytes, the major proportion of the radioactivity in the betaine pool at the end of the pulse period was recovered in the culture medium during the chase period. At the same time, radiolabeled betaine was likely transferred as methyl groups to homocysteine for the synthesis of methionine.
Our results indicated that WCH17 has a higher uptake of CHOL as compared to primary rat hepatocytes, which will contribute to the imaging contrast during the PET imaging of HCC with radiolabeled CHOL tracer. The major metabolites in WCH17 during the chase period was 14C-PCho. Thus, 14C-PCho was believed the major contributor to the uptake contrast within HCC tumor cells seen in PET imaging. On the other hand, choline transported into the primary hepatocytes was rapidly oxidized to betaine or phosphorylated to Pcho. This will increase the background signal in the surrounding hepatic tissue as compared to the HCC region during the PET imaging with CHOL.
92
3.3.4 The effect of oleic acid on the metabolism of [14C]choline in WCH17 cells and primary rat hepatocytes
2.5 PCho A 10000 2 x Bet CHOL 55 1.5 protein) 40 1 25Chase time (dpm/mg
0.5 10 (min)
0 0
Radioactivity 0 102030405060
C 14 Retention time (min) 2.5 PC B 2 10000
x 55 1.5 40 1 Chase time (dpm/mg
25 (min) 0.5 10 protein) 0 0 Radioactivity
0 102030405060 C 14 Retention time (mins)
2.5 C Bet PCh 10000
2 55 x (dpm/mg 1.5 40
1 25 Chase time protein) (min) 0.5 10 Radioactivity
C
14 0 0 0 1020304050
Retention time (min)
93
2.5 PC D 2 55 10000
x (dpm/mg 1.5 40 1 25 Chase time protein) 0.5 10 (min)
Radioactivity 0 0 0 102030405060 Retention time (mins) Figure 3.11 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites in pulse and chase study in oleic acid treated WCH17 cells and primary rat hepatocytes. (A) Water soluble metabolites in oleic acid treated WCH17 cells. (B) Phospholipid metabolites in oleic acid treated WCH17 cells. (C) Water soluble metabolites in oleic acid treated primary rat hepatocytes. (D) Phospholipid metabolites in oleic acid treated primary rat hepatocytes.
7 Total activity Lipid soluble phase A 6
10000 Water Soluble Phase
x 5 Insoluble Phase (DNA/RNA/Protein precipitates) 4 uptake(dpm/mg 3 protein) 2 choline ‐ 1 14C 0 0 10254055 Chase times (mins)
94
8 B 7 10000
x 6 5 Total Media Activity ml)
4 Lipid Soluble Phase 3 Water Soluble Phase 2 (dpm/10 Insoluble Phase (DNA/RNA/Protein precipitates) 1 0 0 102030405060 Radioactivity Chase time (min) 14C 7 Betaine Choline Phosphocholine Phosphatidylcholine C 6 10000
x
protein) 5
4 3 (dpm/mg
2 1 activity
0 14C 0 10254055 Chase time (mins)
7 Total Cellular Activity Lipid Soluble Phase D 10000 6
x Water Soluble Phase 5 Insoluble Phase (DNA/RNA/Protein Precipitates) (dpm/mg 4 3 protein) 2 Radioactivity
1 C
14 0 0 10254055 Chase time (min)
95
8 Total Media Activity E
ml) 7
10000 Water soluble phase
x 6 Lipid soluble phase 5 Insoluble phase (dpm/10 4 3 2 1 Radioactivity
C
14 0 0102540 Chase time (min)
7 Betaine & Betaine aldehyde Choline F
6 Phosphocholine Phosphatidylcholine 10000
x 5 (dpm/mg
4
3 protein) 2 Radioactivity
C 1 14 0 0 10254055 Chase time (min)
Figure 3.12 Effect of oleic acid on the phosphatidylcholine synthesis. Cells were incubated with [methyl-14C]-CHOL in DMEM with 10% FBS for 5 min. The radioactivie media was removed and replaced with DMEM with 10% FBS containing 1%
BSA/1 mM sodium oleated (oleic acid-treated). The cells were then incubated further
(chase). The chase time at 0 represent the radioactivity after the initial 5-min pulse. (A)
Incorporation of [methyl-14C]-CHOL into different phases in WCH17 cells. (B) The efflux of
14C radioactivity into the culture media after 5-min pulse in WCH17 cells with [methyl-14C]-
CHOL. (C) Incorporation of [methyl-14C]-CHOL into different phases in WCH17 cells. (D)
Incorporation of [methyl-14C]-CHOL into different phases in primary rat hepatocytes. (E)
96
The efflux of 14C radioactivity into the culture media after 5-min pulse in primary rat hepatocytes with [methyl-14C]-CHOL. (F) Incorporation of [methyl-14C]-CHOL into different phases in primary rat hepatocytes.
In the pulse and chase study, the addition of 1 mM oleic acid into the chase media increased the incorporation of 14C-CHOL into PC in both cells (Figure. 3.12 vs. Figure 3.10) with a corresponding decrease in the radioactivity in PCho in both WCH17 cells and primary rat hepatocytes (Figure. 3.12 (C), (F)). The total amount of radioactivity in both cells did not change appreciably as compared to untreated cells respectively (Figure. 3.12 vs. Figure 3.10).
Therefore, the results indicate that oleic acid caused an increase in the conversion of Pcho to
PC. In primary rat hepatocytes, betaine was also released from the intracellular content into culture media with the oleic acid treatment (Figure 3.12 (E)). The TLC confirmed that the major component in culture media of primary rat hepatocytes were betaine and CHOL.
Hence, the rate of loss of label from PC appears to govern the rate of accumulation of label in
PC. Since label does not accumulate in CDP-CHOL, the enzyme that converts PC into CDP- choline, CTP:phosphocholine cytidylyltransferase (CCT), must catalyze the rate-limiting step for the incorporation of CHOL into PC.
3.3.5 Kinetics of choline transport system in WCH17 Cells
3.3.5.1 Kinetics parameters of the choline transport system in WCH17 cells
To determine wether WCH17 cells take up CHOL by a carrier-mediated process, we investigated the kinetics of CHOL uptake in WCH17 cells. Uptake rates were measured over a substrate concentration range of 5-640 µM. The dependence of [methyl-14C]-CHOL uptake
97
by WCH17 cells on CHOL concentration is shown in Figure 3.13. The uptake velocity into the cells was fitted to a Michaelis-Menten model that included both facilitative and non- facilitative diffusion transport terms (Eq. 3.1). The facilitative component showed low affinity (KM = 28.59 ± 6.75 µM) (Table 3.1). Transport by nonfacilitative diffusion was also evident and became dominant at choline concentrations greater than 500 µM. Facilitative transport was maintained after replacement of sodium ions with lithium ions in the culture medium from 0-120 µM CHOL (Figure 3.13 (B)). The Vmax value decreased to 0.716 ±
0.067 pmol/mg prot/min, the KM value decreased to 9.558 ± 2.895 uM, indicating that the uptake mechanism was sodium dependent (Figure 3.13).
The time course of [methyl-14C]-CHOL uptake using NaCl media or LiCl media in Figure
3.13 (C) showed WCH17 cells can linearly take up more [methyl-14C]-CHOL in NaCl media as compared to that in LiCl media at least up to 30 min. It was obvious that there was dependence of CHOL uptake on Na+.
4.5 Total A 4 facilitative Non‐facilitative 3.5 Experiment value prot/min) 3 2.5 2 (pmol/mg
1.5 Rate 1 Km = 28.59 ± 6.75 µM Vmax =1.856 ± 0.19 pmol/mg prot/min 0.5 -6
Uptake D = 3.5 ± 0.33 × 10 ml/mg prot./min 0 0 100 200 300 400 500 600 700 [Choline] (µM)
98
5 Total 4.5 Facilitative B 4 Non‐facilitative Experiment value prot/min)
3.5 3 2.5 (pmol/mg 2 Km = 9.558 ± 2.895 uM 1.5
Rate Vmax =0.716 ± 0.067 pmol/mg prot/min
1 D = 6.07 ± 0.16× 10-6 ml/mg prot./min 0.5 Uptake 0 0 100 200 300 400 500 600 700 [Choline] (µM)
2500 NaCl LiCl C y = 62.953x + 141.06
cells) 2000
5 R² = 0.9791
1500 (DPM/3×10 1000
y = 23.599x + 204.96 uptake
500 R² = 0.954 Cell 0 0 5 10 15 20 25 30 35 Time (min)
Figure 3.13 Kinetics of [methyl-14C]-choline uptake in WCH17 cells and effects of lithium-for-sodium replacement. (A) Standard medium containing 141 mM sodium. (B) Lithium medium where sodium was replaced with 141 mM lithium. Lines show the fit of the modified Michaelis-Menten equation (Eq. 4.1) to the data. (C) Time course of [methyl-14C]-CHOL uptake using NaCl media or LiCl media
99
Table 3.1 Parameter estimation by nonlinear least-squares fit to [methyl-14C]-choline uptake in cultured WCH17 cells
HC-3 Non-facilitative Facilitative transport Goodness inhibition diffusion parameters of fit K parameter Conditions I Vmax D × 10-6 (ml/mg r2 (pmol/mg Km (µM) K (µM) I prot./min) prot/min)
Control 1.856 ± 0.19 28.59 ± 6.75 __ 3.5 ± 0.33 0.9843
Li+ for Na+ Replacement 0.716 ± 0.067* 9.558 ± 2.895* __ 6.07 ± 0.16* 0.9954
0-200 µM HC-3 1.339 ± 0.079 15.03 ± 2.877* 23.8 ± 5.472 5.9 ± 0.17* 0.9951
: HC-3: hemicholinium-3; KI: enzyme inhibition constant; KM Michaelis constant; Vmax: maximum velocity. *P<0.05 versus control.
3.3.5.2 Effect of hemicholinium-3 inhibitor on the choline transporter system in
cultured WCH17 cells
The effect of HC-3, a potent inhibitor of transporter, on the CHOL uptake was examined.
The cell culture conditions were the same as above. HC-3 inhibited transporter facilitated
choline uptake in a concentration-dependent fashion (KI = 23.8 ± 5.472 µM) but did not
inhibit free diffusion at higher concentrations of CHOL (Table 3.1 and Figure 3.14).
100
6 Control 5 20 µM HC‐3 prot/min) 4 200 µM HC‐3
3 (pmol/mg
2 Rate
1 Uptake 0 0 100 200 300 400 500 600 700 [Choline] (µM)
Figure 3.14 Inhibition of [methyl-14C]-choline uptake in WCH17 cells by hemicholinium 3. Lines show the fit of the modified Michaelis-Menten equation (Eq 4.1) to the data.
The fitting of our kinetic data to the model of Michaelis-Menten kinetics, competitive inhibition by HC-3, and nonfacilitative diffusion gave a very high r2 values (Table 3.1) that indicated goodness of fit. The resultant KI suggested that the potency of HC-3 to inhibit
CHOL transport in WCH17 cells is intermediate between the higher potency described for hCHT1 inhibition (<1 µM) and the markedly lower potency for hOCT1 or hOCT2 inhibition
(<250 µM) [131-133].
3.3.5.3 Effect of other inhibitors on the choline transporter system in cultured WCH17 cells
To further characterize the mechanism of CHOL transport, it was of interest to determine the sensitivity of transport rates to inhibition of Na+/K+-dependent adenosine triphosphatase
(ATPase) using ouabain (0.2–2 mM) and inhibition of cellular adenosine triphosphate (ATP) production using the oxidative phosphorylation uncoupler dinitrophenol (0.2–2mM) (Table
101
3.2). It was anticipated that a diminishing of the negative membrane potential with ouabain would cause a decrease in CHOL transport rate because the positively charged choline molecule travels in the same direction as the charge gradient across the cell membrane. A second method used to decrease the membrane potential was replacement of sodium ions with potassium ions in the culture medium (Table 3.2). Metabolic inhibition with the uncoupler dinitrophenol was anticipated to also decrease cell membrane CHOL transport, either by decreasing the availability of intracellular ATP needed by an active transport mechanism or by decreased activity of the Na+/K+-dependent ATPase and the consequent effects of this inhibition mentioned above.
WCH17 cells were subjected to the inhibitors during a preincubation period of 5minutes
(unless noted otherwise) followed by an incubation period with [methyl-14C]-CHOL of 5 minutes. CHOL uptake was decreased by replacement of sodium ions with potassium ions or sucrose in the culture medium (P<0.05).
Ouabain can significantly inhibited transporter-facilitated uptake (5 mM CHOL) but not the free diffusion (640 mM CHOL) (P<0.05). The inhibition was dependent on ouabain concentration and was somewhat greater in magnitude at the low concentration of CHOL (5
µM vs. 640 µM CHOL). The sensitivity of low concentration CHOL uptake to ouabain is consistent with the inhibition of uptake of positively charged CHOL molecules as the inwardly directed negative force of the membrane potential is diminished.
102
Metabolic inhibition with the oxidative phosphorylation uncoupler dinitrophenol resulted in a concentration dependent decrease in choline uptake, but the differences were relatively smaller than the inhibition with ouabain.
These data confirmed the importance of a facilitative transport process for radiolabeled
CHOL uptake into tumor cells that was most sensitively inhibited with a reduction in Na/K- dependent ATPase activity by ouabain.
Table 3.2 Effect of other inhibitors on the uptake of [methyl-14C]-choline in cultured WCH17 Cells
Choline Uptake (pmol/mg protein/min) Incubation medium At 5 µM Choline At 640 µM Choline Control medium 0.37 ± 0.01 3.95 ± 0.29
+ + ,a Na replaced by K 0.15 ± 0.01* 4.64 ± 0.31
+ + ,b Na replaced by K after 0.19 ± 0.0015* 4.09 ± 0.25 preincubation for 60 min
Na+ replaced by 0.25 M 0.44 ± 0.04*,a,b 4.59 ± 0.26 surcose + 0.2 mM Ouabain 0.24 ± 0.01*,c 4.10 ± 0.52
+ 2 mM Ouabain 0.03 ± 0.0018*,c 4.28 ± 0.26
+ 0.2 mM 2,4- 0.09 ± 0.0025*,d 4.21 ± 0.12 dinitrophenol + 2 mM 2,4-dinitrophenol 0.03 ± 0.001*,d 4.36 ± 1.10
Control medium: 10mM HEPES/Tris buffer (pH 7.4), 141mM NaCl, 4 mM KCl, 2.8 mM
CaCl2, 1 mM MgCl2, 10 mM glucose. *P <0.05 versus control. a,b,c,d: the pairwise multiple comparison (tukey test), P<0.05
103
3.3.6 Choline kinase activity in WCH17 cells
In order to investigate the association between ChoK activity and HC-3 inhibition, ChoK activity was assayed when the 20 µM HC-3 was added into assay buffer. The cytosolic ChoK activity was decreased by 3 folds in WCH17 cells when 20 µM HC-3 existed (Figure 3.15).
Whole cell ChoK activity was also evaluated by incubating WCH17 cells with [methyl-14C]-
CHOL with or without different concentration of HC-3 and measuring intracellular PCho levels by HPLC. Treatment of WCH17 cells with HC-3 produced a concentration-dependent decrease in PCho production (Figure 3.16).
7
6
10000 WCH17
x 5 WCH17+20uM HC‐3 4 (pmol/mg
3
prot/min) 2 produced 1
Pcho 0
Choline Kinase Figure 3.15 Choline kinase activity in WCH17 cells. WCH17 cells were homogenized and centrifuged to producer cytosolic fraction. The ChoK activity in cytosolic WCH17 cells was assayed with or without adding 20µM HC-3.
104
2 Total uptake Pcho 1.5 CHOL+ BETA 100000
x PC (dpm/mg
mins)
1
0.5 prot/30 0 Radioactivity WCH17 WCH17 + 2µM HC‐ WCH17 + 20µM WCH17 + 200µM 3 HC‐3 HC‐3
Figure 3.16 Whole cell choline kinase activity in WCH17 cells. WCH17 cells were preincubated with different concentration of HC-3. WCH17 cells were incubated 30 min with [methyl-14C]-CHOL containing different concentration of HC-3. After incubation, the radioactive media was removed and WCH17 cells were extracted into different metabolites. The metabolites were identified by HPLC.
3.4 Discussion
It has been demonstrated that malignant transformation of cancer cells is associated with the elevated levels of CHOL metabolites [134, 135]. In addition, CHOL phospholipids and their metabolites have been suggested to be involved in signal transduction and carcinogenesis
[105]. This elevation has been particularly useful for differentiating between malignant and benign lesions using PET imaging with 11C-CHOL tracer, because malignant lesions contain a significantly higher level of CHOL metabolites than the benign or normal tissue [136-140].
However, to fully exploit the potential of early detection of HCC using PET imaging with
11C-CHOL, it is necessary to elucidate the underlying transport mechanism and metabolic fate of radiolabeled CHOL, which has not been investigated. This study characterized the class of 14C-CHOL metabolites and the CHOL transport mechanism in HCC to help define the mechanisms responsible for CHOL uptake seen on the PET images of HCC, which elucidated the role and impact of CHOL in interpreting the PET imaging data.
105
3.4.1 Metabolic fate of radiolabeled choline
PCho is a precursor of PC, an important component of cell membrane. The level of PCho in
WCH17 cells was found significantly higher than in primary rat hepatocytes (Figure 3.8 (A) vs. (B); Figure 3.10 (C) vs. (F); Figure 3.12 (C) vs. (F)). Our data suggested that radiolabeled
CHOL tracer in the WCH17 cells was mainly metabolized along a single pathway (CDP-
CHOL pathway) during the 60 min incubation time (time course study) and 55 min chase time (pulse and chase study). Approximately 90% radioactivity was accumulated as PCho in
WCH17 cells. We found that radiolabel in PC only account for a very small amount within
60 min. The pool of 14C-betaine and 14C-CHOL was much smaller than 14C-PCho. Thus, this suggested that the CCT step was rate-limiting for PC synthesis in HCC. These finding suggested that the major metabolite in HCC cells observed in PET imaging is radiolabeled
PCho.
However, rapid synthesis of betaine and phosphorylation to PCho by basal ChoK activity were found in primary rat hepatocytes despite a significantly lower total uptake of 14C-CHOL in primary rat hepatocytes as compared to WCH17 cells. Betaine is synthesized from CHOL through intermediate betaine aldehyde by the enzymes CHOL dehydrogenase and betaine aldehyde dehydrogenase. Betaine synthesis and metabolism may be important for scavenging methyl groups from CHOL [141, 142]. Sequential demethylation of betaine to form glycine produces one-carbon fragments. The resulting methyl groups are used for regeneration of methionine (Figure 3.1). In the meantime, betaine also appears to have an osmoregulatory effect in the kidney and liver [143, 144].
106
Betaine was released into the cell culture media in primary rat hepatocytes during the pulse and chase study. This corresponds with what is observed in vivo, as a study using TLC and
HPLC of blood samples from patients undergoing [methyl-11C]-CHOL PET scans showed that the major metabolite in the blood was betaine [110]. However, we only found a little amount of 14C-betaine in WCH17 cells as compared to primary rat hepatocytes. It might indicate that the synthesis of betaine was impaired in HCC cells. Taken together, it therefore appears that the phosphorylation of CHOL is augmented and oxidation of CHOL is suppressed in the course of malignant transformation of HCC cells.
On the other hand, after an initial rapid metabolism in the blood circulation, 11C-Betaine produced from 11C-CHOL showed high plasma level and the percentual amounts of 11C-
CHOL and 11C-betaine in human arterial blood plasma remained nearly constant after 20 min
[110]. In this study, WCH17 cells showed much lower uptake of 14C-betaine (Figure 3.6).
Thus, because of peripheral metabolism, the time course of total radioactivity in the arterial blood does not represent the true input function for the mathematical model. Our results suggest that 11C-betaine plays little part in the accumulation of the radioactivity and should be subtracted from total circulation radioactivity in order to get a true input function for liver uptake.
3.4.2 Choline incorporation to lipids
It has been known that fatty acids probably stimulate PC synthesis in the liver [145-147]. The acceleration of this reaction was clearly demonstrated by pulse-chase studies containing
1mM oleic acid with cultured hepatocytes and WCH17 cells correlated with increased
107
biosynthesis of PC. The proposed mechanism for this phenomenon is due to the manipulation of CCT translocation between cytosol and ER by oleic acid.
It is believed that CCT exists in cytosol in an inactive, phosphorylated form [145-147]. The enzyme can be translocated to ER by dephosphorylation or the phosphorylated form could be bound to the membrane in the presence of oleic acid or diacylglycerol. Once bound to membrane the lipids in the membrane cause and activation of CCT. In our study, it is obvious that 1mM oleic acid accelerated the 14C-PCho converted to 14C-PC without 55 min chase time for both WCH17 cells and primary hepatocytes.
3.4.3 Choline transport
The high levels of PCho was believed correlated with up-regulation and increased activity of
ChoK [148]. In the meantime, high choline transport was also suggested as the cause for the elevated levels of PCho in cancer [104]. As an organic cation, CHOL is a substrate for carriers of the family of organic cation transporters, which is a precursor for the hepatic synthesis of phospholipids [149]. Hepatic choline levels are approximately 100-fold greater than blood concentrations, and de novo choline synthesis is minimal, suggesting the existence of a transport mechanism is minimal, suggesting the existence of a transport mechanism [150].
Although there is growing evidence of the importance of CHOL transport in tumor CHOL metabolism as a rate-limiting step for CHOL uptake, the nature and properties of the CHOL transporter(s) in the cell membrane of cancer cells are relatively poorly understood. Much
108
work has been performed to genetically identify and clone the CHOL transporters present in brain, liver, kidney, fibroblasts, and various peripheral tissues [132, 133, 151-153], but there are currently only limited data on the CHOL transport process in tumor cells [154-161].
Preliminary transport kinetic measurements in several human cancer cell lines showed a range of Michaelis constant (KM) values (1–60 µM) for choline that was in the same range as physiologic concentrations of choline in plasma (5–50 µM), with most values falling between the neuronal high-affinity process (hCHT1) and the lower-affinity human choline transporter- like protein (hCTL) 1 and human organic cation transporter (hOCT) family of transporters present in peripheral tissues.
Our study showed at low concentrations in the media CHOL is taken up by WCH17 cells mainly by a facilitative transport procedure. At concentra-tions above 500µM simple diffusion becomes the pre¬dominant mode of entry of choline into the cell. But simple diffusion probably does not play a major role in the uptake of bolus injected radiolabeled
CHOL tracer under natural conditions, since the rate of simple diffusion becomes significant only at relatively high concentrations.
The facilitative transport system has the characteristics of low affinity (KM >10 µM) and sodium dependence, distinguishing it from the high-affinity, sodium-dependent system of the central nervous system (hCHT1) and the low-affinity, sodium-independent systems (hOCTs) in liver and kidney. There are substantial differences reported in the KM values for CHOL transport in various cancer cell lines, although, in general, the KM values are intermediate between those of hCHT1 and the hOCT family of transporters. On the other hand, HC-3
109
inhibits CHOL transport in WCH17 cells with a substantially higher potency than for CHOL transport in Krebs II ascites cells (KI = 160 µM) [154, 158].
Ouabain, an inhibitor of Na/K-dependent ATPase, caused a concentration-dependent reduction in CHOL accumulation in the WCH17 cells. This might because reductions in the amplitude of the negative membrane potential caused by ouabain results in decreased influx
(via facilitative transport and nonfacilitative diffusion) and/or increased efflux of choline cations from the cell [112].
The carrier-mediated transport system for CHOL in non-cholinergic mammalian tissues
(cells) including WCH17 cells and primary rat hepatocytes were summarized in Table 3.3.
The rate of PC biosynthesis does not appear to be influenced significantly by the rate of
CHOL in most nonproliferating cells [147, 162]. Also, for these nonproliferating cells, CDP-
CHOL pathway (phosphorylation to PCho) and oxidization to betaine both exist. However, in both Ehrlich ascites [159] and Novikoff hepatoma cells [156], CHOL was immediately converted to PC without detectable accumulation of other CHOL metabolites (CDP-CHOL pathway alone). Consequently, the apparent Km values for CHOL in PC synthesis were the same as Kt values for CHOL in CHOL transport. Thus, CHOL transport may limit PC biosynthesis in actively proliferating cells. That might be the same situation in WCH17 cells.
In addition, there are also evidences about the existence of a specific CHOL carrier in the inner membrane of normal rat liver mitochondria, which is responsible for the betaine generation from CHOL oxidization [163-165]. The transporter showed saturated kinetics at
110
high membrane potential with a Km of 220 µM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 °C. At physiological concentrations of CHOL, the rate of CHOL uptake by the transporter showed a linear dependence on membrane potential; uptakes distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all nalogues of choline, are high affinity competitive inhibitors of CHOL transport with Ki values of 17, 55, 15, and 127 µM, respectively. The CHOL transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.
Therefore, the significance of the role of CHOL transport also follows from the success of
PET imaging of various cancers with 11C-CHOL or 18F-CHOL (FCH) despite an rapid blood clearance of tracer (2 minutes) [112]. The adequacy of uptake of FCH to allow visualization in PET images of primary and metastatic prostate cancer tumors in the presence of rapid blood clearance of tracer suggested a high dependence of tumor uptake on tumor perfusion and efficient transport from the blood into tumor cells [112, 166, 167]. The radioactivity concentration in tumor at later times (10 minutes post-injection) may reflect intracellular metabolic events. For example, it has been speculated that the maintenance of radioactivity concentration in WCH17 cells in the presence of a slow clearance of radioactivity may reflect lower phosphatase activity in HCC (Figure 3.10 (B)).
111
In this study, we presented an in vitro mechanistic study to compare CHOL transport and metabolism of radiolabeled CHOL in WCH17 well-differentiated HCC cells and primary rat hepatocytes. Active transport and diffusion are major mechanism for the CHOL uptake across the cell membranes. The intracellular metabolism of CHOL in the liver is composed of two major pathways: (1) synthesis of PC via CDP-CHOL pathway; (2) oxidation to produce the methyl donor betaine. CHOL metabolism and CHOL-derived metabolites can undergo alterations during the malignant transformations. Our results suggested that enhanced CHOL transport and augmented synthesis of PCho are dominant mechanisms responsible for the enhanced uptake in HCC cells during PET imaging. In contrast, betaine and PCho were two major metabolites in primary rat hepatocytes. High level of betaine and basal level of PCho will contribute the high background uptake seen in PET imaging of HCC with [methyl-11C]-CHOL. However, incorporation of PCho into PC was slow for both cells.
112
Table 3.3 Characteristics of carrier-mediated choline transport system in mammalian cells
Intracellular fate of choline [when Apparent Cells Specific features and notes incubated at Ref Kt (µM) indicated concentration] Woodchuck 28.59 ± 6.75 Na+ dependent; Competitive PCho (90%) hepatoma inhibition by HC-3, ouabain, CDP-CHOLPC WCH17 dinitrophenol at low [3.6µM] concentration of CHOL; Simple diffusion : [CHOL]> 500 µM.
Rat Kidney ATP and Na+ dependent; Betaine (80%); [168] cortex inhibition by HC-3 and other PCho (10%) [200 structural analogues of µM] CHOL
Rat liver 170 Simple diffusion: Betaine (60%); [169] [CHOL]>300500 µM. in the PCho (30%); perfusate CHOL (10%) [5- 125 µM]
Rat 12 Nonsaturable uptake: Betaine (57%); [170, hepatocytes [CHOL]>40µM in the culture PCho (33%); 171] media; Depression by a CHOL (10%) [40 cAMP analogue or µM] aminophylline
Neuoblastoma 2.1-4.5 Na+ independent, HC-3 PCho (70-95%)[50 [157] cells (Starved); insensitive; temperature µM]; sensitive; 10.2-20.7 CHOL (1-4%) (unstarved) DNP and oubain sensitive
113
Krebs II 36-46 Na+ and temperature PCho (90%) [0.4- [154, ascites cells sensitive; competitive 1mM] 158] inhibition by ethanolamine and HC-3; inhibition by structural analogus of CHOL; ouabain and iodoacetamide sensitive; DNP insensitive; a cyclic transport mechanism
Y-79 0.93 Mixed type inhibition by PCho (90%) [155] retinoblastoma ethanolamine and HC-3; CDP-CHOLPC cells inhibition by monomethyl- [21.5µM] and dimethylethanolamine Novikoff 4-7 Simple diffusion: PCho CDP- [156, hepatoma [CHOL]>20µM in the culture CHOLPC 172] cells media; competitive inhibition [20µM] by phenethylalcohol; uptake PCho (95%) [100 process is rate limiting for PC µM] synthesis with extracellular CHOL concentration below 20µM
Hep G2 11 (Low PCho (95%) [173, concentration CDP-CHOLPC 174] of CHOL)
347 (high concentration of CHOL)
114
Chapter 4 Imaging Lipid Synthesis in Hepatocellular Carcinoma
Correlated with Metabolites Study in vivo
4.1 Introduction
Hepatocellular carcinoma (HCC) is the fifth most common tumor and the third most common cause of cancer death worldwide. The survival of HCC induced by hepatitis B virus (HBV) infection is still dismal (< 3 month). [175]. In U.S.A., it is the tumor with the largest increase in incidence over the last 10 years. This increase is caused, in part, by the epidemic of hepatitis B and C viral infections, which can lead to cirrhosis and HCC. The incidence and death rates are similar indicating the overall poor survival of HCC. Imaging HCC in the early stage may bring patients the benefit of curative treatments.
Positron emission tomography (PET) using 2-[18F]-2-deoxy-D-glucose (FDG) as a probe has been shown to be an accurate technique for tumor detection, staging and monitoring of therapy in a number of malignant tumors [85]. However, FDG does not show uptake in well-differentiated HCC, an early stage of HCC, which led to a high false negative rate. [101, 176-178].
Recently, 31P magnetic resonance spectroscopy (MRS) in vivo and in vitro has revealed an elevated level of phosphatidylcholine (PC) in tumors, which is the most abundant phospholipid in the cell membranes of all eukaryotic cells [134, 179-185]. It is believed that this elevation is due to the increased uptake of choline (CHOL), a precursor of the biosynthesis of PC [104, 186-189]. C-11 labeled CHOL has been reported as a PET probe for tumor detection [190]. Malignant tumors may show a high proliferation and increased metabolism of
115
cell membrane components, which will lead to an increased uptake of choline [191]. In order to investigate the utility of [methyl-11C]-CHOL for imaging well-differentiated HCC and further elucidate the mechanism of tracer accumulation, we studied CHOL PET imaging in woodchuck model of
HCC and the metabolic fate of CHOL in woodchuck model of HCC during PET imaging.
As shown in Figure 4.1, there are three major potential metabolic pathways of 11C-CHOL. After transported into the cells by CHOL transporter, CHOL can be phosphorylated, oxidized or acetylated. In the CDP-CHOL pathway, CHOL can be phosphorylated to phospholcholine (PCho) by choline kinase (ChoK). PCho is then trapped intracellularly. PCho can be converted to CDP-CHOL with the catalysis of CTP:phosphocholine cytidylyltransferase (CCT) and further incorporate into PC
(lecithin). In neuron cells, CHOL can also be coverted to acetylcholine which is an important neurotransmitter. In liver and kidney, CHOL can be oxidized to betaine. Betaine is an osmolyte and a methyl group donor. As an organic osmolyte in the cell, betaine is accumulated or released by cells in order to maintain cell volume homeostasis [108]. It can also provide the methyl group to methionine and further produce s-adenosylmethionine (SAM). SAM can eventually incorporate into PC through reacting with phosphoethanolamine (PE) in the PE methylation pathway. The up-regulation of rates of CHOL uptake and phosphorylation in certain malignancies has motivated the development of C-11 and F-18 labeled CHOL analogues for noninvasive detection of cancer using PET [166, 192].
The increased levels of PCho and ChoK activity were found in tumors [106, 193]. Thus, he increased choline uptake in tumor cells can be explained by up-regulation of CHOL kinase and/or CHOL transporter. However, little radioactivity was observed in the lipids fraction containing PC isolated from cultured cancer cells with 11C or 18F-CHOL [166, 192]. It is 116
not clear if there is the same uptake mechanism for PET imaging of HCC with [11C]-CHOL in vivo. In this study, first of all, we evaluated the utility of [11C]-CHOL for imaging of HCC in comparison to FDG. Secondly, we characterized the class of CHOL metabolites in HCC to explore the mechanisms responsible for CHOL uptake seen on the PET images of HCC, which defines the role and impact of CHOL in interpreting the PET imaging data. PET/CT findings were correlated with metabolites analysis in vivo, which helps to explain the heterogenous uptake of radiolabeled CHOL in HCC.
In this work, the eastern woodchuck (Marmota monax) was used as animal model for the study. WHV infection induced woodchuck model of HCC is an interesting animal model of human HCC. It has been used for preclinical evaluation of efficacy and safety of antiviral drugs, chemoprevention research and assessment of diagnostic imaging technology. For example, it has been effectively for the development of new imaging agents for enhancement of detection of hepatic neoplasms by ultrasound and magnetic resonance imaging (MRI).
Woodchuck Model of HCC harbors a DNA virus - Woodchuck hepatitis virus (WHV).
WHV is a member of the family Hepadnaviridae, genus Orthohepadnavirus, of which human
HBV is the prototype. Like HBV, WHV infects the liver and can cause acute and chronic hepatitis. Furthermore, chronic WHV infection in woodchucks usually leads to development of HCC within the first 2–4 years of life. Thus, the WHV viral infection-induced HCC in the woodchucks is regarded as a natural occurring animal model of human HCC with similar pathology and natural history [52]. A well-differentiated woodchuck HCC cell line WCH17 was also used for the mechanistic study.
117
118
Figure 4.1 Metabolic fate of [methyl-11C]-choline.
118
4.2 Materials and Methods
4.2.1 Materials
All chemical reagents used were obtained from Sigma Chemicals (St. Louis, MO) unless otherwise stated. [Methyl-14C]-CHOL chloride (specific activity 2.035 GBq/mmol) and
[Methyl-14C]-PCho (specific activity 2.035 GBq/mmol) were obtained from American radiochemical Inc. (St. Louis, MO). PC (100mg/ml chloroform, from egg yolk, Type X1-E) and oleic acid were purchased from Sigma Inc. (St. Lois, MO). Liver Perfusion Medium,
Liver Digest Medium, L-15 Medium, Hepatocyte Wash Medium, Hepatocyte Wash Medium,
20% NYCODENZ®, Williams' Medium E, Hepatocyte SFM, Hexobarbital, Dulbecco's
Modified Eagle's Medium, Penicillin Streptomycin were obtained from Invitrogen Co.
(Carlsbad, CA). WCH17 cell line was purchased from American Type Culture Collection
(ATCC) (Manassas, VA). Organic solvents were purchased from Fisher Scientific (Pittsburgh,
PA). Dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylethanolamin (DOPE), lysophosphatidylcholine (lysoPC) were purchased from Avanti Inc. (Birmingham, AL).
4.2.2 Animals
All procedures in this study followed the guidelines and recommendations of the Institutional
Animal Care and Use Committee (IACUC) of Case Western Reserve University (Cleveland,
OH). Fourteen chronic WHV-carrier and one normal (control) woodchucks with HCC obtained from Cornell University (Ithaca, NY) and Northeastern Wildlife, Inc. (Harrison, ID) weighing between 2.3 and 5 kg were used in this study. The animals were fasted for twelve hours before imaging. Otherwise they had access to water and food (Teklad laboratory diets for rodents 8864; Madison, WI) per normal husbandry. The woodchucks were initially
119
sedated with an intramuscular injection of 5 mg/kg of Xylazine and 50 mg/kg of Ketamine. A sublingual catheter was inserted for intravenous injection of the radiolabeled compounds.
When this injection route failed, an incision was made in the upper thigh and a catheter was directly inserted in the femoral vein. Hydration of the animal were maintained with a 0.9% sodium chloride injection (Baxter Healthcare Corporation; Deerfield, IL). Pentobarbital was administered with incremental doses to keep woodchucks under anesthesia.
4.2.3 Radiopharmaceuticals
4.2.3.1 [Methyl-11C]-Choline
Synthesis of CHOL followed the procedure described by Hara et al. [194]. Briefly, 11C-
CHOL was synthesized by 11C-methylation of N,N-dimethylaminoethanol directly on Accell
Plus CM Sep-Pak cartridge at room temperature. The final product was eluted from the cartridge by saline after being washed with ethanol and water and then passed through a 0.2 um sterile filter. The radiochemical purity was greater than 99%. PET images data were acquired dynamically for 60 minutes after bolus injection of 37 MBq of CHOL and the time- frame between 25 and 30 minutes was used to quantify the SUV and tumor-to-liver uptake ratio (T/L).
18 4.2.3.2 2-Deoxy-2[ F]Fluoro-D-Glucose (FDG)
FDG was produced on site. Briefly, 18O water was recovered using ion exchange resin to extract the fluoride. Unreacted fluoride and aminopolyether were removed using a silica cartridge eluted with acetonitrile/ether, and hydrolysis was done using 1N HCl which was then neutralized with NaOH and NaHCO3 to produce a buffered, hypertonic, sterile and
120
pyrogen-free product. Nine woodchucks with HCC and one normal healthy woodchuck were included in the experiments. Each animal was injected with a 37 MBq bolus of 2FDG and dynamic emission scans were acquired for sixty minutes. The time-frame between 50 and 60 minutes was used to quantify the SUV and the tumor-to-liver uptake ratio (T/L).
4.2.3.3 1-11C-acetate (Act)
11 11 Act was produced from [ C]-CO2, which resulted in the [ C] label on carbon 1, using
Grignard reagent [195]. Ten woodchucks with HCC and one normal healthy woodchuck were included in the experiments. Sixty-minutes dynamic emission scans were acquired after bolus injection of 37 MBq of Act and the time-frame between 25 and 30 minutes was used to quantify the SUV and T/L.
4.2.4 Imaging Protocol
Ten woodchucks were scanned on an Allegro PET scanner (Philips Medical Systems, Inc.;
Cleveland, OH). The 3D emission scans were reconstructed using filtered backprojection and attenuation correction was performed using the internal 137Cs source. Prior to scanning woodchucks on the Allegro, scans were performed on a MX 8000 IDT 16 slices CT scanner
(Philips Medical Systems, Inc.; Cleveland, OH) with and without contrast agent (Optiray 300 ioversol injection 64%; Mallinckrodt; Hazelwood, MO) for tumor localization. The pixel size was 0.49 mm × 0.49 mm and slices were 3 mm thick.
Four woodchucks were scanned on a Gemini TF PET/CT scanner (Philips Medical Systems,
Inc.; Cleveland, OH). The 3D emission scans were reconstructed using row-action maximum
121
likelihood algorithm (2 iterations, relaxation factor = 0.045 and blob size = 2.5 pixels) and
CT attenuation correction was used. All reconstructed PET images were 144 × 144 pixels, for a field-of-view of 576 mm, yielding a voxel size of 4 × 4 × 4 mm. At the end of the PET scan, woodchucks had a contrast-enhanced CT scan done, yielding a pixel size of 0.98 mm ×
0.98 mm and the slice thickness was 0.5 mm.
4.2.5 Image Analysis
PET images were first assessed visually, using transaxial, sagittal, and coronal displays. To allow an objective assessment of the amount of tracer uptake, we evaluated the 11C-CHOL uptake by semi-quantitative analysis using the standardized uptake value (SUV) and the tumor-to-liver (T/L) ratio for each abnormal focus. Circular regions of interest with a diameter of 1.2 cm were drawn around tumors (identified on the CT scans) or around the regions of the highest uptake within tumors. Similar regions were drawn around the hepatic tissues surrounding the HCCs. A tumor was considered detected if T/L was greater than 1.2.
Values are reported as mean ± standard deviation. The standardized uptake value (SUV) was calculated as the activity concentration within an ROI normalized by the ratio of the injected dose and the animal weight. SUV is defined as:
where the radioactivity concentration in a pixel (Bq/ml) was to be determined from an apparent pixel count (cps/pixel volume) and a predetermined cofactor.
122
The largest diameter of each tumor was measured on the contrast-enhanced CT images. The intensity within a ROI on the CT images was reported in Hounsfield units
1000 where STP stands for the standard temperature and pressure conditions (0° C and 100 kPa).
4.2.6 Histology
The diagnosis of HCC was also based on routine histological examination. After PET and CT imaging, the animals were euthanized using FatalPlus (Vortech Pharmaceuticals Inc,
Dearborn, MI). For some woodchucks, HCCs and surrounding hepatic tissues were obtained post-mortem and fixed in formalin. Thin sections of paraffin-embedded tissues were prepared and stained with hematoxylin-eosin (H&E). The fat content, presence of necrosis, and the tumor differentiation were assessed from these sections. Cells in well-differentiated tumors have distinct cell membranes, minimal atypia, a moderate amount of finely granular eosinophilic cytoplasm and bile cannaliculi are present. In moderately-differentiated tumors the trabecular structures are three or more cells wide, the cytoplasm is abundant and eosinophilic and the nuclei are round with prominent nucleoli. Multinucleated and giant cells are present in poorly-differentiated tumors and the nuclear/cytoplasm ratio is high and can be noticed by the increased nuclear density. Nuclear atypia is also pronounced [93]. When different levels of differentiation were observed within one tumor, the poorest was reported.
123
4.2.7 Metabolites study in woodchuck model of hepatocellular carcinoma
4.2.7.1 [Methyl-14C]-CHOL metabolism in woodchuck model of hepatocellular carcinoma
After PET/CT imaging, some of the woodchucks which showed higher uptake in HCC during PET scan with 11C-CHOL were chosen for in vivo studies of radiolabeled CHOL tracer metabolism. The animals were anesthetized with pentobarbital. Due to very short physical half live of 11C (20 min as used for labeling CHOL for PET imaging), 14C labeled
CHOL at the imaging tracer dose was used to quantify the different composition of metabolites between HCC and surrounding hepatic tissue. 18.5 MBq of [methyl-14C]-CHOL
(Specific activity: 55 mCi/mmol, American radiochemical Inc., St. Louis, MO) in isotonic saline was bolus intravenous administrated through the same venous catheter used for the
11C-CHOL injection and PET imaging. The woodchucks were euthanized by 12 min or 30 min post 14C injection and the livers were excised immediately. The livers were quickly sliced and the tissue samples from HCC regions and surrounding hepatic tissues were selected for for metabolites extraction. The kidneys were also harvested for a negative control.
4.2.7.2 Extraction of radiolabeled metabolites and lipids using Folch method
Extraction of radiolabled metabolites and lipids from liver tissues was followed the Folch method [196]. 1g tissues from HCC, surrounding hepatic tissue and kidney were homogenized with 20 ml chloroform/methanol (2/1, v/v) containing antioxidant 100 mg/l
Butylated Hydroxytolune (BHT). After dispersion, the whole mixture was vortexed for 15-20 min at room temperature. The homogenate was filtered to separate into liquid phase and
124
insoluble phase. The insoluble phase remained on the filter paper was used for further separation. The liquid phase was washed with 4 ml 0.9% NaCl solution to block the binding of some acidic lipids to denatured lipids. After vortexing some seconds, the mixture was centrifuged at 2500 rpm 10 min to separate the two phases. The upper layer was siphoned and 1ml methanol/water (1/1, v/v) was added to rinse the interface one or two times without mixing the whole preparation. After centrifugation again, the upper phase was combined as water soluble phase. The lower chloroform phase contains lipids class. After evaporation under the nitrogen gas, the lipid soluble phase was redissolved in 1 ml of chloroform/methanol (2/1, v/v); the water soluble phase was redissolved in 1 ml of methanol/water (1/1, v/v). 0.1 ml from each phase was used for measurement of radioactivity. The metabolites in the water soluble and lipid soluble phases were analyzed using high-performance liquid chromatography (HPLC) and thin layer chromatography as described below.
Insoluble phase from filter paper was further seperated to RNA hydrolysate, DNA hydrolysate and protein [197]. The insoluble phase was dissolved in 1ml of 0.3M KOH.
After incubation of the solution at 37C for 1 hour to hydrolyze RNA, 0.32ml of 3N HClO4 was added and the mixture was kept on ice for 5 min. The precipitate was then separated and washed with 1ml of 0.5M HClO4. The combined supernatant was designed as the alkaline- liable fraction containing the RNA hydrolysate. The precipitate was resuspended in 1ml of
0.5M HClO4 and heated at 37C for 15 min to hydrolyze DNA. The solution was kept on ice for 5 min and the precipitate was separated. The supernatant and precipitate were assessed as the acid-labile fraction containing hydrolysates of DNA and protein fraction respectively.
125
The 14C-CHOL incorporated into lipid soluble, water soluble, RNA hydrolysates, DNA hydrolysates and protein fraction were counted in a liquid scientillation counter and normalized to total tissue protein content respectively.
4.2.7.3 Analysis of radiolabeled metabolites
4.2.7.3.1 HPLC system
The HPLC system consisted of HP1050 Instruments (Agilent Technologies Inc., Santa Clara,
CA): 1050 Pumps (isocratic and quaternary), 1050 Diode Array and Multiple UV
Wavelength Detectors, temperature controller (Waters Co., Milford, MA), followed by a radioisotope (14C/3H) detector (Radiomatic 150TR, Flow Scintillation Analyzer;
PerkinElmer, Waltham, Massachusetts, USA) and with computer data acquisition. HPLC analysis was performed on an Adsorbosphere Silica normal phase HPLC column
(250×4.6mm, Prevail Silica 5µ; Grace Davison Discovery Sciences Inc., Deerfield, IL) with an guard column (Guard 7.5×4.6mm, Prevail Silica 5µ; Grace Davison Discovery Sciences
Inc., Deerfield, IL) used as the stationary phase. The TLC technique on the silica gel G plate
(Merck KGaA, Gibbstown, NJ) was used to confirm the HPLC result. The metabolites were identified by comparing the retention time (HPLC) or retention factor (Rf) (TLC) with those of the authentic 14C standard compounds (American radiochemical Inc., St. Louis, MO) or those of authentic standard compounds under UV light.
4.2.7.3.2 Analysis of water soluble phase metabolites
For HPLC, the mobile phase was composed of two buffers: Buffer A, containing acetonitrile/water/ethyl alcohol/aceticacid/0.83 M Sodium acetate (800/127/68/2/3, v/v/v/v),
126
pH 3.6, and buffer B (400/400/68/53/79, v/v/v/v) [124]. A linear gradient from 0 to 100% buffer B, with a slope of 5%/min, was started 15 min after injection. Flow rate was 2.7 ml/min and column temperature was maintained at 45°C. The 14C labeled metabolites: betaine, betaine aldyhyde, choline, CDP-CHOL and PCho were identified in the water- soluble phase via comparing the retention time with the authentic 14C-labeled standards.
For TLC, a solvent system [125] was composed of methanol/0.5% NaCl/aqueous ammonium
(100/100/2, v/v/v). In the solvent system, the water-soluble metabolites were separated and identified via comparing the retention factor (Rf) with the authentic 14C-labeled standards.
The plates were exposed on an imaging storage phosphor screen (Molecular Dynamics,
Sunnyvale, CA, USA) and the exposed plates were scanned with a bio-imaging analyzer
(Typhoon Variable Mode Imager, GE, Piscataway, NJ) to detect radioactivity.
4.2.7.3.3 Lipid soluble phase metabolites analysis
For HPLC, phospholipids and neutral lipids used different mobile phases. phospholipid metabolites were analyzed with the mobile phase: acetonitrile/hexane/methanol/phosphoric acid (918/30/30/7.5,v/v/v/v) [126]. The flow rate was 1.5 ml/min. Neutral lipids metabolites were analyzed with the mobile phase hexane/isopropanol/acetic acid (100/2/0.02, v/v/v)
[127]. The flow rate was 2 ml/min. The non-radioactive phospholipid metabolites were monitored at 206 nm. The 14C-phospholipid metabolites were detected by flow scientillation analyzer and identified via comparing the retention time with the authentic 14C-labeled standards and cold standards under UV light. The PC component from this step were
127
collected for further analyzed the PC species using argentation TLC technique as described below.
For TLC, phospholipid and neutral lipid metabolites were separated with a single step. An aliquot of the fraction was spotted on a silica gel G plate (Merck KGaA, Gibbstown, NJ) and developed the plate in mobile phase: chloroform/methanol water (60/30/5, v/v/v) up to 7cm to separate phospholipids and then change the mobile phase to hexane/dicthyl ether/acetic acid (80/20/1.5, v/v/v) to migrate another 10 cm [128]. The lipid-soluble metabolites were separated and identified via comparing the retention factor (Rf) with the authentic 14C- labeled standards and cold standards.
4.2.8 Contribution of CDP-choline pathway and PE methylation pathway to phosphoatidylcholine synthesis in woodchuck model of hepatocellular carcinoma
4.2.8.1 Argentation TLC to separate different molecular species of PC synthesized from
CDP-choline pathway or PE methylation pathway in woodchuck model of hepatocellular carcinoma
There are different molecular species associated with PC synthesis between CDP-CHOL pathway and PE methylation pathway [198-202]. Molecular species of PC were resolved according to their degree of unsaturation by an argentation TLC method [201]. Individual PC was collected from HPLC elutes of lipid soluble phase of liver tissue described in section
4.2.7 and was spreaded over 20 cm of the 20% argentation TLC plate. The chromatographic system contained chloroform/methanol/water (50/34/7, v/v/v). The retention factor (Rf) from lipid standard (all from Avanti Polar Lipids) were used to identify the positions of species of
PC on the plates, The lower band (Rf = 0.15-0.18) co-chromatographed with a hexaenoic PC 128
standard, dilinolenoyl-PC (18:3, 18:3), i.e. with six double bonds in its fatty acids; The middle band (Rf=0.30-0.35) corresponded to tetraenoic PC standards: 1-palmitoyl-2- arachidonoyl-PC (16:0,20:4), 1-stearoyl-2-rachidonyl-PC (18:0, 20:4) and dilinoleoyl-PC
(18:2, 18:2), i.e. with four double bonds in its fatty acids; The upper band (Rf=0.45-0.50) co- migrated with saturated molecular species of PC, as well as with mono- and di-enes: 1- palmitoyl-2-stearoyl-PC (16:0, 18:0), 1-palmitoyl-2-oleoyl-PC (16:0, 18:1) and dioleoyl-PC
(18:1, 18:1). So the upper band corresponds to the species of PC from CDP-CHOL pathway, the lower two bands correspond to the species of PC from PE methylation pathway. The TLC plates were exposed on an imaging storage phosphor screen (Molecular Dynamics,
Sunnyvale, CA, USA) and then were scanned with a bio-imaging analyzer (Typhoon
Variable Mode Imager, GE, Piscataway, NJ) to detect radioactivity.
4.2.8.2 Verify the contribution of PE methylation pathway to phosphoatidylcholine synthesis in hepatocellular carcinoma
In order to verify the contribution of PE methylation pathway to PC synthesis in HCC, [L- methyl-3H]-methionine (Met) was used to investigate the contribution of PE methylation pathway to the contrast uptake in HCC. For this study, 1 × 107 WCH17 Cells in 75 cm2 corning cell culture flasks were incubated with 10 ml of 370 KBq [methyl-3H]-Met
(American Radiolabeled Chemicals, Inc., St. Louis, MO) in HBSS at 37°C for 5 min (pulse).
After incubation, the radioactive media were removed and the cells were washed three times with ice-cold PBS. And the cells were then incubated with HBSS containing non-radioactive
Met for 10, 25, 40 and 55 min (chase). After chase, the cell layers were rinsed with ice-cold
PBS twice, trypsinised with 3 ml trypsin and neutralised with 3 ml medium. The resultant
129
cell suspension was centrifuged at 2500 rpm for 10 min at 4°C. The supernatants were discarded. Cell pellets were homogenized in 0.4M perchloric acid and separated into acid precipitable (APF) and acid soluble fractions (ASF). The major H-3 components in APF include lipids (PMME, PDME, PC), RNA, DNA, protein etc. In contrast, the major metabolites of ASF include Met, SAM, SAH, Met sulfoxide, Met sulfone and 5'-deoxy-5'- methylthioadenosine etc. APF and ASF were further extracted in chloroform/methonal
(2/1,v/v) respectively and divided into water soluble and lipid soluble fractions. The water soluble and lipid soluble fractions at this step were combined together. The precipitate from
APF was dissolved in 0.3M KOH, incubated at 37C 1 hr and then 3N perchloric acid was added. The mixture kept on ice for 5 min. The supernatant at this step is alkaline-labile fraction containing RNA hydrolysate. The precipitate at this step was resuspended in 0.5M perchloric acid and heated at 90C 15 min. The supernatant at this step was acid-labile fraction containing DNA hydrolysate (no pathway to DNA from Met, this might be derived from basic proteins such as chromosomal histone). The final precipitate is protein. In addition, a protein synthesis inhibitor cycloheximide was also used to pre-treat WCH17 cells
2 hr and then repeat the [L-methyl-3H]-Met pulse and chase study with keeping cycloheximide in the media. The inhibitor cycloheximide was used to divert the pathway to
PE methylation pathway instead of protein synthesis.
4.2.9 Assay of choline kinase activity
The liver samples from HCC regions and surrounding hepatic tissues were also selected for enzyme assay. The livers were excised and frozen immediately and then kept at -80ºC until use. The following procedures were carried out at 2~4 ºC. Livers were homogenized in the
130
homogenized buffer (1g/3ml, g/vol) containing 145 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10 mM NaF, 1 mM EDTA using a hand-holded homogenizer. The homogenate was centrifuged at 10 000 ×g for 15 min and the resulting supernatant was further centrifuged at 100 000 ×g for 60 min at 4C (Beckman L7 ultracentrifuge, Ti90 rotor; Beckman Coulter, Inc., Fullerton,
CA,). The supernatant was used as the crude extract for choline kinase assay.
The procedure of choline kinase assay was performed as described by Ishidate and Nakazawa
[130]. The assay mixture (300 µl) contained 0.1 M Tris-HCl, pH 7.5, 10 mM ATP, 12 mM
MgCl2, 10µM choline, [methyl-14C]-CHOL (0.7 MBq per assay mixture). Samples were incubated at 37°C for 30 min. The reaction was stopped by adding 0.5 ml chloroform/methonal (2/1, v/v) solution. The reaction mix was then separated into two phase by centrifugation. An aliqot (40 µl) of the upper (aqueous) phase from this step was spotted on the silica gel G plate (Merck KGaA, Gibbstown, NJ) using the developing solvent system: methanol/0.5% NaCl/aqueous ammonium (100/100/2, v/v/v).
The PCho produced from CHOL were separated and identified via comparing the retention factor (Rf) with the authentic 14C-labeled standards. In the solvent system, the water soluble metabolites were separated as follows: choline (Rf =0.30), phosphocholine (PC) (Rf=0.43).
The plates were exposed on an imaging storage phosphor screen (Molecular Dynamics,
Sunnyvale, CA, USA) and the exposed plates were scanned with a bio-imaging analyzer
(Typhoon Variable Mode Imager, GE, Piscataway, NJ) to detect radioactivity. The activity of
Chok was normalized to total protein content. One unit of choline kinase will catalyze the phosphorylation of 1.0 µmole of choline to choline phosphate by ATP per minute at pH 8.5 at 25C.
131
4.2.10 Assay of choline-phosphate cytidylyltransferase activity
4.2.10.1 Preparation of cytosolic and microsomal pellet fraction of liver tissue.
The woodchuck liver tissues were placed in 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA,
2 mM dithiothreitol and 0.25% sodium azide buffer (pH 7.4), cut into small pieces, and then homogenized using a hand-holded homogenizer. The homogenate was centrifuged at 10 000
×g for 15 min at 4C and the resulting supernatant was further centrifuged at 100 000 ×g for
60 min at 4C (Beckman L7 ultracentrifuge, Ti90 rotor; Beckman Coulter, Inc., Fullerton,
CA,). The resultant supernatant was designated as the cytosolic fraction. The microsomal pellet fraction was resuspended in 0.5 ml of 0.25 M sucrose, 10 mM Tris-HCI, pH 7.4. The cytosolic and microsomal pellet fraction were divided into small aliquots and stored at -80 °C before use. CTP:phosphocholine cytidylyltransferase (CCT) activity was assayed within one week.
4.2.10.2 Assay of choline-phosphate cytidylyltransferase activity
CCT activity was determined by measuring the formation of radioactive CDP-choline from
[methyl-14C]-phosphocholine [203]. Two fractions, the cytosolic fraction and the microsomal pellet fraction, were incubated at 37°C for 15 min in 100 L of 50 mM imidazole, pH 7.0, containing 150 mM KCl, 2 mM EDTA, 1.5 mM DTT, 15 mM magnesium acetate, 3 mM cytidine triphosphate, 6 mM adenosine diphosphate, 1.6 mM PCho, and 0.1 Ci of [methyl-
14C]-PCho. The reaction mixture was in the absence or presence of 1 mM oleic acid. The reaction was terminated by addition of 100 L of chloroform/methanol (2/1, v/v) solution. After centrifugation, the lower layer only contained 14C-PC. An aliquot of upper layer (40 µl) was applied to the silica gel G plate (Merck KGaA, Gibbstown, NJ, USA) to
132
separate the CDP-CHOL produced from 14C-PCho via TLC method. The solvent system
[125] was composed of methanol : 0.5% NaCl : aqueous ammonium (100:100:2, v:v:v). In the solvent system, the water soluble metabolites were separated as follows: PCho (Rf=0.43) and CDP-CHOL(Rf=0.69). The plates were put on an imaging storage phosphor screen
(Molecular Dynamics, Sunnyvale, CA, USA) and the exposed plates were scanned with a bio-imaging analyzer (Typhoon Variable Mode Imager, GE, Piscataway, NJ) to detect radioactivity. The activity of CCT was normalized to totoal protein content.
4.2.11 Effect of oleic acid on phosphatidylcholine synthesis derived from radiolabeled
choline tracer in cultured WCH17 well-differentiated hepatoma cells
4.2.11.1 Preparation of 1mM oleic acid
The 1mM oleic acid was prepared as described by Van Harken et al. [122]. Sodium oleate
(0.1 mmol) was dissolved in 5.0 ml of warm saline and then mixed this with 5.0 ml of 20% bovine serum albumin (fatty acid-free). The mixture (10.0 ml) was diluted to 100 ml with
DMEM. The final concentration was 1% bovine serum albumin (BSA) containing 1 mM sodium oleate.
4.2.11.2 Pulse and chase study of phosphatidycholine synthesis from [methyl-14C]- choline in cultured WCH17 woodchuck hepatoma cells with 1mM oleic acid treatment
1 × 107 WCH17 Cells in 75 cm2 corning cell culture flasks were incubated in triplicate for 5 min at 37°C with 370 KBq [methyl-14C]-CHOL (specific activity: 2.035 MBq/µmol) (pulse) in Dulbecco's PBS containing CaCl2 and MgCl2. After incubation, the radioactive media were removed and the cells were washed three times with ice-cold PBS. And the cells were
133
then incubated with Dulbecco's PBS containing CaCl2 and MgCl2, 18 µM non-radioactive
CHOL for 10, 25, 40 and 55 min (chase). The DPBS contains either the presence or absence of 1% bovine BSA, 1 mM sodium oleate. After chase, the cell layers were rinsed with ice- cold PBS twice, trypsinised with 3 ml trypsin and neutralised with 3 ml medium. The resultant cell suspension centrifuged at 2500 rpm for 10 min at 4°C. The supernatants were discarded. Cell pellets were resuspended in 1.2 ml water containing antioxidant 100 mg/l
Butylated Hydroxytolune (BHT) and homogenized for the extraction of metabolites and lipids (Folch extraction method) and metabolites analysis of intracellular fate of [methyl-
14C]-CHOL using HPLC and TLC as described above.
4.2.12 Protein assay
Protein content was determined using the by the method of Bradford (Bio-Rad labortaries,
Inc., Hercules, CA) [94].
4.2.13 Liquid scintillation counting
Radioactivity of 14C was determined with a Beckman LS-6500 Liquid Scintillation Counter
(Beckman Coulter Inc., Fullerton, CA) and Bio-safe II (Fisher Scientific, Pittsburgh, PA) as scintillation fluid. Disintegrations per minute (dpm) were obtained by correcting for background activity and efficiency based on calibrated standards. 14C counts were also corrected for quenching effects.
4.2.14 Statistical analysis
All data, unless otherwise stated, are expressed as mean ± standard deviation of the mean
(SD). The data were compared using one way analysis of variance (ANOVA) or ANOVA on 134
ranks when appropriate. All pairwise multiple comparison procedures used Tukey test.
Differences were regarded as statistically significant for p<0.05.
4.3 Results
4.3.1 PET/CT imaging and histology
The tumor/liver ratio from the PET imaging studies are illustrated in Table 4.1. The threshold for tumor detection was set Tumor-to-liver ratio (T/L) 1.2. Based on T/L value larger or smaller than 1.2, the results were divided into six groups. FDG showed an uptake usually in poorly-differentiated HCC not in well-differentiated HCC. In contrast, CHOL and Act showed an uptake in well-differentiated HCC.
Table 4.1 Tumor/Liver ratio from the PET imaging with FDG, 11C-Act, 11C-CHOL
Tumor Highest Tumor Tracers Threshold Diameter tumor/liver Histology† number (cm) ratio (T/L)* FDG T/L >1.2 7 4.53±2.11 1.36±0.13 MD, PD T/L <1.2 6 4.18±2.1 1.01±0.06 WD, MD Act T/L >1.2 15 3.65±1.93 2.02±0.7 WD, MD T/L <1.2 1 3.76 1.17 MD CHOL T/L >1.2 5 2.54 ±1.23 1.63±0.34 WD T/L <1.2 n/a n/a n/a n/a *The threshold for tumor detection was set at T/L >1.2 †WD, well-differentiated HCC; MD, moderate-differentiated HCC; PD, poorly-differentiated HCC
11 11 As shown in Figure 4.2, three tracers, [ C]-CHOL, [ C]-Act and FDG were applied on same woodchuck model of HCC. [11C]-CHOL and [11C]-Act showed promising on detection of HCC. They maintained the cancer to background contrast throughout scan as shown in
Figure 4.2. However, the woodchuck did not take up the FDG tracer. The imaging pattern 135
between CHOL and Act was different. CHOL showed a larger hot spot than Act but its background was a little higher than Act. These findings suggested that the metabolism underlying CHOL and Act might be different due to their different imaging pattern.
Figure 4.2 Coronal PET/CT imaging of well-differentiated HCC with CHOL, Act and FDG (A) [C-11]-CHOL image taken 25 minutes after injection. (B) [C-11]-Act image taken 25 minutes after injection. (C) FDG image taken 50 minutes after injection. The HCC is not detected by FDG. (D) Corresponding CT image where the HCC is indicated by a blue arrow.
P.I.: Post Injection.
HCCs were generally heterogeneous in appearance and composition. An example is illustrated on Figure 4.3. In a woodchuck model of HCC, there were three regions of tumors. 136
[11C]-CHOL can detect all of them but [11C]-Act can only detect two of them. H&E staining showed that that nodulewhich [11C]-Act can not detect is a compact type HCC while the other two nodules were clear cell type of HCC. Even for the same clear cell type of HCC,
[11C]-CHOL and [11C]-Act showed different imaging patterns, suggesting the HCC in one nodule is also heterogeneous. In addition, the surrounding hepatic tissues as indicated by a white arrow have a higher background from [11C]-CHOL as compared to that from [11C]-Act.
Overall, our result suggested that there might be different metabolism between [11C]-CHOL and [11C]-Act although they might share the same end-product, 11C-PC.
Histology also confirmed that HCCs were generally heterogeneous in appearance and composition. An example is illustrated on Figure 4.4. In one region of the tumor (Figure 4.4
(C)), the cells are well-differentiated. Cells in well-differentiated tumors have distinct cell membranes, minimal atypia, a moderate amount of finely granular eosinophilic cytoplasm and bile cannaliculi are present. In moderately-differentiated tumors the trabecular structures are three or more cells wide, the cytoplasm is abundant and eosinophilic and the nuclei are round with prominent nucleoli. Multinucleated and giant cells are present in poorly- differentiated tumors and the nuclear/cytoplasm ratio is high and can be noticed by the increased nuclear density. Nuclear atypia is also pronounced [93]. In another region of the
HCC (Figure 4.4 (E)), macrovesicular steatosis is prominent and very few hepatocytes do not contain fat. Some tumors contained well-differentiated and some moderately-differentiated cells in different lobules within the HCCs.
137
Figure 4.3 PET imaging of a woodchuck model of HCC with [11C]-CHOL and [11C]-Act (A) [11C]-Act image taken 25 minutes 25 minutes after injection. (B) [11C]-CHOL image taken 25 minutes after injection. (C) Exicised liver. (D) H&E stain of clear cell HCCs in the woodchuck. ROI drawn with green dash line. (E) H&E stain of compact type HCC. ROI drawn with blue dash line. The surrounding hepatic tissues indicated as white arrow in A and B.
Figure 4.4 Histology of well-differentiated HCC 138
A. Contrast-enhanced CT image of woodchuck model of HCC. B. Surrounding hepatic tissue. C. Well-differentiated portion of the HCC. Bile cannaliculi are indicated by the arrows (H&E, original magnification × 400). D. Interface between the fatty portion of the HCC (left) and the well-differentiated, trabecular portion of the HCC (right) (H&E, original magnification × 200). E. Fatty portion of the HCC (H&E, original magnification × 400).
4.3.2 Metabolites study in woodchuck model of hepatocellular carcinoma
After PET/CT imaging, some of the woodchucks which showed higher uptake in HCC during PET scan with 11C-CHOL were chosen for in vivo studies of radiolabeled CHOL tracer metabolism. An example is illustrated on Figure 4.5. The woodchucks were intravenous injected [methyl-14C]-CHOL for 12 min or 30 min for metabolites analysis.
Figure 4.5 A woodchuck model of HCC that used for 11C-Choline metabolites study (A) FDG image. (B) [11C]-Acetate image. (C) and (D) [11C]-choline image. There were two tumors in this woodchuck which were indicated by a green dash line and an orange arrow.
139
The uptake of [14C]-CHOL by the woodchuck model of HCC was shown in Figure 4.6.
There was significant difference between HCC and surrounding hepatic tissue at 30 min post-injection. However, no significant difference between them was oberserved at 12 min post-injection. This might be resulted from the fact that the analyzed hepatic tissues surrounding the HCC nodule are consisted mainly of hepatocytes mixed with a low percentage of other materials and cell types including cancerous cells. Similarly, the analyzed
HCC tissues might be heterogeneous and mixed with different stages of HCC cells and normal hepatocytes. At 12 min post-injection, the major radioactivity was accumulated in water-soluble phase of HCC and surrounding hepatic tissue. At 30 min post-injection, the radioactivity shifted to lipid-soluble phase of HCC and surrounding hepatic tissue. Moreover,
HCC showed a significant higher lipid soluble phase at 30 min post-injection as compared to surrounding hepatic tissue. In addition, as a negative control, the majority of radioactivity in kidney was located at water soluble phase at 12 min post-injection and lipid soluble phase at
30 min.
140
Figure 4.6 The distribution of C-14 activity between HCCs and surrounding hepatic tissues. Animals went through PET imaging of [methy-C-11]-CHOL. After imaging, animals were injected with imaging does of [methyl-C-14]-CHOL through same venous catheter used for methyl-11C-11]-CHOL injection and imaging. The woodchucks were euthanized 12 mins or 30 mins post C-14 injection and the liver were excised immediately. HCCs and surrounding hepatic tissues regions were picked for crude extraction using Folch method. Kidneys were also used for a control. P.I.: Post Injection.
The metabolites of 14C-CHOL in the HCC, surrounding hepatic tissue and kidney were quantitated by radio-HPLC (Figure 4.8). The typical chromatograms of radiolabeled CHOL metabolites was shown in Figure 4.7. At 12 min post-injection, the major metabolites in HCC were located in the CDP-CHOL pathway, i.e. PCho and to a lesser extent, PC; In contrast, the major metabolites in surrounding hepatic tissue were betaine and free CHOL. The major metabolites in kidney were betaine and PCho. After 30 min post-injection, PCho was rapidly converted to PC in HCC. However, PCho just started to produce and accumulate in surrounding hepatic tissue, suggesting somewhat slower phosphorylation of CHOL in surrounding hepatic tissue. There was a siginificantly difference of PC content between HCC and surrounding hepatic tissue. At 30 min, the radioactivity incorporated into PC and the amount of betaine and PCho decreased in kidney.
10 Kidney Surrounding hepatic tissue A 8 HCC Thousands
(dpm/mg 6
protein) 4
2 Radioactivity 0 0 102030405060 Retention time (min) 141
1 Kidney Surrounding hepatic tissue B 0.8 HCC Thousands 0.6 (dpm/mg
0.4 0.2 protein) 0
Radioactivity 0 1020304050 Retention time (mins)
18 HCC 16 C 14 Surrounding Hundreds 12 hepatic tissue 10 Kidney (dpm/mg 8 6 protein) 4 2
Radioactivity 0 0 102030405060 Retention time (mins)
Kidney 16 14 Surrounding hepatic tissue D 12 HCC Thousands (dpm/mg 10
8
protein) 6 4 2 Radioactivity 0 0 5 10 15 20 25 30 35 Retention time (mins)
Figure 4.7 Representative HPLC radiochromatograms of [methyl-14C]-choline metabolites in woodchuck model of HCC A. Water soluble metabolites at 12 min post-injection. B. Phospholipid metabolites at 12 min post-injection. C. Water soluble metabolites at 30 min post-injection. D. Phospholipid metabolites at 30 min post-injection.
142
Figure 4.8 Pattern of 14C-choline metabolites in the water- and lipid-soluble fraction from woodchuck model of HCC
PCho is converted to CDP-CHOL and then incorporated into PC. Interestingly, the amounts of 14C-CDP-CHOL in the HCC, surrounding hepatic tissue and kidney were non-detectable by HPLC. Incorporation of 14C-PCho into 14C-PC occurred rapidly in 30 min post-injection in HCC. Thus, it was impossible that 14C-CDP-CHOL to 14C-PC is a rate-limiting step for PC synthesis in HCC. Our data suggested that 14C-PCho to 14C-CDP-CHOL is a rate-limiting step for PC synthesis which catalyzed by key enzyme choline-phosphate cytidydyltransfearse
(CCT) .
14C-beatine was the major product in surrounding hepatic tissue and kidney at 12 min post- injection. At 30 min post-injection, the decrease of 14C-beatine in both tissues suggest that
14C-beatine might release into the blood circulation and/or transfer its methyl group to methionine and incorporate into 14C-PC. 14C-betaine aldehyde, an intermediate in betaine
143
synthesis from CHOL was also identified in some surrounding hepatic tissues and kidney tissues by HPLC.
4.3.3 Contribution of CDP-choline pathway and PE methylation pathway to phosphatidylcholine synthesis
PC is produced in the liver by two metabolic pathways, that is, the CDP-choline pathway and
PE methylation pathway (Figure 4.1). PE methylation pathway involves phospholipid methylation which makes PC by three sequential methylations of PE. It is the only known pathway capable of generating choline moieties de novo.
In order to compare the proportions of the particular molecular subspecies of 14C-PC present in HCC at 12 min and 30 min after injection of 14C-CHOL into the woodchuck model of
HCC. PC was extracted from the liver and kidney tissues, isolated by TLC. and separated by argentation chromatography into three groups of molecular subspecies which differed in their degree of unsaturation. The possible contribution of two pathways to the accumulation of
14C-PC was assessed by comparing the degree of unsaturation of PC species.
Argentation chromatography allowed us to separate liver and kidney PC into three major bands. These represented three major fractions of liver PC that could be distinguished according to the degree of unsaturation in their fatty acids. The lower band (RF = 0.15-0.18) cochromatographed with a hexaenoic PC standard, dilinolenoyl-PC (18:3, 18:3), i.e. with six double bonds in its fatty acids. The middle band (RF =0.30-0.35) corresponded to tetraenoic
PC standards: l-palmitoyl-2-arachidonoyl-PC (16:0, 20:4), l-stearoyl-2-arachidonyl-PC (18:0,
144
20:4) and dilinoleoyl-PC (18:2, 18:2). The upper band (RF =0.45-0.50) comigrated with saturated molecular species of PC, as well as with mono- and di-enes: 1-palmitoyl-2- stearoyl-PC (16:0, 18:0), l-palmitoyl-2-oleoyl-PC (16:0, 18:1) and dioleoyl-PC (18:1, 18:1).
The proportions of radioactivities accumulated in the three major fractions of PC varied depending on the time after injection of radiolabeled CHOL (Table 4.2). At 12 min post- injection, the major PC species in HCC were short chain and unsaturated species. Small amount of six-band fatty acid was also found in HCC. At 30 min post-injection, the short chain and unsaturated species increased in HCC and no six-band fatty acid was found.
However, in surrounding hepatic tissue, PC species almost evenly distributed at saturated species and six-band fatty acid at 12 min post-injectin. At 30 min post-injection, PC species only account for the saturated species, suggesting a rapid turnover rate in PE methylation pathway in surrounding hepatic tissue. In addition, kidney showed a different pattern. At 12 min post-injection, most of the radioactivity was found in the two polyunsaturated fractions of PC- four double bonds and six double bonds. But after 30 min, most of its radioactivity is in more saturated molecular subspecies of PC. Our results suggested that CDP-CHOL pathway is the major metabolic pathway in HCC, which might ; PE methylation pathway is the major pathway in surrounding hepatic tissue at the early time point and it will shift to
CDP-CHOL pathway at the later time point; Kidney has a similar metabolism pattern with surrounding hepatic tissue. This result agreed well with the metabolites analysis in section
4.3.2.
145
In order to further confirm that HCC has an impaired PE methylation pathway, we used [L- methyl-3H]-methionine(Met) to measure how many radioactivity incorporated into PC. Met can incorporate into PE methylation pathway or protein synthesis. We pulsed the WCH17 cells with 370 kBq of L-[methyl-3H]Met followed by incubation WCH17 cells in nonradioactive Met medium different chase period (Figure 4.9(A)). It was found that the major intracellular H-3 activity was located in APF phase (mainly in protein fraction), which is fairly stabilized within 60 minutes chase time with slightly increase. Most importantly, the labeled protein fraction was rapidly increased during the first 5 min pulse time. In addition, in the inhibition study, we used a protein synthesis inhibitor cycloheximide to pre-treat
WCH17 cells 2 hr and then repeat the pulse and chase study while keeping cycloheximide in the media. It is interesting to find that the major H-3 activity was located in ASF (mainly in water soluble phase instead of lipids) and protein fraction stayed at a very low activity due to the effect of inhibition (Figure 4.9(B)). Another noticeable phenomena is that the conversion from water soluble phase to lipid (the major component is 3H-PC) occurs slowly even when the protein synthesis was blocked in WCH17 cells.
146
Table 4.2 Molecular species of 14C-phosphatidylholine formed in woodchuck model of hepatocellular carcinoma receiving [methyl-14C]-choline IV injection % of total 14C-phosphatidylcholine produced
Note: RF= 0.45-0.50 : short chain, saturated species or unsaturated species (two double bonds)
RF= 0.30-0.35: long chain, polyunsaturated species (four double bonds)
RF= 0.15-0.18: long chain, polyunsaturated species (six double bonds)
147
25 A Total Activity 10000
x 20 Acid Soluble Fraction protein) 15 Acid Precipitable Fraction 10 Water Soluble Phase (dpm/mg
Lipid Soluble Phase 5 Alkaline‐labile Phase
Radioactivity (RNA hydrolysate) 0 Acid‐labile Phase (DNA 3H 0102555hydrolysate) Chase time (mins) Protein Phase
25 Total Activity B
10000 Acid Soluble Fraction
x 20 protein)
Acid Precipitable 15 Fraction
(dpm/mg Water Soluble Phase
10 Lipid Soluble Phase 5 Alkaline‐labile Phase Radioactivity (RNA hydrolysate) 3H 0 Acid‐labile Phase (DNA 0102555hydrolysate) Chase time (mins) Protein Phase
Figure 4.9 Pulse and chase study in WCH17 cells using L-[methyl-14C]Methionine. (A) without the protein synthesis inhibitor Cycloheximide (B) with protein synthesis inhibitor Cycloheximide. Chase time 0 means there was only pulse WCH17 cells 5 min and no chase.
4.3.4 Choline kinase activity
After inytravenous injection of 14C-CHOL, 14C-PCho rapidly accumulated in HCC region through metabolites analysis, which is associated with the imaging contrast oberserved
148
during the PET imaging of HCC. Thus, the knowledge about change of the choline kinase activity in HCC is essential to better understand the mechanism of tracer metabolism of 14C-
CHOL. We measured ChoK activity in HCC region as compared to surrounding hepatic tissue in the same woodchuck and normal liver tissue from control tumor-free woodchuck.
As shown in Figure 4.10 , the HCC regions displayed significant increased activity of ChoK
(Figure. 4.10). However, there is no significant difference of ChoK activity between surrounding hepatifc tissue and normail liver tissue.
30
†, ‡ 25 (mU/mg 20 † †† Activity
15 protein) 10 Kinase
5 Choline 0 HCC Surrounding hepatic Normal tissue
Figure 4.10 Choline kinase activity in woodchuck model of hepatocellular carcinoma. Snap-frozen liver tissue samples from HCC regions and surrounding hepatic tissues and from tumor free woodchucks were homogenized and centrifuged. The supernatant was used for enzyme assay and 14C-CHOL was used as the substarte.
4.3.5 Choline-phosphate cytidydyltransfearse
In order to investigate the change in the subcellular distribution and activity of CCT responsible for the different conversion rate from 14C-PCho to 14C-PC between HCC and surrounding hepatic tissue, the activity of CCT was assayed in cytosolic and micosomal
149
fraction of HCC tissues and surrounding hepatic tissues. The activity of this enzyme is located in both cytosolic and microsomal fractions of the live homogenates from HCC regions and surrounding hepatic tissue. To test the effect of oleate on the activation of CCT activity, 1 mM oleate was also added into assay mixture for comparison. No differences in microsomal CCT activity were observed between HCC and surrounding hepatic tissue no matter the assay mixture contains 1mM oleate or not (Figure 4.11). In contrast, the cytosolic
CCT activity was siginificantly higher in HCC than in surrounding hepatic tissue. And the cytosolic CCT activity increased around 1.44-fold in HCC when the assay mixture contained
1mM oleate, and a slight increase was also observed in surrounding hepatic tissue.
Summation of the total activities for the enzyme from microsomes and cytosol shows an overall increase in HCC as compared to surrounding hepatic tissue in both with and without
1 mM oleate condition.
8.00
7.00 ** Control add 1 mM oleic acid 6.00 protein/min) 5.00 * 4.00 ** (dpm/ug
3.00 * 2.00 Activity 1.00 CCT 0.00 Cytosol Microsomes Cytosol Microsomes HCC s Surrounding hepatic tissues
Figure 4.11 Activation of CTP:phosphocholine cytidylyltransferase by 1mM oleic acid. Cytosolic and microsomal cytidylyltransferase activity were measured either in the presence of 1 mM liposomes containing oleic aicd or in the absence of 1mM oleic acid.
150
4.3.6 Effect of oleic acid on phosphatidylcholine synthesis
The effect of oleic acid alone on the PC synthesis through activating CCT was obvious.A
14 pulse-chase experiment was shown in Figure 4.12. After the pulse of C-CHOL for 5 min, the radioactivity media were removed and PBS containing cold CHOL was added into
WCH17 cells for chase different time points up to 55 min. HPLC analysis of PC synthesis during the chase time period showed the oleic acid can significantly increase the PC synthesis during the 55 min chase time. In contrast, without oleic acid, PCho converted to PC slowly within 55 min chase time. Levels of PCho decreased at 1 mM oleate, and the PC level increased dramatically at this concentrations. The ratio of PCho versus PC decreased from 20 to 0.25 upon oleate stimulation, consistent with CT as the regulatory enzyme under this condition.
25 Betaine Choline A Phosphocholine Phosphatidylcholine
10000 20
x (dpm/mg
15
10 protein) metabolites 5
choline 0 ‐ C
14 0 10254055 Chase time (min)
151
25 Betaine Choline B Phosphocholine Phosphatidylcholine 10000 20
x (dpm/mg
15
10
protein) 5 metabolites
0 choline ‐ 0 10254055 C 14 Chase time (min)
Figure 4.12 Effect of oleic acid on the phosphatidylcholine synthesis.
14 Cells were incubated with [methyl- C]-CHOL in PBS with CaCl2 and MgCl2 for 5 min. The radioactivie media was removed and replaced with PBS with CaCl2 and MgCl2 containing 1% BSA/1 mM sodium oleated (oleic acid-treated). The cells were then incubated further (chase). The chase time at 0 represent the radioactivity after the initial 5-min pulse. (A) Incorporation of [methyl-14C]-CHOL into different phases in WCH17 cells. (B) Incorporation of [methyl-14C]-CHOL into different phases in WCH17 cells with the presence of 1mM oleic acid.
4.4 Discussion
4.4.1 PET/CT imaging
The diagnostic accuracy of FDG PET is not satisfactory in the evaluation of HCC, especially in cases of well-differentiated HCC. It was demonstrated that [11C]-CHOL can effectively detect well-differentiated HCC although the surrounding hepatic tissue has a background uptake. The imaging contrast during PET imaging of HCC with [11C]-CHOL seems to be caused by two factors. First of all, HCC tumor cells are characterized by the active incorporation of CHOL for production of PC, a cell membrane constituent, to facilitate rapid cell duplication of tumor cells. Secondly, CHOL incorporates to betaine and to a less extent,
PCho in surrounding hepatic tissues, which contributes to the background signal.
152
Although [11C]-Act has a higher T/L ratio than [11C]-CHOL, the major advantage of 11C-
CHOL for HCC lies in that it can detect both well- and poorly differentiated HCC but 11C-
Act can only detect well- and moderate-differentiated HCC. On the other hand, [11C]-Act has also shown increased uptake in the benign lesions, such as adenoma, hemangioma and focal nodular hyperplasia etc, as compared to the surrounding hepatic tissues [204]. The imaging contrast using [11C]-CHOL in poorly differentiated HCC was reported less than well- differentiated HCC [205]. It remains to be determined what transport mechanism and what metabolism condition are involved in this observation and how it is related to our observation. One of the possibilities is different ChoK expression in well-differentiated HCC and poorly-differentiated HCC.
ChoK seems to be regulated by two alternative, complementary mechanisms, one is post- translational modifications or protein-protein interactions, and the second is transcriptional regulation [206]. In early stages of cancer, Chok is up-regulated by a post-translational mechanism. Thus, under malignant conditions, tumors showed siginificantly higher ChoK activity which is not related to an increase in ChoK protein level. When tumors acquire a more aggressive phenotype such as poorly-differentiated status, ChoK may then be also up- regulated by transcriptional regulation, resulting in an increase in the expression of the protein. This increased ChoK protein level might only have normal level of the enzyme activity. Therefore, well-differentiated HCC might have a higher SUV than poorly- differentiated HCC. In addition, we can not rule out that there might be also different Chok transporter level between well-differentiated and poorly-differentiated HCC.
153
PET imaging using CHOL tracer produced clear images with good contrast at 5 min post injection. The rapid blood clearance of CHOL allows rapid data acquisition, which is convenient for the patient. The rapidity of the uptake process reveals the avidity of the CHOL transport system in HCC and normal liver tissues. Thus, tissue perfusion, transporter density, and ChoK activity are presumably important determinants of tracer uptake and sequestration by tissues. The short residence time of CHOL in the blood may limit the diffusion of tracer into areas that are poorly perfused (e.g. some xenograft animal model that has poor angiogenesis) [166]. The potential effects of perfusion, particularly during interventions that may affect tumor vascularity and vasomuscular tension, should be carefully considered when interpreting the uptake data [166]. On the other hand, the rapid and extensive clearance of tracer also makes the SUV parameter more useful than for a tracer that has a slow (and, therefore, more variable) blood clearance at the time of measurement of tissue uptake [166].
However, the use of an 11C-labelled radiopharmaceutical has a practical restriction, related to the short physical half-life of 11C. This short half-life necessitates the availability of an on- site cyclotron, which may limit the widespread use of CHOL. Theoretically, [18F]- CHOL should be an alternative to [11C]-CHOL.
[11C]-CHOL could be used to image several cancers that have enhanced lipid metabolism, and especially those that accumulate CHOL and its metabolites, as does HCC. Significantly high choline levels in HCC have been detected with proton magnetic resonance spectroscopy by Li et al.[207]. Elevation of the intensity of this peak reflects increased biosynthesis of membrane phospholipids and is a marker for cellular proliferation [208]. PET imaging with
[11C]-CHOL may also be useful for monitoring the therapeutic efficacy of traditional and
154
novel chemotherapeutic agents. The SUV index should be a fairly robust parameter to indicate tracer trapping in neoplasms, but the potential effects of therapy on tumor blood flow need to be carefully considered since the uptake kinetics are likely to be highly dependent on tissue perfusion.
4.4.2 Metabolites study in woodchuck model of hepatocellular carcinoma
Of high interest for investigation is determining the metabolic fate of [11C]-CHOL in the
HCC in comparison to surrounding hepatic tissue. It is known that transformation is accompanied by increases in both choline and PC levels in a variety of cancer types [134,
186, 193, 209, 210]. The activation of the CHOL uptake and phosphorylation seems to be a later event involved in a cascade of intracellular signal transduction events that result in transformation [166], including activation of ras-GTPase-activating protein [211], PI-3 kinase [212], and various protein and tyrosine kinases [213, 214]. Cuadrado et al. [215] showed that PCho triggered DNA synthesis in quiescent NIH3T3 fibroblasts, whereas
CHOL, PS, and PE had no effect. The ChoK inhibitor, HC-3, was found to block proliferation induced by growth factors, but the blockade was bypassed by PCho addition.
Thus, PCho seemed to act as a prerequisite second messenger for mitogenic activity, implicating ChoK activity as a critical step during regulation of cell proliferation by growth factors [215]. If, as suspected, ChoK-mediated phosphorylation of radiolabeled CHOL determines their retention in tissues, these radiotracers may not only be useful as cancer detection probes, but may also be used in experimental studies as unique tools to allow noninvasive monitoring of the regulation of an important signal transduction pathway [166].
155
Radioactive choline in the liver and kidney was metabolized along two pathways during a
30-min. One major pathway for 14C-CHOL was phosphorylation to PCho. I4C-PCho served as a precursor for 14C-PC, which became predominant component in HCC region at 30 min post-injection. A second major pathway for CHOL was oxidation to produce betaine. The betaine further incorporated into PC via PE methylation pathway. Betaine can be also release out of the liver cells in order to maintain the homestatsis of pressure and volume of the cells.
. Phosphorylation of CHOL was quantitatively the major pathway for CHOL metabolism in the HCC region. Approximately half of the radiolabel in the HCC was in PCho after 12 min post-injection. In contrast, the surrounding hepatic tissue was not particularly active in phosphorylating CHOL, since the major radioactivity was found in 14C-betaine. Moreover,
14C-PCho was only slightly accumulated in surrounding hepatic tissue at 30-min post- injection. The major metabolite in kidney was 14C-betaine at 12 min post-injection and switched to 14C-betaine at 30-min post-injection. This suggested that basal oxidation and phosphorylation activity accounted for the background contrast in surrounding hepatic tissue and kidney seen during the PET imaging using radiolabeled CHOL tracer. On the other hand, our data also suggested that there might higher Chok activity in HCC to contribute to the rapid accumulation of 14C-PCho in HCC with a very short time. We also cannot rule out that there might be different CHOL transporter activity between HCC and surrounding hepatic tissue.
The major pathway for PC synthesis in mammalian cells is thought to be the CDP-choline pathway. PCho is converted to CDP-CHOL, which is further synthesized to PC. We found
156
radiolabel in PC only after pools of 14C-PCho had accumulated. The I4C-CDP-CHOL was non-detectable by HPLC and TLC. 14C-PC highly accumulated in HCC at the later time point, 30 min post-injection. Surrounding hepatic tissue has a much slower synthesis of 14C-
PC as compared to HCC. This suggests that the CCT step might be a rate-limiting in normal liver phosphatidylcholine synthesis.
Betaine is synthesized from CHOL through the intermediate betaine aldehyde by the enzymes CHOL dehydrogenase and betaine aldehyde dehydrogenase. The enzyme CHOL dehydrogenase is present in the liver and kidney of many species [216]. The rapid synthesis of betaine in surrounding hepatic tissue and kidney confirmed that liver and kidney are active sites for betaine synthesis. The rapid uptake and clearance of the choline tracers in liver and kidney indicate the importance of tissue perfusion on their biodistribution.
Betaine synthesis and metabolism may be important for scavenging methyl groups from choline [141]. Sequential demethylation of betaine to form glycine produces one-carbon fragments. The resulting methyl groups are used for regeneration of methionine [217].
Betaine also appears to have an osmoregulatory effect in medullary cells of the kidney and in the liver cells, adapting to osmotic stress [144, 218].
Betaine was also identified as an osmolyte in rat liver macrophages Kupffer cells and sinusoidal endothelial cells [108]. Organic osmolytes are compounds which are accumulated or released by the cells in reponse to hyperosmotic cell shrinkage or hypoosmotic cell swelling, respectively, to maintain cell volume homeostasis. During hyperosmotic cell shrinkage, betaine can be accumulated. In contrast, during hypoosmotic cell swelling, betaine 157
is released. In the intact perfused rat liver, betaine derived from choline is released in response to hypoosmotic exposure.
In isolated rate liver cells, choline is taken up by Kupffer cells, sinusoidal endothelial cells and hepatocytes [108]. However, only hepatocytes are able to oxidize choline to betaine. In contrast with hepatocytes, no betaine formation from choline was detectable in Kupffer cells and sinusoidal endothelial cells [108], only metabolites of the choline kinase pathway were detectable (i.e. PCho, CDP-CHOL, and PC). The betaine synthesized in hepatocytes is released and taken up by Kupffer cells and sinusoidal endothelial cells. Osmoregulation of betaine uptake and cell heterogeneity in hepatic betaine synthesis may be similar to the situation in the kidney: in renal medullary cells, hyperosmotic exposure also increases betaine uptake whereas betaine synthesis from choline is low or absent in these cells and is not induced by hyperosmolarity. In contrast, in renal cortex CHOL dehydrogenase activity is high and betaine is released that can be taken up by medullary cells [108].
4.4.3 Contribution of CDP-choline pathway and PE methylation pathway to phosphatidylcholine synthesis
In addition to the CDP-CHOL pathway for PC synthesis, the liver has a unique PE methylation pathway for PC synthesis via three methylations of the ethanolamine moiety of
PE. There is a profound distinction in PC species between the CDP-choline pathway and the
PE methylation pathway [201, 202]. PC molecules produced from the CDP-CHOL pathway were mainly comprised of medium chain, saturated (e.g. 16:0/18:0) species, it may also containe 16:0/18:1, 18:1/18:1. On the other hand, PC molecules from the PE methylation
158
pathway were much more diverse and were comprised of significantly more long chain, polyunsaturated (e.g. 18:0/20:4) species. PC species from the methylation pathway contained a higher percentage of arachidonate amd Docosahexaenoic acid and were more diverse than those from the CDP-choline pathway. This profound distinction of PC profiles may contribute to the different functions of these two pathways in the liver. Our data showed a well agreement with this metabolic context.
The pathophsiological mechanism for the contribution of these two pathways to PC species is still unknowm. Cui et al [219] suggested that inhibition of the CDP-choline pathway in most mammalian cells or overexpression of the hepatic PE methylation pathway in hepatocytes leads to perturbation of PC homeostasis, growth arrest or even cell death. On the other hand,
DeLong et al [220] pointed out that while the contribution to the CDP-CHOL pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to an identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.
In PE methylation pathway, as shown in Figure 4.1, PE reacted with S-adenosylmethionine
(SAM) and converted to PC in liver via the enzyme PEMT. PEMT has two isoforms. An isoform, PEMT2 has been cloned, expressed and localized to a mitochondria-associated
159
membrane in rat liver. Interestingly, PEMT2 expression in rat hepatoma cells can specifically suppresses cell division. It is found that PEMT enzyme activity is required and suppression of cell division is not due to some effect of the protein itself [221]. Other control studies demonstrated that the effect will be not due to depletion of substrate (PE or Met) which might limit cell growth. Transfection of PEMT2 cDNA into Chinese hamster ovary cells had no effect on the rate of division of these cells. Thus, PEMT2 might have a specific role in regulation of hepatoma cell division [221].
There is an inverse relationship between PEMT2 expression and the rate of cell division of hepatoma cells grown in culture and in hepatocytes in intact animals [221, 222]. A PC binding protein (PCBP) is present in hepatocytes that senses the level of PC derived from PE methylation in cellular membranes. When PC derived from PE methylation is high, PCBP binds PE-derived PC and in this form binds to a regulatory element in the CCT gene and down-regulates the expression of CCT. This decreases the level of CCT in the hepatocytes which limits the activity of the CDP-choline pathway. Some product in the CDP-choline pathway appears to be essential for cell division and so the rate is diminished. Surprisingly, it seems that PC derived from PEMT2 cannot substitute for the CDP-choline pathway. That is, the PC levels are unaffected in the PEMT2 transfected hepatoma cells but the rate of cell division is impaired. Thus, it could be that PC derived from the CDP-choline pathway has an unknown function in DNA replication and cell division. Alternatively, the PC species made via PEMT2 might in some unknown fashion inhibit hepatocyte cell division.
160
As shown in Figure 4.9, our data suggest that PEMT activity in WCH17 cells might be much lower than primary hepatocytes, which diverted the metabolic pathway of radiolabeled Met into protein synthesis. Taken together, CDP-CHOL pathway was the major contributor to
PC synthesis from CHOL tracer in HCC.
Figure 4.13 Hypothetical mechanism for the down-regulation of the gene for CCT as a result of over expression of PEMT2 [221]. CHOL: choline, PCho: phosphocholine, CCT: CTP:phosphocholine cytidylyltransferase.
4.4.4 Choline kinase
ChoK has been reported to belong to the cancer-related genes that participate in the regulation of some critical metabolic pathways. Choline kinase (ChoK) is the first enzyme in the Kennedy pathway, responsible for the de novo synthesis of phosphatidylcholine (PC), one of the basic lipid components of membranes. Both ChoK and its product, PCho, have
161
been recently reported as essential molecules in cell proliferation and transformation.
Generation of PCho from ChoK activity has been described as an essential event in growth factor-induced mitogenesis in fibroblasts [218, 223, 224] and has been found to cooperate with several mitogens [225, 226]. Furthermore, overexpression of several oncogenes induces increased levels of ChoK and the intracellular levels of PCho [187, 210, 227]. A strong correlation was established between ChoK activity and cancer onset at least in some human tumors [206]. The results shown in this study indicate that ChoK is higher in HCC region as compared to surrounding hepatic. The results indicate the higher of ChoK catalyzed the uptake 14C-CHOL to 14C-PCho rapidly and then 14C-PC cells to provide higher uptake contrast in HCC regions.
4.4.5 Choline-phosphate cytidydyltransfearse
There are multiple regulatory mechanisms for activation of CCT activity [174]. Oleic acid may promote translocation of cytidylyltransferase from cytosol to membranes (active) but also may activate cytidylyltransferase. The balance between these events could result in a variety of results.
Cytosolic CCT contains two forms, H-form (active) and L-form (inactive). H-form was a lipoprotein complex containing the apoprotein (L-form) along with phospholipids and perhaps other lipids and/or proteins [228, 229]. Active H-form has been preexisting in the cytosol. But it can be also formed from L-form by some of lipids such as oleic acid that can activate enzyme. Also, the active form associated with microsomal membranes was similar, if not identical, to the H-form in cytosol. The conversion of inactive enzyme to H-form
162
enzyme may be the initial event followed by redistribution or equilibration of the newly formed H-form enzyme between cytosol and membranes.
The changes in the amount of H-form could occur either by modulating H-form/L-form interconversions in cytosol, by increasing the binding of L-form to membranes or by converting inactive forms of the enzyme to H-form [174]. The conversion from inactive precursors could occur in either membranes or cytosol with a secondary redistribution be- tween cytosol and membranes. These mechanisms may occur to various degrees in different cell types or under different metabolic conditions. Thus, as an example of modulation of H- form/L-form interconversion, the hormonally induced increases in phosphatidylcholine synthesis in fetal lung preparation was correlated with an increase in cytosolic cytidylyltransferase activity [230, 231]. Increases in membrane activity with corresponding decreases in cytosolic activity may represent a predominant L-form to membrane trans- location process (4-6). Whereas Hep G2 cells as well as some of the other fatty acid effects
(7-10, 16) may involve primarily the release of active enzyme from an inactive precursor.
In our study, cytosolic CCT activity is significantly higher in HCC than surrounding hepatic tissue and addition of 1 mM oleic acid can highly increase the cytosolic CCT activity in
HCC. This indicated that there might be more inactivate L-form CCT in HCC than surrounding hepatic tissue, which can be activated by oleic acid. However, activated cytosolic CCT still stayed at cytosol instead of translocation to membrane of ER. This might be because that CDP CHOL pathway may be organized into multienzyme complexes, associated with the cytoskeleton [232]. The H-form of cytidylyltransferase may be part of a
163
multienzyme complex that is oriented along cytoskeletal elements such that the cytidylyltransferase complex is associated with both the membrane and the cytoskeleton
[233]. When the organized channel is disrupted, some CCT complex (H-form) may remain on the membrane and some may be released into the cytosol. Thus, differences in the distribution of CCT between membrane and cytosol may reflect differences in the stability of the cytoskeleton organization between HCC and normal liver cells.
4.4.6 Effect of oleic acid on phosphatidylcholine synthesis
The major mechanisms for the regulation of PC synthesis has been postulated [234], that is,
CCT activity is sensitive to lipid environment [145, 232, 235-242]. We have demonstrated that PC synthesis in WCH17 cells can be accelerated in the presence of the 1 mM oleic acid
.
However, there might be different mechanism for the activation of CCT activity by fatty acid
(oleic acid) and phospholipids (PC, PE, DOPC, DOPE, lysoPC, lysoPE). One hypothesis is that the CCT has distinct binding sites for phospholipids and fatty acids [234]. These sites may have overlapping specificity. For example, the fatty acid site may bind weakly to phospholipids or neutral lipids, particularly if the lipids contain unsaturated fatty acids. The binding of lipids in the cytosol may cause an interconversion between the L and H forms of the enzyme. In addition, changes in membrane lipid composition may signal the enzyme to reversibly bind to specific membranes. Finally, certain changes in membrane lipid com- position may allow binding of specific phospholipids and fatty acids to the enzyme's lipid binding sites, resulting in activation.
164
In addition, PCho rapidly converted to PC in HCC at 30 min, whose accumulation pattern is different with those in vitro data and the data using xenograft model. The discrepancy between in vitro and in vivo can potentially be attributed to the distinct microenvironment between HCC and surrounding hepatic tissue with changed fatty acid profiles in HCC causing the activation of CCT activity in HCC.
4.4.7 Summary
PET imaging using 11C-CHOL showed promising on the detection of well-differentiated
HCC. Elevated levels of choline uptake and choline kinase activity in HCC relative to surrounding hepatic tissue motivated the use of 11C-CHOL as cancer imaging tracer for early detection of HCC. Phosphorylation of CHOL was the major metabolic fate in HCC.
Oxidative metabolism of radiolabeled choline was a minor pathway in HCC. Incorporation of
14C-CHOL into PC, an important component of cell membrane phospholipids, also increased in very short time, 30 min. Thus, the contrast between HCC and surrounding hepatic tissue on the PET images is associated with the metabolic fate of 11C-Choline. At early time point, the uptake contrast is from the transport and phosphorylation of CHOL; at late time point, the uptake contrast is from increased PC synthesis. Moreover, in contrast to the observations in
HCC cells, radiolabeled CHOL showed extensive oxidation in surrounding hepatic tissue and kidney. The high accumulation of CHOL radiotracers in kidney and surrounding liver tissue reflects the important role of these organs in choline metabolism in the body. Kidney and liver are known to be the major sites for oxidation of choline to betaine, which contributed to the background uptake during PET imaging. Our data suggest that the imaging contrast in
HCC was attributed to the PC synthesis. The discrepancy between in vitro and in vivo can
165
potentially be attributed to the distinct microenvironment between HCC and surrounding hepatic tissue with changed fatty acid profiles in HCC causing the activation of CCT activity in HCC. Future studies with woodchucks on different CHOL metabolism will allow us to image both well- and poorly- differentiated HCCs.
166
Chapter 5 Conclusions and Future Perspectives
5.1 Conclusions
Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Its incidence is increasing, driven by the burden of hepatitis B and C virus infection and cirrhosis [243-246].
The detection of HCC should occur as early as possible because only patients with a small number of nodules and small tumor size may benefit from curative treatments [205]. The current work-up to diagnose HCC includes medical imaging (CT, MRI, US), serum α- fetoprotein (AFP) assay and liver biopsy. When AFP is greater than 200 ng/mL and CT shows arterial hypervascularity, the diagnosis of HCC is most likely. However, the characterization of a hepatic nodule may be difficult in the absence of these criteria, in particular for small lesions (i.e. <2 cm) [205]. Therefore, the unmet needs include early detection of HCC, differentiation of malignant tumors from benign tumors (lesion characterization), accurate preoperative tumor staging etc. The carcinogenesis is frequently associated with the metabolic changes that precede the morphological changes. On the other hand, differentiation of early stage HCC from hepatic adenoma can be difficult even on core biopsy. Therefore, a non-invasive, fast, quantitative technique for detection of HCC is much needed. Positron emission tomography (PET), a molecular imaging technique, holds particular promise for diagnostic imaging of neoplasms. Our long term goal is to develop state-of-the-art PET imaging methods to help setting up successful HCC prognosis and early assessment of treatment efficacy. The focus of this thesis is on the diagnostic utility of PET metabolic imaging on HCC and the mechanisms underlying the imaging using radiolabeled choline (CHOL) as the tracer.
167
PET with 2-Deoxy-2-[18F]-fluoro-D-glucose (FDG) is a noninvasive diagnostic tool for diagnosis, initial staging, and follow up of many malignancies [103]. After intravenous injection, FDG, a non-physiological glucose analog, tends to accumulate mainly in malignant cells due to their high glucose metabolism. The overall sensitivity of FDG-PET in the detection of HCC is low (50-55%). The disappointing results of FDG-PET can be explained by the wide variability in enzyme activity in the individual HCC. In well-differentiated HCC,
FDG metabolism may be similar to that of the surrounding liver, leading to a false negative result, while higher sensitivity was reported in poorly differentiated HCC. Consequently,
FDG-PET is not useful in the diagnosis of small, well-differentiated HCC – the early stage of
HCC. On the contrary, metastatic liver tumors [247] generally show high FDG accumulation as they have both high glucose uptake and very low activity of the glucose-6-phosphatase, which in contrast are highly represented in the surrounding normal liver.
The high false negative rate of FDG emphasizes the need to identify a new metabolic probe of PET for the accurate imaging of HCC. Unique metabolism of CHOL in cancer cells has been used as the basis for molecular imaging with PET. We demonstrated CHOL is an appropriate PET tracer for imaging well-differentiated HCC in that over-expression of choline kinase (ChoK) and choline transporter can be found in HCC.
Transport and phosphorylation of CHOL are responsible for the tracer accumulation during
[11C]-CHOL PET imaging of HCC. Well-differentiated HCC cells incorporate [14C]-CHOL preferentially into Phosphocholine (PCho). Conversion of 14C-PCho into phosphatidylcholine
(PC) occurred slowly in vitro. Moreover, Basal oxidation and phosphorylation activities in
168
surrounding hepatic tissue contribute to the background signal seen in [11C]-CHOL PET images.
Furthermore, PET imaging of lipid synthesis with radiolabeled CHOL is useful in well- differentiated HCC that is not FDG avid. The imaging contrast from [11C]-CHOL in HCC attributed to the PC synthesis in vivo. The discrepancy between in vitro and in vivo can potentially be attributed to the distinct microenvironment between HCC and surrounding hepatic tissue with changed fatty acid profiles in HCC causing the activation of choline- phosphate cytidylyltransferase (CCT) activity in HCC.
PET/CT imaging with radiolabeled CHOL could thus be a very promising diagnostic tool in patients with suspicious liver masses or suspicion of HCC recurrence [205]. Furthermore, radiolabeled CHOL also showed metastases of HCC and could be also very useful for detection of both well-differentiated and poorly-differentiated HCC [205], which could make it a one-stop approach for the pre-therapeutic or restaging work-up of HCC. Future studies with woodchucks on different CHOL metabolism will allow us to image both well- and poorly- differentiated HCCs.
5.2 Future Prospectives
5.2.1 Effect of hypoxia on the tracer metabolism in hepatocellular carcinoma
Generally, hypoxia suppresses the proliferation of cells. However, Hep3B HCC cells do not exhibit orderly G(1)/S arrest in response to severe hypoxia [248, 249]. On the contrary, hypoxia stimulates the growth of HCC via inducing the expression of hexokinase II, an
169
enzyme taking part in the salvage pathway of generating ATP [250]. Hypoxia also up- regulates insulin-like growth factor-2 and thus stimulates the growth of HCC cells [251].
Besides the role of adaptation to hypoxia, Hypoxia induced factor-1 (HIF-1) can also protect cells against apoptosis under hypoxia by over-expressing myeloid cell factor-1, an antiapoptotic protein delaying or blocking the apoptosis of HCC [252].
Moreover, hypoxia enhances the expression of vascular endothelia growth factor (VEGF), which decreases the ratio of Bax/Bcl-2, and consequently leads to apoptosis blocking of HCC
[253]. Hypoxia induces the expression of cyclic AMP-responsive element binding protein, a transcription factor which is activated by multiple extracellular signals and modulates cellular response by regulating the expression of a multitude of genes to control growth, support angiogenesis, and render apoptosis resistance [254].
Taken together, hypoxia enhances the proliferation of HCC, suppresses the differentiation and apoptosis of HCC, and consequently leads to tumor malignancy. The hypothesis is that because of long-term hypoxia, HCC cells develop the ability to survive and proliferate in a hypoxia microenvironment. It is important to understand how the tracer metabolism changes in such challenging environmental conditions.
5.2.2 Ethanolamine and N, N'-dimethyl ethanolamine as probes for early detection of hepatocellular carcinoma
CHOL is a substrate for the synthesis of PC, which is the principal phospholipid component of mammalian cell membrane. Phosphorous-31 MRS data has established that phospholipid
170
precursor phosphomonoesters (PME), particularly phosphorylethanolamine (PEt) and phosphorylcho-line (PCho) are highly elevated in most human tumors compared with most normal tissues [255]. This has been attributed to the high proliferative rate of tumors, leading to an increased biosynthesis of phospholipid membranes. Therefore, imaging with radiolabeled CHOL by PET, which is more sensitive and has much higher spatial resolution than 3IP MRS, has provided a facile method for detecting tumors [256].
However, recent advances in MRS demonstrate that there may be better tracers than radiolabeled CHOL tor detecting phospholipid biosynthesis and hence proliferation in tumors
[256]. The development of in vivo 1H-decoupled, Nuclear Overhauser Enhancement (NOE)- enhanced 3IP-Magnetic Resonance Spectroscopic Imaging (MRSI) enabled the resolution or the resonances of phosphorylethanolamine (PE) and PCho peaks that normally overlap under a broad PME peak. This advance led to the discovery that PE concentrations in tumor are significantly greater then PC levels. Thus, ethanolamine or N, N'-dimethyl ethanolamine may be a more sensitive marker for HCC detection and prediction of proliferative capacity (Figure
5.1).
171
Figure 5.14 Comparison of metabolic pathway of choline and ethanolamine.
172
Bibliography
1. Hendee WR, Ritenour ER: Medical imaging physics. New York: Wiley-Liss Inc.;
2002.
2. Massoud TF, Gambhir SS: Molecular imaging in living subjects: seeing fundamental
biological processes in a new light. Genes Dev 2003, 17(5):545-580.
3. Weissleder R, Mahmood U: Molecular imaging. Radiology 2001, 219(2):316-333.
4. Plathow C, Weber WA: Tumor cell metabolism imaging. J Nucl Med 2008, 49 Suppl
2:43S-63S.
5. Bombardieri E, Bonadonna G, Gianni L: Breast Cancer: Nuclear Medicine in
Diagnosis and Therapeutic Options. Berlin Heidelberg: Springer-Verlag, Berlin-
Heidelberg-New York; 2008.
6. Herschman HR: Molecular imaging: looking at problems, seeing solutions. Science
2003, 302(5645):605-608.
7. Jaffer FA, Weissleder R: Molecular imaging in the clinical arena. JAMA 2005,
293(7):855-862.
8. Glunde K, Pathak AP, Bhujwalla ZM: Molecular-functional imaging of cancer: to
image and imagine. Trends Mol Med 2007, 13(7):287-297.
9. Rudin M, Weissleder R: Molecular imaging in drug discovery and development. Nat
Rev Drug Discov 2003, 2(2):123-131.
10. Rodes J, Benhamou J, Blei A, Reichen J, Rizzetto M: The textbook of hepatology:
from basic science to clinical practice, 3rd Ed. Malden: Blackwell Publishing, Inc.;
2007.
11. Gharib AM, Thomasson D, Li KC: Molecular imaging of hepatocellular carcinoma.
173
Gastroenterology 2004, 127(5 Suppl 1):S153-158.
12. Bailey DL, Townsend DW, Valk PE, Maisey MN: Positron emission tomography:
basic sciences. London: Springer-Verlag.
13. S D, C.E. C, W.R. K, H. P: On the Angular Distribution of Two-Photon Annihilation
Radiation. Phys Rev 1950, 77(2):205 - 212.
14. Levin CS, Hoffman EJ: Calculation of positron range and its effect on the
fundamental limit of positron emission tomography system spatial resolution. Phys
Med Biol 1999, 44(3):781-799.
15. Ollinger JM, Fessler JA: Positron-emission tomography. Ieee Signal Processing
Magazine 1997, 14(1):43-55.
16. Raylman RR, Hammer BE, Christensen NL: Combined MRI-PET scanner: A Monte
Carlo evaluation of the improvements in PET resolution due to the effects of a static
homogeneous magnetic field. Ieee Transactions on Nuclear Science 1996,
43(4):2406-2412.
17. Takigawa A, Nishikawa Y, Kubota H, Ogata Y, Matsumoto M, Kanamori H:
Measurement of scattered X-ray spectrum for the evaluation of radiographic contrast.
Journal of Photographic Science 1996, 44(1):31-34.
18. Lyamshev LM, Chelnokov BI: Theory of Sound Radiation by the Absorption of
Modulated Penetrating Radiation in a Solid Waveguide. Soviet Physics Acoustics-
Ussr 1983, 29(4):301-306.
19. Lyamshev LM, Chelnokov BI: Effect of the Penetrating Radiation Absorption
Peculiarities on the Sound Generation in Condensed Media. Zhurnal Tekhnicheskoi
Fiziki 1983, 53(11):2238-2241.
174
20. Hubbell JH: Review of photon interaction cross section data in the medical and
biological context. Physics in Medicine and Biology 1999, 44(1):R1-R22.
21. Hall H: The Theory of Photoelectric Absorption for X-Rays and γ-Rays. Reviews of
Modern Physics 1936, 8(4):358-397.
22. Tai YC, Chatziioannou AF, Yang YF, Silverman RW, Meadors K, Siegel S, Newport
DF, Stickel JR, Cherry SR: MicroPET II: design, development and initial
performance of an improved microPET scanner for small-animal imaging. Physics in
Medicine and Biology 2003, 48(11):1519-1537.
23. Humm JL, Rosenfeld A, Del Guerra A: From PET detectors to PET scanners.
European Journal of Nuclear Medicine and Molecular Imaging 2003, 30(11):1574-
1597.
24. Budinger TF: Time-of-Flight Positron-Emission Tomography Status Relative to
Conventional Pet - Reply. Journal of Nuclear Medicine 1984, 25(1):137-138.
25. Budinger TF: Time-of-Flight Positron Emission Tomography - Status Relative to
Conventional Pet. Journal of Nuclear Medicine 1983, 24(1):73-76.
26. Terpogossian MM, Ficke DC: Time-of-Flight Positron-Emission Tomography Status
Relative to Conventional Pet. Journal of Nuclear Medicine 1984, 25(1):136-137.
27. Lewellen TK: Time-of-Flight PET. Seminars in Nuclear Medicine 1998, 28(3):268-
275.
28. Kuhn A, Surti S, Karp JS, Muehllehner G, Newcomer FM, VanBerg R: Performance
assessment of pixelated LaBr3 detector modules for time-of-flight PET. Ieee
Transactions on Nuclear Science 2006, 53(3):1090-1095.
29. Rust TC: Advances in rapid multi-tracer positron emission tomography. Ph.D. United
175
States -- Utah: The University of Utah; 2007.
30. Introduction to PET Physics. http://deptswashingtonedu/nucmed/IRL/pet_intro/.
31. Kalman S: Introduction to PET Instrumentation. J Nucl Med Technol 2002, 30(2):63;
author reply 63.
32. Mazoyer BM, Roos MS, Huesman RH: Dead time correction and counting statistics
for positron tomography. Phys Med Biol 1985, 30(5):385-399.
33. Alessio AM, Kinahan PE, Cheng PM, Vesselle H, Karp JS: PET/CT scanner
instrumentation, challenges, and solutions. Radiologic clinics of North America 2004,
42(6):1017-1032, vii.
34. Kadrmas DJ: LOR-OSEM: statistical PET reconstruction from raw line-of-response
histograms. Phys Med Biol 2004, 49(20):4731-4744.
35. Yamaya T, Hagiwara N, Obi T, Yamaguchi M, Ohyama N, Kitamura K, Hasegawa T,
Haneishi H, Yoshida E, Inadama N et al: Transaxial system models for jPET-D4
image reconstruction. Phys Med Biol 2005, 50(22):5339-5355.
36. Brooks RA, Di Chiro G: Principles of computer assisted tomography (CAT) in
radiographic and radioisotopic imaging. Phys Med Biol 1976, 21(5):689-732.
37. Shepp LA, Vardi Y: Maximum likelihood reconstruction for emission tomography.
IEEE Trans Med Imaging 1982, 1(2):113-122.
38. Lange K, Carson R: EM reconstruction algorithms for emission and transmission
tomography. J Comput Assist Tomogr 1984, 8(2):306-316.
39. Hudson HM, Larkin RS: Accelerated image reconstruction using ordered subsets of
projection data. IEEE Trans Med Imaging 1994, 13(4):601-609.
40. Brasse D, Kinahan PE, Lartizien C, Comtat C, Casey M, Michel C: Correction
176
methods for random coincidences in fully 3D whole-body PET: impact on data and
image quality. J Nucl Med 2005, 46(5):859-867.
41. Bailey DL, Meikle SR: A convolution-subtraction scatter correction method for 3D
PET. Phys Med Biol 1994, 39(3):411-424.
42. Cherry SR, Meikle SR, Hoffman EJ: Correction and characterization of scattered
events in three-dimensional PET using scanners with retractable septa. J Nucl Med
1993, 34(4):671-678.
43. Grootoonk S, Spinks TJ, Sashin D, Spyrou NM, Jones T: Correction for scatter in 3D
brain PET using a dual energy window method. Phys Med Biol 1996, 41(12):2757-
2774.
44. Koike K, Tsutsumi T, Fujie H, Shintani Y, Kyoji M: Molecular mechanism of viral
hepatocarcinogenesis. Oncology 2002, 62 Suppl 1:29-37.
45. Hino O, Kajino K, Umeda T, Arakawa Y: Understanding the hypercarcinogenic state
in chronic hepatitis: a clue to the prevention of human hepatocellular carcinoma. J
Gastroenterol 2002, 37(11):883-887.
46. Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K:
Comprehensive analysis of microRNA expression patterns in hepatocellular
carcinoma and non-tumorous tissues. Oncogene 2006, 25(17):2537-2545.
47. Peng Z, Zhang Y, Gu W, Wang Z, Li D, Zhang F, Qiu G, Xie K: Integration of the
hepatitis B virus X fragment in hepatocellular carcinoma and its effects on the
expression of multiple molecules: a key to the cell cycle and apoptosis. Int J Oncol
2005, 26(2):467-473.
48. Singh M, Kumar V: Transgenic mouse models of hepatitis B virus-associated
177
hepatocellular carcinoma. Rev Med Virol 2003, 13(4):243-253.
49. Tai PC, Suk FM, Gerlich WH, Neurath AR, Shih C: Hypermodification and immune
escape of an internally deleted middle-envelope (M) protein of frequent and
predominant hepatitis B virus variants. Virology 2002, 292(1):44-58.
50. Momosaki S, Hsia CC, Nakashima Y, Kojiro M, Tabor E: Integration of hepatitis B
virus containing mutations in the core promoter/X gene in patients with
hepatocellular carcinoma. Dig Liver Dis 2003, 35(11):795-800.
51. Ikeda K, Saitoh S, Koida I, Arase Y, Tsubota A, Chayama K, Kumada H, Kawanishi
M: A multivariate analysis of risk factors for hepatocellular carcinogenesis: a
prospective observation of 795 patients with viral and alcoholic cirrhosis. Hepatology
1993, 18(1):47-53.
52. Tennant BC, Toshkov IA, Peek SF, Jacob JR, Menne S, Hornbuckle WE, Schinazi
RD, Korba BE, Cote PJ, Gerin JL: Hepatocellular carcinoma in the woodchuck model
of hepatitis B virus infection. Gastroenterology 2004, 127(5 Suppl 1):S283-293.
53. Korba BE, Wells FV, Baldwin B, Cote PJ, Tennant BC, Popper H, Gerin JL:
Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks:
presence of viral DNA in tumor tissue from chronic carriers and animals serologically
recovered from acute infections. Hepatology 1989, 9(3):461-470.
54. Wei Y, Fourel G, Ponzetto A, Silvestro M, Tiollais P, Buendia MA: Hepadnavirus
integration: mechanisms of activation of the N-myc2 retrotransposon in woodchuck
liver tumors. J Virol 1992, 66(9):5265-5276.
55. Jacob JR, Sterczer A, Toshkov IA, Yeager AE, Korba BE, Cote PJ, Buendia MA,
Gerin JL, Tennant BC: Integration of woodchuck hepatitis and N-myc rearrangement
178
determine size and histologic grade of hepatic tumors. Hepatology 2004, 39(4):1008-
1016.
56. Su Q, Benner A, Hofmann WJ, Otto G, Pichlmayr R, Bannasch P: Human hepatic
preneoplasia: phenotypes and proliferation kinetics of foci and nodules of altered
hepatocytes and their relationship to liver cell dysplasia. Virchows Arch 1997,
431(6):391-406.
57. Murakami Y, Saigo K, Takashima H, Minami M, Okanoue T, Brechot C, Paterlini-
Brechot P: Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV
related hepatocellular carcinomas. Gut 2005, 54(8):1162-1168.
58. Ferber MJ, Montoya DP, Yu C, Aderca I, McGee A, Thorland EC, Nagorney DM,
Gostout BS, Burgart LJ, Boix L et al: Integrations of the hepatitis B virus (HBV) and
human papillomavirus (HPV) into the human telomerase reverse transcriptase
(hTERT) gene in liver and cervical cancers. Oncogene 2003, 22(24):3813-3820.
59. Lupberger J, Hildt E: Hepatitis B virus-induced oncogenesis. World J Gastroenterol
2007, 13(1):74-81.
60. Bruni R, Conti I, Villano U, Giuseppetti R, Palmieri G, Rapicetta M: Lack of WHV
integration nearby N-myc2 and in the downstream b3n and win loci in a considerable
fraction of liver tumors with activated N-myc2 from naturally infected wild
woodchucks. Virology 2006, 345(1):258-269.
61. Taketa K: Alpha-fetoprotein: reevaluation in hepatology. Hepatology 1990,
12(6):1420-1432.
62. Camaggi V, Piscaglia F, Bolondi L: Recent advances in the imaging of hepatocellular
carcinoma. From ultrasound to positron emission tomography scan. Saudi Med J
179
2007, 28(7):1007-1014.
63. Cheson BD, Pfistner B, Juweid ME, Gascoyne RD, Specht L, Horning SJ, Coiffier B,
Fisher RI, Hagenbeek A, Zucca E et al: Revised response criteria for malignant
lymphoma. J Clin Oncol 2007, 25(5):579-586.
64. Kelloff GJ, Hoffman JM, Johnson B, Scher HI, Siegel BA, Cheng EY, Cheson BD,
O'Shaughnessy J, Guyton KZ, Mankoff DA et al: Progress and promise of FDG-PET
imaging for cancer patient management and oncologic drug development. Clin
Cancer Res 2005, 11(8):2785-2808.
65. von Schulthess GK, Steinert HC, Hany TF: Integrated PET/CT: current applications
and future directions. Radiology 2006, 238(2):405-422.
66. Pirotte B, Goldman S, Massager N, David P, Wikler D, Vandesteene A, Salmon I,
Brotchi J, Levivier M: Comparison of 18F-FDG and 11C-methionine for PET-guided
stereotactic brain biopsy of gliomas. J Nucl Med 2004, 45(8):1293-1298.
67. Menendez JA, Lupu R: Fatty acid synthase and the lipogenic phenotype in cancer
pathogenesis. Nat Rev Cancer 2007, 7(10):763-777.
68. Swinnen JV, Roskams T, Joniau S, Van Poppel H, Oyen R, Baert L, Heyns W,
Verhoeven G: Overexpression of fatty acid synthase is an early and common event in
the development of prostate cancer. Int J Cancer 2002, 98(1):19-22.
69. Van de Sande T, Roskams T, Lerut E, Joniau S, Van Poppel H, Verhoeven G,
Swinnen JV: High-level expression of fatty acid synthase in human prostate cancer
tissues is linked to activation and nuclear localization of Akt/PKB. J Pathol 2005,
206(2):214-219.
70. Swinnen JV, Ulrix W, Heyns W, Verhoeven G: Coordinate regulation of lipogenic
180
gene expression by androgens: evidence for a cascade mechanism involving sterol
regulatory element binding proteins. Proc Natl Acad Sci U S A 1997, 94(24):12975-
12980.
71. Hochachka PW, Rupert JL, Goldenberg L, Gleave M, Kozlowski P: Going malignant:
the hypoxia-cancer connection in the prostate. Bioessays 2002, 24(8):749-757.
72. Yoshimoto M, Waki A, Obata A, Furukawa T, Yonekura Y, Fujibayashi Y:
Radiolabeled choline as a proliferation marker: comparison with radiolabeled acetate.
Nucl Med Biol 2004, 31(7):859-865.
73. Delbeke D, Meyerowitz C, Lapidus RL, Maciunas RJ, Jennings MT, Moots PL,
Kessler RM: Optimal cutoff levels of F-18 fluorodeoxyglucose uptake in the
differentiation of low-grade from high-grade brain tumors with PET. Radiology 1995,
195(1):47-52.
74. Kotzerke J, Prang J, Neumaier B, Volkmer B, Guhlmann A, Kleinschmidt K,
Hautmann R, Reske SN: Experience with carbon-11 choline positron emission
tomography in prostate carcinoma. Eur J Nucl Med 2000, 27(9):1415-1419.
75. Beasley RP, Hwang LY, Lin CC, Chien CS: Hepatocellular carcinoma and hepatitis
B virus. A prospective study of 22 707 men in Taiwan. Lancet 1981, 2(8256):1129-
1133.
76. Huang GT, Sheu JC, Yang PM, Lee HS, Wang TH, Chen DS: Ultrasound-guided
cutting biopsy for the diagnosis of hepatocellular carcinoma--a study based on 420
patients. J Hepatol 1996, 25(3):334-338.
77. Jeng LB, Changlai SP, Shen YY, Lin CC, Tsai CH, Kao CH: Limited value of 18F-2-
deoxyglucose positron emission tomography to detect hepatocellular carcinoma in
181
hepatitis B virus carriers. Hepatogastroenterology 2003, 50(54):2154-2156.
78. Sheu JC, Sung JL, Chen DS, Yu JY, Wang TH, Su CT, Tsang YM: Ultrasonography
of small hepatic tumors using high-resolution linear-array real-time instruments.
Radiology 1984, 150(3):797-802.
79. Yoshida T, Matsue H, Okazaki N, Yoshino M: Ultrasonographic differentiation of
hepatocellular carcinoma from metastatic liver cancer. J Clin Ultrasound 1987,
15(7):431-437.
80. Flier JS, Mueckler MM, Usher P, Lodish HF: Elevated levels of glucose transport and
transporter messenger RNA are induced by ras or src oncogenes. Science 1987,
235(4795):1492-1495.
81. Monakhov NK, Neistadt EL, Shavlovskil MM, Shvartsman AL, Neifakh SA:
Physicochemical properties and isoenzyme composition of hexokinase from normal
and malignant human tissues. J Natl Cancer Inst 1978, 61(1):27-34.
82. Knox WE, Jamdar SC, Davis PA: Hexokinase, differentiation and growth rates of
transplanted rat tumors. Cancer Res 1970, 30(8):2240-2244.
83. Di Chiro G, DeLaPaz RL, Brooks RA, Sokoloff L, Kornblith PL, Smith BH, Patronas
NJ, Kufta CV, Kessler RM, Johnston GS et al: Glucose utilization of cerebral gliomas
measured by [18F] fluorodeoxyglucose and positron emission tomography.
Neurology 1982, 32(12):1323-1329.
84. Di Chiro G, Oldfield E, Wright DC, De Michele D, Katz DA, Patronas NJ, Doppman
JL, Larson SM, Ito M, Kufta CV: Cerebral necrosis after radiotherapy and/or
intraarterial chemotherapy for brain tumors: PET and neuropathologic studies. AJR
Am J Roentgenol 1988, 150(1):189-197.
182
85. Gambhir SS, Czernin J, Schwimmer J, Silverman DH, Coleman RE, Phelps ME: A
tabulated summary of the FDG PET literature. J Nucl Med 2001, 42(5 Suppl):1S-93S.
86. Wahl RL, Quint LE, Greenough RL, Meyer CR, White RI, Orringer MB: Staging of
mediastinal non-small cell lung cancer with FDG PET, CT, and fusion images:
preliminary prospective evaluation. Radiology 1994, 191(2):371-377.
87. Vitola JV, Delbeke D, Sandler MP, Campbell MG, Powers TA, Wright JK, Chapman
WC, Pinson CW: Positron emission tomography to stage suspected metastatic
colorectal carcinoma to the liver. Am J Surg 1996, 171(1):21-26.
88. Vitola JV, Delbeke D, Meranze SG, Mazer MJ, Pinson CW: Positron emission
tomography with F-18-fluorodeoxyglucose to evaluate the results of hepatic
chemoembolization. Cancer 1996, 78(10):2216-2222.
89. Hepatocellular Carcinoma Screening, Diagnosis, and Management.
90. Palma LD: Diagnostic imaging and interventional therapy of hepatocellular
carcinoma. Br J Radiol 1998, 71(848):808-818.
91. Gouillat C, Manganas D, Zoulim F, Vitrey D, Saguier G, Guillaud M, Ain JF, Duque-
Campos R, Jamard C, Praves M et al: Woodchuck hepatitis virus-induced carcinoma
as a relevant natural model for therapy of human hepatoma. J Hepatol 1997,
26(6):1324-1330.
92. Summers J, Smolec JM, Snyder R: A virus similar to human hepatitis B virus
associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci U S A
1978, 75(9):4533-4537.
93. Qiu W, David D, Zhou B, Chu PG, Zhang B, Wu M, Xiao J, Han T, Zhu Z, Wang T
et al: Down-regulation of growth arrest DNA damage-inducible gene 45beta
183
expression is associated with human hepatocellular carcinoma. Am J Pathol 2003,
162(6):1961-1974.
94. Bradford MM: A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
1976, 72:248-254.
95. Baginski ES, Foa PP, Zak B: Microdetermination of inorganic phosphate,
phospholipids, and total phosphate in biologic materials. Clin Chem 1967, 13(4):326-
332.
96. Ashmore J, Cahill GF, Jr., Hastings AB, Zottu S: Studies on carbohydrate metabolism
in rat liver slices. IX. Ionic and hormonal effects on phosphorylase and glycogen. J
Biol Chem 1957, 224(1):237-250.
97. Burchell A, Burchell B: Identification and purification of a liver microsomal glucose
6-phosphatase. Biochem J 1982, 205(3):567-573.
98. Shibata T, Yamamoto K, Hayashi N, Yonekura Y, Nagara T, Saji H, Mukai T,
Konishi J: Dynamic positron emission tomography with 13N-ammonia in liver
tumors. Eur J Nucl Med 1988, 14(12):607-611.
99. Chen S, Feng D: Noninvasive quantification of the differential portal and arterial
contribution to the liver blood supply from PET measurements using the 11C-acetate
kinetic model. IEEE Trans Biomed Eng 2004, 51(9):1579-1585.
100. Shinagawa T, Ohto M, Kimura K, Tsunetomi S, Morita M, Saisho H, Tsuchiya Y,
Saotome N, Karasawa E, Miki M et al: Diagnosis and clinical features of small
hepatocellular carcinoma with emphasis on the utility of real-time ultrasonography. A
study in 51 patients. Gastroenterology 1984, 86(3):495-502.
184
101. Okazumi S, Isono K, Enomoto K, Kikuchi T, Ozaki M, Yamamoto H, Hayashi H,
Asano T, Ryu M: Evaluation of liver tumors using fluorine-18-fluorodeoxyglucose
PET: characterization of tumor and assessment of effect of treatment. J Nucl Med
1992, 33(3):333-339.
102. Oka H, Kurioka N, Kim K, Kanno T, Kuroki T, Mizoguchi Y, Kobayashi K:
Prospective study of early detection of hepatocellular carcinoma in patients with
cirrhosis. Hepatology 1990, 12(4 Pt 1):680-687.
103. Rigo P, Paulus P, Kaschten BJ, Hustinx R, Bury T, Jerusalem G, Benoit T, Foidart-
Willems J: Oncological applications of positron emission tomography with fluorine-
18 fluorodeoxyglucose. Eur J Nucl Med 1996, 23(12):1641-1674.
104. Katz-Brull R, Degani H: Kinetics of choline transport and phosphorylation in human
breast cancer cells; NMR application of the zero trans method. Anticancer Res 1996,
16(3B):1375-1380.
105. Zeisel SH: Choline phospholipids: signal transduction and carcinogenesis. FASEB J
1993, 7(6):551-557.
106. Ishidate K: Choline/ethanolamine kinase from mammalian tissues. Biochimica et
biophysica acta 1997, 1348(1-2):70-78.
107. Wright PS, Morand JN, Kent C: Regulation of phosphatidylcholine biosynthesis in
Chinese hamster ovary cells by reversible membrane association of CTP:
phosphocholine cytidylyltransferase. The Journal of biological chemistry 1985,
260(13):7919-7926.
108. Wettstein M, Weik C, Holneicher C, Haussinger D: Betaine as an osmolyte in rat
liver: metabolism and cell-to-cell interactions. Hepatology (Baltimore, Md 1998,
185
27(3):787-793.
109. Wajda IJ, Manigault I, Hudick JP, Lajtha A: Regional and subcellular distribution of
choline acetyltransferase in the brain of rats. J Neurochem 1973, 21(6):1385-1401.
110. Roivainen A, Forsback S, Gronroos T, Lehikoinen P, Kahkonen M, Sutinen E, Minn
H: Blood metabolism of [methyl-11C]choline; implications for in vivo imaging with
positron emission tomography. Eur J Nucl Med 2000, 27(1):25-32.
111. Zeisel SH: Dietary choline: biochemistry, physiology, and pharmacology. Annu Rev
Nutr 1981, 1:95-121.
112. Hara T, Bansal A, DeGrado TR: Choline transporter as a novel target for molecular
imaging of cancer. Mol Imaging 2006, 5(4):498-509.
113. Gillies RJ, Morse DL: In vivo magnetic resonance spectroscopy in cancer. Annu Rev
Biomed Eng 2005, 7:287-326.
114. Moller-Hartmann W, Herminghaus S, Krings T, Marquardt G, Lanfermann H, Pilatus
U, Zanella FE: Clinical application of proton magnetic resonance spectroscopy in the
diagnosis of intracranial mass lesions. Neuroradiology 2002, 44(5):371-381.
115. Smith TA, Eccles S, Ormerod MG, Tombs AJ, Titley JC, Leach MO: The
phosphocholine and glycerophosphocholine content of an oestrogen-sensitive rat
mammary tumour correlates strongly with growth rate. Br J Cancer 1991, 64(5):821-
826.
116. Ackerstaff E, Pflug BR, Nelson JB, Bhujwalla ZM: Detection of increased choline
compounds with proton nuclear magnetic resonance spectroscopy subsequent to
malignant transformation of human prostatic epithelial cells. Cancer Res 2001,
61(9):3599-3603.
186
117. Mori N, Delsite R, Natarajan K, Kulawiec M, Bhujwalla ZM, Singh KK: Loss of p53
function in colon cancer cells results in increased phosphocholine and total choline.
Mol Imaging 2004, 3(4):319-323.
118. Evelhoch JL, Keller NA, Corbett TH: Response-specific adriamycin sensitivity
markers provided by in vivo 31P nuclear magnetic resonance spectroscopy in murine
mammary adenocarcinomas. Cancer Res 1987, 47(13):3396-3401.
119. Guengerich FP: Isolation and purification of cytochrome P-450, and the existence of
multiple forms. Pharmacol Ther 1979, 6(1):99-121.
120. Berry MN, Friend DS: High-yield preparation of isolated rat liver parenchymal cells:
a biochemical and fine structural study. J Cell Biol 1969, 43(3):506-520.
121. Nilsson A, Sundler R, Akesson B: Biosynthesis of fatty acids and cholesterol in
isolated rat-liver parenchymal cells. Effect of albumin-bound fatty acids. Eur J
Biochem 1973, 39(2):613-620.
122. Van Harken DR, Dixon CW, Heimberg M: Hepatic lipid metabolism in experimental
diabetes. V. The effect of concentration of oleate on metabolism of triglycerides and
on ketogenesis. J Biol Chem 1969, 244(9):2278-2285.
123. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 1959, 37(8):911-917.
124. Liscovitch M, Freese A, Blusztajn JK, Wurtman RJ: High-performance liquid
chromatography of water-soluble choline metabolites. Anal Biochem 1985,
151(1):182-187.
125. Yavin E: Regulation of phospholipid metabolism in differentiating cells from rat
brain cerebral hemispheres in culture. Patterns of acetylcholine phosphocholine, and
187
choline phosphoglycerides labeling from (methyl-14C)choline. J Biol Chem 1976,
251(5):1392-1397.
126. Arduini A, Peschechera A, Dottori S, Sciarroni AF, Serafini F, Calvani M: High
performance liquid chromatography of long-chain acylcarnitine and phospholipids in
fatty acid turnover studies. J Lipid Res 1996, 37(3):684-689.
127. Hamilton JG, Comai K: Separation of neutral lipids and free fatty acids by high-
performance liquid chromatography using low wavelength ultraviolet detection. J
Lipid Res 1984, 25(10):1142-1148.
128. Kupke IR, Zeugner S: Quantitative high-performance thin-layer chromatography of
lipids in plasma and liver homogenates after direct application of 0.5-microliter
samples to the silica-gel layer. J Chromatogr 1978, 146(2):261-271.
129. Bansal A, Shuyan W, Hara T, Harris RA, Degrado TR: Biodisposition and
metabolism of [(18)F]fluorocholine in 9L glioma cells and 9L glioma-bearing fisher
rats. Eur J Nucl Med Mol Imaging 2008, 35(6):1192-1203.
130. Ishidate K, Nakazawa Y: Choline/ethanolamine kinase from rat kidney. Methods
Enzymol 1992, 209:121-134.
131. Bansal A, Shuyan W, Hara T, Harris RA, Degrado TR: Biodisposition and
metabolism of [(18)F]fluorocholine in 9L glioma cells and 9L glioma-bearing fisher
rats. European journal of nuclear medicine and molecular imaging 2008.
132. Zhang L, Dresser MJ, Gray AT, Yost SC, Terashita S, Giacomini KM: Cloning and
functional expression of a human liver organic cation transporter. Mol Pharmacol
1997, 51(6):913-921.
133. Gorboulev V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U,
188
Quester S, Baumann C, Lang F, Busch AE, Koepsell H: Cloning and characterization
of two human polyspecific organic cation transporters. DNA Cell Biol 1997,
16(7):871-881.
134. Negendank W: Studies of human tumors by MRS: a review. NMR Biomed 1992,
5(5):303-324.
135. Preul MC, Caramanos Z, Collins DL, Villemure JG, Leblanc R, Olivier A, Pokrupa R,
Arnold DL: Accurate, noninvasive diagnosis of human brain tumors by using proton
magnetic resonance spectroscopy. Nat Med 1996, 2(3):323-325.
136. Cecil KM, Schnall MD, Siegelman ES, Lenkinski RE: The evaluation of human
breast lesions with magnetic resonance imaging and proton magnetic resonance
spectroscopy. Breast Cancer Res Treat 2001, 68(1):45-54.
137. Yeung DK, Cheung HS, Tse GM: Human breast lesions: characterization with
contrast-enhanced in vivo proton MR spectroscopy--initial results. Radiology 2001,
220(1):40-46.
138. Roebuck JR, Cecil KM, Schnall MD, Lenkinski RE: Human breast lesions:
characterization with proton MR spectroscopy. Radiology 1998, 209(1):269-275.
139. Kvistad KA, Bakken IJ, Gribbestad IS, Ehrnholm B, Lundgren S, Fjosne HE,
Haraldseth O: Characterization of neoplastic and normal human breast tissues with in
vivo (1)H MR spectroscopy. J Magn Reson Imaging 1999, 10(2):159-164.
140. Jagannathan NR, Kumar M, Seenu V, Coshic O, Dwivedi SN, Julka PK, Srivastava A,
Rath GK: Evaluation of total choline from in-vivo volume localized proton MR
spectroscopy and its response to neoadjuvant chemotherapy in locally advanced
breast cancer. Br J Cancer 2001, 84(8):1016-1022.
189
141. Rafter JE, Chalmers RA, Iles RA: Conversion of choline to betaine in young rats: a
1H n.m.r. spectroscopic study. Biochem Soc Trans 1989, 17(6):1130-1131.
142. Brotschi EA, Hilbinger CL, Kahl EA, Vaules WA, Midis NA, Blusztajn JK, Zeisel
SH: Radioactive choline metabolism in guinea pig gallbladder. Is there measurable
acetylcholine release? Dig Dis Sci 1995, 40(9):1982-1989.
143. Moriyama T, Garcia-Perez A, Olson AD, Burg MB: Intracellular betaine substitutes
for sorbitol in protecting renal medullary cells from hypertonicity. Am J Physiol 1991,
260(4 Pt 2):F494-497.
144. Grossman EB, Hebert SC: Renal inner medullary choline dehydrogenase activity:
characterization and modulation. The American journal of physiology 1989, 256(1 Pt
2):F107-112.
145. Pelech SL, Pritchard PH, Brindley DN, Vance DE: Fatty acids promote translocation
of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and
stimulate rat hepatic phosphatidylcholine synthesis. The Journal of biological
chemistry 1983, 258(11):6782-6788.
146. Pelech SL, Pritchard PH, Vance DE: cAMP analogues inhibit phosphatidylcholine
biosynthesis in cultured rat hepatocytes. The Journal of biological chemistry 1981,
256(16):8283-8286.
147. Pelech SL, Vance DE: Regulation of phosphatidylcholine biosynthesis. Biochim
Biophys Acta 1984, 779(2):217-251.
148. Hernandez-Alcoceba R, Fernandez F, Lacal JC: In vivo antitumor activity of choline
kinase inhibitors: a novel target for anticancer drug discovery. Cancer Res 1999,
59(13):3112-3118.
190
149. Zeisel SH, Da Costa KA, Franklin PD, Alexander EA, Lamont JT, Sheard NF, Beiser
A: Choline, an essential nutrient for humans. FASEB J 1991, 5(7):2093-2098.
150. Pomfret EA, daCosta KA, Schurman LL, Zeisel SH: Measurement of choline and
choline metabolite concentrations using high-pressure liquid chromatography and gas
chromatography-mass spectrometry. Anal Biochem 1989, 180(1):85-90.
151. Okuda T, Haga T, Kanai Y, Endou H, Ishihara T, Katsura I: Identification and
characterization of the high-affinity choline transporter. Nat Neurosci 2000, 3(2):120-
125.
152. Schloss P, Mayser W, Niehuis A, Betz H: Na(+)-dependent high-affinity uptake of
choline into cultured fibroblasts. Biochem Biophys Res Commun 1994, 199(3):1320-
1325.
153. Michel V, Yuan Z, Ramsubir S, Bakovic M: Choline transport for phospholipid
synthesis. Exp Biol Med (Maywood) 2006, 231(5):490-504.
154. Ribbes G, Hamza M, Chap H, Douste-Blazy L: Carrier-mediated choline uptake by
Krebs II ascites cells. Biochim Biophys Acta 1985, 818(2):183-190.
155. Yorek MA, Dunlap JA, Spector AA, Ginsberg BH: Effect of ethanolamine on choline
uptake and incorporation into phosphatidylcholine in human Y79 retinoblastoma cells.
J Lipid Res 1986, 27(11):1205-1213.
156. Plagemann PG: Choline metabolism and membrane formation in rat hepatoma cells
grown in suspension culture. 3. Choline transport and uptake by simple diffusion and
lack of direct exchange with phosphatidylcholine. J Lipid Res 1971, 12(6):715-724.
157. Lanks K, Somers L, Papirmeister B, Yamamura H: Choline transport by
neuroblastoma cells in tissue culture. Nature 1974, 252(5483):476-478.
191
158. Lloveras J, Hamza M, Chap H, Douste-Blazy L: Action of hemicholinium-3 on
phospholipid metabolism in Krebs II ascites cells. Biochem Pharmacol 1985,
34(22):3987-3993.
159. Haeffner EW: Studies on choline permeation through the plasma membrane and its
incorporation into phosphatidyl choline of Ehrlich-Lettre-ascites tumor cells in vitro.
Eur J Biochem 1975, 51(1):219-228.
160. Doppler W, Hofmann J, Maly K, Grunicke H: Amiloride and 5-N,N-
dimethylamiloride inhibit the carrier mediated uptake of choline in Ehrlich ascites
tumor cells. Biochem Pharmacol 1987, 36(10):1645-1649.
161. Walum E: Uptake and release of choline in cultures of human glioma cells. Cell Mol
Neurobiol 1981, 1(4):389-399.
162. Nuwayhid SJ, Vega M, Walden PD, Monaco ME: Regulation of de novo
phosphatidylinositol synthesis. Journal of lipid research 2006, 47(7):1449-1456.
163. Porter RK, Scott JM, Brand MD: Choline transport into rat liver mitochondria.
Characterization and kinetics of a specific transporter. J Biol Chem 1992,
267(21):14637-14646.
164. de Ridder JJ: The uptake of choline by rat liver mitochondria. Biochim Biophys Acta
1976, 449(2):236-244.
165. Tyler DD: Transport and oxidation of choline by liver mitochondria. Biochem J 1977,
166(3):571-581.
166. DeGrado TR, Coleman RE, Wang S, Baldwin SW, Orr MD, Robertson CN, Polascik
TJ, Price DT: Synthesis and evaluation of 18F-labeled choline as an oncologic tracer
for positron emission tomography: initial findings in prostate cancer. Cancer Res
192
2001, 61(1):110-117.
167. Morse DL: Choline metabolites as diagnostic and therapeutic response indicators for
breast cancer. Ph.D. United States -- Arizona: The University of Arizona; 2004.
168. Sung CP, Johnstone RM: Evidence for active transport of choline in rat kidney cortex
slices. Can J Biochem 1965, 43(7):1111-1118.
169. Zeisel SH, Story DL, Wurtman RJ, Brunengraber H: Uptake of free choline by
isolated perfused rat liver. Proc Natl Acad Sci U S A 1980, 77(8):4417-4419.
170. Pelech SL, Pritchard PH, Vance DE: Prolonged effects of cyclic AMP analogues of
phosphatidylcholine biosynthesis in cultured rat hepatocytes. Biochim Biophys Acta
1982, 713(2):260-269.
171. Pritchard PH, Vance DE: Choline metabolism and phosphatidylcholine biosynthesis
in cultured rat hepatocytes. Biochem J 1981, 196(1):261-267.
172. Plagemann PG, Roth MF: Permeation as the rate-limiting step in the phosphorylation
of uridine and choline and their incorporation into macromolecules by Novikoff
hepatoma cells. Competitive inhibition by phenethyl alcohol, persantin, and
adenosine. Biochemistry 1969, 8(12):4782-4789.
173. Sinclair CJ, Chi KD, Subramanian V, Ward KL, Green RM: Functional expression of
a high affinity mammalian hepatic choline/organic cation transporter. Journal of lipid
research 2000, 41(11):1841-1848.
174. Weinhold PA, Charles L, Rounsifer ME, Feldman DA: Control of
phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity
and immunoreactive content of choline phosphate cytidylyltransferase. The Journal of
biological chemistry 1991, 266(10):6093-6100.
193
175. Carr BI: Hepatocellular cancer: diagnosis and treatment. Totawa: Humana Press Inc.;
2005.
176. Delbeke D, Martin WH, Sandler MP, Chapman WC, Wright JK, Jr., Pinson CW:
Evaluation of benign vs malignant hepatic lesions with positron emission tomography.
Arch Surg 1998, 133(5):510-515; discussion 515-516.
177. Khan MA, Combs CS, Brunt EM, Lowe VJ, Wolverson MK, Solomon H, Collins BT,
Di Bisceglie AM: Positron emission tomography scanning in the evaluation of
hepatocellular carcinoma. J Hepatol 2000, 32(5):792-797.
178. Trojan J, Schroeder O, Raedle J, Baum RP, Herrmann G, Jacobi V, Zeuzem S:
Fluorine-18 FDG positron emission tomography for imaging of hepatocellular
carcinoma. Am J Gastroenterol 1999, 94(11):3314-3319.
179. Narayan P, Jajodia P, Kurhanewicz J, Thomas A, MacDonald J, Hubesch B,
Hedgcock M, Anderson CM, James TL, Tanagho EA et al: Characterization of
prostate cancer, benign prostatic hyperplasia and normal prostates using transrectal
31phosphorus magnetic resonance spectroscopy: a preliminary report. J Urol 1991,
146(1):66-74.
180. Narayan P, Kurhanewicz J: Magnetic resonance spectroscopy in prostate disease:
diagnostic possibilities and future developments. Prostate Suppl 1992, 4:43-50.
181. Daly PF, Cohen JS: Magnetic resonance spectroscopy of tumors and potential in vivo
clinical applications: a review. Cancer Res 1989, 49(4):770-779.
182. Shinkwin MA, Lenkinski RE, Daly JM, Zlatkin MB, Frank TS, Holland GA, Kressel
HY: Integrated magnetic resonance imaging and phosphorus spectroscopy of soft
tissue tumors. Cancer 1991, 67(7):1849-1858.
194
183. Smith TA, Bush C, Jameson C, Titley JC, Leach MO, Wilman DE, McCready VR:
Phospholipid metabolites, prognosis and proliferation in human breast carcinoma.
NMR Biomed 1993, 6(5):318-323.
184. Merchant TE, van der Ven LT, Minsky BD, Diamantis PM, Delapaz R, Galicich J,
Glonek T: 31P NMR phospholipid characterization of intracranial tumors. Brain Res
1994, 649(1-2):1-6.
185. Aiken NR, Gillies RJ: Phosphomonoester metabolism as a function of cell
proliferative status and exogenous precursors. Anticancer Res 1996, 16(3B):1393-
1397.
186. Macara IG: Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells
arises from increased choline kinase activity, not from phosphatidylcholine
breakdown. Mol Cell Biol 1989, 9(1):325-328.
187. Teegarden D, Taparowsky EJ, Kent C: Altered phosphatidylcholine metabolism in
C3H10T1/2 cells transfected with the Harvey-ras oncogene. J Biol Chem 1990,
265(11):6042-6047.
188. Ronen SM, Rushkin E, Degani H: Lipid metabolism in T47D human breast cancer
cells: 31P and 13C-NMR studies of choline and ethanolamine uptake. Biochim
Biophys Acta 1991, 1095(1):5-16.
189. Aiken NR, Szwergold ES, Kappler F, Stoyanova R, Kuesel AC, Shaller C, Brown TR:
Metabolism of phosphonium choline by rat-2 fibroblasts: effects of mitogenic
stimulation studied using 31P NMR spectroscopy. Anticancer Res 1996,
16(3B):1357-1363.
190. Hara T, Kosaka N, Kishi H: PET imaging of prostate cancer using carbon-11-choline.
195
J Nucl Med 1998, 39(6):990-995.
191. Aboagye EO, Bhujwalla ZM: Malignant transformation alters membrane choline
phospholipid metabolism of human mammary epithelial cells. Cancer Res 1999,
59(1):80-84.
192. DeGrado TR, Baldwin SW, Wang S, Orr MD, Liao RP, Friedman HS, Reiman R,
Price DT, Coleman RE: Synthesis and evaluation of (18)F-labeled choline analogs as
oncologic PET tracers. J Nucl Med 2001, 42(12):1805-1814.
193. Nakagami K, Uchida T, Ohwada S, Koibuchi Y, Suda Y, Sekine T, Morishita Y:
Increased choline kinase activity and elevated phosphocholine levels in human colon
cancer. Jpn J Cancer Res 1999, 90(4):419-424.
194. Hara T, Kosaka N, Shinoura N, Kondo T: PET imaging of brain tumor with [methyl-
11C]choline. J Nucl Med 1997, 38(6):842-847.
195. Kihlberg T, Valind S, Langstrom B: Synthesis of [1-11C], [2-11C], [1-11C](2H3) and
[2-11C](2H3)acetate for in vivo studies of myocardium using PET. Nucl Med Biol
1994, 21(8):1067-1072.
196. Folch J, Lees M, Sloane Stanley GH: A simple method for the isolation and
purification of total lipides from animal tissues. J Biol Chem 1957, 226(1):497-509.
197. Ishiwata K, Kubota K, Murakami M, Kubota R, Senda M: A comparative study on
protein incorporation of L-[methyl-3H]methionine, L-[1-14C]leucine and L-2-
[18F]fluorotyrosine in tumor bearing mice. Nucl Med Biol 1993, 20(8):895-899.
198. Glenn JL, Austin W: The conversion of phosphatidyl ethanolamines to lecithins in
normal and choline-deficient rats. Biochim Biophys Acta 1971, 231(1):153-160.
199. LeKim D, Betzing H: Synthesis of labelled phosphatidyl-N,N-dimethylethanolamine
196
and phosphatidylcholine. Hoppe Seylers Z Physiol Chem 1973, 354(10-11):1490-
1492.
200. LeKim D, Betzing H, Stoffel W: Studies in vivo and in vitro on the methylation of
phosphatidyl-N,N-dimethylethanolamine to phosphatidylcholine in rat liver. Hoppe
Seylers Z Physiol Chem 1973, 354(4):437-444.
201. Lakher MB, Wurtman RJ: Molecular composition of the phosphatidylcholines
produced by the phospholipid methylation pathway in rat brain in vivo. Biochem J
1987, 244(2):325-330.
202. DeLong CJ, Shen YJ, Thomas MJ, Cui Z: Molecular distinction of
phosphatidylcholine synthesis between the CDP-choline pathway and
phosphatidylethanolamine methylation pathway. The Journal of biological chemistry
1999, 274(42):29683-29688.
203. Adibhatla RM, Hatcher JF, Dempsey RJ: Cytidine-5'-diphosphocholine affects CTP-
phosphocholine cytidylyltransferase and lyso-phosphatidylcholine after transient
brain ischemia. J Neurosci Res 2004, 76(3):390-396.
204. Ho CL, Yu SC, Yeung DW: 11C-acetate PET imaging in hepatocellular carcinoma
and other liver masses. J Nucl Med 2003, 44(2):213-221.
205. Talbot JN, Gutman F, Fartoux L, Grange JD, Ganne N, Kerrou K, Grahek D,
Montravers F, Poupon R, Rosmorduc O: PET/CT in patients with hepatocellular
carcinoma using [(18)F]fluorocholine: preliminary comparison with [(18)F]FDG
PET/CT. Eur J Nucl Med Mol Imaging 2006, 33(11):1285-1289.
206. Ramirez de Molina A, Gutierrez R, Ramos MA, Silva JM, Silva J, Bonilla F, Sanchez
JJ, Lacal JC: Increased choline kinase activity in human breast carcinomas: clinical
197
evidence for a potential novel antitumor strategy. Oncogene 2002, 21(27):4317-4322.
207. Li CW, Kuo YC, Chen CY, Kuo YT, Chiu YY, She FO, Liu GC: Quantification of
choline compounds in human hepatic tumors by proton MR spectroscopy at 3 T.
Magn Reson Med 2005, 53(4):770-776.
208. Ruiz-Cabello J, Cohen JS: Phospholipid metabolites as indicators of cancer cell
function. NMR Biomed 1992, 5(5):226-233.
209. Kano-Sueoka T, Watanabe T, Miya T, Kasai H: Analysis of cytosolic
phosphoethanolamine and ethanolamine and their correlation with prognostic factors
in breast cancer. Jpn J Cancer Res 1991, 82(7):829-834.
210. Ratnam S, Kent C: Early increase in choline kinase activity upon induction of the H-
ras oncogene in mouse fibroblast cell lines. Arch Biochem Biophys 1995, 323(2):313-
322.
211. Molloy CJ, Bottaro DP, Fleming TP, Marshall MS, Gibbs JB, Aaronson SA: PDGF
induction of tyrosine phosphorylation of GTPase activating protein. Nature 1989,
342(6250):711-714.
212. Galetic I, Andjelkovic M, Meier R, Brodbeck D, Park J, Hemmings BA: Mechanism
of protein kinase B activation by insulin/insulin-like growth factor-1 revealed by
specific inhibitors of phosphoinositide 3-kinase--significance for diabetes and cancer.
Pharmacol Ther 1999, 82(2-3):409-425.
213. Cuadrado A, Issing W, Fleming TP, Molloy CJ: Uneven distribution of protein kinase
C-alpha and -beta isozymes in human sarcomas and carcinomas. J Cell Physiol 1994,
159(3):434-440.
214. Kypta RM, Goldberg Y, Ulug ET, Courtneidge SA: Association between the PDGF
198
receptor and members of the src family of tyrosine kinases. Cell 1990, 62(3):481-492.
215. Cuadrado A, Carnero A, Dolfi F, Jimenez B, Lacal JC: Phosphorylcholine: a novel
second messenger essential for mitogenic activity of growth factors. Oncogene 1993,
8(11):2959-2968.
216. Haubrich DR, Gerber NH: Choline dehydrogenase. Assay, properties and inhibitors.
Biochem Pharmacol 1981, 30(21):2993-3000.
217. Zeisel SH: Importance of methyl donors during reproduction. Am J Clin Nutr 2009,
89(2):673S-677S.
218. Pocard JA, Bernard T, Smith LT, Le Rudulier D: Characterization of three choline
transport activities in Rhizobium meliloti: modulation by choline and osmotic stress.
J Bacteriol 1989, 171(1):531-537.
219. Cui Z, Houweling M: Phosphatidylcholine and cell death. Biochim Biophys Acta
2002, 1585(2-3):87-96.
220. DeLong CJ, Hicks AM, Cui Z: Disruption of choline methyl group donation for
phosphatidylethanolamine methylation in hepatocarcinoma cells. J Biol Chem 2002,
277(19):17217-17225.
221. Vance DE, Houweling M, Lee M, Cui Z: Phosphatidylethanolamine methylation and
hepatoma cell growth. Anticancer Res 1996, 16(3B):1413-1416.
222. Cui Z, Houweling M, Vance DE: Expression of phosphatidylethanolamine N-
methyltransferase-2 in McArdle-RH7777 hepatoma cells inhibits the CDP-choline
pathway for phosphatidylcholine biosynthesis via decreased gene expression of
CTP:phosphocholine cytidylyltransferase. Biochem J 1995, 312 ( Pt 3):939-945.
223. Jimenez B, del Peso L, Montaner S, Esteve P, Lacal JC: Generation of
199
phosphorylcholine as an essential event in the activation of Raf-1 and MAP-kinases
in growth factors-induced mitogenic stimulation. J Cell Biochem 1995, 57(1):141-149.
224. Rameh LE, Cantley LC: The role of phosphoinositide 3-kinase lipid products in cell
function. The Journal of biological chemistry 1999, 274(13):8347-8350.
225. Kiss Z, Chung T: Choline phosphate and phorbol ester potentiate the mitogenic effect
of insulin by competitive mechanisms in NIH 3T3 fibroblasts. Biochem Biophys Res
Commun 1996, 218(2):505-509.
226. Chung T, Crilly KS, Anderson WH, Mukherjee JJ, Kiss Z: ATP-dependent choline
phosphate-induced mitogenesis in fibroblasts involves activation of pp70 S6 kinase
and phosphatidylinositol 3'-kinase through an extracellular site. Synergistic mitogenic
effects of choline phosphate and sphingosine 1-phosphate. The Journal of biological
chemistry 1997, 272(5):3064-3072.
227. Ramirez de Molina A, Penalva V, Lucas L, Lacal JC: Regulation of choline kinase
activity by Ras proteins involves Ral-GDS and PI3K. Oncogene 2002, 21(6):937-946.
228. Weinhold PA, Rounsifer ME, Charles L, Feldman DA: Characterization of cytosolic
forms of CTP: choline-phosphate cytidylyltransferase in lung, isolated alveolar type
II cells, A549 cell and Hep G2 cells. Biochimica et biophysica acta 1989,
1006(3):299-310.
229. Feldman DA, Rounsifer ME, Charles L, Weinhold PA: CTP:phosphocholine
cytidylyltransferase in rat lung: relationship between cytosolic and membrane forms.
Biochimica et biophysica acta 1990, 1045(1):49-57.
230. Rooney SA, Dynia DW, Smart DA, Chu AJ, Ingleson LD, Wilson CM, Gross I:
Glucocorticoid stimulation of choline-phosphate cytidylyltransferase activity in fetal
200
rat lung: receptor-response relationships. Biochimica et biophysica acta 1986,
888(2):208-216.
231. Chu AJ, Rooney SA: Stimulation of cholinephosphate cytidylyltransferase activity by
estrogen in fetal rabbit lung is mediated by phospholipids. Biochimica et biophysica
acta 1985, 834(3):346-356.
232. Sleight R, Kent C: Regulation of phosphatidylcholine biosynthesis in cultured chick
embryonic muscle treated with phospholipase C. The Journal of biological chemistry
1980, 255(22):10644-10650.
233. Rao RH, Mansbach CM, 2nd: Purification and partial characterization of intestinal
acid lipase. Biochimica et biophysica acta 1990, 1046(1):19-26.
234. Sleight RG, Dao HN: Synergistic activation of CTP:phosphocholine
cytidylyltransferase by phosphatidylethanolamine and oleic acid. Lipids 1990,
25(2):100-107.
235. Sleight R, Kent C: Regulation of phosphatidylcholine biosynthesis in mammalian
cells. II. Effects of phospholipase C treatment on the activity and subcellular
distribution of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary
and LM cell lines. The Journal of biological chemistry 1983, 258(2):831-835.
236. Choy PC, Vance DE: Lipid requirements for activation of CTP: phosphocholine
cytidylyltransferase from rat liver. The Journal of biological chemistry 1978,
253(14):5163-5167.
237. Sleight R, Kent C: Regulation of phosphatidylcholine biosynthesis in mammalian
cells. III. Effects of alterations in the phospholipid compositions of Chinese hamster
ovary and LM cells on the activity and distribution of CTP:phosphocholine
201
cytidylyltransferase. The Journal of biological chemistry 1983, 258(2):836-839.
238. Weinhold PA, Rounsifer ME, Feldman DA: The purification and characterization of
CTP:phosphorylcholine cytidylyltransferase from rat liver. The Journal of biological
chemistry 1986, 261(11):5104-5110.
239. Fiscus WG, Schneider WC: The role of phospholipids in stimulating
phosphorylcholine cytidyltransferase activity. The Journal of biological chemistry
1966, 241(14):3324-3330.
240. Feldman DA, Brubaker PG, Weinhold PA: Activation of CTP : phosphocholine
cytidylyltransferase in rat lung by fatty acids. Biochimica et biophysica acta 1981,
665(1):53-59.
241. Feldman DA, Dietrich JW, Weinhold PA: Comparison of the phospholipid
requirements and molecular form of CTP : phosphocholine cytidylyltransferase from
rat lung, kidney, brain and liver. Biochimica et biophysica acta 1980, 620(3):603-611.
242. Pelech SL, Vance DE: Regulation of rat liver cytosolic CTP: phosphocholine
cytidylyltransferase by phosphorylation and dephosphorylation. The Journal of
biological chemistry 1982, 257(23):14198-14202.
243. Kim WR, Brown RS, Jr., Terrault NA, El-Serag H: Burden of liver disease in the
United States: summary of a workshop. Hepatology 2002, 36(1):227-242.
244. El-Serag HB: Hepatocellular carcinoma: recent trends in the United States.
Gastroenterology 2004, 127(5 Suppl 1):S27-34.
245. Yoshizawa H, Tanaka J, Miyakawa Y: National prevention of hepatocellular
carcinoma in Japan based on epidemiology of hepatitis C virus infection in the
general population. Intervirology 2006, 49(1-2):7-17.
202
246. Davila JA, Morgan RO, Shaib Y, McGlynn KA, El-Serag HB: Hepatitis C infection
and the increasing incidence of hepatocellular carcinoma: a population-based study.
Gastroenterology 2004, 127(5):1372-1380.
247. Park JW, Kim JH, Kim SK, Kang KW, Park KW, Choi JI, Lee WJ, Kim CM, Nam
BH: A Prospective Evaluation of 18F-FDG and 11C-Acetate PET/CT for Detection
of Primary and Metastatic Hepatocellular Carcinoma. J Nucl Med 2008, 49(12):1912-
1921.
248. Wu XZ, Xie GR, Chen D: Hypoxia and hepatocellular carcinoma: The therapeutic
target for hepatocellular carcinoma. J Gastroenterol Hepatol 2007, 22(8):1178-1182.
249. Li Y, Jenkins CW, Nichols MA, Xiong Y: Cell cycle expression and p53 regulation
of the cyclin-dependent kinase inhibitor p21. Oncogene 1994, 9(8):2261-2268.
250. Gwak GY, Yoon JH, Kim KM, Lee HS, Chung JW, Gores GJ: Hypoxia stimulates
proliferation of human hepatoma cells through the induction of hexokinase II
expression. J Hepatol 2005, 42(3):358-364.
251. Kim KW, Bae SK, Lee OH, Bae MH, Lee MJ, Park BC: Insulin-like growth factor II
induced by hypoxia may contribute to angiogenesis of human hepatocellular
carcinoma. Cancer Res 1998, 58(2):348-351.
252. Piret JP, Minet E, Cosse JP, Ninane N, Debacq C, Raes M, Michiels C: Hypoxia-
inducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic
cells against tert-butyl hydroperoxide-induced apoptosis. J Biol Chem 2005,
280(10):9336-9344.
253. Baek JH, Jang JE, Kang CM, Chung HY, Kim ND, Kim KW: Hypoxia-induced
VEGF enhances tumor survivability via suppression of serum deprivation-induced
203
apoptosis. Oncogene 2000, 19(40):4621-4631.
254. Abramovitch R, Tavor E, Jacob-Hirsch J, Zeira E, Amariglio N, Pappo O, Rechavi G,
Galun E, Honigman A: A pivotal role of cyclic AMP-responsive element binding
protein in tumor progression. Cancer Res 2004, 64(4):1338-1346.
255. Cohen JS: Phospholipid and energy metabolism of cancer cells monitored by 31P
magnetic resonance spectroscopy: possible clinical significance. Mayo Clin Proc
1988, 63(12):1199-1207.
256. Mintz A, Wang L, Ponde DE: Comparison of radiolabeled choline and ethanolamine
as probe for cancer detection. Cancer Biol Ther 2008, 7(5):742-747.
204