An Investigation on the Anti-tumor Activities of Sophoraflavanone G on Human Myeloid Leukemia Cells
LIU, Xiaozhuo
A Thesis Submitted in Partial Fulfillment
of the Requirements for the Degree of
Master of Philosophy
in
Biochemistry
• The Chinese University of Hong Kong
September 2008
The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School. 統系餘t圖 |( 2 0 1 雇)l) ^^ UNIVERSITY /M ^^S^UBRARY SYSTEMX^ Declaration
I declare that the assignment here submitted is original except for source material explicitly acknowledged. I also acknowledge that I am aware of University policy and regulations on honesty in academic work, and of the disciplinary guidelines and procedures applicable to breaches of such policy and regulations, as contained in the website http://www.cuhk.edu.hk/policy/academichonesty/
16/09/2008 Signature Date
LIU Xiaozhuo 06148340 Name Student ID
Thesis of M.Phil, in Biochemistry Title: An Investigation on the Anti-tumor Activities of Sophoraflavanone G on Human Myeloid Leukemia Cells Course code Course title Thesis/ Assessment Committee
Professor S. K. Kong (Chair)
Professor K. N. Leung (Thesis Supervisor)
Professor K. P. Fung (Thesis Supervisor)
Professor C. K. Wong (Committee Member)
Professor Ricky N. S. Wong (External Examiner) Abstract
Abstract
During the last one and a half centuries, our basic understanding of leukemia has
expanded greatly, from the first description as "white blood" in the year of 1845 to
the recognition of leukemia as disorders of hematopoietic cell differentiation and
apoptosis. Nevertheless, there is still an urgent need for developing novel approaches
for leukemia therapy since conventional methods of leukemia treatment, including
chemotherapy and radiotherapy, have a number of limitations and are often
accompanied with many adverse side effects. Natural products, especially traditional
Chinese medicine (TCM), as an important resource for discoveries of drugs against
cancer, have extensively caught the sight of many scientists.
Kushen, the dried roots of Sophora flavescens Aiton, is a TCM herb commonly
used to treat gastrointestinal disorders, skin ulcers, inflammatory diseases and cancer.
Previous studies from our laboratory had found that Sophoraflavanone G, a flavanone
isolated from Kushen, exhibited potent anti-tumor effect on human hepatoma cells
via induction of apoptosis. However, the modulatory effects of this compound on
human myeloid leukemia cells and its possible anti-leukemic mechanisms remain
• poorly understood.
In the present study, Sophoraflavanone G was examined for its modulatory
effects on the proliferation, apoptosis and differentiation of human acute
promyelocytic leukemia HL-60 cells. Our in vitro studies showed that
Sophoraflavanone G could suppress the proliferation of HL-60 cells in a dose- and
time-dependent manner, with an estimated IC50 of 20 |j.M at 48 hours of incubation.
On the other hand, Sophoraflavanone G showed no significant direct cytotoxicity
towards the human peripheral blood leukocytes and murine bone marrow cells at its
-i - Abstract
growth-inhibitory concentrations for HL-60 cells. The suppressive effect of
Sophoraflavanone G on the tumorigenicity of the HL-60 cells in BALB/c nude mice
was also examined. The results show that pretreatment of HL-60 cells with
Sophoraflavanone G in vitro could significantly inhibit the in vivo growth of
leukemia cells in nude mice. Moreover, as suggested by Western blotting analysis
and cell cycle analysis, Sophoraflavanone G was capable of down-regulating the
protein expression of CDK-6 and triggering cell cycle arrest at the GQ/GI phase in the
HL-60 cells after 24 hours of treatment. Furthermore, Sophoraflavanone G was also
shown to inhibit the proliferation of the multidrug-resistant (MDR) leukemia
HL-60/MX2 cells and induce cell cycle arrest at the GQ/GI phase. Apart from
suppressing the proliferation of the HL-60 cells by cell cycle arrest, we found that
Sophoraflavanone G could also induce cell death through triggering apoptosis in the
HL-60 cells. DNA fragmentation, which is a hallmark of apoptosis, was induced in
Sophoraflavanone G-treated HL-60 cells in a dose-dependent manner. This apoptosis
inducing effect was further confirmed by PI/Annexin V-GFP double staining. The
results of activation of caspase-3 and caspase-9, as well as induction of
mitochondrial membrane depolarization, suggest that the mitochondria-mediated
“ apoptotic pathway, also known as intrinsic apoptotic pathway, may be involved in the
Sophoraflavanone G-induced apoptosis. Western blotting experiments also indicated
that Sophoraflavanone G might activate the intrinsic apoptotic pathway by
up-regulating the pro-apoptotic Bcl-2 family members Bax and Bak proteins, and
down-regulating the anti-apoptotic member Bcl-2 protein. Interestingly, our
preliminary results show that Sophoraflavanone G failed to trigger terminal
differentiation of HL-60 cells under the prescribed experimental conditions, as
judged by morphological analysis on cytospin preparations of Sophoraflavanone
-ii - Abstract
G-treated HL-60 cells and functional assessment of differentiation by the nitroblue tetrazolium reduction assay. Our results, when taken together, suggest that
Sophoraflavanone G may exert its anti-leukemic effect through induction of cell cycle arrest as well as triggering apoptosis of the leukemia cells. More in-depth investigations are needed to reveal the molecular and signaling mechanisms underlying the anti-tumor activities of Sophoraflavanone G before it can be exploited for the therapeutic treatment of some forms of human myeloid leukemia.
-iii - Abstract in Chinese (摘數
摘要
人類對白血病（血癌）的認識已逾一個半世紀。白血病是由於造血幹細胞、
造血祖母細胞發生惡性改變’失去進一步分化、成熟的能力，使細胞阻滯在不
同的造血階段，從而導致的一組異質性造血系統惡性腫瘤。儘管傳統的化療藥
物已經治癒了不少白血病病人，但是由於這些以工業合成爲主的化療藥物除了
殺死癌細胞，正常細胞也會被破壞，令科學家們把開發新治療藥物的目光投到
了天然藥物上，特別是當中被長期廣泛應用到中醫學的中草藥。科學家們希望
以抑制癌細胞體外增殖活性爲指標餘選出中草藥中的活性成分。在本課題小組
早期的硏究中，我們選定了在藥典上具有抗癌作用記載的苦參作爲硏究的對
象，並以抑制肝癌細胞增殖活性爲指標，分離篩選出一系列活性化合物。其中，
槐屬二氫黃酮G (Sophoraflavanone G)具有最好的抑制肝癌細胞HepG2增殖
的活性，也是分離產率最高的活性化合物之一。槐屬二氫黃酮G是一種分佈於豆
科槐屬的異戊稀基黃酮類化合物，已經有硏究指出它具有抗菌、抗炎，以及抗
腫瘤作用，但是它是如何抑制人類骨髓性白血病細胞體外增殖的機制則仍然未
有深入的硏究。
在本硏究課題中，我們將會從抑制生長、誘導调亡、誘導分化三方面探討
槐屬二氫黃酮G對人類骨髓性白血病細胞HL-60的抗腫瘤能力。在抑制生長方
面’槐屬二氫黃酮G對白血病細胞HL-60的生長抑制呈現劑量和時間依賴關
係，而對人外周血細胞及小鼠骨髓細胞不具有明顯的細胞毒性作用。與此同時，
體內實驗亦顯示槐屬二氫黃酮G有效地抑制白血病細胞H L-60在BALB/C裸
鼠體內的生長。使用流式細胞術分析細胞分裂週期發現槐屬二氫黃酮(3能使白
血病細胞停留在Go/Gi的時相，P且滯細胞進行分裂。從西方蛋白質印跡實驗來
-iv - Abstract in Chinese (摘動
看，槐屬二氣黃酮G可能是通過下調細胞週期蛋白激酶-6 (CDK-6)的表達水準
來阻止細胞週期的前進°另一方面，經槐屬二氫黃酮G處理的白血病細胞HL-60
產生了脫氧核糖核酸的斷裂和細胞膜上憐脂醯絲氨酸的外翻，說明槐屬二氣黃 .
酮G能誘發白血病細胞调亡。此外，在调亡的白血病細胞中’我們還觀察到半
胱氨酸天冬氣酸蛋白酶-3和-9的活性有顯著提高，並伴隨有錢粒體膜的去極
化，這說明槐屬二氫黃酮G誘發的调亡可能是以綫粒體爲信號傳遞媒介(亦即
是，依賴於綫粒體的凋亡途徑)產生的。我們利用西方蛋白質印跡實驗去硏究调
亡相關蛋白BC1-2家族成員的表達水準，結果顯示’經槐屬二氫黃酮G處理的
白血病細胞HL-60中促進細胞调亡的Bax和Bak蛋白表達水準有所提高，而
對抗細胞调亡的Bcl-2蛋白表達水平則減少。這說明槐屬二氫黃酮G通過破壞
Bcl-2家族成員之間蛋白表達水準的平衡，啓動了依賴於綫粒體途徑的调亡。在
硏究槐屬二氫黃酮G誘導白血病細胞HL-60分化能力方面，通過觀察細胞形態
和檢測細胞內超氧陰離子的産生水平，我們的實驗結果顯示在現階段的實驗條
件下槐屬二氫黃酮G不能誘導白血病細胞HL-60的終極分化。
綜上所述，我們的硏究發現槐屬二氫黃酮G能有效地抑制人類骨髓性白血
‘ 病細胞的生長，這抗腫瘤作用可能是通過槐屬二氫黃酮G阻滯細胞進行分裂’
誘導細胞调亡產生的。儘管如此，細胞如何協調各種不同的分子機制以最終產
生调亡仍然未被揭示，有待作更深入的硏究。
-V - A cknowledgm ents
Acknowledgments
First of all, I would like to give my sincere thank to my supervisors, Prof. Kwok
Nam Leung and Prof. Kwok Pui Fung, for giving me a valuable opportunity to do
research under their supervisions for my M.Phil, project and encouragement
throughout the whole learning process. Besides, they have also encouraged me to see
how big the world is, by letting me to attend overseas conferences; therefore, I've
been to so many places that I couldn't ever hope for. This is a pleasant experience.
Many thanks to my friends and colleagues, including Ms. Ada Kong, Ms. Avis
Chan, Ms. Lilian Ho, Dr. Fai-hang Lo and Mr. Michael Yip in Prof. Leung's research
group; Ms. Anita Yiu, Dr. Linda Zhang, Dr. Ming Lam, Ms. Judy Chan, Ms. Kitman
Tsang, Ms. Linli Zhou, Ms. Ngoc Ha Bui Xuan, Ms. Sandy Hoi, Dr. Patrick Tang and
Mr. Jonney Jiang in Prof. Fung's research group; Ms. Julia Lee, Mr. P.M. Hon and Mr.
Franlcy Choi in the Institute of Chinese Medicine, The Chinese University of Hong
Kong.
Last but not least, I would also like to thank my family for their unconditional
love and care. They are the most important part of my life.
-vi - List of Abbreviations
List of Abbreviations
°C Degree Celsius jag Microgram jil Microlitre
|iM Micromolar
Aij/m Mitochondrial Membrane Potential
% Percentage
^H-TdR [Methy-^H] Thymidine; Tritiated Thymidine
Ac-DEVD-AMC Acetyl-Asp-Glu-Val-Asp-7-Amido^4-Methylcoumarin
Ac-DEVD-CHO Acetyl-Asp-Glu-Val-Asp-CHO
Ac-IETD-AMC N-Acetyl-Ile-Glu-Thr-Asp-7-Amido-4-Methylcoumarin
Ac-IETD-CHO N-Acetyl-Ile-Glu-Thr-Asp-CHO
Ac-LEHD- AMC N-Acetyl-Leu-Glu-His-Asp-7-Amido-4-Methylcoumarin
Ac-LEHD-CHO N-Acetyl-Leu-Glu-His-Asp-CHO
ACK Ammonium Chloride-Potassium
AIF Apoptosis Inducing Factor
ALL Acute Lymphocytic Leukemia
AMC 7-Amino-4-Methylcoumarin
AML Acute Myeloid Leukemia
ANT Adenine Nucleotide Translocator
Apaf-1 Apoptotic Protease Activating Factor-1
APL Acute Promyelocytic Leukemia
APS Ammonium Persulphate
AS2O3 Arsenic Trioxide
ATCC American Type Culture Collection
ATO Arsenic Trioxide
ATP Adenosine Triphosphate
ATRA All-Trans Retinoic Acid
-vii - List of Abbreviations
Bad BC1-XL/BC1-2 Associated Death Promoter
Bak Bcl-2 Antagonist/Killer
Bax Bcl-2 Associated X Protein
Bcl-2 B-Cell Leukemia/Lymphoma-2
BC1-XL/S B Cell Lymphoma Protein Long/Short Isoform
Bid BH-3 Interacting Domain Death Agonist
BH Bcl-2 Homology
BSA Bovine Serum Albumin
CAD Caspase-Activated Deoxyribonuclease
Caspase Cysteinyl Aspartic Acid-Protease •
CDK Cyclin Dependent Kinase
Ci Curie(s)
CK Creatine Kinase
CKI Cyclin Dependent Kinase Inhibitor
CLL Chronic Lymphocytic Leukemia
CM Complete Medium
CML Chronic Myeloid Leukemia
CO2 Carbon Dioxide
COX Cyclooxygenase
CR Complete Remission
CSF Colony Stimulating Factor
Cyt c Cytochrome c
Da Dalton
DAG Diacylglycerol
dATP Deoxyadenosine Triphosphate
DHE Dihydroethidium
DIABLO Direct lAP Binding Protein with Low pi
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic Acid
-viii - List of Abbreviations
DSMZ Deutsche Sammlimg von Mikroorganismen und
Zellkulturen GmbH
DTT Dithiothieitol
ECL Enhanced Chemiluminescence
EDTA Ethylene-Diamine-Tetra-Acetic Acid
EGCG Epigallocatechin-3 -Gallate
EL Eosinophilic Leukemia
Endo G Endonuclease G
EPA Eicosapentaenoic Acid
EPO Erythropoietin
ER Endoplasmic Reticulum
EtBr Ethidium Bromide
EtOAc Ethyl Acetate
EtOH Ethanol
FAB French-American-British
FACS Fluorescence-Activated Cell Sorter
FADD Fas-Associated Death Domain
Fas Death Receptor CD 95 or Apo-1
FasL Fas Ligand
FBS Fetal Bovine Serum
FDA Food and Drug Administration
FITC Fluorescein Isothiocyanate
FSC Forward Scatter
GAPDH Glyceraldehyde-3-phosphate Dehydrogenase
G-CSF Granulocyte-Colony Stimulating Factor
GFP Green Fluorescent Protein
GM-CSF Granulocyte-Macrophage Colony Stimulating Factor
GSH Glutathione
GST Glutathione S-transferase
-ix - List of Abbreviations
H2O Water
H2O2 Hydrogen Peroxide
HCl Hydrochloric Acid
HDACi Histone Deacetylase Inhibitor •
HEPES N-2-hydroxy-ethyl-piperazine-N'-2-ethane-sulfonic Acid
HI-FBS Heat Inactivated Fetal Bovine Serum
HLA Human Leukocyte Antigen
HMBA Hexamethylene Bisacetamide
HPLC High Performance Liquid Chromatography
HRP Horseradish Peroxidase ‘
HSC Hematopoietic Stem Cell
HSCT Hematopoietic Stem Cell Transplantation
HTLV-1 Human T-lymphotropic Virus Type 1
IC 50 50% Inhibitory Concentration
ICAD Inhibitor of Caspase-Activated Deoxyribonuclease
IFN Interferon
IL Interleukin
Ig Immunoglobulin i.p. Intraperitoneal
IP3 Inositol Triphosphate
JC-1 5,5’，6，6，-Tetrachloro-l,r’3,3，-
Tetraethylbenzimidazolylcarbocyanine Iodide
kb Kilo-Base
KCl Potassium Chloride
kDa Kilo-Dalton
KS-Fs Kushen Flavonoids
L Litre
LPS Lipopoly saccharide
LOX Lipooxygenase
-X - List of Abbreviations
MAPK Mitogen-Activated Protein Kinase
M-CSF Macrophage-Colony Stimulating Factor
MDR Multidrug Resistance
MgCl2 Magnesium Chloride
MHC Major Histocompatibility Complex
mg Milligram
MMP Mitochondrial Membrane Potential
MPT Mitochondrial Permeability Transition
MS Mass Spectrometry
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- •
diphenyltetrazoliumbromide
mM Millimolar
NaHCOs Sodium Bicarbonate
NaOAc Sodium Acetate
NaOH Sodium Hydroxide
NaPB Sodium Phenylbutyrate
NK Natural Killer
NBT Nitroblue Tetrazolium
NF-KB Nuclear Factor Kappa B
NMR Nuclear Magnetic Resonance
“ NO Nitric Oxide
O2 " Superoxide Anion
OD Optical Density
OMM Outer Mitochondrial Membrane
p P Value
PARP Poly(ADP-ribose) Polymerase
PBL Peripheral Blood Leukocyte
PBS Phosphate Buffered Saline
PG Prostaglandin
-xi - List of Abbreviations
P-gp P-glycoprotein
PHA Phytohemagglutinin
PI Propidium Iodide
PIP2 Phosphatidylinositol Bisphosphate
PKC Protein Kinase C
PLC Phospholipase C
PMA Phorbol 12-Myristate 13-Acetate
PML-RARa Promyelocytic Leukemia-Retinoic Acid Receptor a
PMSF Phenylmethyl-sulphonyl Fluoride
PS Phosphatidylserine
PSF Penicillin-Streptomycin-Fungizone
P SN Penicillin-Streptomycin-Neomycin
PTP Permeability Transition Pore
PUMA P53 Upregulated Modulator of Apoptosis
PVDF Polyvinylidene Difluoride
Rb Retinoblastoma Protein
RNA Ribonucleic Acid
RNase A Ribonuclease A
ROS Reactive Oxygen Species
RPMI Roswell Park Memorial Institute r.t. Room Temperature
SAHA Suberoylanilide Hydroxamic Acid
SAR Structure-Activity Relationship
SC Solvent Control
SCT Stem Cell Transplantation
S.D. Standard Deviation
SDA State Drug Administration
SDS Sodium Dodecyl Sulfate
SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis
-xii - List of Abbreviations
S.E. Standard Error (Mean)
SSC Side Scatter
TAE Tris-Acetate-EDTA
TBE Tris-Borate-EDTA tBid Truncated Bid
TBS-T Tris-Buffered Saline-Tween-20
TCM Traditional Chinese Medicine
TEMED N,N,N',N'-Tetra-Methylethylenenidamine
TG Thioglycollate
TNF Tumor Necrosis Factor .
TPA Tetradecanoylphorbol Acetate
TRAIL TNF-Related Apoptosis Inducing Ligand
TRAILR TNF-Related Apoptosis Inducing Ligand Receptor
Tris Tris[hydroxylmethyl]-amino-methane
U Unit
UV Ultraviolet v/v Volume by Volume
VDAC Voltage-Dependent Anion Channel w/v Weight by Volume
-xiii - Table of Contents
Table of Contents
Abstract i
Abstract in Chinese (摘要） iv
Acknowledgments vi
List of Abbreviations vii
Table of Contents xiv
Chapter One: General Introduction
1.1 Hematopoiesis and Leukemia 1 1.1.1 An Overview on Hematopoiesis 1 1.1.2 Leukemia 6 1.1.2.1 An Overview of Leukemia 6 1.1.2.2 Classification and Epidemiology of Leukemia 8 1.1.2.3 Conventional Approaches to Leukemia Therapy 12 1.1.2.4 Novel Approaches to Leukemia Therapy 15
1.2 Sophoraflavanone G: A Bioactive Compound Isolated from 18 Kushen - 1.2.1 An Overview of Kushen: A Traditional Chinese Medicine 19 1.2.2 An Overview of Lavandulyl Flavanones 22 1.2.3 Historical Development and Occurrence of 24 Sophoraflavanone G 1.2.4 Biological Activities of Sophoraflavanone G 25 1.2.4.1 Anti-microbial and Insecticidal Activities 25 1.2.4.2 Anti-tumor Activities 26 1.2.4.3 Pharmacodynamics of Sophoraflavanone G 27
1.3 Objectives and Scopes of the Present Study 30
-xiv - Table of Contents
Chapter Two: Materials and Methods
2.1 Materials 32 2.1.1 Animals 32 2.1.2 Cell lines 32 2.1.3 Cell Culture Medium, Buffers and Other Reagents 34 2.1.4 Reagents and Buffers for Flow Cytometry 37 2.1.5 Reagents for DNA Extraction 39 2.1.6 Reagents for Measuring Caspase Activity 40 2.1.7 Reagents, Buffers and Materials for Western Blotting 43
2.2 Methods 48 2.2.1 Extraction and Isolation of Sophoraflavanone G from Kushen 48 2.2.2 Culture of Tumor Cell Lines 49 2.2.3 Isolation, Preparation and Culturing of Human Peripheral 50 Blood Leukocytes and Murine Bone Marrow Cells 2.2.4 Assays for Anti-proliferation and Cytotoxicity 51 2.2.5 Determination of Anti-leukemic Activity In Vivo {In Vivo 52 Tumorigenicity Assay) 2.2.6 Cell Cycle Analysis by Flow Cytometry 53 2.2.7 Measurement of Apoptosis-induced Activities 54 2.2.8 Protein Expression Study 59 ‘ 2.2.9 Assessment of Differentiation-associated Characteristics 64 2.2.10 Statistical Analysis 65
-xix - Table of Contents
Chapter Three: Studies on the Anti-proliferative Effect of Sophoraflavanone G on Human Myeloid Leukemia Cells
3.1 Introduction 66
3.2 Results 69 3.2.1 Structure Identification of Sophoraflavanone G Isolated from 69 Sophora flavescens 3.2.2 Anti-proliferative Activity of Sophoraflavanone G on Various 72 Myeloid Leukemia Cell Lines 3.2.3 Effect of Sophoraflavanone G on the Viability of the Human 80 Promyelocytic Leukemia HL-60 Cells 3.2.4 Cytotoxic Effect of Sophoraflavanone G on Primary Normal 83 Cells In Vitro 3.2.5 Kinetic and Reversibility Studies of the Anti-proliferative 85 Effect of Sophoraflavanone G on the Human Promyelocytic Leukemia HL-60 Cells 3.2.6 Effect of Sophoraflavanone G on the In Vivo Tumorigenicity 88 of the HL-60 Cells 3.2.7 Effect of Sophoraflavanone G on the Cell Cycle Profile of the 90 HL-60 cells In Vitro 3.2.8 Effect of Sophoraflavanone G on the Expression of Cell 93 ‘ Cycle-regulatory Proteins in the HL-60 Cells 3.2.9 Anti-proliferative Effect of Sophoraflavanone G on 95 Multidrug-resistant (MDR) Leukemia Cell Line HL-60/MX2 Cells
3.3 Discussion 101
-xvi - Table of Contents
Chapter Four: Studies on the Apoptosis- and Differentiation-inducing Activities of Sophoraflavanone G on Human Myeloid Leukemia Cells
4.1 Introduction 109
4.2 Results 114 4.2.1 Induction of DNA Fragmentation in the Human Promyelocytic 114 Leukemia HL-60 Cells by Sophoraflavanone G 4.2.2 Induction of Phosphatidylserine Extemalization in the Human 116 Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G as Detected by Annexin V-GFP and PI Double Staining Method 4.2.3 Effects of Sophoraflavanone G on the Caspase Activities in 119 the Human Promyelocytic Leukemia HL-60 Cells 4.2.4 Induction of Mitochondrial Membrane Depolarization in the 124 Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G 4.2.5 Involvement of Bcl-2 Family Members in Sophoraflavanone 128 G-induced Apoptosis in the Human Promyelocytic Leukemia HL-60 Cells 4.2.6 Effects of Sophoraflavanone G on the Induction of Reactive 131 Oxygen Species in the Human Promyelocytic Leukemia - HL-60 Cells 4.2.7 Effect of Sophoraflavanone G on the Intracellular Ca?^ Level 134 in the Human Promyelocytic Leukemia HL-60 Cells 4.2.8 Morphological Studies on the Sophoraflavanone G-treated 136 Human Promyelocytic Leukemia HL-60 Cells 4.2.9 Effect of Sophoraflavanone G on the NBT Reducing Activity 138 of the Human Promyelocytic Leukemia HL-60 Cells
4.3 Discussion 140
-xvii - Table of Contents
Chapter Five: 148 Conclusions and Future Perspectives
References 156
-xviii - Chapter One
General Introduction Chapter 1 General Introduction
1.1 Hematopoiesis and Leukemia
1.1.1 An Overview on Hematopoiesis
Blood participates in many important biological processes of human life, ranging
from the transport of oxygen to the production of antibodies. Everyday the human
body produces up to 5x10^^ blood cells in normal tissue turnover and for body
defense against foreign invasion (Leung et al, 2005). The term "hematopoiesis" is
used to describe this highly dynamic and coordinated process of blood cell formation
and development (Smith, 2003). Hematopoiesis obtains its name from the ancient
Greek words haima and poiesis, meaning "blood" and "forming", respectively. In
this section, several aspects of hematopoiesis in humans, including the site switching
of hematopoiesis, the process of hematopoiesis, and the regulation of this life process
by various hematopoietic cytokines, will be discussed.
In humans, blood development system involves multiple switching in the sites of
hematopoiesis. Generally, it can be divided into two phases: the transient embryonic
("primitive") phase and the definitive ("adult") phase (Orkin and Zon, 2002). During
early embryogenesis, hematopoiesis has already begun to take place in the mesoderm
. and two waves of hematopoietic stem cell generation occur in the first month of
human gestation. The first one occurs in the yolk sac, generating myeloid precursor
cells devoid of lymphoid potential. On the other hand, the second wave started from
the 27th day of gestation, taking place on the ventral endothelium of the dorsal aorta
and vitelline artery of the embryo, producing the first multipotent, lympho-myeloid
progenitor cells in human ontogeny (Peault and Tavian, 2003). The site of
hematopoiesis is then migrated and this blood development system continues in the
liver and spleen at mid-gestation. Finally, from the sixth to seventh month of fetal
life and thereafter, the red bone marrow gradually becomes the major organ for the
-1 - Chapter 1 General Introduction production and development of all peripheral blood hematopoietic cells (Leung et al.,
2005; Hoffbrand et al, 2006).
Human blood contains many types of mature blood cells, e.g. T and B lymphocytes, erythrocytes, monocytes and granulocytes, with very different morphologies and functions. All blood cells, however, generated ultimately from a common "ancestor" - the so called hematopoietic stem cells (HSC) (Leung et al.,
2005). The pluripotent HSC are capable of self-renewal and of producing all cell lineages found in the hematopoietic system through a series of downstream hematopoietic progenitors (Jones et al, 1996). HSC proliferate and generate common myeloid and lymphoid progenitors, which differentiate into immature progenitor cells of the various blood cell lineages and further lead to accomplish the process of hematopoiesis. As depicted in Figure 1.1, the common myeloid progenitor is the precursor of four distinct cell lineages: erythrocytic and megakaryocytic, monocytic and granulocytic lineages. The erythrocytic and megakaryocytic lineages generate red blood cells and blood platelets, respectively. In addition, the monocytic lineage gives rise to monocytes in the circulation and macrophages in the tissues.
Furthermore, the granulocytic lineage produces granulocytes, including basophils, eosinophils, and neutrophils. On the contrary, the common lymphoid progenitor is responsible for generation of B and T lymphocytes through B and T cells development pathways, respectively (Leung et al., 2005). Therefore, hematopoiesis involves the irreversible differentiation commitment of the progenitor cells to one or another lineage and the progressive maturation of cells within that lineage (Metcalf,
1998).
Hematopoietic cell development is tightly regulated by a number of cytokines,
collectively known as the hematopoietic cytokines. The hematopoietic cytokines,
-2- Chapter 1 General Introduction including the colony-stimulating factors (CSF) and interleukins (IL), is a broad family of proteins or glycoproteins. In addition, these hematopoietic cytokines, produced by a wide variety of hematopoietic and non-hematopoietic cells, function
as mediators both positively and negatively signaling the hematopoietic cell to
undergo differentiation, proliferation, quiescence, and apoptosis (Leung et al, 2005).
Generally speaking, hematopoietic cytokines function by binding to their specific
receptors and this binding further triggers a variety of signaling pathways, including
the protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways
(Vitale et al, 2002; Lentzsch et al., 2004). The inducing signals then initiate a
sequential genetic program of transcriptional activation, resulting in production of
specific cell types. So far four CSF have been characterized, including macrophage
colony-stimulating factor (M-CSF)，granulocyte macrophage colony-stimulating
factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and the
multi-colony-stimulating factor [multi-CSF, also known as interleukin-3 (IL-3)]
(Metcalf, 1988; Crosier and Clark, 1992). These four CSF can induce proliferation
and differentiation of normal myeloid precursor cells to form colonies. For example,
GM-CSF promotes non-lymphoid cells formation at the early developmental stage.
M-CSF and G-CSF, relatively lineage-specific, trigger formation of monocytes and
granulocytes, respectively (Comalada et al., 2005). On the other hand, IL-7 induces
the lymphoid progenitors to differentiate into B cell progenitors and T cell
progenitors (Fry and Mackall, 2005). Furthermore, instead of having limited effects
on a specific cell lineage，most of hematopoietic cytokines are involved in the
development of different cell types and in various developmental stages.
Nevertheless, each hematopoietic cytokine has unique functions that cannot be
completely compensated by other cytokines. It appears that immature myeloid
-3- Chapter 1 General Introduction progeny responds mainly to stimulation by multiple cytokines whereas more mature myeloid progenitor cells respond to stimulation by single cytokine (Leung et al,
2005).
To conclude, hematopoiesis is a complex network and highly coordinated
process. In normal hematopoiesis, there is a subtle balance between cell proliferation
and differentiation on one hand and apoptosis and cell senescence on the other hand.
Catastrophes will be resulted when the "balance" is broken down in abnormal
hematopoiesis. The disorders include aplastic anemia, neutropenia,
thrombocytopenia, myelodysplasia, and hematologic malignancies like lymphoma
and leukemia (Smith, 2003; Leung et al., 2005). Among all these disorders, leukemia
will be discussed in detail in the next section.
-4- Chapter 1 General Introduction
Hematopoietic Stem Cell (pluripotent and capable of extensive self-renewal)
Myeloid Lymphoid Progenitor Cell Progenitor Cell
Early Early Myeloblast Pre-B Cell Pre-T Cell Erythroid Cell| Megakaryocyte| / Promyelocyte
Late Promonocyte Erythroid Cell Megakaryocyte] Myelocyte
Monocyte / \ IB Cell I IT Cell Reticulocyte / N^
Basophil Eosinophil
Red Blood Cell] | Platelet | Neutrophil
Fig. 1.1 Diagrammatic representation of hematopoiesis. The hematopoietic network begins with pluripotent hematopoietic stem cells (HSC), through intermediate progenitors, all cell lineages found in the hematopoietic system. HSC proliferate and generate the common myeloid and lymphoid progenitors, which then differentiate into immature progenitor cells of the various blood cell lineages. Generally speaking, throughout the whole development process, the proliferative ‘ potential of the hematopoietic cells decreases with increasing degree of differentiation. (Leung et al，2005)
-5- Chapter 1 General Introduction
1.1.2 Leukemia
As discussed in Section 1.1.1，in normal hematopoiesis, hematopoietic progenitor cells can successfully differentiate into functional blood cells. In leukemia, a disease of abnormal hematopoiesis, these progenitor cells continue to proliferate but fail to differentiate into functional mature blood cells. Leukemia may result from disturbance or abnormalities in any stages of hematopoietic cell development (Figure
1.1). In this section, the classification, the current and novel treatments for leukemia will be discussed.
1.1.2.1 An Overview of Leukemia
Leukemia was first described in 1845 by the British pathologist John Hughes
Bennett and the German pathologist Rudolf Virchow and later leukemia was named by Virchow (Stone et al, 2003). Virchow described leukemia as "white blood"
because he observed that the number of red blood cells was very few compared to the
"colorless" corpuscles or the white blood cells (leukocytes) under microscope
(Leung et al, 2005). In fact, apart from leukocyte leukemia, leukemia also involves
erythrocyte leukemia and megakaryocyte leukemia though they are quite rare.
Leukemia can be regarded as a cancer of the blood cells, representing a large
proportion of hematological malignancies (Tsiftsoglou et al., 2003). In leukemia,
early hematopoietic progenitor cells are irresponsive to the external stimuli within
the bone marrow microenvironment and fail to differentiate into discrete types of
blood cells (Tsiftsoglou et al, 2003). These cells acquire immortality, self-perpetuate,
grow in high numbers in the bone marrow at the expense of other cells and then spill
into the blood circulation, eventually reach the secondary lymphoid organs such as
lymph nodes and spleen, and the central nervous system, as well as to other organs
-6- Chapter 1 General Introduction including liver, lungs, testicles，ovaries and kidneys (Tsiftsoglou et al., 2003; Leung era/., 2005).
Leukemia produces specific symptoms corresponding to the type of leukemia and organs involved as well as the condition of the patient. However, leukemia patients do have generalized symptoms such as fatigue, loss of appetite, unexplained weight loss, bone and back pain, and easy bruising. Moreover, anemia, neutropenia and thrombocytopenia are usually the consequences of bone marrow failure, which can lead to infection and hemorrhage (Leung et al., 2005). Certain diagnostic tests, including blood cell counts and bone marrow tests through bone marrow aspiration or biopsy, are generally used to find out if leukemia is present, what type of leukemia it is, and how well the disease is responding to the treatments. Furthermore, more and more precise laboratory tests have been developed and applied to diagnose and classify leukemia, and these include cytochemistry, immunocytochemistry, cytogenetics and molecular genetic studies, and flow cytometry (American Cancer
Society, 2008).
-7- Chapter 1 General Introduction
1.1.2.2 Classification and Epidemiology of Leukemia
There are two basic criteria to consider in classifying leukemia, one is the degree of maturity of the leukemia cells together with their onset rate, and the other factor is
developmental lineage of the leukemia cells. Therefore, leukemia can be classified
by the first criterion into acute leukemia or chronic leukemia. In acute leukemia, the
leukemia cells stop differentiation in an earlier stage of hematopoiesis and begin to
proliferate rapidly. Therefore, this type of leukemia has a sudden onset with rapid
progression, leading to accumulation of immature and fimctionless cells in the
marrow and blood. On the other hand, the cells of chronic leukemia appear more
mature but they usually have abnormal functions. However, the lifespan of chronic
leukemia cells is much longer than that of normal leukocytes and result in
over-infiltration of mature granulocytes or lymphocytes in bone marrow. The chronic
leukemia usually progresses slower than the acute one and with a prolonged clinical
course (Leung et al, 2005).
Leukemia can also be classified into myeloid or lymphoid, according to the
second criterion. Myeloid leukemia develops from precursor cells of the granulocytic,
monocytic, erythrocytic or megakaryocytic lineage while lymphocytic leukemia
develops from precursor cells of the lymphocytic lineage (Leung et al, 2005).
Therefore, taking the two criteria together, most cases of leukemia can be briefly
classified into one of the four major types: acute myeloid leukemia (AML), acute
lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic
lymphoblastic leukemia (CLL). However, classifying leukemia is difficult and
controversial when the morphology of the leukemia cells is not so clear cut. To have
a better classification, the French-American-British (FAB) system was developed to
classify the subtypes of different acute leukemia. The subtypes of different acute
-8- Chapter 1 General Introduction leukemia are summarized in Table 1.1.
According to the statistical data and information from the American. Cancer
Society (2008)，the key statistics and the risk factors for the four major types of leukemia are summarized in Table 1.2. In the United States, leukemia accounts for approximately 3% of all cancer diagnoses with the death rate around 4% (American
Cancer Society, 2008). In China, the occurrence of leukemia is high, which is around
4 in 100,000 and leukemia ranks as No.6 high incidence in all cancer diagnoses
(Chen et al, 2005). In Hong Kong, childhood cancer is the second leading cause of death among children. Among different types of childhood malignancy, leukemia has the highest incidence in Hong Kong, accounting for approximately 36% of total cancer diagnoses as reported in the most recent ten-year (1990-2000) review
(Children's Cancer Foundation, Hong Kong, 2008).
-9- Chapter 1 General Introduction
Table 1.1 The French-American-British (FAB) system for classification of acute leukemia
TVpe Subtype • MO: undifferentiated myeloblastic • Ml: myeloblastic with minimal maturation • M2: myeloblastic with granulocytic maturation • M3: promyelocytic with many granules Acute myeloid leukemia • myelomonocytic with eosinophilia (AML) • m5a: monoblastic • M5B: promonoblastic • M6: erythroblastic • M7: megakaryoblastic • LI: T cell or pre-B cell; homogeneous small blasts with little cytoplasm Acute lymphoblastic • 丁 cell or pre-B cell; heterogeneous large I A"! 1pm 1 o blasts with variable amounts of cytoplasm (ALL) , L3: B cell; homogeneous large blasts with vacuolated basophilic cytoplasm
(American Cancer Society, 2008)
-10- Chapter 1 General Introduction
Table 1.2 Key statistics and risk factors for the four major types of leukemia
AML ALL CML CLL
Estimated new cases in 2008 in 13,290 5,430 4,830 15,110 the US alone
Estimated no. of death in 2008 in 8,820 1,460 450 4,390 the US alone Average age of 幻 • 67 72 patients
Ratio of adult to Adult: 90% Adult: 67% Adult children Children: 10% Children: 33% (mostly) Adult only
* Five-year relat 二二 2O.40/0 65.2% 42.3% 74.2�/� rate (1996-2002, in the US)
Male Male Sex prevalence ^^^ghtly higher) (slightly higher) _ “
• Smoking • High-dose • High-dose • Herbicides • Long-term radiation radiation (e.g. Agent exposure to • Viral infection Orange) Risk factors high levels of (e.g. HTLV-1) • Insecticides benzene • High-dose radiation • Viral infection (e.g. HTLV-1)
* The five-year relative survival rate refers to the percentage of patients who live at least 5 years after their cancer is diagnosed. It varies according to the type of leukemia and the age of the patient.
(American Cancer Society, 2008)
-11 - Chapter 1 General Introduction
1.1.2.3 Conventional Approaches to Leukemia Therapy
Nowadays, chemotherapy, radiotherapy and hematopoietic stem cell transplantation are the conventional methods of leukemia treatment. Treatment options are based on the leukemia subtypes and disease stages.
Chemotherapy is the most common approach for leukemia therapy. In this approach, leukemia patients are treated with a combination of different kinds of cytotoxic drugs through mouth, veins, muscle, or cerebrospinal fluid (American
Cancer Society, 2008). The dosage and the type of drugs used depend on the
leukemia subtypes and progression. Moreover, the chemotherapeutic treatment can
be generally divided into three stages: 1) induction therapy: using high dosage of
drugs, such as vincristine and L-asparaginase, to rapidly kill the leukemia cells in
blood and bone marrow; 2) intensive consolidation therapy: using more drugs, such
as 6-mercaptopurine, vincristine, prednisone, and methotrexate, and higher dosages
to kill the hidden leukemia cells in other organs; 3) maintenance therapy: using low
dosage of drug, such as 6-mercaptopurine and methotrexate, to prevent the relapse of
leukemia. The patient is commonly regarded to be completely cured if there is no
relapse within five years. Chemotherapy has an advantage of direct and rapid
delivery of drug to kill circulating leukemia cells (Leung et al, 2005). However,
chemotherapy is the double-edged sword of modem medicine. Due to the low
selectivity and high cytotoxicity, most of the chemotherapeutic drugs
non-specifically kill all rapidly dividing cells involving not just leukemia cells but
also normal rapidly growing cells such as the hematopoietic cells in the bone marrow,
hair follicular cells and gastrointestinal epithelial cells (American Cancer Society,
2008). As a result, chemotherapy can cause serious side effects in patients. Reduced
production of blood platelets contributes to easy bruising and profuse bleeding,
-12- Chapter 1 General Introduction growth inhibition of hair follicular cells and gastrointestinal epithelial cells result in loss of hair, diarrhea and vomiting, respectively. Apart from the side effects, multidrug resistance (MDR) is also a major concern in the chemotherapeutic agents against cancer (Ullah, 2008). Although the resistance is usually developed from a single chemotherapeutic substance, it will further lead to multiple resistance to numerous substances characterized by different mechanisms of action and by different chemical structures (Stavrovskaya, 2000). Many common anti-cancer drugs, such as doxorubicin, mitoxantrone, etoposide，vincristine, have already been reported to cause MDR in cancer cells (Stavrovskaya, 2000; Ullah, 2008).
In radiotherapy, X-rays and gamma-rays are employed to kill leukemia cells. It is often used with chemotherapy and is an essential part of treatment before bone marrow transplantation. Radiotherapy is also applied when leukemia cells have
spread to the brain and spinal fluid or to the testicles. However, radiation may also
affect the function of the treated organs. For instance, children who receive radiation
to the brain may cause problems in learning and coordination, and radiation to the
testicles may develop problems in both fertility and hormone production (Leung et
a/., 2005).
Hematopoietic stem cell transplantation (HSCT) includes both bone marrow
transplantation, which involves the collection of stem cells from bone marrow, and
peripheral blood stem cell transplantation, which is the collection of stem cells from
peripheral blood. HSCT therapy involves two major steps: 1) elimination of the
patient's hematopoietic system by chemotherapy and/or radiotherapy; 2) replacement
with normal hematopoietic stem cells. While in the second step, the source of normal
hematopoietic stem cells could be either from another individual with same tissue
type as the patient, namely allogeneic transplantation, or from the patient's own
-13 - Chapter 1 General Introduction hematopoietic stem cells which were frozen previously, namely autologous transplantation (Leung et al, 2005). Nowadays, HSCT has been developed into a well-established therapy not only for leukemia but also some inherited severe blood cell diseases. It has reported that HSCT is the most successful curative treatment for
AML and can even cure relapsed AML (Rubnitz et al, 2008). Nevertheless, bone marrow transplantation has the limitations of higher cost and difficulty of finding human leukocyte antigen (HLA)-compatible donors for the leukemia patients. In addition, the hematopoietic stem cells for the donor may react against the patient's normal cells. This leads to graft-versus-host disease, which can make the patients very sick (Leung et al., 2005; American Cancer Society, 2008).
-14- Chapter 1 General Introduction
1.1.2.4 Novel Approaches to Leukemia Therapy
During the last one and a half centuries, our basic understanding of leukemia has been greatly explored, from the first description as "white blood" in the year of 1845 to the recognition of leukemia as disorders of hematopoietic cell differentiation and apoptosis (Tsiftsoglou et al, 2003). This development of understanding stepwise pushes the treatment of leukemia becoming more effective with fewer side effects.
In the earlier development of leukemia therapy, the treatment mainly targeted on the leukemia characteristic of rapid growth since at that time leukemia was only recognized as diseases of uncontrolled cellular growth. The earlier treatments include radiotherapy and chemotherapy. The conventional chemotherapeutic agents involve alkylating agents, anti-metabolites, anti-tumor antibiotics, folate analogues as enzyme inhibitors, blockers of mitosis, cell cycle cytotoxic inhibitors, nitrosourea derivatives, and so on (Tsiftsoglou et al., 2003). Unequivocally, cancer chemotherapy has been successful in effectively curing a few malignant neoplasms
(e.g. Burkitt's lymphoma and choriocarcinoma) and useful in treating symptoms and
prolonging life for several years in some other cases like childhood leukemias
(Tsiftsoglou et al, 2003). However, conventional chemotherapy has not been able to
eradicate hematological malignancies. One report has shown that prior exposure to
radiation and chemotherapy such as alkylating agents (cyclophosphamide) and
intercalating topoisomerase II inhibitors (etoposide) may trigger secondary AML
(Rubnitz et al, 2008). As summarized by Tsiftsoglou et al. (2003), the major
problems leading to the failure of conventional cancer chemotherapy include: 1)
enormous diversity in morphology and physiology of neoplasms; 2) extensive
cellular heterogeneity and heterogeneity in molecular abnormalities in malignant
tumors; 3) inadequate delivery of anti-cancer agents to the targeted tumor sites; 4)
-15- Chapter 1 General Introduction lack of selective anti-tumor activity and broad-spectrum toxicity to normal tissues; 5) genome instability; and 6) development of MDR (Tsiftsoglou et al., 2003).
With the development of modem biology such as molecular biology and molecular genetics, apart from uncontrolled cellular proliferation, leukemia has been recognized as disorders of differentiation and apoptosis (Tsiftsoglou et al., 2003).
Based on this updated understanding, many novel exploitable targets for drug development have been revealed over the past several years. These include genes and unique proteins regulating apoptosis and cell differentiation in malignant cells, specific proteins driving the cell cycle machinery, cell lineage-specific transcription factors and angiogenesis factors (Tsiftsoglou et al, 2003). For instance, Genasense®, a clinical grade Bcl-2 antisense oligonucleotide, was advanced into clinical trials in patients with leukemia (Schimmer, 2008). Bcl-2 is a key intracellular anti-apoptotic protein. Reduced expression of Bcl-2 protein by Bcl-2 antisense oligonucleotide can induce apoptosis in preclinical studies (Schimmer, 2008). Apart from Bcl-2 protein, more key regulators involving in apoptosis signaling pathways, such as death receptors, caspases as well as transcription factors (e.g. nuclear factor kappa B), have been used as the targets of chemotherapeutic agents. In addition to inducing apoptosis, triggering differentiation is another novel approach to leukemia treatment.
All-trans retinoic acid (ATRA), which is able to induce cellular differentiation, has been clinically used to treat acute promyelocytic leukemia (APL). APL, an unique subtype of AML (classified as M3 of AML), is characterized by the blockage of granulocytic differentiation at the promyelocytic stage and specific reciprocal chromosomal translocation t(15;17)(q22;ql2). This chromosomal translocation produces a fusion gene, resulting in the formation of fusion protein PML-RARa
(promyelocytic leukemia-retinoic acid receptor a). This protein blocks
-16- Chapter 1 General Introduction differentiation of myeloid cells and inhibits apoptosis of the cells. ATRA can degrade the fusion protein and restore the normal differentiation of the promyeloid progenitor cells and inhibit their replicative capacity (Chen et al,’ 2005). Apart from these two examples of "molecularly targeted oncotherapy", cellular therapy with haploidentical natural killer cells has been reported to exhibit anti-tumor activity with
minimal toxicity in patients who have relapsed AML (Rubnitz et al, 2008).
Our understanding of leukemia continues to expand, leading to more and more
discoveries of novel targets to leukemia therapy. Nevertheless, most of these targets
are "locked" thus need to be activated by the "keys". Where can we find such keys?
Many of natural products, such as green tea catechin and soybean isoflavonoid, have
been reported to show anti-tumor activities (Kampa et al, 2007). In particular,
traditional Chinese medicine (TCM), with well-characterized medicinal effects, is an
important resource for discoveries of drugs against cancer. According to the
pharmacopeia of TCM, a number of TCM with historical clinical application on
leukemia treatment can be initially selected. The active component candidates of the
TCM may be isolated using bioassay-guided fractionation. It is essential to reveal the
molecular mechanisms of the active component against leukemia cells before
opening the "locked" target. Arsenic trioxide (ATO), one of the TCM, has been
approved by the State Drug Administration (SDA) in China and the Food and Drug
Administration (FDA) of the United States for clinical usage (Zhang and Demain,
2005). The development and use of ATO, a potent inducer which activates apoptosis
via down-regulation of the anti-apoptotic Bcl-2 protein and triggers differentiation
through breakdown of differentiation-inhibitory protein PML-RARa, as an effective
agent for treatment of APL patients represents a successful example of molecularly
targeted oncotherapeutic agent derived from TCM (Zhang and Demain, 2005).
-17- Chapter 1 General Introduction
1.2 Sophoraflavanone G: A Bioactive Compound Isolated from Rush en
As discussed in Section 1.1.2.4，natural products, especially traditional Chinese medicine (TCM), as an important resource for discoveries of drugs against cancer have extensively caught the sight of scientists. Cumulating evidence demonstrated that herb-based TCM could be used for treatment of cancer. It has been reported that
some Chinese herbs can directly kill cancer cells, such as Qing Dai (青黛){Indigo
naturalis), Ya Dan Zi 膽子){Brucea Javanica), and She Xiang (窮香){Moschus
moschiferus). In addition, some can inhibit cancer cell growth, including Jiang
Huang (薑黃){Curcuma aromatica) and Chang Chun Teng (長春藤){Hedera helix).
Furthermore, more than 100 kinds of Chinese herbs can stimulate the patient's
immune system to attack cancer cells (Zhang and Demain, 2005). As mentioned at
the end of Section 1.1.2.4, many active components can be isolated and characterized
from the medicinal herbs using bioassay-guided purification. In a previous research
program in our laboratory, Ku Shen (苦參）�Sophor Radix),a or Kushen (this form of
presentation will be used in this dissertation), which has been clinically applied for
cancer treatment as recorded in TCM history, was chosen and screened for the
bioactive components against hepatoma. Among different bioactive compounds
isolated from Kushen, Sophoraflavanone G had the highest yield of purification and
it was found to exhibit the most potent anti-proliferative effect on the human
hepatocellular carcinoma HepG2 cells (Tsang, 2006). Additionally,
Sophoraflavanone G was also reported to demonstrate high cytotoxic activity against
the human myeloid leukemia HL-60 cells among different flavonoids isolated from
Kushen (Ko et al, 2000). Therefore, the modulatory effects of Sophoraflavanone G
-18- Chapter 1 General Introduction on human myeloid leukemia cells were further investigated in the present research project. In the second part of this chapter, a brief introduction will be given to
Kushen and Sophoraflavanone G.
1.2.1 An Overview of Kushen: A Traditional Chinese Medicine
Kushen is the dried roots of Sophora flavescens Aiton, which is a perennial shrub from the leguminous plant family (De Naeyer et al,, 2004). Sophora flavescens Aiton is an evergreen shrub reaching approximately 1 to 1.5 meters with leaves of various shapes, greenish-yellow flowers and brown seed pods (Figure 1.2 A). The root ranges from 10 to 30 centimeters in length and 2 to 3 centimeters in diameter (Figure 1.2 B).
The plant is widely cultivated in northeast Asia, including China, Japan, and the
Korean peninsula. Sophora flavescens Aiton can live in light (sandy), medium
(loamy), or heavy (clay) soils but it requires well-drained soil. It can be cultivated on acidic, neutral, and basic (alkaline) soils. However, it cannot grow in the shade (De
Naeyer et al, 2004; Bagchi and Preuss, 2005).
The Chinese characters for Kushen, i.e. “苦參"，which literally means "the bitter root", point to the fact that although this root may taste bitter, it has superior medicinal qualities. Kushen was first described in "Shen Nong Ben Cao Jing"(神農
本草,經)，a famous Chinese pharmacopeia with more than two thousand years of history (Sun et al,, 2007). The roots are prepared for herbal use by bundling and
cutting them cross-wise into slices which are then dried under sunlight. The
traditional use of Kushen includes the decoction and the powder of dried plant roots
(Sun et al, 2007). Kushen has been applied frequently in traditional Chinese
medicine as a diuretic, stomachic, anti-pyretic, analgesic, anti-helmintic drug
whereas it also has been used to treat rashes externally (De Naeyer et al, 2004).
-19- Chapter 1 General Introduction
Recently, using modem phytochemical and biochemical techniques, different groups of researchers have tried to elucidate the bioactive compounds leading to the medicinal effects of Kushen. Up to now, the major chemical constituents of Kushen consist of alkaloids (3.3%) and flavonoids (1.5%) (Sun et al, 2007). Matrine and oxymatrine, contributing approximately 20% of the total alkaloids, are the main alkaloid components. The reported bioactivities of alkaloids in Kushen include anti-arrhythmic, anti-ulcer, anti-asthmatic, and anti-tumor activities (Tang and
Eisenbrand, 1992). On the other hand, flavonoids, another major component in
Kushen, also demonstrated pleiotropic bioactivities in vitro (Sun et al, 2007).
-20- Chapter 1 General Introduction 醒 W (B) 10 “ 9 ’，
丨 2，
5| 4 OH 0 (C) 5,7,2',4'-tetrahydroxy-8-lavandulyl-flavanone
Fig. 1.2 Photos of Sophora flavescens (A. Sophora flavescens & B. root of Sophora flavescens) and chemical structure of Sophoraflavanone G (C). Figure A and B were adapted from Volume 4 of Chinese Herbal Medicines (中華本草）（1998)， Figure 1 and 2 of Kushen (No.3383) respectively on page 635. Figure C was modified from Shirataki et al (1988).
-21 - Chapter 1 General Introduction
1.2.2 An Overview of Lavandulyl Flavanones
Flavonoids comprise the most common group of plant polyphenols and provide much of the flavor and color to the plants. Chemically, flavonoids possess a common structure of diphenylpropanes (C6-C3-C6), consisting of two aromatic rings linked through three carbons (Figure 1.3). Based on various substitution patterns in the heterocyclic C-ring, flavonoids can be further subdivided into six major subclasses including flavones, flavonols, flavanones, catechins, anthocyanidins, and isoflavones
(Ross and Kasum, 2002).
Among the flavonoids in Kushen，lavandulyl flavanones, a sub-family of prenylated flavanones, are attracting more and more attention (Botta et al., 2005).
Lavandulyl flavanones have a relatively narrow distribution in the plant kingdom and they are mainly found in Sophora species (Kim et al, 2004). It has been reported that the bioactivities of flavaonoids is correlated with the position, the number of hydroxyl groups, and the structure of side chains (Kuntz et al, 1999). The lavandulyl side chain, as suggested by accumulating evidences from structure-activity relationship (SAR) studies, is essential for the pleiotropic bioactivities of lavandulyl flavanones, such as in vitro growth-inhibitory activity on tumor cells, tyrosinase-inhibitory and cyclooxygenase-inhibitory activities (Ko et al, 2000; Chi era/.�2001 So; n era/., 2003).
-22- Chapter 1 General Introduction
Polyphenols
Flavonoids (2-phenyl-chromone)
Flavanois Flavonols Flavanonols r^i n rt ^^^Oy^ x^J^^Y'^
‘
Flavones Flavanones o Isoflavones O r n r H r T T C T T ^^ ^^ 5 U o o
Lavandulvl flavanones 1(1 “ <)’’ HjCvyjCH^
'Sf'^r^ ' OH
OH 0
Fig. 1.3 Classification of flavonoids. Flavonoids are the largest class of polyphenols, with a common structure of diphenylpropanes (C6-C3-C6), consisting of two aromatic rings linked through three carbons. Based on various substitution patterns in the heterocyclic C-ring, flavonoids can be further subdivided into six major subclasses including flavones, flavanois, flavanones, flavonols, isoflavones, and flavanonol (Ross and Kasum, 2002).
-23- Chapter 1 General Introduction
1.2.3 Historical Development and Occurrence of Sophoraflavanone G
“A new flavanone, named Sophoraflavanone G ..., was isolated from the root of
Sophora moorcroftiana Benth. Ex Baker (Leguminosae)....“
——Yoshiaki Shirataki, 1988.
Sophoraflavanone G, i.e. 5,7,2',4'-tetrahydroxy-8-lavandulyl-flavanone
(chemical formula: C25H28O6)，is one of the members of lavandulyl flavanones
(Figure 1.2 C). Sophoraflavanone G, also known as Vexibinol, was first reported as a new member of flavonoids family in 1988 by Shirataki and his colleagues. After its initial discovery in the root of Sophora moorcroftiana (Shirataki et al, 1988)，
Sophoraflavanone G was reported to mainly exist in Sophora species (Leguminosae),
such as Sophora leachiano (linuma et al, 1990)，Sophora alopecuroides (linuma et
al., 1995) and Sophora flavescens (Ryu et al, 1996).
Apart from isolation from the natural plants, Sophoraflavanone G was reported
to be biosynthesized in cell suspension cultures from callus tissue of Sophora flavescens. After this first description of biosynthesis of Sophoraflavanone G in 1991,
Yamamoto's group also found that the addition of cork tissue increased the
production of biosynthesized Sophoraflavanone G, three- to five-fold higher than that
of the cells cultured alone (Zhao et al, 2003; Zhao et al., 2004). Additionally, using
molecular biology techniques, it was found that the biosynthetic route producing
Sophoraflavanone G was mainly regulated by three enzymes: naringenin
8-dimethylallytransferase, 8-dimethylallylnaringenin 2'-hydroxylase, and
leachianone G 2"-dimethylallytransferase (Zhao et al., 2004). Therefore, it is hoped
that more in-depth molecular studies on the action mechanisms of these regulatory
enzymes can enhance the production of Sophoraflavanone G from cell cultures.
-24- Chapter 1 General Introduction
1.2.4 Biological Activities of Sophoraflavanone G
After its first isolation by Shirataki in 1988, Sophoraflavanone G has been frequently isolated as an active component with different bioactivities from Sophora species in various bioassay-guided researches. These biological effects include anti-microbial and insecticidal activities, anti-tumor activities and some other pharmacodynamics activities, involving multiple inhibitory effects on phospholipase
Cyl, cyclooxygenases, lipooxygenases, tyrosinases, as well as glycosidases.
1.2.4.1 Anti -microbial and Insecticidal Activities
In the nature, Sophoraflavanone G and other members of flavonoids have been suggested to play important roles in protecting the host plants from attack by parasites such as insect pests, fungi, bacteria and nematodes (Rao, 1990). In the
laboratories, different researchers have demonstrated the anti-microbial and
insecticidal activities of Sophoraflavanone G.
Yamaki et al. (1990) reported that Sophoraflavanone G exhibited anti-bacterial
effects against Staphylococcus aureus {S. aureus) and Streptococcus mutans.
Moreover, Sophoraflavanone G showed strong anti-bacterial activity against
methicillin-resistant S. aureus with minimum inhibitory concentration of 3.13-6.25
mg/ml (Sohn et al, 2004). These results suggest that Sophoraflavanone G has high
potential as an anti-bacterial agent. Tsuchiya and linuma (2000) suggested that the
possible mechanism underlying the anti-bacterial activities of Sophoraflavanone G
was that this flavanone, with a hydrophobic lavandulyl side chain, can target on the
bacterial membrane and further reduce the membrane fluidity.
Besides anti-bacterial effects, Sophoraflavanone G was reported to have in vitro
anti-malarial activities against Plasmodium falciparum which is the most widespread
-25- Chapter 1 General Introduction etiological agent of human malaria (Kim et al., 2004). In addition, Sophoraflavanone
G, as demonstrated by Zheng et al. (1999), was found to possess strong insecticidal and fungicidal activities, and these activities were shown to decrease after methylation of the 5-hydroxy 1 group. Moreover, Matsuo et al (2003) suggested that
Sophoraflavanone G exhibited trypanocidal activity with the minimum lethal concentration of 3.7 against Trypanosoma cruzi which is the etiological agent of
Chagas disease. On the other hand, naringenin, a flavanone without lavandulyl and
2'-hydroxyl groups, showed only a very weak trypanocidal activity (minimum lethal concentration = 370 |iM). These results suggest that the lavandulyl group is essential
for the trypanocidal activity.
1.2.4.2 And -tumor Activities
Earlier studies by Ryu et al (1997)，using a cytotoxicity-guided procedure,
found that Sophoraflavanone G was one of the most potent anti-tumor flavonoids
from Kushen and this active compound exhibited high cytotoxic activities against
various human tumor cell lines, including the lung cancer A549 cells, ovarian cancer
SK-OV-3 cells, skin cancer SK-MEL-2 cells, central nervous system cancer XF498
cells, and colon cancer HCT-15 cells. In the same year, 1997, it was also reported
that Sophoraflavanone G had similar potent anti-tumor activities against human
cervical cancer HeLa cells and human leukemia K-562 cells, but showed mild
activity against the mouse leukemia L1210 cells (Kim et al, 1997). In addition,
similar in vitro anti-tumor effects of Sophoraflavanone G were also shown on the
human leukemia HL-60 cells and human hepatocarcinoma HepG2 cells (Kang et al.’
2000; Ko et al, 2000).
Apart from screening the in vitro anti-tumor effects of Sophoraflavanone G
-26- Chapter 1 General Introduction against various tumor cell lines, some attempts were made to elucidate the possible mechanisms leading to its anti-tumor effects. Kang et al. (2000) demonstrated that four lavandulyi flavanones isolated from Kushen, including Sophoraflavanone G, were able to induce apoptosis in the human leukemia HL-60 cells and human hepatocarcinoma HepG2 cells, as judged by detection of DNA fragmentation.
Nevertheless, this study didn't elucidate the molecular mechanisms leading to the
apoptosis-inducing effects in the HL-60 cells and HepG2 cells. Recent studies from
our laboratory showed that Sophoraflavanone G was able to trigger apoptosis in
HepG2 cells and multidrug resistant RHepG2 cells via death receptor pathway,
mitochondria pathway and caspase-independent pathway (Tsang, 2006).
1.2.4.3 Pharmacodynamics of Sophoraflavanone G
Growing evidences suggested that Sophoraflavanone G was able to interact with
the key enzymes involved in several important biological processes, such as cellular
signaling, fatty acid metabolism, lipid metabolism and carbohydrate metabolism.
Phospholipase C (PLC), an important enzyme in cellular signaling, is able to
catalyze the hydrolysis of phosphatidylinositol bisphosphate (PIP2) into two
important second messenger molecules, the inositol triphosphate (IP3) and
diacylglycerol (DAG). IP3 and DAG will further alter cell responses such as
proliferation, differentiation, apoptosis, and ion channel conductance. Lee et al.
(1997) suggested that Sophoraflavanone G, together with other 11 prenylated
flavonoids from Kushen, had relatively strong inhibitory activity on PLCyl. Analysis
of structure-activity relationship (SAR) showed that the presence of lavandulyi
moiety is crucial for the inhibitory effect since hydration of the C4"-C5" double bond
of the lavandulyi group will lead to complete loss of the inhibitory activity (Lee et al.,
-27- Chapter 1 General Introduction
1997;Botta etal^imS),
Cyclooxygenase (COX) and lipooxygenase (LOX) are two key enzymes involved in the arachidonic acid metabolism. COX, including COX-1 and COX-2, catalyzes the formation of prostaglandins (PG) from arachidonic acid (Kampa et al，
2007). Three lipooxygenases (5-LOX, 12-LOX, and 15-LOX) have been reported to be present in human tissues and all of them can oxidize polyunsaturated fatty acids.
Their oxidation products were demonstrated to be important in inflammation and carcinogenesis (Kampa et al, 2007). Sophoraflavanone G is known to possess relatively strong inhibitory effects on COX-1 and 5-LOX, but show weak inhibitory effect on 12-LOX (Chi et al, 2001; Kim et al, 2002). In addition, Kim et al. (2002) also demonstrated that Sophoraflavanone G was able to alleviate the in vivo inflammation in animal models. These findings indicate a high potential for
Sophoraflavanone G to be used as an anti-inflammatory agent (Chi et al, 2001; Kim et al, 2002; Sohn et a/., 2004).
Melanin formation, due to oxidation of tyrosine, is the most important determinant of the color of mammalian skin. Therefore, the inhibition of melanin formation may result in a reduction in skin darkness. Since tyrosinase is the rate-limiting enzyme catalyzing the oxidation of tyrosine, tyrosinase inhibitors may have potential for treating abnormal pigmentation disorders and to be used as a skin-whitening agent in the cosmetic industry (Kim et al., 2003). It has been reported that Sophoraflavanone G (IC50 = 6.6 |iM) possesses more potent tyrosinase-inhibitory activity as compared with kojic acid (IC50 = 20.5 ^iM), a widely used skin-whitening agent in Northeast Asia. Moreover, with regard to the structure-activity relationship, the presence of A-ring C-8 lavandulyl and B-ring
2'.4'-dihydroxyl moiety is important for the considerable tyrosinase-inhibitory
-28- Chapter 1 General Introduction
activity. These findings suggest that Sophoraflavanone G and certain other prenylated flavonoids may have potential for use as skin-whitening agents (Kim et
a/., 2003; Zhao et a/.，2004).
In recent years, certain flavonoids, such as genistein and luteolin, have been
reported to show potent inhibitory effects for glycosidases. Since glycosidases
participate in several important biological processes, such as digestion, biosynthesis
of glycoproteins and lysosomal catabolism of glycoconjugates, glycosidase inhibitors
are potential therapeutics for type 2 diabetes, viral infection and cancer (Kim et al.,
2006). In a screening of methanol extract of Kushen, most flavanones with A-ring
C-8 lavandulyl group, including Sophoraflavanone G, potently inhibited the
a-glucosidase and p-amylase activities (Kim et al., 2006).
-29- Chapter 1 General Introduction
1.3 Objectives and Scopes of the Present Study
Sophoraflavanone G is a lavandulyl flavonoid that mainly exists in Sophora species (Leguminosae) and it has been demonstrated to possess multiple biological and pharmacological effects as described in Section 1.2.3. Until recently, the anti-tumor activities and action mechanisms of Sophoraflavanone G on human myeloid leukemia cells remain elusive. Therefore, in our present study, we aimed at investigating the anti-tumor activity and possible cellular and molecular mechanisms of Sophoraflavanone G against human myeloid leukemia.
In the present study, the human promyelocytic leukemia HL-60 cells, a well-characterized cell line, was chosen as the major leukemia model to investigate the anti-tumor effect of Sophoraflavanone G. Three other human myeloid leukemia cell lines, including the acute promyelocytic leukemia (NB-4), eosinophilic leukemia
(EoL-1), chronic myelogenous leukemia (K-562), were used to demonstrate that the growth-inhibitory effect of Sophoraflavanone G was not restricted only to one single leukemia cell line. The kinetics and reversibility of Sophoraflavanone G with respect to its anti-proliferative effect on HL-60 cells were also examined. Apart from leukemia cells, the cytotoxic effect on normal cell models, including human peripheral blood leukocytes and murine bone marrow cells, was also investigated.
The anti-proliferative and cytotoxic effects of Sophoraflavanone G were assessed by
MTT reduction assay, ^H-thymidine incorporation assay and trypan blue exclusion assay. Additionally, the cell cycle profile and cell cycle-regulatory proteins of HL-60 cells after Sophoraflavanone G treatment were assayed by flow cytometry and
Western blot analysis, respectively. Moreover, the apoptosis-inducing ability of
Sophoraflavanone G was measured by the DNA fragmentation assay and Annexin
V-GFP/PI double staining assay. Furthermore, the mitochondrial membrane potential
-30- Chapter 1 General Introduction and enzymatic activities of caspase-3, -8，and -9 were monitored to clarify the possible apoptotic pathway(s) that might be involved. The expression of apoptosis-associated proteins was also analyzed to elucidate the possible action mechanism(s) of Sophoraflavanone G by the Western blotting. In addition to in vitro studies, the effect of Sophoraflavanone G on the in vivo tumorigenicity of HL-60 cells was also investigated in BALB/c nude mice. Finally, the differentiation-inducing ability of Sophoraflavanone G on HL-60 cells was studied by morphological analysis on cytospin preparations and functional assessment of superoxide anion production by the NBT reduction assay.
-31 - Chapter Two
Materials and Methods Chapter 2 Materials and Methods
2.1 Materials
2.1.1 Animals
Inbred female BALB/c (haplotype H-2'^) mice and inbred female BALB/c nude mice aged 6 to 8 weeks old were bred at the University Laboratory Animal Services
Centre of The Chinese University of Hong Kong under specific pathogen-free condition. They were fed with a pelleted standard chow diet (Prolab RMH 2500
Rodent diet #5P14, PMI Nutrition International) with free access to tap water.
2.1.2 Cell Lines
1) EoL-1
EoL-1 is a human eosinophilic leukemia cell line which was kindly provided by
Prof. C. K. Wong, Department of Chemical Pathology, The Chinese University of
Hong Kong. It was established from the peripheral blood of a patient with
Philadelphia chromosome-negative eosinophilic leukemia (EL) (Saito et al.’ 1985).
The cells were maintained in plain RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and incubated at 37� Cin a humidified incubator supplied with 5% CO2.
2) HL-60
HL-60 is a human acute promyelocytic leukemia cell line purchased from the
American Type Culture Collection (ATCC) with the cell line code ATCC number
CCL-240. It was established from the peripheral blood of a 36-year-old Caucasian
female with acute promyelocytic leukemia (Collins et al., 1977). The cells were
-32- Chapter 2 Materials and Methods maintained in plain RPMI 1640 medium supplemented with 20% (v/v) fetal bovine serum (Invitrogen) and incubated at 37 °C in a humidified incubator supplied with
5% CO2.
3) HL-60/MX2
HL-60/MX2 is a mitoxantrone resistant derivative of the HL-60 cell line
(Harker et al, 1991) purchased from ATCC (ATCC number CRL-2257).
Maintenance of HL-60/MX2 cells is the same as HL-60 cells.
4) K-562
K-562 is a human chronic myelogenous leukemia cell line purchased from
ATCC (ATCC number CCL-243). It was originated from the pleural effusion of a
3 5-year-old female with chronic myelogenous leukemia in terminal blast crises
(Lozzio and Lozzio, 1975). Maintenance of K-562 cells is the same as EoL-1 cells.
5) NB-4
NB-4 is a human acute promyelocytic leukemia cell line purchased from
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ,
German Collection of Microorganisms and Cell Cultures) with the cell line code
DSMZ number ACC 207. It was established from the bone marrow of a 23-year-old
female with acute promyelocytic leukemia in the second relapse (Lanotte et al.,
1991). Maintenance of NB-4 cells is the same as EoL-1 cells.
-33 - Chapter 2 Materials and Methods
2.1.3 Cell Culture Medium, Buffers and Other Reagents
1) RPMI fRoswell Park Memorial Institute) 1640 Medium
A pack of RPMI 1640 powder (GIBCO BRL Life Technologies Inc.) with 2 mM
L-glutamine and 25 mM N-2-hydroxy-ethyl-piperazine-N'-2-ethane-sulfonic acid
(HEPES) was dissolved in one litre (L) of deionized water. Two grams of sodium bicarbonate (NaHCOs) (Sigma Chemical Co.) was added and the pH was adjusted to
7.2 by either 1 M HCl or 1 M NaOH (Sigma Chemical Co.). The medium was then sterilized by membrane filtration with 0.22 \xm bottle-top filter (Millipore).
Sterilized medium was stored at 4°C until use.
"Plain RPMI 1640 medium" was prepared by supplementing the sterilized
RPMI 1640 medium with 1% (v/v) antibiotics (PSF). "Complete RPMI 1640 medium" was prepared by supplementing plain RPMI 1640 medium with 10% or
20% (v/v) fetal bovine serum (GIBCO BRL Life Technologies Inc.). Heat inactivated fetal bovine serum (HI-FBS) was used to supplement plain RPMI 1640 medium when bioassays were performed.
2) Heat-inactivated fetal bovine serum
Heat-inactivated fetal bovine serum (HI-FBS) was prepared by heating the fetal bovine serum at 56°C for 30 minutes in a water bath and then kept frozen at -20°C
until use.
3) Antibiotic mixture solutions
Antibiotic-Antimycotic or Penicillin-Streptomycin-Fungizone (PSF) stock
solution (100 ml, lOOx) contains 10,000 units/ml penicillin G (sodium salt), 10,000
|ig/ml streptomycin sulphate, and 25 |ig/ml amphotericin B as Fungizone in 0.85%
-34- Chapter 2 Materials and Methods
(w/v) saline. The stock solution was purchased from Invitrogen Life Technologies
Inc. and was stored at -20°C as 5 ml aliquots until use.
4) Phosphate-Buffered Saline (PBS)
PBS (Sigma Chemical Co.) was prepared by dissolving 8 g NaCl (137 mM), 0.2
g KCl, 0.2 g KH2PO4 (1.4 mM) and 1.15 g NaaHPCU (4.3 mM) in one litre deionized
water. The chemicals were purchased from Sigma Chemical Co. The pH of the
solution was adjusted to 7.2 by either 1 M HCl or 1 M NaOH. It was then sterilized
by autoclaving at 121� Cfor 20 minutes.
5) Ammonium chloride-potassium (ACK) buffer
ACK buffer consists of 150 mM NH4CI, 1 mM KHCO3 and 0.001 mM EDTA.
The pH was adjusted to 7.2 - 7.4 by adding either 1 M HCl or 1 M NaOH. After
being sterilized by filtration, the ACK buffer was kept at 4°C until use. NH4CI and
KHCO3 were purchased from Sigma Chemical Co. while EDTA was bought from
USB Corporation.
6) Methvlthiazoletetrazolium (MTT)
MTT was purchased from USB Corporation in powdered form. The powder was
dissolved in sterile PBS to prepare the stock solution at a concentration of 5 mg/ml.
In each analysis, 30 ^iL of MTT solution was applied to each well of 96-well
flat-bottomed microtiter plate. The stock was stored at 4°C until use.
7) rMethvl-^H1 Thymidine (F^HI-TdR)
The stock solution (Amersham Life Science Ltd.) with specific activity of 2
Ci/mmol was stored as 500 ^il aliquots at 4°C. It was freshly diluted with complete
RPMI medium to give a working solution of 25 fiCi/ml before use. In each analysis,
-35- Chapter 2 Materials and Methods
20 |j.l of working solution was added to each well of the 96-well flat-bottomed microtiter plate.
8) Thioglvcollate (TG) Broth
Thioglycollate (TG) broth (3% w/v) was prepared by adding 3 g dehydrated thioglycollate powder (Difco Lab.) in 100 ml deionized water. The solution was then heated until the powder was completely dissolved and was sterilized by autoclaving at 121°C for 20 minutes. It was stored in dark for at least one month at room temperature before use.
9) Dye Solutions a) Hemacolor® Staining Solutions
Hemacolor® staining solutions (Merck Biosciences) include three distinct solutions were used to stain cells. Hemacolor® Solution 1 is a fixation solution,
Solution 2 is a buffered red color reagent and Solution 3 is a buffered blue color reagent. All solutions were kept in dark, tightly closed glass bottles at room temperature. b) Trypan Blue Solution
Trypan blue solution was purchased from Gibco BRL Life Technologies Inc. It contains 0.4% (w/v) trypan blue dissolved in a solution of 0.817% (w/v) NaCl and
0.06% (w/v) KH2PO4. The solution was stored at room temperature. c) Nitroblue Tetrazolium (NBT)
NBT powder was purchased from Sigma-Aldrich Co. NBT-PMA mix solution contains 0.02 g NBT powder in 10 ml RPMI with 1 jig/ml phorbol 12-myristate
13-acetate (PMA) (Sigma Chemical Co.).
-36- Chapter 2 Materials and Methods d) Safranin
Safranin powder was purchased from South East Chemicals and Instruments
Ltd. The staining solution contains 0.1 g safranin dissolved in 10 ml of deionized water.
10) Sophoraflavanone G
Sophoraflavanone G used in the present study was isolated from Kushen (a traditional Chinese medicinal herb) by my previous labmate Ms. Tsang Kit Man with the collaboration of Institute of Chinese Medicine, The Chinese University of Hong
Kong. The purification method of Sophoraflavanone G was briefly summarized in
Section 2.2.1 of this chapter. Before use, Sophoraflavanone G powder was dissolved in absolute ethanol to make the final stock solutions with the concentration of 10 mg/ml and then sterilized by filtration. All stock solutions were stored at - 20°C. The concentration of ethanol used as the solvent control in each experiment was corresponding to the highest concentration of Sophoraflavanone G used in that experiment.
2.1.4 Reagents and Buffers for Flow Cytometry
1) Propidium Iodide (PI) DNA Staining Buffer
The PI DNA staining buffer was freshly prepared by adding 400 |ig/mL ribonuclease A (RNase A) (Boehringer Mannheim), 50 |ag/mL propidium iodide
(Boehringer Mannheim), 10 mM EDTA, pH 7.4 (Sigma Chemical Co.), 0.1% trisodium citric acid (Sigma Chemical Co.) and 0.1% Triton X-100 (Sigma Chemical
Co.). The PBS and PI (stored in dark) stock solution were kept at 4°C while the
RNase A stock solution was kept at -20°C.
-37- Chapter 2 Materials and Methods
2) AnnexinV-GFP
Annexin V-GFP was kindly supplied by Prof. S.K. Kong, Department of
Biochemistry, The Chinese University of Hong Kong. The 50x reagent was stored in dark at -20°C and was freshly diluted with Annexin V binding buffer before use.
3) Annexin V Binding Buffer
Annexin V Binding Buffer was made up of 140 mM NaCl, 2.5 mM CaCb, 10 mM HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid) at pH 7.4. The chemicals were purchased from USB Corporation. The buffer was filtered and then stored at 4°C until use.
4) JC-1 Staining Solution
5,5',6,6'-tetrachloro-l,r,3,3'-tetraethylbenzimidazolyl-carbocyanine iodide
(JC-1) was purchased from Molecular Probes Inc. The stock solution of JC-1 was prepared by dissolving the compound in DMSO to a concentration of 5 mg/ml and was stored in dark at -20°C. The dye was stored at -20°C and diluted to 10 ^M in warm PBS before use.
5) Dihvdroethidium (DHE)
DHE, also known as hydroethidine, was ordered from Molecular Probes Inc.
The 10 mM DHE stock solution was prepared by dissolving 1 mg DHE powder in
DMSO and was kept at -20°C in dark. The working concentration of DHE was 10
I^M.
6) Fluo-3/AM
Fluo-3/AM was purchased from Molecular Probes, Inc. The 10 mM Fluo-3/AM
-38- Chapter 2 Materials and Methods
Stock solution was prepared by dissolving the powder in DMSO and stored at -20°C in dark. The working concentration of Fluo-3/AM was 10 |j.M.
7) 75% (vM Ethanol
The 75% (v/v) ethanol was prepared by adding 75 ml absolute ethanol
(Analytical grade) (Merck Biosciences) to 25 ml autoclaved deionized water. It was stored at 4°C and used for fixing cells in cell cycle analysis.
8) 1% fw/v) Paraformaldehyde
Paraformaldehyde was purchased from Sigma Chemical Co. in powdered form.
One gram paraformaldehyde powder was completely dissolved in 50 ml deionized water by heating to 60°C with constant stirring for 1 hour in a fume hood. A few drops of 1 M NaOH were added to facilitate the dissolution until a clear solution was obtained. After cooling down, 1% paraformaldehyde in Ix PBS was obtained by adding 50 ml of 2x PBS. The solution was stored in dark at 4� Cuntil use.
2.1.5 Reagents for DNA Extraction
1) IGEPAL CA-630 Lysis Buffer
This lysis buffer (Sigma Chemical Co.) was prepared in 50 mM Tris
[hydroxyImethyl] amino methane (Tris)-HCl, pH 7.5 with 3% non-ionic detergent
IGEPAL CA-630 ((Octylphenoxy) polyethoxyethanol) and 20 mM EDTA. The lysis
buffer was stored at room temperature. "IGEPAL" is a registered trademark of
Phone-Poulenc, Inc.
-39- Chapter 2 Materials and Methods
2) TIOEM Buffer
The buffer was prepared by 10 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA in deionized water. It was stored at room temperature.
3) Proteinase K
Proteinase K was purchased from USB Corporation. The stock solution (20 mg/ml) was prepared by dissolving the powder of proteinase K in autoclaved TioEo.i buffer and it was stored as 500 \x\ aliquots at -20°C.
4) RNase A
RNase A, a bovine pancreatic RNase A, was purchased from Boehringer
Mannheim in a powdered form. The stock solution was prepared at a concentration of 10 mg/ml by dissolving the RNase powder in 10 mM Tris-HCl (pH 7.5) and 15 mM NaCl. The solution was then boiled for 15 minutes to denature any contaminated
DNase. It was stored as 500 \x\ aliquots at -20°C.
5) Sodium Acetate Solution (NaOAc)
The 3M NaOAc stock solution was prepared by dissolving 24.61 g sodium
acetate (Sigma Chemical Co.) in 100 ml deionized water. It was then sterilized by
autoclaving at 121°C for 20 minutes. The solution was stored at room temperature.
2.1.6 Reagents for Measuring Caspase Activity
1) Cell Lysis Buffer
This lysis buffer contains 1% (v/v) IGEPAL CA-630 (Sigma Chemical Co.),
150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA and one tablet of protease inhibitor
-40- Chapter 2 Materials and Methods cocktail (Roche Molecular Biochemicals) in 50 ml deionized water. The pH was adjusted to 7.5. The buffer was kept at 4°C until use. Each tablet of complete protease inhibitor cocktail when dissolved in 50 mL lysis buffer yielded a mixture of several protease inhibitors with a broad spectrum of activity on serine-, cysteine- and metallo-proteases and calpains.
2) Reaction Buffer
The reaction buffer was made up of 10 mM HEPES-KOH, 40 mM P- glycerophosphate, 50 mM NaCl, 2 mM MgCh, 5 mM EGTA, 0.1% CHAPS, 100 l_ig/ml BSA (Sigma Chemical Co.) and 10 mM DTT (Invitrogen Life Technologies
Inc.). The pH was adjusted to 7.0. The buffer was kept at 4°C until use.
3) Dithiothreitol (DTT)
DTT was purchased from Invitrogen Life Technologies Inc. It was dissolved in deionized water as 1 M stock and kept at -20°C. It was added to the reaction buffer to a final concentration of 10 mM and was used to stabilize the enzymes with free sulfhydryl groups to give full activity of the enzymes.
4) Bradford Reagent
It is a 5x solution purchased from Bio-Rad Laboratories. When use in
quantification of protein, one volume of Bradford reagent was added to four volumes
of protein sample. The Bradford solution was stored at 4°C until use.
5) Bovine Serum Albumin (BSA)
BSA (Sigma Chemical Co.) powder was dissolved in sterilized deionized water
and made up to 2 mg/ml stock aliquots. The powder was kept at 4°C while the
aliquots were stored at -20°C. The BSA standard curve was constructed by using 0
-41 - Chapter 2 Materials and Methods
Hg/ml, 2 |j.g/ml, 4 jig/ml, 6 |ig/ml, 8 |ig/ml and 10 |ig/ml filtered water diluted-BSA solutions.
6) Substrates, Inhibitors, and Calibrator
Substrates, inhibitors and calibrator for caspase activities determination are listed in Table 2.1. They were dissolved in DMSO as stock solutions and stored at
-20� Cuntil use.
Table 2.1 List of caspase substrates, inhibitors and calibrator for measuring caspase activities
Stock Item Chemical name Company concentration Cas ase 3 Acetyl-Asp-Glu-Val-Asp-7-amido-4 g. ^^ aspase- -methylcoumarin 2 mM r , „ rate (Ac-DEVD-AMC) Chemical Co.
Caspase-3 Acetyl-Asp-Glu-Val-Asp-CHO ALEXIS Inhibitor (Ac-DEVD-CHO) 川 mM Biochemicals ^ o N-Acetyl-Ile-Glu-Thr-Asp-7-amido-4 ^ ^ …t� Casp,e;8 -methylcoumarin 1 mM 口 严 Substrate (Ac-IETD-AMC) Biochemicals
Caspase-8 N-Acetyl-Ile-Glu-Thr-Asp-CHO ALEXIS Inhibitor (Ac-IETD-CHO) iUmM Biochemicals
(^asp二 -methylcoumarin 5 mM ^ ALEXIS 她 strate (Ac-LEHD-AMC) Biochemicals
Caspase-9 N-Acetyl-Leu-Glu-His-Asp-CHO ALEXIS Inhibitor (Ac-LEHD-CHO) 川 mM Biochemicals
AMC 7-Amino-4-methylcoumarin Sigma Calibrator (AMC) mM chemical Co.
All chemicals were dissolved in DMSO as stock solutions and stored at -20°C until use.
-42 - Chapter 2 Materials and Methods
2.1.7 Reagents, Buffers and Materials for Western Blotting
1) Cell Lysis Buffer for Total Protein Extraction
The preparation of cell lysis buffer for total protein extraction is the same as that of cell lysis buffer for measuring caspase activity (see Section 2.1.6 for details).
2) Bradford Reagent
The Bradford reagent, purchased from Bio-Rad Laboratories, is a 450 ml ready-to-use solution. It was kept at 4°C until use.
3) Bovine Serum Albumin (BSA)
BSA (Sigma Chemical Co.) powder was dissolved in sterilized deionized water
and made up to 2 mg/ml stock aliquots. The powder was kept at 4°C while the
aliquots were stored at -20°C. The BSA standard curve was constructed by using 0
|ig/ml, 2 ^g/ml, 4 |^g/ml, 6 ^ig/ml, 8 |ig/ml and 10 |ag/ml filtered water diluted-BSA
solutions.
4) Lower Buffer for Separating Gel
The lower buffer, a Tris-HCl buffer, was prepared by dissolving 1.5 M Tris
(Sigma Chemical Co.) in deionized water to prepare the buffer. The pH was adjusted
to 8.8. The buffer was then kept at 4°C until use.
5) Upper Buffer for Stacking Gel
The upper buffer, a Tris-HCl buffer, was prepared by dissolving 0.5 M Tris
(Sigma Chemical Co.) in deionized water to prepare the buffer (pH had to be
adjusted to 6.8). It was stored at 4�C.
-43 - Chapter 2 Materials and Methods
6) 10% Sodium Dodecvl Sulfate (SDS) Solution
SDS was purchased from Sigma Chemical Co. in a powdered form. 10% SDS solution (w/v) was prepared by dissolving 10 g SDS in 100 ml deionized water and stored at room temperature.
7) Acrylamide Stock Solution
The ready-to-use acrylamide stock solution, also known as 30% (w/v) acrylamide/ bis stock solution was purchased from Bio-Rad Laboratories. The 500 ml solution contains 146.1 g acrylamide and 3.9 g N,N'-methylene-bis-acrylamide for a total monomer to cross-linker ratio of 37.5:1. It was kept at 4°C.
8) 10% Ammonium Persulfate (APS)
APS was purchased from Bio-Rad Laboratories in powdered form. The 10%
APS solution (w/v) was prepared by dissolving 0.5 g APS in 5 ml deionized water.
The solution was stored in 500 |il aliquots at -20°C.
9) N,N,N,N'-Tetra-Methvl-ethvlenediamine (TEMED)
The ready-to-use TEMED stock solution was bought from Bio-Rad
Laboratories and was stored at 4°C until use.
10) 4x SDS Sample Loading Buffer
This loading buffer (8 ml) was prepared by mixing 0.4 ml 0.05% bromophenol
blue, 2 ml upper buffer, 2 ml glycerol, 2 ml 10% SDS, 0.2 ml 2-mercaptoethanol and
1.4 ml deionized water together. The loading buffer was then kept at 4°C until use.
All reagents were purchased from Sigma-Aldrich Co.
-44 - Chapter 2 Materials and Methods
11) SDS Running Buffer (lOx)
This buffer, also known as Tris-Glycine-SDS Electrophoresis Buffer (lOx), contains 0.25 M Tris-HCl (pH 8.6), 1.92 M glycine (USB Corporation) and 1% SDS in deionized water. The lOx buffer was stored at 4°C. The working buffer (Ix) was freshly diluted 10-fold from the 1 Ox stock solution.
12) Tris-Glvcine Buffer (IQx)
This lOx Tris-glycine buffer was prepared by dissolving 0.25 M Tris-HCl (pH
7.5) and 1.92 M glycine in deionized water. The buffer was stored at 4°C.
13) Transfer Buffer (Ix)
This buffer, also known as Tris-Glycine-Methanol Transfer Buffer (Ix), was
prepared by mixing 100 ml methanol with 50 ml Tris-Glycine Buffer (lOx) and 350
ml deionized water. The buffer was stored at 4°C.
14) Washing Buffer (IQx)
This buffer, also known as TBS-Tween washing buffer (lOx), was made up of
100 mM Tris (pH 7.5)，1 M NaCl and 1% Tween 20 (Sigma Chemical Co.) in
deionized water. The buffer was stored at 4°C. The working buffer (Ix) was freshly
prepared by diluting the stock solution 10-fold.
15) Kaleidoscope Prestained Standards
These prestained standards (Bio-Rad Laboratories) are mixtures of
well-characterized proteins that are used as a reference to determine the molecular
weight of proteins of interest identified by the antibody probes. They consist of seven
colored proteins, including myosin (199,000 Da), P-galactosidase (128,000 Da),
-45 - Chapter 2 Materials and Methods bovine serum albumin (85,000 Da), carbonic anhydrase (41,700 Da), soybean trypsin inhibitor (32,100 Da), lysozyme (18,300 Da) and aprotinin (7,500 Da). These protein standards were prepared in 33% (v/v) glycerol, 3% SDS, 10 mM Tris (pH 7), 10 mM
DTT, 2 mM EDTA and 0.01% NaNa and stored at -20°C.
16) Polyvinylidene Fluoride fPVDF) Western Blotting Transfer Membrane
The PVDF membrane with pore size of 0.45 jim was purchased from Roche
Applied Science. It was cut into 8.5 cm x 5.5 cm sized transfer membranes for
ready-to-use and stored at room temperature.
17) Filter Paper
Chromatography paper (3 mm thick) (Whatman International Ltd.) was used as
filter paper. It was cut into 8.5 cm x 5.5 cm sized filter paper for ready-to-use.
18) 5% (w/v) Skimmed Milk Solution (Blocking Solution)
Blocking solution was prepared by dissolving skimmed milk powder (Nestle
Company) in washing buffer (1 x) to give a final concentration of 5% (w/v).
19) Western Blotting Luminol Reagents
The Western blotting luminol reagents (Santa Cruz Biotechnology Inc.) consist
of two reagents: reagent A (125 ml) and B (125 ml). An equal volume of reagent A
and B was mixed freshly before use and a final volume of 0.125 mL/cm^ membrane
was required. The reagents were stored in dark at 4°C.
20) Fuji Medical X-ray Film
The film was bought from Fuji Photo Film Co. Ltd. It was stored in dark at 4°C.
-46 - Chapter 2 Materials and Methods
21) Primary and Secondary Antibodies
Primary and secondary antibodies for Western blot analysis are listed in Table
2.2. All the primary antibodies, except anti-p-actin antibody (stored at -20�C)，were stored at 4°C. All the secondary antibodies were stored at 4°C.
Table 2.2 List of the primary and secondary antibodies used for Western blotting experiment
Antibody Name Source Company
Anti-p-actin Mouse Sigma Chemical Co.
Anti-CDK-4 Rabbit StressGen Biotechnologies Corp.
Primary Anti-CDK-6 Rabbit StressGen Biotechnologies Corp.
antibody Anti-Bax Mouse Santa Cruz Biotechnology
Anti-Bak Rabbit Santa Cruz Biotechnology
Anti-Bcl-2 Mouse Santa Cruz Biotechnology
Anti-mouse-HRP conjugate Goat Amersham Pharmacia Biotech Secondary antibody ” Anti-rabbit-HRP conjugate Donkey Amersham Pharmacia Biotech
-47 - Chapter 2 Materials and Methods
2.2 Methods
2.2.1 Extraction and Isolation of Sophoraflavanone G from Kushen
Sophoraflavanone G used in the present study was isolated from Kushen by my previous labmate Ms. Tsang Kit Man with the collaboration of Institute of Chinese
Medicine, The Chinese University of Hong Kong. No further purification was carried out. The purification method of Sophoraflavanone G was described in detail by
Tsang (2006) and briefly summarized as follows:
Kushen was purchased from Guangzhou with the specimen voucher number
2004-2531 and stored in the Institute of Chinese Medicine of The Chinese University of Hong Kong. The specimen was morphologically authenticated by Dr. Chak Yin
Lee, South China Botanical Garden, The Chinese Academy of Sciences. All experiments were carried out using this batch of specimen. One kg of sliced and air-dried Kushen was refluxed with 8 L of 95% ethanol for 2 hours to prepare 2 kg ethanol extract. The ethanol extract was then filtered, and the filtrate was further evaporated under reduced pressure to giving syrup. After partition of the syrup between 1 L of water (H2O) and 400 ml of ethyl acetate (EtOAc), the ethyl acetate
extract was collected and the water extract was further extracted with 300 ml fresh
EtOAc twice. The ethyl acetate extracts collected from three separate extracts were
combined and washed with 200 ml of 10% hydrochloric acid and H2O to remove the
alkaloid groups. After adjusted to pH 7, the pH-adjusted ethyl acetate fraction was
subjected to silica gel column chromatography and eluted with a solvent of hexane
and EtOAc at the ratio of 1:1. The eluents were further isolated by silica gel column
-48 - Chapter 2 Materials and Methods using hexane and EtOAc at the ratio of 2:1 to obtain the Compound 106-1 (190 mg).
The Compound 106-1 powder was stored in dark at room temperature.
The chemical structure of Compound 106-1 was identified as Sophoraflavanone
G by spectral analysis with Mass Spectrometry (MS), Nuclear Magnetic Resonance
(^H-NMR and ^^C-NMR) and by direct comparison of spectroscopic data with those reported in the literature (Shirataki et al, 1988). The purity was measured by High
Performance Liquid Chromatography (HPLC).
2.2.2 Culture of Tumor Cell Lines
In the present study, three human leukemia cell lines (NB-4, EoL-1, K-562) were cultured in 10% complete RPMI medium while HL-60 and HL-60/MX2 cells
were cultured using RPMI medium supplemented with 20% FBS. All cells were
cultured in tissue culture flasks (25 or 75 cm^) and incubated at 37°C in a humidified
incubator supplied with 5% carbon dioxide (CO2). According to the doubling time of
each cell line, cells were sub-cultured every 2 or 3 days to maintain a desirable cell
population and prevent over-growth. Cells in exponential growth phase were either
chosen for performing assays or for long-term storage of cell lines by
cryopreservation in liquid nitrogen (the composition of cryopreservation medium
was 50% FBS, 40% RPMI medium and 10% DMSO).
-49 - Chapter 2 Materials and Methods
2.2.3 Isolation, Preparation and Culturing of Human Peripheral Blood
Leukocytes and Murine Bone Marrow Cells
1) Human Peripheral blood Leukocytes (PBL)
A package of human peripheral blood from healthy donors was purchased from the Prince of Wales Hospital, Hong Kong. Dead cells and red blood cells were then removed by centrifugation on Ficoll-paque gradient. The harvested human PBL cells were washed twice with plain RPMI medium and finally resuspended in 10%
HI-FBS RPMI medium. The human PBL cells were then ready for assays.
2) Murine Bone Marrow Cells
To obtain the mouse bone marrow cells, 6 to 8 weeks old BALB/c mice were
sacrificed by cervical dislocation. The skin of mice was sterilized with 70% ethanol.
The femurs were removed with aseptic techniques and put into a petri dish
containing 10% complete RPMI medium. After cutting two ends of the bones, the
cells inside were flushed out using a 2 ml syringe fitted with a 25-Gauge needle
containing 10% complete RPMI medium. In order to remove the contaminating red
blood cells, the flushed-out cells were resuspended in 10 mL ACK buffer and
co-incubated at room temperature for 5 minutes to lyse the red blood cells. The
isolated bone marrow cells were then resuspended in complete RPMI medium and
ready for assays.
-50 - Chapter 2 Materials and Methods
2.2.4 Assays for Anti-proliferation and Cytotoxicity
1) Growth-inhibitory Effect as Assayed by MTT Reduction Assay
To study the growth-inhibitory effect of Sophoraflavanone G on different cancer cell lines as well as normal primary cells, MTT reduction assay was performed. In this assay, different types of cells, including leukemia cells (1x10^ cells/ml), human
PBL cells (1x10^ cells/ml) and murine bone marrow cells (1x10^ cells/ml) were first incubated with either solvent control (ethanol) or different concentrations of
Sophoraflavanone G in 96-well flat-bottomed microtiter plates at 37°C for 24, 48 or
72 hours in a humidified 5% CO2 incubator. Thirty microlitres MTT (5 mg/ml) was
then added to the drug-treated cells for 4 hours. After centrifuging the plate at 300 x
g for 10 minutes, the medium was removed and 100 jj,l DMSO was added to dissolve
the blue crystal formed inside the cells or acted as the blank. The absorbance was
measured at 540 nm by a microplate reader (Bio-Rad Laboratories). The results were
expressed as the mean percentage cell viability 士 standard error (S.E.) of
quadruplicate samples, using solvent-treated cells as a control. The percentage of cell
viability was calculated as follows:
Viability (%) = [OD540 of test sample / OD540 of control] x 100%
2) Determination of Cell Proliferation by [^H1-TdR Incorporation Assay
Leukemia cells (1x10^ cells/ml) were seeded and incubated with either solvent
control (ethanol) or different concentrations of Sophoraflavanone G in 96-well
flat-bottomed microtiter plates at 37� Cfor 24, 48 or 72 hours in a humidified 5%
CO2 incubator. The drug-treated cultures were then pulsed with 0.5 ^iCi [^H]-TdR (in
20 )j.l complete medium) for 6 hours. The cells were subjected to a freeze and thaw
process before being harvested onto glass microfiber filters using a cell harvester.
-51 - Chapter 2 Materials and Methods
The radioactivity in the filters was measured by the liquid scintillation analyzer
[(Microplate Scintillation and Luminescence Counter (Topcount NXT™)]. The percentage inhibition of [^H]-TdR incorporation was calculated as follows:
Inhibition of [^H]-TdR incorporation (%)
=[(cpm of control - cpm of test sample)/cpm of control] x 100%
3) Determination of Cell Viability by Trypan Blue Exclusion Assay
Trypan blue exclusion assay was used to determine cell viability. The viability is
distinguished by the integrity of plasma membrane and the ability of the viable cells
to exclude the trypan blue dye. Viable cells with intact membrane are visible as clear
cell bodies while the dead cells are stained in blue. The leukemia HL-60 cells (1x10^
cells/ml) were incubated with either solvent control or different concentrations of
Sophoraflavanone G at 37°C. The viability of cells was determined after 24, 48 and
72 hours of incubation. Ten microlitres cell suspension was mixed with 10 |J.1 0.4%
trypan blue solution, and 10 |j.l mixture was then loaded onto a hemocytometer for
counting the numbers of viable and dead cells. The results were expressed as the
mean percentage of cell viability 士 S.E. of triplicate cultures. The percentage
viability was calculated by the following equation:
Viability (%) = (number of viable cells/number of total cells counted) x 100%
2.2.5 Determination of Anti-leukemic Activity In Vivo {In Vivo
Tumorigenicity Assay)
Human leukemia HL-60 cells (1x10^ cells/ml) were incubated with either
solvent control (0.13% ethanol) or 30 |j.M Sophoraflavanone G for 16 hours at 37°C.
The drug-treated cells were harvested and washed once with PBS. Viable HL-60
-52 - Chapter 2 Materials and Methods cells (1x10^ cells in 100 PBS) were injected subcutaneously on the back of each
BALB/c nude mouse in groups of twenty. The tumor weight was measured at the end of the experiment, and the tumor volume was measured twice weekly with a vernier caliper. The tumor volume was calculated according to the formula [volume = height
X length X width x 7c/6]. Maximum tumor dimensions were measured on sagittal
(vertical height and anterior-posterior length) and coronal (width) views.
2.2.6 Cell Cycle Analysis by Flow Cytometry
There were 2 major steps in this assay:
a) Cell Preparation
Leukemia HL-60 cells and HL-60/MX2 cells (1x10^ cells/ml) were first
synchronized in RPMI medium supplemented with 0.5% HI-FBS for 12 hours at
37°C. After that, the leukemia cells were then resuspended in RPMI medium
supplemented with 20% HI-FBS and incubated with different concentrations of
Sophoraflavanone G at 37°C for 24 hours. The cells were harvested and washed once
with PBS and centrifuged at 400 x g for 5 minutes. Next, cells were fixed with 1 ml
70% ethanol at 4°C for 30 minutes.
b) PI Staining and Cell Cycle Analysis
Before staining the DNA contents within cells, the ethanol had to be removed
completely. That was achieved by first centrifuging the samples at 400 x g for 10
minutes to remove most of the ethanol and then washing the cells once again with
PBS and centrifuging the cells down at the same conditions. Each sample was mixed
with 1 ml freshly-prepared PI staining solution in dark at 37°C for 30 minutes. The
stained cells were subjected to fluorescence-activated cell sorter (FACS) analysis
using the FACSCanto flow cytometer (Becton Dickinson). The excitation wavelength
-53 - Chapter 2 Materials and Methods for PI was at 488 nm and the emission wavelength was at 590 nm for measuring red fluorescence intensity. Ten thousands events were collected for each sample determination. The percentage distribution in GQ/GI, S and G2/M phase of the cell cycle was calculated by the MODFIT program (Verity Software House).
2.2.7 Measurement of Apoptosis-induced Activities
1) Detection of DN A FraRmentation by Agarose Gel Electrophoresis
There were 3 major steps in this assay: a) Cell Preparation
HL-60 cells (1x10^ cells/ml) were incubated with various concentrations of
Sophoraflavanone G for 24 hours at 37°C. The cells were harvested and washed once with cold PBS by centrifuging at 400 x g for 5 minutes. Cell counting with 0.4% trypan blue solution was performed to determine the cell number and cell viability. b) Extraction of DNA Fragments
DNA fragmentation is the hallmark of apoptosis. By analyzing the genomic
DNA extracted from apoptotic cells in a 2% agarose gel, the occurrence of "DNA ladder" can be observed. HL-60 cells (2x10^ cells) were collected after cell counting and treated with 200 [i\ IGEPAL CA-630 lysis buffer for 10 minutes at 37°C. The
samples were then centrifuged at 6,000 x g for 5 minutes so as to collect the
supernatants that containing the apoptotic DNA fragments. The supematants were
transferred to new microcentrifuge tubes and 50 |j.l 5% SDS was added to each
sample. Ten microlitres DNase-free RNase A (10 mg/ml) was added and incubated
with the samples at 56°C for 90 minutes to remove the cellular RNA. Afterwards,
twenty microlitres proteinase K (20 mg/ml) was added and incubated with the
-54 - Chapter 2 Materials and Methods samples at 56°C for another 90 minutes to remove all proteins. The DNA fragments were precipitated with 0.1 volume (30 |j.l) of 3 M sodium acetate and 2.5 volumes
(750 1^1) of 4°C absolute ethanol. After centrifugation at 20,000 x g at 4°C for 30 minutes, the DNA pellets were washed with 4°C 70% ethanol by resuspension. The pellets were centrifuged down at 20,000 x g at 4°C for 5 minutes and the supernatants were discarded to remove ions. In order to remove the remaining water, the DNA samples were washed with cold absolute ethanol 4°C) and the DNA samples were obtained by centrifugation at 20,000 x g at 4°C for 5 minutes. After washing, the DNA pellets were air-dried for about 10 minutes and a milky layer should be observed on the walls of microcentrifuge tubes. The milky layer of the
DNA fragments was dissolved in 20 fj,l TioEo.i buffer, c) Detection of the DNA Fragments by Agarose Gel Electrophoresis
The DNA samples were incubated at 65°C for 5 minutes to denature any DNA secondary structures before performing agarose gel electrophoresis. After mixing with 5 loading dye, each sample, with a final volume of 25 fj.1, was subjected to
2% agarose gel electrophoresis at 90 volts. After electrophoresis, the gel was stained with ethidium bromide (1 [•xg/ml) for 3 minutes and destained with deionized water for 15 minutes. The DNA bands were detected under UV illumination by the Gel
Doc 2000 UV transilluminator (Bio-Rad Laboratories).
2) Detection of Early Apoptosis by Annexin V-GFP and PI Double Staining
HL-60 cells (1x10^ cells/ml) were incubated with either solvent control (0.13%
ethanol) or various concentrations of Sophoraflavanone G for 12 hours at 37°C. The
cells (1x10^ cells) were harvested and washed once with PBS by centrifuging at 400
X g for 5 minutes. Reaction buffer (500 pi/sample) was prepared by mixing 5 ^il
-55 - Chapter 2 Materials and Methods
Annexin V-GFP and 2 |il PI (50 |ig/ml) with 496 |il Annexin V binding buffer. The
PBS-washed cells were kept on ice in dark for 15 minutes after resuspending with
500 |il reaction buffer. The stained cells were subjected to BD FACSCanto flow cytometer for measuring green (FL-1 channel) and red (FL-2 channel) fluorescence intensities simultaneously. The data, expressed as the percentage of early apoptotic cells, were calculated by the WinMDI software (Version 2.8).
3) Measurement of Caspase Activities
There were three major steps involved in measuring the caspase activities: a) Treatment of Cells and Extraction of Proteins
HL-60 cells (1x10^ cells/ml) were incubated with either solvent control (0.13% ethanol) or various concentrations of Sophoraflavanone G for 24 hours at 37°C. The cells were harvested and washed twice with PBS by centrifuging at 400 x g for 5 minutes at 4°C. After cell counting by trypan blue exclusion test, the cell pellet was resuspended in lysis buffer (50 cells) and vortexed vigorously. The cell lysates were incubated on ice for 10 minutes to free the cytoplasmic proteins. After incubation, the cell lysates were then centrifuged at 20,000 x g for 10 minutes at 4°C.
The caspases-containing supematants were collected for the next step. b) Quantification of Protein
The protein concentration of the cell lysate was measured by the Bradford protein assay. Bovine serum albumin (BSA) standard solutions were prepared by
diluting BSA stock solution (2 mg/ml) with deionized water to 2, 4，6, 8 and 10
|ig/ml. Protein samples (3 |j.l) were added to 2400 |j.l of deionized water for dilution.
The BSA standard solutions (800 |J,1) and the diluted samples (800 pi) were
-56 - Chapter 2 Materials and Methods thoroughly mixed with 200 pii Bradford reagent in cuvettes. After thorough mixing, the 1 ml mixtures were allowed to stand at room temperature for 3 minutes and the absorbance at 595 nm was measured using 200 }il Bradford reagent in 800 [i\ deionized water as the blank. A standard curve was constructed by the
BioPhotometer (Eppendorf) and the protein concentrations of the cell lysates were
determined by the machine automatically.
c) Fluorometric Measurement of Caspase Activities
Fluorescent signal-generating caspase specific substrates were used for the
detection of caspase activities. Here the measurement of caspase-3 activity was
chosen as an example to introduce the basic principle. Ac-DEVD-AMC, a caspase-3
specific fluorescent AMC conjugated substrate, can be cleaved by active caspase-3 in
the protein extract. The amount of fluorescent AMC released from the substrate by
the action of caspase-3 is directly proportional to its enzymatic activity. Therefore,
the activity of caspase-3 can be determined by measuring the fluorescent intensities
generated by the cleaved substrate. To ensure the signal detected was due to the
caspase activities, special control was set up with the presence of the specific caspase
inhibitor. The control was set up by incubating 50 cell lysate and 50 )il reaction
buffer with 1 of specific caspase inhibitor in a 96-well flat-bottomed microliter
plate for 30 minutes at 37°C. Meanwhile, 50 [i\ of cell lysate was incubated 50 yd
reaction buffer without caspase inhibitor. All the samples, with or without caspase
inhibitor, were incubated with 1 of caspase-specific substrate for 1 hour at 37°C.
The fluorescence intensity generated by the cleaved products was quantified by the
Polarizon fluorescent plate reader (Tecan Group). Caspase activity was then
converted from the fluorescence intensities using a standard curve. The standard
-57 - Chapter 2 Materials and Methods curve was made by diluting the AMC solution with lysis buffer to 4 |a.M, 2 foM, 1 |iM,
0.5 |IM and 0.25 |J.M concentrations.
4) Detection of Mitochondrial Membrane Potential Changes
JC-1 staining method is a common method to determine the mitochondrial
membrane potential (A\|/m) of cells. HL-60 cells (1x10^ cells/ml) were incubated
with either solvent control (0.13% ethanol) or various concentrations of
Sophoraflavanone G for 12 and 24 hours at 37�C Th. e cells (1x10^ cells) were
harvested and washed once with PBS by centrifuging at 400 x g for 5 minutes. The
cells were resuspended in 1 ml JC-1 working solution obtained by mixing 1.3 JC-1
stock solution (5 mg/ml in DMSO) with warm PBS. The mixtures were incubated for
15 minutes at 37°C. The excitation wavelength of JC-1 dye was at 488 run and the
emission wavelengths were 525 nm (for green fluorescence) and 590 run (for red
fluorescence). After staining, the cells were measured for green (FL-1 channel) and
red (FL-2 channel) fluorescence intensities simultaneously using the BD FACSCanto
flow cytometer. The percentage cell population of A\|/m dissipation was calculated by
the WinMDI software (Version 2.8).
5) Detection of Intracellular Reactive Oxygen Species (ROS) Generation
The intracellular ROS level was measured by staining the cells with
dihydroethidium (DHE), an oxygen radical sensor dye specifically converted by
superoxide anions (O2 ') to highly fluorescent ethidium. After treated with different
concentrations of Sophoraflavanone G for 24 or 48 hours, the HL-60 cells (1x10^
cells) were collected and resuspended in 0.5 ml staining solution containing 10 |j,M
DHE for 30 minutes at 37°C. After staining, the samples were then subjected to the
BD FACS Canto flow cytometer to detect the generation of ROS by measuring the
-58 - Chapter 2 Materials and Methods change of red fluorescence intensity. The excitation wavelength was 488 nm and the emission wavelength was 590 nm. The data from flow cytometry were analyzed by the WinMDI software (Version 2.8).
6) Detection of Intracellular Calcium ion (Ca^"^) Level
The intracellular Ca^"^ level was measured by staining the cells with stained with
Fluo-3/AM (C51H50CI2N2O23). The excitation wavelength of Fluo-3/AM dye was 488 nm and the emission wavelength was 525 nm (green fluorescence). HL-60 cells
(1x10^ cells/ml) were either incubated with solvent control (0.17% ethanol) or
Sophoraflavanone G (20 \iM and 30 [M) for 24 and 48 hours at 37�C Th. e cells were harvested and washed once with PBS by centrifuging at 400 x g for 5 minutes. The harvested cells (1x10^ cells) were then stained with Fluo-3/AM and subjected to a flow cytometer (BD FACSCanto) to measure the intracellular Ca^^ level by detecting the green fluorescence intensity. The data from flow cytometry were analyzed by the
WinMDI software (Version 2.8).
2.2.8 Protein Expression Study
The method of protein expression study involves four major steps: a) treatment of cells and extraction of protein, b) quantification of protein, c) Western Blotting analysis, and d) ECL detection. The first two steps a) and b) have been described previously in Section 2.2.7 "Measurement of Caspase Activities". The last two steps c) and d) are briefly described as follows: c) Western Blotting Analysis
Western blotting analysis includes three major steps: 1) SDS-polyacrylamide gel
-59 - Chapter 2 Materials and Methods electrophoresis (SDS-PAGE), 2) Protein transfer by electroblotting, 3)
Immunodetection of target protein using specific antibody.
To perform SDS-PAGE, a gel was set first. The gel was made up of two parts: the separating gel that positioned at the lower region for separating proteins according to their molecular size; and the stacking gel that located at the upper region for sample loading and for the formation of highly concentrated protein bands.
The percentage of acrylamide in the separating gel was determined by the size of protein being examined. A protein with higher molecular weight needs a lower percentage of acrylamide gel and vice versa. The composition of the chemicals for setting the separating gels is summarized in Table 2.3. The protein sizes and their corresponding percentages of acrylamide used for separation are shown in Table 2.4.
-60 - Chapter 2 Materials and Methods
Table 2.3 Composition of the SDS-Polyacrylamide Gel
Stacking Gel (3 ml) Separating Gel (5 ml)
Gel percentage 5% 7.5% 10% 12% 15% Deionized Water 1.65 ml 2.4 ml 2 ml 1.65 ml 1.15 ml Lower Buffer — 1.25 ml 1.25 ml 1.25 ml 1.25 ml Upper Buffer 0.75 ml … --- … Acrylamide Stock 0.5 ml 1.25 ml 1.67 ml 2 ml 2.5 ml 10% SDS 30 ^il 25 ^il 25 ^il 25 |il 25 p,! 10% APS 4075 75 75 \i\ 75 \i\ TEMED 3 2 III 2^1 2 2 \i\
Table 2.4 Protein sizes and their corresponding percentages of acrylamide used for separation
Protein Molecular Mass Separating Gel (% Acrylamide) (kPa) — 12.5% 15% P-actin 42 " CDK-4 33 Z CDK-6 40 Z Bax 25 Z Bak 30 / Bcl-2 26 /
-61 - Chapter 2 Materials and Methods
The protein concentration of each sample was adjusted to 1 i^ig/jjl with lysis buffer based on the result from the Bradford protein assay. Twenty microlitres of protein samples (1 fig/^il) were mixed with 5 (j.1 protein loading dye (4x) to make up a total volume of 25 |JJL The mixed samples were boiled for 10 minutes and subjected to SDS-poly acrylamide gel with 5% stacking gel. Kaleidoscope prestained protein standards (8 jj-l) were loaded and the gel was run under constant voltage of 100 volts for about 2 hours.
Following electrophoresis, the stacking gel was removed by cutting with spacer and the protein containing separating gel was immersed into deionized water so as to wash away any SDS which might hinder protein transfer. After that, the washed gel
was soaked in transfer buffer (Ix) for 5 minutes. Before the electroblotting, the
PVDF membrane was initially activated with 100% methanol and then soaked in
transfer buffer (Ix) while six pieces of 3 mm filter papers were directly soaked in
transfer buffer (Ix). Three pieces of filter papers were taken out first and placed on
the clean surface of a semi-dry electroblotter (Bio-Rad Laboratories). The PVDF
membrane was then placed on the top of the filter papers. The separating gel was
carefully placed on the membrane for protein transfer and finally covered by another
3 pieces of wet filter papers. Air bubbles were excluded by rolling a glass tube on the
surface of wet filter papers and the surrounding buffer should be dried before
electroblotting. A voltage of 16 volts was applied for 30 minutes to force the proteins
to transfer from the gel to the PVDF membrane. After electroblotting, the membrane
was washed with washing buffer (Ix) for 10 minutes and then incubated with 5%
skimmed-milk at room temperature for at least 1 hour with rotation to block the
non-specific sites for probing. Primary antibody for the target protein was added into
the milk solution at a ratio of 1:1,000. After incubating with the primary antibody at
-62 - Chapter 2 Materials and Methods room temperature for an extra 1 hour, the membrane was washed with washing buffer (Ix) for 5 minutes for 3 times. Subsequently, the PVDF membrane was incubated with the corresponding horseradish peroxidase-linked secondary antibody at a 1:1，000 dilution in washing buffer for 1 hour at room temperature with rotation.
The membrane was then washed 3 times with washing buffer (Ix) and subjected to
ECL detection. d) ECL Detection
The Western blotting luminol reagents include reagent A and reagent B. An equal volume of reagent A and B was mixed freshly before ECL detection and a final volume of 0.125 mL/cm^ membrane was required. The mixture of reagent A and B was then poured onto the PVDF membrane. After removing the excessive solution, the membrane was sandwiched between transparent plastic sheets and an X-ray film was placed on top of the membrane in a dark room. The film cassette was closed for
different exposure time periods, ranging from 15 seconds to 5 minutes. Finally, the
film was developed and the protein bands were visualized on the film. The protein
size for the corresponding band w as determined by comparing with the protein
standards, so as to verify the identity of protein being detected. The intensity of the
bands was quantified by the ImageQuant software (Molecular Dynamics). After
normalization with respect to the house-keeping protein p-actin, the relative intensity
of each protein band was compared with that of the solvent control.
-63 - Chapter 2 Materials and Methods
2.2.9 Assessment of Differentiation-associated Characteristics
1) Morphological Detection of Cell Differentiation on Cytospin Preparation
The effect of Sophoraflavanone G on the differentiation of HL-60 cells was assessed by preparing cytospin smears. HL-60 cells (5x10"^ cells/ml) were either incubated with solvent control (0.13% ethanol) or different concentrations of
Sophoraflavanone G at 37°C for 3 days. The solvent-treated cells were harvested and washed twice, and finally resuspended in 1 ml cold plain RPMI medium. On the other hand, the Sophoraflavanone G-treated cells were allowed to stand at the room temperature for a while before harvesting the cells. This procedure gives a short cold
shock for the differentiated cells to detach from the flask. The harvested cells were then washed twice with cold plain RPMI medium and resuspended in 1 ml cold plain
RPMI medium. Cell morphology was then examined on the preparation of cytospin
smears. Harvested cells treated with either solvent or Sophoraflavanone were fixed
onto a clean microscopic glass slide by cyto-centrifugation at 500 rpm for 5 minutes
using the Shandon Cytospin 3 centrifuge (Shandon Scientific Ltd.). The cells were
air-dried and subsequently stained with Hemacolor staining solution in the order of
Solution 1, 2 and 3. Each step involved staining with Hemacolor staining solution for
15 seconds and followed by destaining with deionized water. Finally, the slides of
cytospin smears were mounted with a neutral mounting medium, Canada Balsam
(Sigma Chemical Co.) and the cell morphology was observed and examined under
the Axioskop Plus II light microscope (Carl Zeiss, Inc).
2) Functional Assessment of Differentiation by Nitroblue Tetrazolium (NBT)
Reduction Assay
When HL-60 cells are induced to differentiate along the granulocytic or
-64 - Chapter 2 Materials and Methods monocytic lineage, the conversion of oxygen to superoxide anions will be markedly increased upon exposure to phorbol 12-myristate 13-acetate (PMA) due to induction of respiratory burst activity (Collins, 1987). The production of superoxide anions can be monitored by the reduction of nitroblue tetrazolium (NBT). HL-60 cells (5x10"^ cells/ml) were incubated with different concentrations of Sophoraflavanone G at
37°C for 3 days. Then, NBT-PMA mix solution (100 ^1) was added into the cultures and co-incubated at 37°C for 4 hours. The cells were then harvested and washed twice with cold plain RPMI medium with vigorous shaking. Afterwards, the cells were fixed onto a clean microscopic glass slide by cyto-centrifugation at 500 rpm for
5 minutes and counter-stained with 1% safranin in methanol for 30 seconds and then gently washed with running water. The cells producing superoxide anions will be stained with dark blue deposits while the cells which fail to undergo functional differentiation will only be stained red (Newburger et al, 1979).
2.2.10 Statistical Analysis
Each experiment was performed at least two to three times and the results of
only one representative experiment were presented in the thesis. The data were
expressed as arithmetic mean 士 standard error (S.E.). Microsoft Excel program was
used to analyze the experimental data. The Student's t-test was used for
determination of the confidence limits in group comparison. Normally, P-value (p) <
0.05 was regarded as statistical significance.
-65 - Chapter Three
Studies on the Anti-proliferative Effect of Sophoraflavanone G on Human Myeloid Leukemia Cells Chapter 2 A nti-proliferative Effects
3.1 Introduction
Sophoraflavanone G was firstly reported by Shirataki and his colleagues in
1988. Earlier studies by other groups found that it was able to inhibit the growth of a variety of human tumor cell lines derived from lung (A549), ovary (SK-OV-3), skin
(SK-MEL-2), central nervous system (XF498), colorectal region (HCT-15), peripheral blood (HL-60) and liver (HepG2) (Ryu et al, 1997; Ko et al., 2000).
Recent studies from our laboratory showed that Sophoraflavanone G exhibited potent anti-tumor effect on human hepatoma HepG2 cells as well as its multidmg-resistant variant RHepG2 cells via induction of apoptosis. However, the modulatory effects of this compound on human myeloid leukemia cells and its possible anti-leukemic mechanisms remain poorly understood.
In this chapter, Sophoraflavanone G was examined for its modulatory effects on the proliferation and survival of human myeloid leukemia cells. Sophoraflavanone G is still commercially unavailable so the compound used in the present study was isolated from Sophora flavescens by my previous labmate Ms. Tsang Kit Man. At the beginning of this study, further characterization of this purified compound was carried out by Mass Spectrometry (MS), Nuclear Magnetic Resonance (^H-NMR and
13C-NMR) and High Performance Liquid Chromatography (HPLC).
The human promyelocytic leukemia HL-60 cells were selected as the major leukemia model in the present study. HL-60 cell line is a well-characterized and
subcloned leukemia cell model. Previous studies showed that HL-60 cells were able to differentiate into granulocytic and monocytic cells in response to different
inducers, like retinoic acid and vitamin D3 (Collins, 1987). Moreover, HL-60 cells
can form localized subcutaneous tumors in athymic (nude) mice (Gallagher et al,
-66 - Chapter 2 A nti-proliferative Effects
1979). In addition, Harker et al (1989) established an in vitro multidrug-resistant model HL-60/MX2 cell line, which was selectively developed from HL-60 cells. Due to these properties, HL-60 cells were chosen in the present study to investigate the anti-leukemic effect of Sophoraflavanone G on different aspects, including in vitro anti-proliferative activity, differentiation- and apoptosis-inducing properties, in vivo effect on tumorigenicity and in vitro multidrug resistant studies.
Initially, the anti-proliferative effect of Sophoraflavanone G was screened on different human myeloid leukemia cell lines, including the acute promyelocytic leukemia (HL-60, NB-4), eosinophilic leukemia (EoL-1) and chronic myelogenous leukemia (K-562). After the screening, HL-60 cells were chosen for the subsequent mechanistic studies. In addition to leukemia cells, the cytotoxic effect of
Sophoraflavanone G on normal cell models, including human peripheral blood leukocytes and murine bone marrow cells, was also investigated. The MTT reduction assay, ^H-thymidine incorporation assay and trypan blue exclusion assay were used to evaluate the anti-proliferative and cytotoxic effects of Sophoraflavanone G. Apart from the in vitro studies, the effect of Sophoraflavanone G on the in vivo tumorigenicity of HL-60 cells in xenogeneic inbred female BALB/c nude mice was
also examined. Moreover, attempts were made to examine the underlying
mechanisms for the in vitro anti-proliferative effect of Sophoraflavanone G on the
HL-60 cells. These include the study on cell cycle profile distribution and the
expression of the cell cycle regulatory proteins, using flow cytometry and Western
blot analysis respectively. Moreover, the development of multidrug resistance (MDR)
continues to be a major reason for the failure of chemotherapeutic agents in the
effective treatment against cancer (Ullah, 2008). Therefore, at the end of this chapter,
in order to mimic the situation of MDR in vitro, the anti-leukemic effect of
-67 - Chapter 3 Anti-proliferative Effects
Sophoraflavanone G on HL-60/MX2 cells, a well-characterized multidrug resistant leukemia cell model, was also investigated.
-68 - Chapter 2 A nti-proliferative Effects
3.2 Results
3.2.1 Structure Identification of Sophoraflavanone G Isolated from Sophora
flavescens
Compound 106-1 was successfully isolated from Sophora flavescens by the purification method described in Chapter Two (Section 2.2.1). The purity of the
Compound 106-1 was measured by High Performance Liquid Chromatography
(HPLC). The chemical structure of Compound 106-1 was further characterized by spectral analysis with Mass Spectrometry (MS) and Nuclear Magnetic Resonance
(IH-NMR and ^^C-NMR). The NMR measurement was done by Mr. C.C. Lee,
Department of Chemistry, The Chinese University of Hong Kong. The characterization and analysis of Compound 106-1 were done with the help of Mr.
P.M. Hon, Mr. Franky Choi and Dr. D.M. Zhang, Institute of Chinese Medicine, The
Chinese University of Hong Kong.
The purity of Compound 106-1 was estimated to be 93% as measured by HPLC.
It is a white crystal with melting point ranges from 176 to 178，UV nm: 295; EI MS m/z (rel. int): 424(20), 406(18)，301(60)，283(100), 219(20)，165(60), 69(50).
Spectroscopic data of 'H-NMR and ^^C-NMR spectra are summarized in Table 3.1.
By direct comparison of our spectral data with the published values (Shirataki et al,
1988), Compound 106-1 was identified as Sophoraflavanone G, i.e.
5,7,2',4'-tetrahydroxy-8-lavandulyl-flavanone (chemical formula: C25H28O6). The
established chemical structure of Compound 106-1 is shown in Figure 3.1.
(Unpublished data done by Mr. P.M. Hon)
-69 - Chapter 2 A nti-proliferative Effects
Table 3.1 NMR spectral data of Compound 106-1 in DMS0-d6
Position Compound 106-1 No. IH-NMR 13C-NMR 2 5.75，dd (13.5,2.7) 73.80 3 3.17，dd (13.5，2.7) 41.42 2.89，dd (16.8，2.7) 4 197.21 5 161.12 6 6.10，s 95.09 7 164.72 8 106.41 9 160.78 10 101.61 1' 115.84 2， 155.48 3’ 6.52, s 102.34 4’ 158.35 5， 6.54, d (7.8) 106.29 6’ 7.49，d (7.8) 127.61 1" 2.66, m;2.18,m 26.56 2" 2.78，m 46.35 3" 2.18, m; 1.81, m 30.74 4" 5.13,m 123.34 5" 130.66 6" 1.82, s 17.59 7" 1.76，s 25.49 8" 147.78 9" 1.66，s 110.80 10" 4.71, d (2.3); 18.56 4.77，d (2.3) Compound 106-1 (10 mg powder) was dissolved in 1 ml DMS0-d6 for ^H-NMR measurement. On the other hand, 35 mg powder of Compound 106-1 was dissolved in 1 ml DMS0-d6 for ^^C-NMR measurement. NMR measurement was done by Mr. C.C. LEE, Department of Chemistry, The Chinese University of Hong Kong. Remarks: s repr esents singlet; d represent doublet; dd represents double doublet; m represents multilet.
-70 - Chapter 2 A nti-proliferative Effects
10，， 9，，
6” 4” r 5，
‘ 2，
5| 4 OH C
Fig. 3.1 Chemical structure of the Compound 106-1 isolated from Sophora flavescens. Based on the spectroscopic data obtained from UV, MS and NMR spectra, the chemical structure of the Compound 106-1 was established and identified as Sophoraflavanone G, i.e. 5,7,2',4'-tetrahydroxy-8-lavandulyl-flavanone (chemical formula: C25H28O6； molecular weight: 424).
-71 - Chapter 2 A nti-proliferative Effects
3.2.2 Anti-proliferative Activity of Sophoraflavanone G on Various Myeloid
Leukemia Cell Lines
Initially four well-characterized human leukemia cell lines, including the eosinophilic leukemia EoL-1 cells, the acute promyelocytic leukemia HL-60 and
NB-4 cells, and chronic myelogenous leukemia K-562 cells, were chosen for examining the in vitro anti-proliferative effect of Sophoraflavanone G on myeloid leukemia using the MTT reduction test. The principle of this assay is that the MTT salt is metabolized into a colored water-insoluble formazan by the mitochondrial dehydrogenases in metabolically active cells. After it is solubilized, the amount of formazan formed, which reflects normal cell functioning, and hence cell proliferation
(Lau et al., 2004)，can be measured and quantified by the ELISA plate reader. The percentage of cell viability after treatment with Sophoraflavanone G can be calculated by comparing with the control cells. In the present study, EoL-1, HL-60,
K-562 and NB-4 cells were incubated with various concentrations of
Sophoraflavanone G for 48 hours. As shown in Figure 3.2, the percentages of cell viability in the four leukemia cell lines were decreased with increasing concentrations of Sophoraflavanone G. Table 3.2 summarized the estimated 50% inhibitory concentration (IC50) of Sophoraflavanone G on four leukemia cell lines.
As shown in Table 3.2, the IC50 values at 48 hours of incubation for the EoL-1,
HL-60, K-562 and NB-4 cells were very similar and were estimated to be around 20
pM.
Besides using the MTT reduction test, the anti-proliferative effects of
Sophoraflavanone G on the above four leukemia cell lines were also examined by the
tritiated thymidine ([^H]-TdR) proliferation assay, which is a sensitive method for
measuring the incorporation of ^H-labeled thymidine into the DNA of proliferating
-72 - Chapter 2 A nti-proliferative Effects cells. The mechanism behind this assay is that the uptake rate of ^H-TdR, a radioactive labeled DNA precursor, into the DNA of the cells is directly proportional to the rate of cell proliferation. By measuring the incorporated radioactivity of the cells treated with different concentrations of Sophoraflavanone G, the anti-proliferative effect of this compound can be determined. In this study, EoL-1,
HL-60, K-562 and NB-4 cells were incubated with various concentrations of
Sophoraflavanone G for 48 hours. Figure 3.3 shows that Sophoraflavanone G inhibited the proliferation of four human myeloid leukemia cells in a dose-dependent manner. As summarized in Table 3.3, the estimated IC50 values of Sophoraflavanone
G at 48 hours of incubation on four leukemia cell lines EoL-1, HL-60, K-562 and
NB-4 cells were 19 jiM，20 |j,M, 14|a,M and 14 p.M respectively, as determined by the
^H-TdR incorporation test.
-73 - Chapter 2 A nti-proliferative Effects
A. I ？。-� H \
I 40 - \
2�l
0 ‘ ‘ ‘ ‘ ‘"“I 0 10 20 30 40 50 Concentration of Sophoraflavanone G (^iM) B. 100
冬 80 -
•I 6� - \
I 40 _
20 - \j-
0 L L 1 1 ^ 7 0 10 20 30 40 50 Concentration of Sophoraflavanone G (^iM)
-74 - Chapter 2 A nti-proliferative Effects
c. 1
100
f 60. \ .2 40 - \
0 ‘ ‘ ‘ ‘ ‘ 0 10 20 30 40 50 Concentration of Sophoraflavanone G (pM)
D. 100 ?� • \
|6� ’ \ .2 40 - \
0 ‘ ‘ ‘ ‘ ‘ 0 10 20 30 40 50 Concentration of Sophoraflavanone G (|iM)
Fig. 3.2 Growth-inhibitory effect of Sophoraflavanone G on various myeloid leukemia cell lines as assayed by the MTT reduction test. EoL-1 (A), HL-60 (B) K-562 (C) and NB-4 (D) cells (1x10^ cells/ml) were either incubated with solvent control (0.2% ethanol) or treated with different concentrations of Sophoraflavanone G for 48 h at 37°C. After incubation, MTT reduction test was carried out as described in Chapter Two. Results were expressed as percentage of viability, using solvent-treated cells as a control. Each point represents the mean 士 S.E. of quadruplicate cultures.
-75 - Chapter 2 A nti-proliferative Effects
Table 3.2 The estimat ed IC50 values of Sophoraflavanone G on various types of leukemia cell lines at 48 hour incubation as assayed by the MTT reduction test
Leukemia cell line Estimated IC50 value at 48 h (^iM)
EoL-1 �19
HL-60 �20
K-562 �18
NB-4 ~ 20
IC50 value is the estimated concentration of Sophoraflavanone G that causes 50% inhibition on the in vitro growth of the leukemia cells under the specified experimental conditions.
-76 - Chapter 2 A nti-proliferative Effects
& r~ :100 - .2
I 80 - / ^
I 60 -
I 40 - ^^ |2�/ S 0 ^ ‘ ‘ 1 ^ 0 20 40 60 80 Concentration of Sophoraflavanone G (^xM)
B.冬 � -100 - ^^
I 0 80 - 1 / .S 60 - /
1 / 『/
2 0 ^ ‘ ' -__I 0 20 40 60 80
Concentration of Sophoraflavanone G (^M)
-77 - Chapter 2 A nti-proliferative Effects
£"d 100 - » I 8 � ^ jS 60 - /
S 40 - /
fl 20 - / 1 / 2 0 » ‘ ‘ ^ ^ 0 20 40 60 80 Concentration of Sophoraflavanone G (pM)
D-? [ ::zi:-iiz： g 100 - • “
% 80 h / I / I 60 - /
I 40 _ /
I 20 - J I 乂 % 0 ‘ ‘ ‘ 0 15 30 45 60 Concentration of Sophoraflavanone G (|JM)
Fig. 3.3 Anti-proliferative effect of Sophoraflavanone G on various myeloid leukemia cell lines as assayed by the [^H]-TdR incorporation test. EoL-1 (A), HL-60 (B) K-562 (C) and NB-4 (D) cells (1x10^ cells/ml) were either incubated with solvent control (0.3% ethanol) or treated with different concentrations of Sophoraflavanone G for 48 h at 37°C. After incubation, [^H]-TdR incorporation test was carried out as described in Chapter Two. Results were expressed as percentage inhibition of ^H-TdR incorporation, using solvent-treated cells as a control. Each point represents the mean 士 S.E. of quadruplicate cultures.
-78 - Chapter 2 A nti-proliferative Effects
Table 3.3 The estimated IC50 values of Sophoraflavanone G on various types of leukemia cell lines at 48 hour incubation as assayed by the [^H]-TdR incorporation test
Leukemia cell line Estimated IC50 value at 48 h (jiM)
EoL-1 �19
HL-60 ~ 20
K-562 �14
NB-4 �14
ICso value is the estimated concentration of Sophoraflavanone G that causes 50% inhibition on the in vitro growth of the leukemia cells under the specified experimental conditions.
-79 - Chapter 2 A nti-proliferative Effects
3.2.3 Effect of Sophoraflavanone G on the Viability of the Human Promyelocytic
Leukemia HL-60 Cells
In order to verify whether the observed anti-proliferative activity of
Sophoraflavanone G was due to its cytotoxic or cytostatic effect, the trypan blue exclusion assay was used to examine for its direct cytotoxicity. After treatment with
Sophoraflavanone G for 24，48 and 72 hours, viable HL-60 cells were counted by the trypan blue exclusion assay in which non-viable cells took up the dye and turned blue. Figure 3.4 shows that Sophoraflavanone G exerted minimal, if any, direct cytotoxicity on the HL-60 cells at the concentrations tested, and the percentage of viable cells remained over 85% after 72 hours of incubation with 23 jiM
Sophoraflavanone G. On the other hand, Figure 3.5 shows that in the presence of
Sophoraflavanone G, the actual numbers of viable HL-60 cells declined in a dose-
and time-dependent manner. The results indicate that the growth-inhibitory effect
Sophoraflavanone G on HL-60 cells could not be attributed to its direct cytotoxicity
on the leukemia cells.
-80 - Chapter 2 A nti-proliferative Effects
千 ：^
80 -
^ 60 -
ct > 40 - • Solvent control —•~~ 12 nM S ophoraflavanone G 20 • k 17 — S ophoraflavanone G >(23 fiM Sophoraflavanone G
0 ‘ ‘ 0 12 3 Time of incubation (day)
Fig. 3.4 Effect of Sophoraflavanone G on the viability of the human promyelocytic leukemia HL-60 cells as assayed by trypan blue exclusion test. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.13% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 h, 48 h and 72 h at 37°C. The viable and non-viable cell numbers were counted by trypan blue exclusion test using a hemocytometer. The percentage of cell viability was calculated as a ratio between the number of viable cells and the number of total cells. Results were expressed as mean percentage of viability 士 S.E. of triplicate cultures.
-81 - Chapter 2 A nti-proliferative Effects
8
—•— Solvent control -•— 5 |iM Sophoraflavanone G A 6 - -k-10 pM Sophoraflavanone G ® 20 一 Sophoraflavanone G y/
~~ ^
0 ‘ ‘ ‘
0 12 3 Time of incubation (day)
Fig. 3.5 Growth-inhibitory effect of Sophoraflavanone G on the human promyelocytic leukemia HL-60 cells as assayed by trypan blue exclusion test. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.13% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 h, 48 h and 72 h at 37°C. The number of viable cells was counted by trypan blue exclusion test using a hemocytometer. Results were expressed as mean percentage of viable cells 士 S.E. of triplicate cultures.
-82 - Chapter 2 A nti-proliferative Effects
3.2.4 Cytotoxic Effect of Sophoraflavanone G on Primary Normal Cells In Vitro
Apart from studying the anti-proliferative effect of Sophoraflavanone G on leukemia cells, in vitro cytotoxicity studies on normal hematopoietic cells were also conducted to evaluate the potential toxic effect of this compound on normal cells.
The human peripheral blood leukocytes and murine bone marrow cells, selected as the normal cell models in the study, were treated with various concentrations of
Sophoraflavanone G for 24 hours. The viability of the cells was measured by the
MTT assay. Figure 3.6 shows that at the concentration of 20 to 30 [iM of
Sophoraflavanone G which inhibited the growth of HL-60 leukemia cells by over
50%, only a slight cytotoxic effect (< 20% cytotoxicity) was observed on these two
normal cell types. However, around 40% cytotoxicity was observed in these normal
cells when they were incubated with a higher concentration (45 \iM) of
Sophoraflavanone G. Therefore, Sophoraflavanone G is relatively non-toxic to the
normal hematopoietic cells at concentrations which effectively inhibit the
proliferation of myeloid leukemia cells.
-83 - Chapter 2 A nti-proliferative Effects
A. — I 1��
^ 80 - ^^^^ I 6�
40 - 1> tg 20 -
pii 0 ‘ 1 L. 1
0 10 20 30 40 50
Concentration of Sophoraflavanone G (fiM) B . ? 100 ^^^^^^
I 80 I 6� - ^^^ I 40 -
tg 20 -
S tf 0 1 1 ) 1 0 10 20 30 40 50
Concentration of Sophoraflavanone G (|iM)
Fig. 3.6 Effect of Sophoraflavanone G on the viability of normal human peripheral blood leukocytes (PBL) and normal murine bone marrow cells as assayed by the MTT reduction test. Human peripheral blood leukocytes (PBL) were isolated from the peripheral blood of healthy donors. Murine bone marrow cells were prepared from female BALB/c mice. The isolation methods of these normal cells were described in Chapter Two. (A) Human PBL cells (1x10^ cells/ml) and (B) Murine bone marrow cells (2x10^ cells/ml) were either incubated with solvent control (0.2% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 h at 37°C. After treatment, MTT reduction test was carried out as described in Chapter Two. Results were expressed as percentage of relative viability, using solvent-treated cells as a control. Each point represents the mean 士 S.E. of quadruplicate cultures.
-84 - Chapter 2 A nti-proliferative Effects
3.2.5 Kinetic and Reversibility Studies of the Anti-proliferative Effect of
Sophoraflavanone G on the Human Promyelocytic Leukemia HL-60 Cells
The ^H-TdR incorporation assay was used for studying the kinetics of
Sophoraflavanone G on the human promyelocytic leukemia HL-60 cells. The HL-60 cells were cultured with various concentrations of Sophoraflavanone G at 37 °C for
24, 48 and 72 hours. Figure 3.7 shows the kinetic studies of the anti-proliferative
effect of Sophoraflavanone G on HL-60 cells. The results indicate that
Sophoraflavanone G inhibited the in vitro growth of HL-60 cells in a dose- and
time-dependent manner.
The above experiments demonstrated the growth-inhibitory effect of
Sophoraflavanone G on the HL-60 cells. A question was raised: is the inhibitory
effect reversible? To answer this question, the reversibility study was also performed
by the ^H-TdR incorporation test. HL-60 cells were firstly treated with either solvent
control (0.2% ethanol) or Sophoraflavanone G at 12.5 ^iM, 25 |iM and 50 for 12,
24，or 36 hours. After washing away the drug-containing medium, the cells were then
further incubated with drug-free fresh complete medium up to a total of 48 hours. As
a positive control, HL-60 cells were incubated for 48 hours in the presence of
Sophoraflavanone G. Figure 3.8 indicates that the reversibility of the
anti-proliferative effect on HL-60 cells was correlated with the duration of drug
exposure and the concentration of drug used. The results show that the
growth-inhibitory effect of Sophoraflavanone G was less than 40% after 12 hours
and 24 hours of drug exposure at lower concentrations (12.5 |iM and 25 jiM),
whereas more than 50% inhibition was seen at 50 ^iM even after 12 hours of drug
exposure. It is clear that the growth-inhibitory effect of Sophoraflavanone G was
partially reversible at lower concentrations (12.5 ^iM and 25 |xM) and became almost
-85 - Chapter 3 Anti-proliferative Effects irreversible when a higher concentration (50 |xM) of drug was used.
？ 100 rTHHi __• ^
o 0 ^ ^ ‘ ‘ ‘ ‘ ‘ ‘—— 1P ly 20 30 40 50 60 70 t -25 ——^ ^ Concentration of Sophoraflavanone G (|iM)
Fig. 3.7 Kinetic studies on the anti-proliferative effect of Sophoraflavanone G on the human promyelocytic leukemia HL-60 cells as assayed by the [^H]-TdR incorporation test. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.3% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 h, 48 h and 72 h at 37°C. After treatment, ^H-TdR incorporation test was carried out as described in Chapter Two. Results were expressed as percentage inhibition of ^H-TdR incorporation, using solvent-treated cells as a control. Each point represents the mean 士 S.E. of quadruplicate cultures.
-86 - Chapter 2 A nti-proliferative Effects
？ 本 w • 12hr 0 24hr 0 36hr • 48hr i 1 0 * * • .•5 I 1 « 100 - I * * �I [—I 1 so - m •I — n I T3 gQ * • * T ； ； ；； to- ^ TJ m I-J1 il III 12.5 25 50
Concentration of Sophoraflavanone G (^iM) Fig. 3.8 Reversibility study on the growth-inhibitory effect of Sophoraflavanone G on the human promyelocytic leukemia HL-60 cells. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.2% ethanol) or treated with four different concentrations of Sophoraflavanone G at 37°C. Drugs were removed and replaced by fresh culture medium at different time periods (16 h, 24 h or 36 h after drug addition) and all cultures were incubated up to 48 hours after initial drug addition. The growth-inhibitory effect was then measured by ^H-TdR incorporation test as described in Chapter Two. Results were expressed as percentage inhibition of ^H-TdR incorporation, using solvent-treated cells as a control. Each point represents the mean 士 S.E. of quadruplicate cultures. Remarks: * p<0.05, ** p<0.005, *** p<0.001, significantly different from corresponding 48h-treatment samples.
-87 - Chapter 2 A nti-proliferative Effects
3.2.6 Effect of Sophoraflavanone G on the In Vivo Tumorigenicity of the HL-60 Cells
In addition to the in vitro studies, animal experiments were performed to investigate the anti-proliferative effect of Sophoraflavanone G on HL-60 cells in vivo.
HL-60 cells were incubated with 30 |LIM Sophoraflavanone G for 16 hours at 37°C.
The cells were harvested, washed and 1x10^ viable cells (in 100 |il PBS) were injected subcutaneously into the back of each BALB/c nude mouse in groups of twenty. The tumor weight was measured at the end of experiments, and the tumor volume was measured twice weekly with a vernier caliper up to one month. As shown in Figure 3.9, pretreatment of the leukemia HL-60 cells in vitro with
Sophoraflavanone G markedly reduced the in vivo tumorigenicity of the HL-60 cells in the human tumor xenograft model.
-88 - Chapter 2 A nti-proliferative Effects
A.
• solvent control ^ 2000 • 30^M of Sophoraflavanone G jJL|
T 1500 ^ • I
11�� � r^ i h “� ^ ^ m I I 0 ^^ I , m^ I m^ , • I I • I , • I
16 19 22 27 30 33 36
Days after tumor implantation B. C. 40 O 25 OJD • solvent control t • solvent control R 30 - DBOHM of Sophoraflavanone G fe ^ " • 30一 of Sophoraflavanone G
蓋一 "I
Fig. 3.9 Effect of in vitro pretreatment of the human promyelocytic leukemia HL-60 cells with Sophoraflavanone G on their tumorigenicity in BALB/c nude mice. (A) Size of tumor (expressed as tumor volume); (B) Body weight on Day 36 (last day for the experiment) after tumor implantation; (C) Percentage of final tumor to body weight on Day 36. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.13% ethanol) or treated with 30 \iM Sophoraflavanone G for 16 h at 37°C. The cells were washed once with PBS. Viable HL-60 cells (1x10^ cells in 100 |il PBS) were injected subcutaneously on the back of each nude mouse in groups of twenty. The tumor weight was measured at the end of the experiment, and the tumor volume was measured twice weekly with a vernier caliper. Tumor volume was calculated according to the formula [volume = height x length x width x 71/6] • Remarks: 1. Sample size for graph B & C (n = 8) is different from graph A (n = 20). At the end of the experiment, eight mice were randomly chosen from each of solvent control group and drug-pretreated group, sixteen mice in total. Their bearing tumors were dissected. The body weight of the mice and the weight of the dissected tumor were measured. 2. * p<0.05, **p<0.005, significantly different from solvent control.
-89 - Chapter 2 A nti-proliferative Effects
3.2.7 Effect of Sophoraflavanone G on the Cell Cycle Profile of the HL-60 cells
In Vitro
Previous results as shown in Figure 3.5 indicate that the doubling time of HL-60 cells was increased with increasing concentrations of Sophoraflavanone G， suggesting that Sophoraflavanone G may regulate the cell cycle progression. In the present study, the modulatory effect of Sophoraflavanone G on the cell cycle profile of the HL-60 cells was examined by propidium iodide (PI) staining followed by flow
cytometric analysis.
Cellular division, or cell cycling, is composed of four sequential phases: Gi, S,
G2 and M phases. After preparation for DNA synthesis in Gi phase, the diploid cells
enter S phase and start to replicate the DNA content. When the DNA content is
doubled, the cells reach G2 phase and prepare for entering mitosis (M phase).
Therefore, measurement of DNA contents in the cells can give a picture of the cell
cycle. Propidium iodide (PI), a DNA staining fluorescent dye, can bind to DNA by
intercalating into double-stranded nucleic acids of permeabilized cells without any
sequence preference. Once the dye is bound to DNA, its fluorescence is enhanced
20- to 30-fold. The intensity of emitted fluorescence, which represents the DNA
contents of the cells, can be measured by flow cytometry.
In the present study, HL-60 cells were treated with Sophoraflavanone G at 20
p-M and 30 \iM for 24 hours. The results of cell cycle profiles are shown in Figure
3.10. The percentage distribution of each phase is summarized in the lower panel
of Figure 3.10, which indicated that Sophoraflavanone G increased the proportion of
HL-60 cells in the GQ/GI phase while decreased the percentage of treated cells in the
S phase. It can be seen that in solvent-treated HL-60 cells, -43% cells were in the S
phase and -47% cells were in GQ/GI phase. In contrast, the majority (-75%) of
-90 - Chapter 2 A nti-proliferative Effects
Sophoraflavanone G-treated (30 |iM) HL-60 cells were arrested in the GQ/GI phase.
Therefore, Sophoraflavanone G might exert its anti-proliferative effect on HL-60 cells through triggering cell cycle arrest at the GQ/GI phase.
-91 - Chapter 2 A nti-proliferative Effects
ijuii^，Lu
Concentration of Cell cycle distribution (%) Sophoraflavanone G GQ/GI phase S phase G2/M phase Control 46.77 % 42.59 % 10.64% 20 nM 69.05 % 22.19% 8.76 %
30>IM 74.46% 15.04% 10.49%
Fig. 3.10 Cell cycle profile of HL-60 cells after the treatment with Sophoraflavanone G for 24 hours. HL-60 cells (1x10^ cells/ml) were synchronized by starving in RPMI medium containing 0.5% serum for 12 hours and were incubated with (A) solvent control (0.13% ethanol); (B) 20 \iM of Sophoraflavanone G; and (C) 30 |LIM of Sophoraflavanone G for another 24 hours. The control and Sophoraflavanone G-treated cells were fixed by ethanol and stained by PI staining solution. The PI stained samples were analyzed by flow cytometry to obtain cell cycle profile. Cell cycle distribution was calculated by the MODFIT program. The cell cycle profiles are shown in the upper panel. The table in the lower panel shows the percentage distribution of GQ/GI, S and G2/M phases of the cell cycle.
-92 - Chapter 2 A nti-proliferative Effects
3.2.8 Effect of Sophoraflavanone G on the Expression of Cell Cycle-regulatory
Proteins in the HL-60 Cells
Cell cycle progression is highly regulated by a network of cell cycle regulatory protein complexes, which include cyclins and cyclin-dependent kinases (CDK). In
Section 3.2.7, the results show that Sophoraflavanone G could induce GQ/GI phase
arrest in the HL-60 cells. It is of interest to find out how Sophoraflavanone G could
affect the expression of the cell cycle-regulatory proteins that might contribute to
GQ/GI arrest. The activated cyclin/CDK complex can phosphorylate the
retinoblastoma protein (Rb). After phosphorylation, Rb will release the
transcriptional factor E2F which will further promote the progression from G1 to S
phase. The main cyclin/CDK complexes responsible for Gi/S progression are cyclin
A/CDK-1 or -2，cyclin D/CDK-4 or -6，and cyclin E/CDK-2 (Shapiro, 2006). In the
present study, the protein expression of cyclin-dependent kinases CDK-4 and CDK-6
was studied by Western blot analysis. HL-60 cells (4x10^) were treated with various
concentrations of Sophoraflavanone G for 24 hours. The cells were then harvested
and the total cell lysate was isolated and subjected to Western blot analysis. As
shown in Figure 3.11, Sophoraflavanone G slightly down-regulated the expression of
CDK-4 and CDK-6 after 24 hours of treatment.
-93 - Chapter 2 A nti-proliferative Effects
Concentrations of Sophoraflavanone G
CDK-4 __ •書 33 kDa
CDK-6 mmm ^mmmm 麵�峰 40 kDa
-actin ."*^^^H!iiiw*iHPPPP.:*iH|iiiiP�^IBilP^ : ^^ Kua
Relative Band Intensity Control 10 ^iM 20 ^iM 40 ]iM CDK-4 ^ ^ ^ CDK-6 ^ ^ 0.66
Figure 3.11 Effect of Sophoraflavanone G on the expression of cyclin-dependent kinase (CDK) in HL-60 cells. HL-60 cells (2x10^) were serum starved for 24 h and then treated with Sophoraflavanone G (0 - 40 ^M) for another 24 h. Proteins were extracted from total cell lysate and separated by 12.5% SDS gel. After that, Western blot analysis was performed as described in Chapter Two. The relative band intensities, shown in the bottom table, were calculated after normalization with respect to p-actin, and comparison was made with the corresponding control.
-94 - Chapter 2 A nti-proliferative Effects
3.2.9 Anti-proliferative Effect of Sophoraflavanone G on Multidrug-resistant
(MDR) Leukemia Cell Line HL-60/MX2 Cells
Multidrug resistance (MDR) is the defence of tumor cells against many
chemotherapeutic drugs with different structures and action mechanisms. MDR has
been recognized as a major cause for the failure of cancer chemotherapy. The
mechanisms of cellular MDR may be classified as classical (transport-dependent)
and non-classical (transport-independent) mechanisms (Ullah, 2008). The
transport-dependent mechanism involves the efflux of different drugs from the cell
by various energy-dependent membrane transport proteins, such as P-glycoprotein,
therefore decreasing drug accumulation inside the cell. The mechanisms of
transport-independent may include a) activation of the enzymes of the glutathione
detoxification system; b) alteration of drug targets or by enhancement of target repair,
such as reduction in the activity of topoisomerase II and c) alteration of the genes and
the proteins in the regulation of apoptosis, such as p53 and Bcl-2 (Ullah, 2008;
Stavrovskaya, 2000).
Previous studies in our laboratory on human hepatoma cells showed that the
resistant RHepG2 cells with P-glycoprotein over-expression were still sensitive to
Sophoraflavanone G (Tsang, 2005). In the present study, we further compared the
anti-proliferative effect on the sensitive HL-60 cells with that of the resistant
HL-60/MX2 cells (in the absence of P-glycoprotein over-expression).
HL-60/MX2 is a multidrug-resistant variant of HL-60 cells. MX here stands for
mitoxantrone resistance. Apart from mitoxantrone, it was also found that
HL-60/MX2 is cross-resistant to several anti-cancer agents, including doxorubicin,
etoposide, teniposide and amsacrine. The resistance is not due to P-glycoprotein but
caused by reduced expression of DNA topoisomerase II p isoform.
-95 - Chapter 2 A nti-proliferative Effects
The anti-proliferative effect of Sophoraflavanone G on HL-60/MX2 was examined by the MTT reduction test (Figure 3.12) and [^H]-TdR proliferation assay
(Figure 3.13). Kinetic studies on anti-proliferative effect of Sophoraflavanone G were also measured by the [^H]-TdR proliferation assay (Figure 3.14). The results clearly show that Sophoraflavanone G exhibited potent anti-proliferative effect on this resistant leukemia cell line HL-60/MX2 in a time- and dose-dependent manner.
The estimated IC50 value is around 8 |iM as determined by the [^H]-TdR proliferation assay (Figure 3.13).
Cell cycle profile analysis on the Sophoraflavanone G-treated HL-60/MX2 cells was also performed. The results were similar to those obtained from its parental cell line HL-60 cells. As shown in Figure 3.15, Sophoraflavanone G also triggered cell cycle arrest in HL-60/MX2 cells at GQ/GI phase.
-96 - Chapter 3 A nti-proliferative Effects
100 ！;-
h 60 I- \ I V I 40 -
2�
0 ‘ ‘ ‘
0 10 20 30 40 Concentration of Sophoraflavanone G (jiM) Fig. 3.12 Growth-inhibitory of Sophoraflavanone G on the multidrug-resistant human promyelocytic leukemia HL-60/MX2 cells as assayed by the MTT reduction test. HL-60/MX2 cells (1x10^ cells/ml) were either incubated with solvent control (0.17% ethanol) or treated with different concentrations of Sophoraflavanone G for 48 h at 37°C. After incubation, MTT reduction test was carried out as described in Chapter Two. Results were expressed as percentage of viability, using solvent-treated cells as a control. Each point represents the mean 士 S.E. of quadruplicate cultures.
-97- Chapter 3 Anti-proliferative Effects
？ 1�� [ ^ ^ H厂 • 6� - / 夢 40 — /
！ 20 7 I -/ . • . . — Q ^ I I 1 I 1
0 10 20 30 40 Concentration of Sophoraflavanone G (|JM)
Fig. 3.13 Anti-proliferative effect of Sophoraflavanone G on the multidrug-resistant human promyelocytic leukemia HL-60/MX2 cells as assayed by the [^H]-TdR incorporation test. HL-60/MX2 cells (1x10^ cells/ml) were either incubated with solvent control (0.17% ethanol) or treated with different concentrations of Sophoraflavanone G for 48 h at 37°C. The growth-inhibitory effect was then measured by ^H-TdR incorporation test as described in Chapter Two. Results were expressed as percentage inhibition of ^H-TdR incorporation, using solvent-treated cells as a control. Each point represents the mean 士 S.E. of quadruplicate cultures.
-98- Chapter 2 A nti-proliferative Effects
I 1 J^^^ I I： / I 40 /y I JJ i 20 - /W "••~48hr
0 10 20 30 40 Concentration of Sophoraflavanone G (|iM)
Fig. 3.14 Kinetic studies on the anti-proliferative effect of Sophoraflavanone G on the multidrug-resistant human promyelocytic leukemia HL-60/MX2 cells as assayed by the [^H]-TdR incorporation test. HL-60/MX2 cells (1x10^ cells/ml) were either incubated with solvent control (0.17% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 h, 48 h and 72 h at 37�C .The growth-inhibitory effect was then measured by ^H-TdR incorporation test as described in Chapter Two. Results were expressed as percentage inhibition of ^H-TdR incorporation, using solvent-treated cells as a control. Each point represents the mean 土 S.E. of quadruplicate cultures.
-99 - Chapter 3 A nti-prol iferative Effects
A A. iC.
1:; I i:j I N i
60 100 ISO < SO 1&0 ite 30 、{jo ISO Ch«nn®l» (PE-A) Channals (PE-A) “ Ctivirwt* (PE^)
Concentration of Cell cycle distribution (%) Sophoraflavanone G GQ/GI phase S phase Gj/M phase Control 52.25 % 33.62 % 14.13 % 5jiM 64.58 % 23.56 % 11.86% 10 nM 71.06% 18.84% 10.10%
Fig. 3.15 Cell cycle profile of multidrug-resistant human promyelocytic leukemia HL-60/MX2 cells after treatment with Sophoraflavanone G for 24 hours. HL-60/MX2 cells (1x10^ cells/ml) were synchronized by starving in RPMI medium containing 0.5% serum for 12 hours and were incubated with (A) solvent control (0.04% ethanol); (B) 5 [iM of Sophoraflavanone G; and (C) 10 ^iM of Sophoraflavanone G for another 24 hours. The control and Sophoraflavanone G-treated cells were fixed by ethanol and stained by PI staining solution. The PI stained samples were analyzed by flow cytometry to obtain the cell cycle profile. Cell cycle distribution was calculated by the MODFIT program. The cell cycle profiles are shown in the upper panel. The table in the lower panel shows the percentage distribution of GQ/GI, S and G2/M phases of the cell cycle.
-100 - Chapter 2 A nti-proliferative Effects
3.3 Discussion
In this Chapter, the chemical structure and purity of Sophoraflavanone G were
first identified. Subsequently, Sophoraflavanone G was further examined for its
anti-proliferative effect against various human myeloid leukemia cell lines.
Sophoraflavanone G used in the present study was isolated from Kushen by our
previous labmate Ms. Tsang Kit Man with the collaboration of the Institute of
Chinese Medicine, The Chinese University of Hong Kong. No further purification
was carried out. By direct comparison of our spectral data (MS, ^H-NMR, and
^^C-NMR) with the published values (Shirataki et al, 1988), the isolated compound,
with the purity of 93% as measured by HPLC, was identified as Sophoraflavanone G,
i.e. 5,7,2',4'-tetrahydroxy-8-lavandulyl-flavanone (chemical formula: C25H28O6). It
has been reported that many synthetic and naturally occurring flavonoids can exhibit
anti-proliferative effect in human cancer cell lines and the cytotoxic potency of
flavonoids is correlated with the position, the number of hydroxyl groups, and the
structure of side chains (Kuntz et al” 1999). Sophoraflavanone G is a flavanone with
one lavandulyl side chain and four hydroxyl groups. In particular, the lavandulyl side
chain, as suggested by accumulating evidences from structure-activity relationship
(SAR) studies, is essential for the pleiotropic bioactivities of lavandulylflavonoids,
such as in vitro growth-inhibitory activity on tumor cells, tyrosinase-inhibitory and
cyclooxygenase-inhibitory activities (Ko et al., 2000; Chi et a/.,2001; Son et al.,
2003). However, the modulatory effects of Sophoraflavanone G on human myeloid
leukemia cells and its possible anti-leukemic mechanisms remain poorly understood.
In the present study, the anti-proliferative effect of Sophoraflavanone G on
human myeloid leukemia cells was investigated. Using the MTT reduction test,
-101 - Chapter 3 A nti-prol iferative Effects
Sophoraflavanone G was found to inhibit the growth of human acute promyelocytic leukemia HL-60 cells in a dose-dependent manner, with an estimated IC50 value of approximately 20 |J.M following 48 hours of treatment. Similar growth-inhibitory effect of Sophoraflavanone G was also shown on three other human myeloid leukemia cell lines, including the acute promyelocytic leukemia NB-4 cells, eosinophilic leukemia EoL-1 cells, and chronic myelogenous leukemia K-562 cells.
The results from ^H-thymidine incorporation test further confirmed the anti-proliferative ability of Sophoraflavanone G on these four human myeloid
leukemia cell lines. Our results are in line with earlier studies done by other
researchers. In the study by Ko et al. (2000), the IC50 value of Sophoraflavanone G
on HL-60 cells after 96 hours of treatment was reported to be 12.5 fiM, which is
comparable to the results obtained in our present study (20 |JM after 48 hours of
treatment). Similarly, Sophoraflavanone G was found to exhibit the anti-proliferative
effect on the human skin melanoma SK-MEL-2 cells, human colorectal carcinoma
HCT-15 cells, human lung carcinoma A549 cells, and human ovarian cancer
SK-OV-3 cells with IC50 values of 3.9，5.7, 6.4 and 7.9 |iM respectively (Ryu et al,
1997). In addition, certain members of lavandulylflavonoids, such as Kurarinone,
Leachianone A, and 2 ‘ -methoxykurarinone, have been shown to inhibit the growth of
human hepatoma HepG2 cells and human breast cancer MCF-7/6 cells, with
estimated IC50 values ranged from 11.3 to 22.2 (Ko et al, 2000; De Naeyer et aL,
2004). These findings further support the important role of lavandulyl side chain in
exerting the anti-proliferative activity on human tumor cells.
Kinetic studies of the anti-proliferative effect showed that Sophoraflavanone G
was able to inhibit the proliferation of HL-60 cells in a time- and dose-dependent
manner. The estimated IC50 values after 24，48，and 72 hours of treatment with
-102- Chapter 3 A nti-prol iferative Effects
Sophoraflavanone G on HL-60 cells were found to be 26, 20 and 17 }iM respectively.
Interestingly, such anti-proliferative effect was partially reversible when the HL-60 cells were exposed to Sophoraflavanone G at lower concentrations (12.5 and 25 )iM) and for shorter incubation periods (12 and 24 hours), although this effect was almost irreversible when a higher concentration (50 jiM) of drug was used. These results indicate that Sophoraflavanone G may exert its anti-proliferative effect on HL-60
cells within the first 24 hours and that longer duration of treatment would result in
irreversible changes.
In order to distinguish whether the observed growth-inhibitory activity of
Sophoraflavanone G on the leukemia HL-60 cells was due to its cytotoxic or
cytostatic effect, the trypan blue exclusion assay was carried out to test for its direct
cytotoxicity on the leukemia cells. Our results show that Sophoraflavanone G exerted
no significant cytotoxic effect on HL-60 cells at its growth-inhibitory concentrations,
suggesting that the anti-proliferative effect of Sophoraflavanone G on HL-60 cells
could not be solely attributed to its direct cytotoxicity. Moreover, from the results of
the growth curve of Sophoraflavanone G-treated HL-60 cells, it can be seen that the
doubling time of drug-treated HL-60 cells was increased with increasing
concentrations of Sophoraflavanone G, implying that Sophoraflavanone G may
modulate the cell cycle progression of the treated HL-60 cells.
This speculation was further confirmed by cell cycle profile analysis. The results
demonstrated that Sophoraflavanone G could increase the percentage of HL-60 cells
in the GQ/GI phase dose-dependently which was accompanied by a corresponding
decrease in the percentage of treated cells in the S phase. The percentage of cell
population in S phase reflects the proportion of proliferating cells. Figure 3.10
demonstrated that the cell cycle distribution in S phase at 30 p,M of
-103 - Chapter 3 A nti-prol iferative Effects
Sophoraflavanone G was ~ 35% of the solvent-treated cells in S phase. As shown in
Figure 3.7，kinetic studies illustrated that 30 pM of Sophoraflavanone G can inhibit approximate 70% of cell growth following 24 hours of incubation. Therefore, our results of cell cycle redistribution are in line with our previous anti-proliferative study as measured by the [^H]-TdR incorporation test. Until now, the ability of
Sophoraflavanone G to induce cell cycle arrest in human myeloid leukemia cells, as well as the underlying action mechanisms, have not yet been reported. Progression through the cell cycle is governed by a network of cell cycle regulatory protein
complexes, which include cyclins and cyclin-dependent kinases (CDK). The main
cyclin/CDK complexes responsible for Gi/S progression are cyclin A/CDK-1 or -2，
cyclin D/CDK-4 or -6, and cyclin E/CDK-2. The activated cyclin/CDK complexes
can phosphorylate the retinoblastoma protein (Rb). After phosphorylation, Rb will
release the transcriptional factor E2F, which will further promote the progression
from Gi to S phase (Shapiro, 2006). In the present study, our results show that
Sophoraflavanone G can down-regulate the expression of CDK-6 after 24 hours of
treatment which may affect the assembling of cyclin D/CDK-6 complex and thus
activate the Gi checkpoint arrest. Interestingly, Kim et al (2003) reported that the
aqueous extracts of Sophora flavescens can increase the in vitro proliferation of
normal human oral mucosal fibroblasts through induction of cyclin E and CDK-2.
Nonetheless, detailed information regarding the effects of Sophoraflavanone G on
the cell cycle regulatory proteins are still lacking. Therefore, more intensive
molecular studies on other cyclin/CDK complexes and their corresponding CDK
inhibitors should be carried out so as to reveal the underlying mechanisms of cell
cycle arrest.
-104- Chapter 3 A nti-prol iferative Effects
Since Sophoraflavanone G was found to exhibit anti-proliferative effect and triggered cell cycle arrest at GQ/GI phase in the HL-60 cells, the suppressive effect of
Sophoraflavanone G on the tumorigenicity of the HL-60 cells in xenogeneic BALB/c
nude mice was also examined. The results show that pretreatment of HL-60 cells
with Sophoraflavanone G in vitro was able to reduce the in vivo tumorigenicity of the
subcutaneous-injected leukemia cells in the nude mice. On the other hand, Sun et al
(2007) reported that KS-Fs, an extract of flavonoid mixture isolated from Kushen, in
combination with Taxol was able to suppress the tumor growth of human lung cancer
H460 and esophageal cancer Eca-109 xenograft models in vivo. This flavonoids
mixture KS-Fs was identified to be rich in lavandulyIflavonoids. Moreover,
Leachianone A, another lavandulylflavonoid isolated from Kushen, was shown to
inhibit the tumor growth of HepG2 cells in vivo after intravenous injection (Cheung
et al,’ 2007). Nevertheless, the in vivo anti-leukemic activity of Sophoraflavanone G,
which can be administered orally or intravenously into tumor-bearing mice, still
awaits further investigation using the human HL-60 xenografted tumor model.
On the other hand, a major problem encountered in conventional cancer
chemotherapy is the adverse side effects due to lack of selective anti-tumor activity
and broad-spectrum toxicity to normal tissues (Tsiftsoglou et al, 2003). One of the
toxic effects of anti-leukemic chemotherapeutic agents is myelosuppression. In the
present study, Sophoraflavanone G showed minimal, if any, in vitro cytotoxic effect
on human peripheral leukocytes and murine bone marrow cells at concentrations
which inhibit the growth of HL-60 leukemia cells by -50%. There is still lack of
information on the toxicity studies of Sophoraflavanone G in animal models and on
other types of normal cells, although it has been reported that the extract of Kushen
could be processed as an injection for clinical usage (Ren and Zhang, 2008). Kim et
-105- Chapter 3 A nti-prol iferative Effects al (2003) also reported that the aqueous extracts of Sophora flavescens can enhance the in vitro proliferation of normal human oral mucosal fibroblasts. In addition, by measuring the activities of specific enzymes for heart and liver such as creatine
kinase, alanine transaminase and aspartate transaminase, Cheung et al (2007)
showed that Leachianone A, a lavandulylflavonoid also isolated from Kushen, did
not produce apparent toxicity to the nude mice at dose of 30 mg/kg/day. In contrast,
Sophoraflavanone G was also observed to demonstrate in vitro growth-inhibitory
effect on some fast-dividing normal cells, such as human hepatocyte-like WRL-68
cells (Tsang, 2006) and human foreskin fibroblast Hs-68 cells (data not shown).
Therefore, more studies should be carried out so as to clarify its possible toxic side
effects. For example, the in vitro cytotoxicity of Sophoraflavanone G on different
types of normal cells should also be investigated. In addition, toxicity assessment of
Sophoraflavanone G on animal models, such as measurement of the serum levels of
the tissue-specific enzyme activities for heart, liver and kidney etc., should be
performed to elucidate the toxicity of Sophoraflavanone G on normal tissues in vivo.
Another major cause for the failure of cancer chemotherapy is development of
multidrug resistance (MDR). MDR is the self-protection of tumor cells against many
chemotherapeutic drugs. Over-expression of P-glycoprotein, an energy-dependent
multidrug efflux pump, is one of the well-characterized mechanisms for MDR
development (Tsiftsoglou et al, 2003). Previous studies in our laboratory on human
hepatoma cells had demonstrated that the resistant RHepG2 cells with
over-expression P-glycoprotein were still sensitive to Sophoraflavanone G, which is
similar to its parental cell line HepG2 (Tsang, 2006). Apart from over-expression of
energy-dependent membrane transport proteins, alteration of the biochemical activity
of drug targets is another mechanism resulting in MDR (Stavrovskaya, 2000).
-106- Chapter 3 A nti-prol iferative Effects
Topoisomerase is an essential nuclear enzyme involved in DNA replication, thus it is an important target for many of the clinically used anti-cancer agents, such as mitoxantrone, doxorubicin and etoposide (Harker et al, 1995). By reducing the expression of DNA topoisomerase II p isoform, multidrug-resistant leukemia
HL-60/MX2 cells are cross-resistant to the topoisomerase II poisons as shown in
Table 3.4. In the present study, HL-60/MX2 cells, together with HL-60 cells, were used to assess the anti-proliferative effect of Sophoraflavanone G and its modulatory effect on the cell cycle progression. Surprisingly, similar to its effects on HL-60 cells,
Sophoraflavanone G was found to be able to inhibit the growth of HL-60/MX2 cells in a dose- and time-dependent manner, as well as triggering cell cycle arrest at the
GQ/GI phase. The susceptibility of HL-60/MX2 to Sophoraflavanone G suggests that topoisomerase II may not represent the essential cellular target for Sophoraflavanone
G. Alternatively, it is also possible that the expression level of topoisomerase II p isoform may affect the action(s) of Sophoraflavanone G on leukemia cell lines because leukemia cell line HL-60/MX2 with reducd expression of topoisomerase II p isoform was found to be more sensitive to Sophoraflavanone G when compared to leukemia cell lines with normal expression of topoisomerase II p isoform (e.g. EoL-1,
HL-60, K-562 and NB-4 cells).
Our results, when taken together, suggest that Sophoraflavanone G can exhibit
anti-proliferative effect on various human myeloid leukemia cell lines. Further
investigation on HL-60 cells demonstrated that induction of cell cycle arrest and
down-regulation of CDK-6 expression could be one of the possible mechanisms
contributing to their anti-proliferative activity. Other possible mechanisms, such as
induction of apoptosis and differentiation of the leukemia cells would be addressed
in Chapter Four.
-107- Chapter 3 A nti-prol iferative Effects
Table 3.4 Cross-resistant patterns of HL-60/MX2 cells comparing with its parental cell line HL-60 cells
Drug Degree of resistance®
Mitoxantrone 35^'
Etoposide 1
Doxorubicin
Vincristine
Sophoraflavanone G 0.4 a Expressed as a ratio of the drug concentration (HL-60/MX2 : HL-60) inhibiting cell growth by 50% (IC50) as measured by the MTT reduction assay, b Adapted from Harker et al. (1991)，Table I，p. 9955.
-108- Chapter Four
Studies on the Apoptosis- and Differentiation-inducing Activities of Sophoraflavanone G on Human Myeloid Leukemia Cells Chapter 4 Apoptosis- and Differentiation-inducing Effects
4.1 Introduction
In Chapter Three, the anti-proliferative effect of Sophoraflavanone G on various human myeloid leukemia cells was examined. The results demonstrated that
Sophoraflavanone G can inhibit the growth of various human myeloid leukemia cell lines in vitro and down-regulate the expression of CDK-6 in the HL-60 cells which may further trigger cell cycle progression arrest at GQ/GI phase. These findings are in line with a previous report investigating the anti-proliferative effect of
Sophoraflavanone G on various human tumor cell lines, such as A549, SK-OV-3, and
SK-MEL-2 (Ryu et al, 1997).
Apart from proliferating independently of growth signals, tumor cells are also characterized as capable of avoiding appropriate cell death (Roderick and Cook,
2008). Therefore, it is of interest to further investigate whether Sophoraflavanone G can lead to cell death in the treated leukemia cells. Cell death is a vital cellular process which may include up to 11 proposed pathways occurring in mammals
(Melino et al., 2005). Among these 11 pathways, necrosis, apoptosis and autophagy
are three major forms of cell death (Belizario et al, 2007). Accumulating evidence
has suggested that evasion of apoptosis is a major cause in the development and
progression of cancer, thus many chemotherapeutic agents nowadays have targeted
on the apoptotic pathways to induce cancer cell death (Russo et al,, 2006).
Morphologically, apoptosis is a process characterized by cell shrinkage, membrane
blebbing and nuclear condensation. In molecular term, apoptosis is triggered by
"death" signals and is a complex series of events with multiple positive and negative
feedback loops (Schimmer, 2008). Based on the original source of "death" signals,
apoptosis can be classified into two pathways: extrinsic pathway, which responds
mainly to extracellular stimuli, and the other intrinsic pathway, which is activated by
-109 - Chapter 4 Apoptosis- and Differentiation-inducing Effects modulators within the cell itself (Russo et al, 2006).
In the extrinsic pathway, also known as receptor-mediated death pathway, death ligands such as TRAIL and FasL induce apoptosis by activating the death receptors
(DR4/TRAILR1 /Apo2 and Fas/Apol, etc) at the cell surface. The activated death receptors will oligomerize, which leads to recruitment of the adaptor proteins such as
FADD (Fas-associated protein with death domain) and further chain activation of the caspase-8 and -3 (Russo et al., 2006; Schimmer, 2008; Han et al, 2008).
The intrinsic pathway, which is also known as mitochondria-mediated death pathway, is initiated by damage to the mitochondria which results in the release of a series of proteins into cytoplasm, including cytochrome c. When released into cytoplasm, cytochrome c complexes with Apaf-1 and this complex further activates caspase-9 and ultimately caspase-3. Although, these two pathways are separated from each other at least at the beginning, they crosstalk to each other by certain proteins (Bid and tBid) and finally converge in a key event which is conversion of procaspases into "executioner" caspases, such as caspase-3 and caspase-7 (Russo et
al., 2006; Belizario et al, 2007; Schimmer, 2008; Han et al., 2008).
Bcl-2 family of proteins, in particular, play a central role in the regulation of the
intrinsic pathway (Belizario et al., 2007). Members of the Bcl-2 family can be
divided into two subfamilies: one includes the pro-apoptotic proteins such as Bak,
Bax, and Bid; another includes the anti-apoptotic proteins such as Bcl-2 and Bcl-xL
(Han et al., 2008). Recent studies have reported that induction of pro-apoptotic
proteins and inhibition of anti-apoptotic proteins, triggered by certain anti-cancer
agents, will directly alter the permeability of mitochondrial membrane and further
induce the apoptosis signaling pathway (Jeong et al, 2008).
In the present study, the apoptosis-inducing ability of Sophoraflavanone G on
-110 - Chapter 4 Apoptosis- and Differentiation-inducing Effects the human promyelocytic leukemia HL-60 cells was investigated. In the first part of this Chapter, the apoptosis-inducing activity of Sophoraflavanone G on the HL-60 cells was examined by detecting DNA fragmentation in agarose gel electrophoresis and quantified by the Annexin V-GFP and PI double staining. The change of mitochondrial membrane potential was monitored by the mitochondria-selective
JC-1 dye staining method. To reveal the possible apoptotic pathway(s) has/have been induced by Sophoraflavanone G, the activities of caspase-3, -8 and -9 were measured using specific fluorometric substrates. In addition, since Bcl-2 family proteins play an active role in the apoptosis signaling pathway (Belizario et al., 2007)，thus the expression levels of the Bcl-2 family proteins were investigated by Western blot analysis. Moreover, it is known that apoptosis can also be initiated by over-generation of reactive oxygen species (ROS) or by alteration of Ca^"^ homeostasis (Dirsch et al, 1998; Roderick, 2008). In the present study, using flow
cytometric analysis, the ROS production and the level of intracellular calcium were
measured by dihydroethidine staining and Fluo-3/AM staining, respectively.
In addition to proliferate independently of growth signals and by avoiding
appropriate cell death, another abnormality of leukemia cells is their inability to
differentiate into functional mature cells (Tsiftsoglou et al., 2003; Roderick and Cook,
2008). The degree of differentiation is inversely related to their malignancy and
terminal differentiation is usually accompanied by the cessation of cell proliferation
(Tsiftsoglou et al, 2003). The observation of Friend and his colleagues in early
1970s that MEL leukemia cells can be induced by dimethylsuloxide (DMSO) to
differentiate into non-dividing mature erythroid cells sparked extensive exploration
and led to the idea of differentiation therapy of cancer, which is inducing terminal
differentiation of neoplastic cells by various agents (Friend et al., 1971; Tsiftsoglou
-Ill - Chapter 4 Apoptosis- and Differentiation-inducing Effects et al., 2003). During the last three decades, a wide variety of agents (chemical, natural, pharmacological, and biological) have been found and proved to induce leukemic cell differentiation (Tsiftsoglou et al., 2003). Some of them have been applied in clinical trials for hematologic malignancies, which were summarized and listed in Table 4.1. All-trans-rttmoic acid (ATRA), one of the most well-known inducers, was demonstrated to induce HL-60 cells differentiate into granulocytic cells by Breitman et al in 1980. Nowadays, either alone or in combination with other anti-cancer drugs like arsenic trioxide (ATO, AS2O3), ATRA therapy has been recognized as the most successful treatment for acute promyelocytic leukemia (APL)
(Leung et al., 2005; Rubnitz et al., 2008). Apart from ATRA, HL-60 cells can also be induced to differentiate in response to different inducers, such as camptothecin and vitamin D3 (Tsiftsoglou et al, 2003). Moreover, recent studies reported that some members of flavonoids family, such as genistein and apigenin, can trigger HL-60
cells to undergo differentiation (Takahashi et al., 1998; Kawaii et al., 1999). Thus, it
is possible that Sophoraflavanone G，a member of the flavonoids family, can also
induce leukemic cell differentiation. Terminal differentiation of HL-60 cells can be
monitored by changes in morphological, biochemical and immunological properties
(Kawaii et al., 1999). In the second part of this Chapter, the differentiation-inducing
ability of Sophoraflavanone G was evaluated by examining the morphological and
functional changes in the drug-treated HL-60 cells.
-112 - Chapter 4 Apoptosis- and Differentiation-inducing Effects
Table 4.1 Common differentiation inducers used in clinical trials for hematologic malignancies
• •• • I • • ——^―——^ - ‘ • ‘ ———— • ‘ —— Differentiation inducers Examples
^— ——^^―—— Arsenic trioxide (AS2O3) ,.p . Cytosine arabinoside Anti-proliferative agents ,,, ^ Hydroxyurea Interferons
_ , 8-chloro-cyclic adenosine 3', 5' monophosphate Cyclic AMP analogs (S-Cl-cAMP)
Demethylating agents Aza-cytidine
. Depsipeptide ir^l^L/c^Sl^Si^ Sodium phenylbutyrate (NaPB) Suberoylanilide hydroxamic acid (SAHA) Erythropoietin (EPO) Granulocyte-colony stimulating factor (G-CSF) .. ,. Granulocyte-macrophage-colony stimulating Hematopoietic cytokines factor (GM-CSF) Interleukin-3 (IL-3) Interleukin-6 (IL-6) Alkylophospholipids . Gangliosides Short-chain fatty acids Sodium butyrate
PKC agonists and antagonists Tetradecanoylphorbol acetate (TPA)
Polar-planar compounds Hexamethylene bisacetamide (HMBA)
Retinoic acid and its derivatives Vitamin analogs . ta Vitamin D3 and its derivatives
(Modified from Miller and Waxman, 2002)
-113 - Chapter 4 Apoptosis- and Differentiation-inducing Effects
4.2 Results
4.2.1 Induction of DNA Fragmentation in the Human Promyelocytic Leukemia
HL-60 Cells by Sophoraflavanone G
The induction of apoptosis is accompanied by a number of biochemical changes.
One of the biochemical changes is DNA fragmentation, which is the hallmark of apoptosis. By analyzing the genomic DNA extracted from apoptotic cells in a 2% agarose gel, the occurrence of "DNA ladder", which is a characteristic of DNA fragments in multiples of 180-200 base pairs, can be observed (Robertson et al.,
2000).
In this study, HL-60 cells were either incubated with solvent control or treated with different concentrations of Sophoraflavanone G for 24 hours. Genomic DNA of treated-HL-60 cells were extracted and analyzed by electrophoresis on 2% agarose gel stained with ethidium bromide. Figure 4.1 shows that Sophoraflavanone G induced DNA fragmentation in HL-60 cells after 24 hours of incubation in a dose-dependent response manner.
-114 - Chapter 4 Apoptosis- and Differentiation-inducing Effects
•Hil • J
Lane 1: 100 bp DNA marker Lane 2: HL-60 cells treated with solvent control Lane 3: HL-60 cells treated with 5 |iM Sophoraflavanone G Lane 4: HL-60 cells treated with 10 [xM Sophoraflavanone G Lane 5: HL-60 cells treated with 20 |JM Sophoraflavanone G Lane 6: HL-60 cells treated with 30 [xM Sophoraflavanone G
Fig. 4.1 Dose response study for the induction of DNA fragmentation in the human promyelocytic leukemia HL-60 cells treated with Sophoraflavanone G. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.13% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 hours at 37°C. Apoptotic DNA fragments were extracted by mild detergent IGEPAL CA-630 lysis buffer, and analyzed by electrophoresis on 2% agarose gel stained with ethidium bromide.
•
-115 - Chapter 4 Apoptosis- and Differentiation-inducing Effects
4.2.2 Induction of Phosphatidylserine Externalization in the Human
Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G as Detected
by Annexin V-GFP and PI Double Staining Method
Apart from DNA fragmentation, apoptosis is also characterized by phosphatidylserine (PS) externalization from inner plasma membrane to outer plasma membrane. Therefore, PS was exposed to the extracellular environment (van
Engeland et al., 1998). Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for PS. Thus, it is a sensitive marker to detect PS externalization (Vermes et al., 1995). Annexin V conjugated with a fluorophore such as Green Fluorescence Protein (GFP) can identify apoptotic cells by binding to PS when it is exposed to the outer leaflet. However, Annexin V can also bind to necrotic cells due to the loss of membrane integrity. Thus, another dye in this assay, propidium iodide (PI) was used to differentiate the apoptotic cells from necrotic cells because PI can be excluded by viable and early apoptotic cells with intact plasma membrane. Therefore, viable cells are Annexin V-GFP and PI double negative. Early apoptotic cells are Annexin V-GFP positive and PI negative. Necrotic and/or late apoptotic cells are Annexin V-GFP positive and PI positive. The distribution of these subpopulations can be measured and quantified by flow cytometry analysis (Figure
4.2).
In this study, after incubation with Sophoraflavanone G for 12 hours, HL-60
cells were harvested and stained with GFP-conjugated Annexin V and propidium
iodide (PI). By flow cytometric analysis, the early apoptotic cells can be
distinguished from the late apoptotic and/ or necrotic cells.
As shown in Figure 4.3, after 12 hours of treatment of HL-60 cells with
Sophoraflavanone G, there was a dose-dependently increase of cell population in
-116 - Chapter 4 Apoptosis- and Differentiation-inducing Effects population of early apoptotic cells. The result further confirms that Sophoraflavanone
G is capable of inducing apoptosis in HL-60 cells with detectable PS translocation from the inner to the outer plasma membrane.
PI+/Ann(3Xin V+
o “ Dead cells (Necrotic cells or 6 Late apoptotic cells) PI
_ Pl'/Annexin V" Pf/Annexin V^ B
丨 Viable Early apoptotic cells cells
10� 10' 10: 10' Annexin V-GFP
Fig. 4.2 Detection of phosphatidylserine extemalization by Annexin V-GFP and
PI double staining method. The green fluorescence emitted by Annexin V-GFP and the red fluorescence emitted by PI are detected by flow cytometry. The lower left quadrant (LL) represents viable cells without PS translocation; the lower right
quadrant (LR) represents early apoptotic cells with PS translocation and the upper
right quadrant (UR) represents necrotic and/or late apoptotic cells.
-117 - Chapter 4 Apoptosis- and Differentiation-inducing Effects
A n • OR* 5 1,0% °
专 • . .最 • • _- •• • < < •
1 :B! '10, _ G • ‘ ‘ '10' 10^ 10^ W 0 � ‘ _ 10^ "LO^ 10' 2 91.3% FITC-A 2.8% 83.8% FITC-A 5.1% i c. 0^% 22.9%D. 0.1% 36_6% O ° °
< !•. • ••• < . • • •：" r^.
10" 10' 10= 10' 10‘ LB''" '"""^LO'' , .10: 10' 10' 63.4% FITC-A 13.5% 41 6% FITC-A 21.6%
Green fluorescence (FL-1)
Concentration of Distribution of subpopulation (%) Sophoraflavanone G viable cells Early Late apoptotic cells apoptotic cells and/or necrotic cells Solvent Control (A) 91.3% 5^8% 10 抖M(B) 83.8% 5.1% 11.0% 20 fiM (C) 63.4% 13.5% 22.9% 30 ^M (D) 41.6% 21.6% 36.6%
Fig. 4.3 Induction of phosphatidylserine externalization in the human promyelocytic leukemia HL-60 cells by Sophoraflavanone G as detected by Annexin V-GFP and PI double staining method. HL-60 cells (1x10^ cells/ml) were incubated with (A) solvent control (0.13% ethanol); (B) 10 |iM of Sophoraflavanone G; (C) 20 ^iM of Sophoraflavanone G and (D) 30 |iM of Sophoraflavanone G for 12 hours at 37°C. The HL-60 cells were harvested and stained with Annexin V-GFP and PI as described in Chapter Two. The stained cells were subjected to BD FACSCanto flow cytometer for measuring green (FL-1 channel) and red (FL-2 channel) fluorescence intensities simultaneously. The percentage distribution of three subpopulations, as shown in the Table in the lower panel, is calculated by WinMDI software (Version 2.8).
-118 - Chapter 4 Apoptosis- and Differentiation-inducing Effects
4.2.3 Effects of Sophoraflavanone G on the Caspase Activities in the Human
Promyelocytic Leukemia HL-60 Cells
As demonstrated in Section 4.2.1 and 4.2.2, DNA fragmentation and phosphatidylserine externalization, typical biochemical changes occur in the process of apoptosis, were detected in the Sophoraflavanone G-treated HL-60 cells. These biochemical changes are mainly caused by an enzyme family, known as caspases
(cysteinyl-aspartate-specific proteases) (Belizario et al, 2007). At least 14 members have been identified in this cysteine protease family. All of them are homologous to
each other and they cleave their substrate proteins specifically at sites next to
aspartate residues. They are synthesized constitutively and are normally present as
inactive proenzymes, also known as procaspases, which have very weak protease
activity. Cleavage at specific internal aspartate residues, also known as caspase
activation, is required for inducing full enzymatic activity, which is vital for the
occurrence of apoptotic characteristic. The simplest way to activate a procaspase is to
expose it to a previously activated caspase member. This "caspase cascade" strategy
of caspase activation is extensively used in apoptotic cells (Saraste and Pulkki, 2000).
Based on their point of entry into the cascade, apoptotic caspases can further
classified into upstream "initiator" caspases (caspase-2, -8，-9 and -10) and
downstream "executioner" caspases (caspase-3, -6 and -7) (Belizario et al” 2007).
For instance, activation of upstream "initiator" caspase-8 or caspase-9 by
oligomerization and self-cleavage of its monomeric procaspases lead to proteolytic
activation of the downstream "executioner" caspase-3. Activated caspase-3 will
further cleave critical intracellular proteins and thereby induce the final stage of cell
death (Schimmer, 2008). Thus, it is essential to monitor the level of caspase-3
activation in Sophoraflavanone G-treated HL-60 cells, which may further confirm
-119 - Chapter 4 Apoptosis- and Differentiation-inducing Effects that Sophoraflavanone G is able to trigger apoptosis in vitro. Apart from caspase-3, the activation level of caspase-8 and caspase-9 were be also investigated because caspase-8 and caspase-9 are the hallmark "initiator" caspases for the extrinsic and intrinsic pathway of apoptosis, respectively (Chowdhury et al, 2006).
In the present investigation, the levels of caspase-3, -8 and -9 activation were monitored by enzymatic activity studies. HL-60 cells were treated with either solvent control or different concentrations of Sophoraflavanone G for 24 hours. The protein of each sample was extracted and the activities of caspase-3, -8 and -9 were measured by fluorometric assay. Specific fluorescent AMC conjugated caspase substrates and specific caspase inhibitors were used. For example, for the measurement of caspase-3 activity, the protein extract was incubated with caspase-3 specific fluorescent AMC conjugated substrate Ac-DEVD-AMC for one hour.
Ac-DEVD-AMC was then cleaved by active caspase-3. The amount of fluorescent
AMC released from the substrate by the action of caspase-3 is directly proportional to its enzymatic activity. To make sure the correlation between the fluorescence
signal and caspase-3 activity, caspase-3 specific inhibitor Ac-DEVD-CHO was
incubated with the protein sample before the addition of substrate. The results are
shown in Figure 4.4 - 4.6. It was observed that the activities of caspase-3 and -9 were
significantly enhanced in the Sophoraflavanone G-treated HL-60 cells in a
dose-dependent manner (Figure 4.4 and Figure 4.5). On the other hand, the caspase-8
activity was markedly elevated when HL-60 cells were incubated with 50 fiM
Sophoraflavanone G for 24 hours, but no significantly changes were detected at
lower concentrations (20 \iM and 30 )j.M) of Sophoraflavanone G (Figure 4.6). The
specificity of the caspase activity assays was supported by the evidence that the three
caspase-specific inhibitors markedly suppressed the activities of caspases in all cases.
-120 - Chapter 4 Apoptosis- and Differentiation-inducing Effects
氺氺氺 4 Q � .e • Sophoraflavanone G ^ •Softeaflavanone G +Ac-DEVD-CHQ Fig. 4.4 Fluorometric detection of caspase-3 activity in the Sophoraflavanone G-treated human promyelocytic leukemia HL-60 cells. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.2% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 hours at 37°C. Cytosolic extracts were prepared and assayed for caspase-3 activity with caspase-3 specific fluorescent AMC conjugated substrate Ac-DEVD-AMC with or without caspase-3 inhibitor Ac-DEVD-CHO. The samples were excited at 430 nm and the fluorescence emitted at 465 nm was measured by the fluorescence plate reader Polarizon (Tecan). Results were expressed as fluorescence units of AMC release, which was estimated by comparing sample AMC fluorescence units with the standard curve of free AMC molecules. Each point represents the mean 士 S.E. of quadruplicate cultures. The caspase-3 activity corresponded to the amount of AMC release. *** Significantly different compared with the solvent control, p < 0.001 -121 - Chapter 4 Apoptosis- and Differentiation-inducing Effects ^ * * * *S 10 - 口 Sophoraflavanone G 工 ‘ • Sophoraflavanone G +Ac-ETD-CHO G 0.8 I- U C« U 0 0.6 h S S 1 0.4 - i- Hi [h rb • Solvent control 20 30 50 Concentration of Sophoraflavanone G (_) Fig. 4.5 Fluorometric detection of caspase-8 activity in the Sophoraflavanone G-treated human promyelocytic leukemia HL-60 cells. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.2% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 hours at 37°C. Cytosolic extracts were prepared and assayed for caspase-8 activity with caspase-8 specific fluorescent AMC conjugated substrate Ac-IEID-AMC with or without caspase-8 inhibitor Ac-IEID-CHO. The samples were excited at 430 nm and the fluorescence emitted at 465 nm was measured by the fluorescence plate reader Polarizon (Tecan). Results were expressed as fluorescence units of AMC release, which was estimated by comparing sample AMC fluorescence units with the standard curve of free AMC molecules. Each point represents the mean 士 S.E. of quadruplicate cultures. The caspase-8 activity corresponded to the amount of AMC release. *** Significantly different compared with the solvent control, p < 0.00J -122- Chapter 4 Apoptosis- and Differentiation-inducing Effects 口 5.0 厂 *** • Sophoraflavanone G ^ 3 • Sophoraflavanone G + Ac-LEHD-CHO S 4.0 c CU 1 3.0 [ *** O R^ s 山山山 C 氺氺氺 (U 2.0 H [— ii^filLISolvent control 20 30 L50 Concentration of Sophoraflavanone G (|iM) Fig. 4.6 Fluorometric detection of caspase-9 activity in the Sophoraflavanone G-treated human promyelocytic leukemia HL-60 cells. HL-60 cells (1x10^ cells/ml) were either incubated with solvent control (0.2% ethanol) or treated with different concentrations of Sophoraflavanone G for 24 hours at 37°C. Cytosolic extracts were prepared and assayed for caspase-9 activity with caspase-9 specific fluorescent AMC conjugated substrate Ac-LEHD-AMC with or without caspase-9 inhibitor Ac-LEHD-CHO. The samples were excited at 430 run and the fluorescence emitted at 465 nm was measured by the fluorescence plate reader Polarizon (Tecan). Results were expressed as fluorescence units of AMC release, which was estimated by comparing sample AMC fluorescence units with the standard curve of free AMC molecules. Each point represents the mean 士 S.E. of quadruplicate cultures. The caspase-9 activity corresponded to the amount of AMC release. *** Significantly different compared with the solvent control, p < 0.001 -123 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 4.2.4 Induction of Mitochondrial Membrane Depolarization in the Human Promyelocytic Leukemia HL-60 Cells by Sophoraflavanone G As shown in Figure 4.6, the activity of caspases-9 was significantly enhanced in Sophoraflavanone G-treated HL-60 cells in a dose-dependent manner. It is known that caspase-9, as an "initiator" caspase of the intrinsic pathway, carries an apoptotic signal from mitochondrion which leads to the activation of caspase-3 and causing apoptosis (Bratton et al, 2001). Therefore, it is of interest to further investigate the possible roles of mitochondria in Sophoraflavanone G-induced apoptosis. One of the early events of apoptosis is the loss of mitochondrial transmembrane potential (A\};m), which can be measured by means of JC-1 staining. JC-1, chemically known as 5,5',6,6'-tetrachloro-l,r,3,3'- tetraethylbenzimidazolyl-carbocyanine iodide, is a cell permeant cationic fluorescent probe which selectively accumulates in the mitochondria. It is a sensitive marker for the detection of changes in Ax^/m. In the presence of intact polarized mitochondrial membrane (with high A\}/m), JC-1 molecules form J-aggregates (dimeric JC-1) with the maximum fluorescence emission at 590 run (red fluorescence) after excitation at 488 nm. When the mitochondrial membrane is depolarized (with reduced A\|;m), the dyes remain in a monomeric form and emits green fluorescence at 525 nm upon the same excitation wavelength. Therefore, depolarization of mitochondrial membrane can be indicated by a fluorescence emission shift from red to green (Figure 4.7). In the present study, HL-60 cells were treated with various concentrations of Sophoraflavanone G for 12 or 24 hours. After stained with JC-1 dye, the control and treated HL-60 cells were analyzed by flow cytometry. The data were expressed as a density plot in Figure 4.8. The X-axis of the plot represents the green fluorescence intensity of monomeric JC-1 while the Y-axis represents the red fluorescence -124 - Chapter 4 Apoptosis- and Differentiation-inducing Effects intensity of dimeric JC-1. The lower right region R1 indicated the cell population with higher green to red fluorescence ratio which reflects break-down of A\|/m. After the treatment of Sophoraflavanone G for 12 and 24 hours, the proportion of HL-60 cells with higher green to red fluorescence ratio was increased time- and dose-dependently (Figure 4.8). The results suggest that Sophoraflavanone G may induce mitochondrial membrane depolarization in HL-60 cells in a time- and dose-dependent manner. -125 - Chapter 4 Apoptosis- and Differentiation-inducing Effects % / • r? Polarized Z I / H Mitochondria 丨 / ^ Membrane Z. w aggregates^W • £ � _ _ g / ^ Depolarized 2 / Mitochondrial p^ Z Membrane / Monomeric • JC-1 (525 nm) ^ Green Fluorescence (FL-1) Fig. 4.7 Detection of mitochondrial membrane depolarization by JC-1 staining method. JC-1 molecules can form aggregates due to the physiological membrane potential of mitochondria. This aggregation changes the fluorescence properties of JC-1 leading to a shift from green to red fluorescence. Intact living cells stained with JC-1 therefore exhibit a distinct red fluorescence of mitochondria, which is detectable by flow cytometric analysis (in the FL-2 channel). Apoptosis results in a breakdown of the mitochondrial membrane potential and a subsequent decrease of the red fluorescence and an increase of the green fluorescence (in the FL-1 channel). In this graph, the X-axis represents the intensity of green fluorescence while the Y-axis represents the intensity of red fluorescence. Upper left region represents cells with normal polarized mitochondrial membrane which emit red fluorescence. Lower right region represents cells with depolarized mitochondrial membrane which emit green fluorescence. -126 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 12hr-treatment 24hr-treatment A. Control (0,13% ethanol) A，. Control (0.13% ethanol) Z Rl:2% Z Rl： 2 % % • 圓 & 『10。 10, 10= 10' “ 10" 10' 10， 10^ 10' "tc-a fitc-a ^ B. 20 /jM of Sophoraflavanone G B\ 20 piM of Sophoraflavanone G � o ° I ' ‘ ^ I F -WrF' p W^ E Z Rl:4% Z Rl:7% M "10� 1� ' 10' 10' "10� 10' 10: 10， 10* 逆 "^tc-a fitc-a C 30 fjM of Sophoraflavanone G C\ 30 fjM of Sophoraflavanone G :d ： 4 Rl： 29 % Z Rl: 36 % & ______10 � 10' 10= 10' 10. 10" 10' 10: 10' 10. "tc-a fitc-a Green Fluorescence (FL-1) Fig. 4.8 Effect of Sophoraflavanone G on the mitochondrial membrane depolarization in the human promyelocytic leukemia HL-60 cells as detected by JC-1 staining method. HL-60 cells (1x10^ cells/ml) were incubated with (A & A’） solvent control (0.13% ethanol); (B & B’）20 ^iM of Sophoraflavanone G; (C & C,) 30 |aM of Sophoraflavanone G for 12 and 24 hours. The cells were then stained with JC-1 dye as described in Chapter Two. The stained cells were subjected to BD FACSCanto flow cytometer for measuring green (FL-1 channel) and red (FL-2 channel) fluorescence intensities simultaneously. The percentage cell population in the lower right region Rl, representing cells with higher green to red fluorescence ratio which reflects Ai|/m dissipation, was calculated by the WinMDI software (Version 2.8). -127 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 4.2.5 Involvement of Bcl-2 Family Members in Sophoraflavanone G-induced Apoptosis in the Human Promyelocytic Leukemia HL-60 Cells Results in the previous section show that Sophoraflavanone G can trigger mitochondria membrane depolarization in the HL-60 cells. It has been reported that the permeability of the mitochondrial outer membrane is modulated by the Bcl-2 family of proteins, which include at least 17 or more members in mammalian cells (Cory and Adams, 2002; Han et al, 2008). Bcl-2 proteins share at least one conserved Bcl-2 homology (BH) domain. Based on the BH domains and function, Bcl-2 family can be classified into three groups. The anti-apoptotic members, such as Bcl-2 and Bcl-xL (Bcl-x protein long form), typically possess four BH domains (BHl to BH4). The pro-apoptotic Bcl-2 members, which promote apoptosis, can be further divided into two groups, one is BH3-only proteins which include Bid, Bim， Bad, Noxa, and PUMA, while the other group members have three BH domains (BHl to BH3)，such as Bak and Bax. In healthy cells, Bak anchors at the site of the mitochondrial outer membrane, whereas Bax resides in the cytosol. Anti-apoptotic members will bind to and antagonize Bak and Bax so as to block the function of Bak and Bax. When Bak and Bax are stimulated by death signals, they can form homo- and/or hetero-oligomers in the mitochondrial outer membrane to permeabilize the outer membrane, which leads to the release of mitochondrial intermembrane space proteins, such as cytochrome c and AIF, to the cytosol (Belizario et al., 2007; Han et fl/.,2008; Jeong era/., 2008). To elucidate the possible involvement of Bcl-2 family proteins in Sophoraflavanone G-induced apoptosis of HL-60 cells, the expression of several proteins of the Bcl-2 family, including Bax, Bak and Bcl-2, was examined by Western blotting analysis. HL-60 cells were treated with either solvent control or -128 - Chapter 4 Apoptosis- and Differentiation-inducing Effects different concentrations of Sophoraflavanone G for 24 hours. The cells were then harvested and the total cell lysate was isolated and further subject to Western blot analysis. The levels of pro-apoptotic proteins Bax and Bak were increased while the level of anti-apoptotic proteins Bcl-2 was decreased (Figure 4.9). -129 - Chapter 4 Apoptosis- and Differentiation-inducing Effects Concentration of Sophoraflavanone G Bax ：丨纖_ ^^^tlgm "丨丨丨___| 25 kDa Bak I .一 暴尋導 I 30 kDa 3-actin ri*—| 42 kDa Bcl-2 mmm mm' mmm mmmm... 26 kDa 3-actin _歐.WKm mmmm mimKI^^ 42 kDa =二二 10 ^M 20 ]iM 50 jiM ITO LOO L^ \32 \M Bak 1.00 1.30 1.52 1.37 Bcl-2 1.00 1.01 0.96 0.55 Fig. 4.9 Effect of Sophoraflavanone G on the expression of Bcl-2 family proteins in the human promyelocytic leukemia HL-60 cells. HL-60 cells (2x10^) were treated with different concentrations of Sophoraflavanone G (0 - 50 |aM) for 24 hours at 37°C. Proteins were extracted from total cell lysate and separated by 12.5% SDS gel. After that, Western blot analysis was performed as described in Chapter Two. Relative band intensities, shown in the bottom Table, were calculated after normalization with respect to P-actin, and comparison was made with the corresponding control. -130 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 4.2.6 Effect of Sophoraflavanone G on the Induction of Reactive Oxygen Species in the Human Promyelocytic Leukemia HL-60 Cells In Section 4.2.4 we demonstrated that Sophoraflavanone G may trigger mitochondrial membrane depolarization in HL-60 cells. A recent report shows that disruption of the mitochondria transmembrane potential is usually associated with increased reactive oxygen species (ROS) production (Ishii et al, 2002). ROS includes a variety of molecules and free radicals (chemical species with one unpaired electron) derived from molecular oxygen but possessing higher reactivities than molecular oxygen, such as superoxide anion (O2,)，hydrogen peroxide (H2O2) and hydroxyl radical (0H") (Martindale et al.’ 2002; Turrens, 2003). At the physiological level, ROS, whether generated intracellularly or derived from exogenous sources, can act as second messengers in various signal transduction pathways or as byproducts of normal aerobic metabolism. Transient fluctuations in ROS serve important regulatory functions, but when present above a threshold and/or sustained levels, ROS can randomly attack DNA, proteins and lipids, and interfere with vital pathways to cause cell death either by apoptosis or necrosis (Martindale et al, 2002; Belizario et al, 2007). To investigate the accumulation of ROS in Sophoraflavanone G-treated HL-60 cells, dihydroethidine, an oxygen radical sensor dye while can be specifically converted by superoxide anion (O2,) to highly fluorescent ethidium, was used to measure the intracellular ROS level by flow cytometric analysis. After treatement with different concentrations of Sophoraflavanone G for 24 or 48 hours, the HL-60 cells were stained with dihydroethidium and further analyzed by flow cytometry. The data were expressed as a histogram graph as shown in Figure 4.10. The X-axis of the graph represents the red fluorescence intensity. The right region Ml represents the -131 - Chapter 4 Apoptosis- and Differentiation-inducing Effects cell population with higher red fluorescence which reflects higher intracellular superoxide anion (O2') production level. The results in Figure 4.10 show that superoxide anion (O2') production was markedly enhanced in Sophoraflavanone G-treated HL-60 cells at a higher concentration (40 fiM) of the drug and a longer incubation time (48 hours). -132 - Chapter 4 Apoptosis- and Differentiation-inducing Effects h ^Solvent Control /• Solvent Control UM '20 MM 二 'Mzz ^ ^o.^KL^ -•(0� 10' 10= 10* 10' 10= 10' PE-A Pe-A 24hr-treatment 48hr-treatment Red Fluorescence (FL-2) Distribution of Concentration of Sophoraflavanone G Ml region (%) Solvent Control 20 ^iM 40 ^iM 24hr-treatment 2.98% 5.24% 15.06% 48hr-treatment 2.12% 3.23% 39.07% Fig. 4.10 Effect of Sophoraflavanone G on the production of superoxide anions in the human promyelocytic leukemia HL-60 Cells. HL-60 cells (1x10^ cells/ml) were incubated with solvent control (0.17% ethanol) (red line), 20 pM of Sophoraflavanone G (black line) or 40 |iM of Sophoraflavanone G (green line) for 24 and 48 hours. The cells were then stained with oxygen radical sensor dye dihydroethidium as described in Chapter Two. The stained cells were subjected to BD FACSCanto flow cytometer for measuring red fluorescence intensity (FL-2 channel). The percentage distribution of the right region Ml, as shown in the Table of the lower panel, represents cells with higher red fluorescence intensity which reflects higher intracellular superoxide anion (O2') production level. The results of percentage distribution of Ml are calculated by WinMDI software (Version 2.8). -133 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 4.2.7 Effect of Sophoraflavanone G on the Intracellular Ca^^ Level in the Human Promyelocytic Leukemia HL-60 Cells The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+，plays a pivotal role in the cell survival and death (Belizario et al., 2007). Cytosolic free calcium [Ca ]i increases have been observed during apoptotic cell death and have been shown to be required for apoptosis to take place (Szado et al, 2008; Roderick and Cook, 2008). The results in previous sections show that several biochemical changes related to apoptosis, such as DNA fragmentation, caspase activation and mitochondrial membrane depolarization, can be observed in Sophoraflavanone G-treated HL-60 cells. Therefore, it is of interest to examine whether the intracellular Ca^^ level is increased in Sophoraflavanone G-treated HL-60 cells. In this study, HL-60 cells were incubated with different concentrations of Sophoraflavanone G for 24 or 48 hours. The harvested cells were then stained with Fluo-3/AM (C51H50CI2N2O23) and further analyzed by flow cytometry. The data were expressed as a histogram graph as shown in Figure 4.11. The X-axis of the graph represents the green fluorescence intensity. The right region Ml represents indicated the cell population with higher green fluorescence which reflects higher intracellular Ca^^ level. The results in Figure 4.11 show that the intracellular Ca^^ level of Sophoraflavanone G-treated HL-60 cells was increased dose-dependently after 24 hours of treatment. In addition, it was also found that the intracellular Ca^^ level was higher in the 24hr-treatment than in the 48hr-treatment. -134 - Chapter 4 Apoptosis- and Differentiation-inducing Effects ^ Solvent Control S /^-Solvent Control 賽j -20 u M u M /~• 40 nM 10� 10 � FI;C� A ' � 1� ‘ IO^ ~TO> FITC-A 24hr-treatment 48hr-treatment Green Fluorescence (FL-1) Distribution of Concentration of Sophoraflavanone G Ml region (%) —Solvent Control 20 ^M 40 ^M — 24hr-treatment 2.74% 9.40% 43.07% 48hr-treatment 2.13% 6.57% 10.13% Fig. 4.11 Effect of Sophoraflavanone G on the intracellular Ca^^ level in the human promyelocytic leukemia HL-60 Cells. HL-60 cells (1x10^ cells/ml) were incubated with solvent control (0.17% ethanol) (red line), 20 jiM of Sophoraflavanone G (black line) or 40 |iM of Sophoraflavanone G (green line) for 24 and 48 hours. The cells were then stained with Fluo-3/AM as described in Chapter Two. The stained cells were subjected to BD FACSCanto flow cytometer for measuring green fluorescence intensity (FL-1 channel). The percentage distribution of the right region Ml, as shown in the Table of the lower panel, represents cells with higher green fluorescence intensity which reflects higher intracellular Ca^^ level. The percentage distribution of Ml was calculated by the WinMDI software (Version 2.8). -135 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 4.2.8 Morphological Studies on the Sophoraflavanone G-treated Human Promyelocytic Leukemia HL-60 Cells Human promyelocytic leukemia HL-60 cells can be induced to differentiate into four general types of cells: granulocytes, monocytes, macrophage-like cells and eosinophils by various agents (Collins, 1987). These differentiations are usually accompanied by morphological changes including general enlargement of the cells, decrease in the ratio of nucleus to cytoplasm and/or increase in vacuolation and cellular granularity. To investigate the differentiation-inducing potential of Sophoraflavanone G on the human promyelocytic leukemia HL-60 cells, HL-60 cells were incubated with different concentrations of Sophoraflavanone G (10 - 30 |_LM) at 37°C for 72 hours. Cytospin preparations of harvested HL-60 cells were made on microscopic slides and were further stained with Hemacolor 1，2 and 3 solutions as described in Chapter Two. The cell morphology was observed by light microscopy. The morphological changes of Sophoraflavanone G-treated HL-60 cells are shown in Figure 4.12. It was observed that Sophoraflavanone G-treated HL-60 cells did not have the typical morphologies of terminally differentiated cells, like cell enlargement and increases in cytoplasm to nucleus ratio and vacuolation. The Sophoraflavanone G-treated cells (Figure 4.12 B - D) only showed similar morphology to the solvent control-treated cells (Figure 4.12 A). These results indicate that Sophoraflavanone G failed to induce the terminal differentiation of the HL-60 cells under the given experimental conditions. -136 - Chapter 4 Apoptosis- and Differentiation-inducing Effects r务一 J例 til 气公.ri p .全 Fig. 4.12 Cellular morphology of the Sophoraflavanone G-treated human promyelocytic leukemia HL-60 cells. HL-60 cells (1x10^ cells/ml) were treated with (A) solvent control (0.13% ethanol); (B) 10 ^M of Sophoraflavanone G; (C) 20 fiM of Sophoraflavanone G; (D) 30 ^M of Sophoraflavanone G for 72 hours at ITC. The treated cells were harvested and further cytocentrifuged onto microscopic glass slides and stained with Hemacolor solutions as described in Chapter Two. The cell morphology was observed by light microscopy. The scale bars represent 20 jim and the magnification is 400x. -137 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 4.2.9 Effect of Sophoraflavanone G on the NBT-Reducing Activity of the Human Promyelocytic Leukemia HL-60 Cells Apart from examining the differentiation-inducing effect by morphological criteria, functional characteristics assessment, like the NBT-reducing activity, is also a widely used method. When HL-60 cells undergo differentiation into granulocytes or monocytes, the conversion of oxygen to superoxide anions will enhance upon exposure to phorbol myristate acetate (PMA), due to induction of respiratory burst activity (Collins, 1987; Ishii et al, 2002). Nitroblue tetrazolium (NBT), an electron acceptor, can be used to monitor the production of superoxide anions. NBT can be reduced by superoxide anions from the water-soluble form into water-insoluble, dark blue nitroblue diformazan deposits. Therefore, the cells producing superoxide anions will be stained with these dark blue deposits (Newburger et al, 1979). In the present study, HL-60 cells were incubated with different concentrations of Sophoraflavanone G at 37°C for 72 hours. The treated cells were then incubated with NBT-PMA solution. The cells were then cytocentrifuged onto microscopic slides and counter-stained with 1% safranin in methanol as described in Chapter Two. As shown in Figure 4.11，Sophoraflavanone G-treated HL-60 cells were stained red, which were similar to the solvent-treated control cells. Therefore, Sophoraflavanone G failed to enhance the NBT reduction activity of HL-60 cells under the prescribed experimental conditions. -138 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 72 hours treatment _ Fig. 4.13 Effect of Sophoraflavanone G on the NBT-reducing activity of the human promyelocytic leukemia HL-60 cells. HL-60 cells (1x10^ cells/ml) were treated with (A) solvent control (0.04% ethanol); (B) 5 [iM of Sophoraflavanone G; (C) 10 ^iM of Sophoraflavanone G for 72 hours at 37^C. The 1:1 (v/v) mixture of cell suspension (2x10^ cells/lOOp,!) was incubated with freshly prepared NBT-PMA solution for 4 hours at 37°C in the dark. The cells were then cytocentrifuged onto microscopic slides and counter-stained with 1% safranin in methanol as described in Chapter Two. The cells were examined by light microscopy (400x magnification). Differentiated cells will be stained in dark brown color, due to formation of intracellular dark blue diformazan, instead of red color. -139 - Chapter 4 Apoptosis- and Differentiation-inducing Effects 4.3 Discussion In addition to proliferating independently of growth signals, leukemia cells are characterized as capable of avoiding appropriate cell death and fail to differentiate into functional and mature cells. Most of the strategies for cancer therapy are targeting on these three aberrant characteristics. In Chapter Three, Sophoraflavanone G was shown to exhibit potent anti-proliferative effect on human promyelocytic leukemia HL-60 cells via cell cycle arrest at GQ/GI phase. Previous studies in our laboratories had found that Sophoraflavanone G could inhibit the growth as well as inducing apoptosis in the human hepatocellular carcinoma HepG2 cells (Tsang, 2006). In addition, both cell cycle arrest and apoptosis are known to occur in a simultaneous or sequential manner (Shapiro, 2006; Blagosklonny, 2007). Moreover, it has been reported that genistein, also a member of flavonoids, could trigger HL-60 cells differentiation (Takahashi et al, 1998). Therefore, investigation on whether Sophoraflavanone G could induce apoptosis and/or cell differentiation of human myeloid leukemia HL-60 cells was performed. The induction of apoptosis is accompanied by a number of biochemical changes, such as DNA fragmentation and phosphatidylserine (PS) extemalization. In order to examine the hypothesis that Sophoraflavanone G could induce apoptosis, DNA fragmentation assay and phosphatidylserine extemalization analysis were initially carried out. In DNA fragmentation assay, as detected by agarose gel electrophoresis, it was observed that 24 hour treatment of HL-60 cells with 10 to 30 |iM of Sophoraflavanone G triggered the formation of "DNA ladder" in a dose-dependent manner, which is a characteristic of DNA fragments in multiples of 180-200 base pairs. The apoptosis-inducing ability of Sophoraflavanone G was further confirmed by PS extemalization analysis, which was performed by Annexin V-GFP and PI -140 - Chapter 4 Apoptosis- and Differentiation-inducing Effects double staining. The co-staining method helps to differentiate and quantify the early apoptotic cells from total cell population. The data showed that the proportion of HL-60 cells in early apoptotic stage was increased from 3% in solvent-treated cells to 22% in cells treated with 30 |_LM of Sophoraflavanone G for 12 hours. This finding is in line with a previous report showing that four lavandulylflavonoids isolated from Kushen, including Sophoraflavanone G, could induce DNA fragmentation in the HL-60 cells as detected by gel electrophoresis and sandwich enzyme immunoassay (Ko et al, 2000). Nevertheless, the underlying action mechanisms for the apoptosis-inducing activity of Sophoraflavanone G have not been studied. To further confirm the apoptosis-inducing ability of Sophoraflavanone G in HL-60 cells, the activities of caspases were measured. It has been well documented that apoptosis triggered by "death" signals will culminate in the activation of caspases which will further cleave apoptotic proteins and hence induce the final stages of cell death (Schimmer, 2008). Multiple pathways are known to lead to caspase activation. These pathways can be divided into two main pathways: extrinsic pathway, which responds mainly to extracellular stimuli and is mediated by caspase-8, and the other intrinsic pathway, which is mainly initiated by modulators within the cell itself and subsequently mediated by caspase-9 (Russo et al., 2006). Activation of upstream "initiator" caspase-8 or -9 lead to further activation of the downstream "executioner" caspase-3 (Schimmer, 2008). Therefore, in the present study, the activities of caspase-3, -8 and -9 were measured using specific fluorometric substrates. The results demonstrated that, after 24 hours of incubation with Sophoraflavanone G，the activities of caspase-3 and -9 were significantly increased in the HL-60 cells in a dose-dependent manner, whereas the caspase-8 activity was only significantly induced at a higher concentration of -141 - Chapter 4 Apoptosis- and Differentiation-inducing Effects Sophoraflavanone G (i.e. 50 \iM). These findings suggest that Sophoraflavanone G at lower concentrations may trigger caspase-dependent apoptosis in the HL-60 cells mainly through the intrinsic pathway, although activation of caspase-8, mediator of extrinsic pathway, was also observed when HL-60 cells were treated with higher concentrations of Sophoraflavanone G. In the intrinsic pathway, the activation of caspase-9 is triggered by upstream apoptotic proteins released from damaged mitochondria and the release of such mitochondrial intermembrane space proteins results from collapse of the mitochondrial membrane potential (Bratton et al, 2001). Therefore, the change of mitochondrial transmembrane potential (A\|/m) was monitored by JC-1 staining in the present study. The results suggest that Sophoraflavanone G may induce mitochondrial membrane depolarization in HL-60 cells in a time- and dose-dependent manner. These findings confirmed our speculation that Sophoraflavanone G may activate the intrinsic apoptotic pathway in HL-60 cells. The intrinsic pathway, also known as mitochondria-mediated death pathway, is initiated by damage to the mitochondria which results in the release of mitochondrial intermembrane space proteins into cytoplasm, including cytochrome c, apoptosis inducing factor (AIF), and endonuclease G. When released into cytoplasm, cytochrome c binds to an adaptor protein Apaf-1 and this complex further recruits procaspase-9 to form an apoptosome. The formation of apoptosome leads to autocatalytic activation of procaspase-9 to caspase-9 and subsequently activates the downstream "executioner" caspase-3. During apoptotic execution, activated caspase-3 is able to cleave a number of critical proteins to inhibit survival pathways as well as promote death activities. Poly(ADP-ribose) polymerase (PARP), one of the caspase-3 substrates, is responsible for DNA repair, transcription and chromosomal -142 - Chapter 4 Apoptosis- and Differentiation-inducing Effects Stability, which are important for cell survival. However, cleavage of PARP caused by caspase-3 during apoptosis leads to stop any pro-survival functions of PARP. Moreover, caspase-activated DNase (CAD), also known as caspase-activated nuclease, is one of the death-promoting factors. CAD resides in cytosol and is normally inactivated by binding to its inhibitor iCAD. During apoptosis, activated caspase-3 cleaves the iCAD to release active CAD which then enters the nucleus to degrade chromosomal DNA into fragments (Enari et al., 1998; Tang and Kidd, 1998). To get a more complete picture on the downstream intrinsic pathway, more studies should be carried out to investigate the differential expression of apoptotic regulatory proteins. When look back to the upstream pathway, it has been reported that the disruptions leading to mitochondrial membrane depolarization involve the mitochondrial membrane permeability transition pore (PTP). This mitochondrial PTP is regulated by Bcl-2 family proteins, such as Bcl-2, Bcl-xL, Bax and Bak, through the interaction of the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT) (Jeong et al, 2008). Bax and Bak, the pro-apoptotic members, will physically interact with one another and form the homo- and/or hetero-oligomers which can move onto the mitochondrial outer membrane and cause its permeabilization. Anti-apoptotic members Bcl-2 and Bcl-xL can block the function of Bax and Bak by binding to and antagonizing Bax and Bak (Belizario et al, 2007; Han et al.’ 2008). The interaction between anti-apoptotic members and Bax/Bak is reported to be associated with "BH3 (Bcl-2 homology 3)-only proteins", another subgroup of Bcl-2 family pro-apoptotic members. The BIB-only proteins, such as Bim, Bid, Bad, Noxa, and PUMA, respond to different forms of cellular stress, resulting in activation of Bax and Bak. BH3-only proteins can bind to Bcl-2 -143 - Chapter 4 Apoptosis- and Differentiation-inducing Effects anti-apoptotic proteins, releasing Bax and Bak to promote cell death. Additionally, it is suggested that certain BH3-only proteins can also bind and activate Bax and Bak after their dissociation from anti-apoptotic proteins (Roderick and Cook, 2008). Therefore, in order to elucidate the possible involvement of Bcl-2 family in Sophoraflavanone G-induced apoptosis, the expression level of several proteins in Bcl-2 family, including Bax, Bak and Bcl-2, was first examined. It was found that the levels of pro-apoptotic proteins Bax and Bak were up-regulated while the level of anti-apoptotic protein Bcl-2 was down-regulated. However, more studies should be performed on other Bcl-2 family proteins so as to elucidate the detailed mechanisms modulated by the Bcl-2 family proteins in Sophoraflavanone G-induced apoptosis of HL-60 cells. The disruption of mitochondria transmembrane potential, modulated by Bcl-2 family proteins, is usually associated with reactive oxygen species (ROS) overproduction and the onset of oxidative stress (Giovannini et al., 2007). ROS is a collective term for a variety of molecules and free radicals (chemical species with one unpaired electron) such as superoxide anion (O2,), hydrogen peroxide (H2O2) and hydroxy 1 radical (OH"). Among these free radicals, superoxide anion (O2 ') is the precursor of most ROS (Martindale et al, 2002; Turrens, 2003). On the other hand, Sophoraflavanone G, as a member of flavonoids, was reported to be a potent antioxidant which is able to scavenge the ROS (Piao et al., 2006; Jung et al,, 2008). It seems to be paradoxical that some of flavonoids can act as an antioxidant releasing the intracellular oxidative stress and show potent pro-apoptotic effect at the same time. Giovannini et al (2007) suggested that the actions of flavonoids exerting in the cancer cells depend on the concentration of flavonoids and the stage of the degenerative progress (such as oxidative stress). Under certain experimental -144 - Chapter 4 Apoptosis- and Differentiation-inducing Effects conditions, flavonoids can paradoxically have pro-oxidant effects and generate ROS. It has been observed that low or high concentration of the same flavonoid compound is responsible for antioxidant and pro-oxidant activity, respectively (Giovannini et al., 2007; Vailo et al, 2007). Apart from having the role of antioxidants, it is known that flavonoids, even in low concentration, can directly interact with specific proteins responsible for the regulation of apoptotic signaling and thus lead to the apoptotic cascade in the cancer cells (Giovannini et al, 2007). In agreement with these speculations, our study found that the intracellular level of superoxide anion (O2,) in the HL-60 cells was markedly enhanced only at a higher concentration of Sophoraflavanone G (i.e. 40 |IM) after 24 hours and 48 hours of incubation. However, whether the loss of membrane integrity is a direct result of mitochondrial membrane permeability transition pore (PTP) formed by pro-apoptotic Bcl-2 family members or due to ROS destroying membrane components remains to be determined. Growing evidence has shown that, apart from the oxidative stress, Ca^"^ as an initiator or mediator in various apoptotic signaling pathways, plays an important role in the activation and execution of cell death (Belizario et al., 2007; Roderick and Cook, 2008). Ca2+ is mainly stored in the endoplasmic reticulum (ER). Increase of intracellular Ca^^ level has been observed as one of the important events during apoptotic cell death (Roderick and Cook, 2008). Furthermore, it has been demonstrated that the flow of Ca^^ released through inositol-1,4,5-triphosphate receptor (IP3R) of ER can cause mitochondrial permeability transition and further activate the apoptotic cascade (Hanson et al., 2004). Interestingly, our results show that the intracellular Ca^"^ level in Sophoraflavanone G-treated HL-60 cells is higher in the 24 hour treatment than in the 48 hour treatment, which may indicate that Ca^"^ release into cytosol is an early event during apoptosis induced by Sophoraflavanone -145 - Chapter 4 Apoptosis- and Differentiation-inducing Effects G. However, more extensive studies, such as monitoring the intracellular Ca^^ level at different time points and investigating the activities of InsPs receptor, should be carried out so as to elucidate the possible role of Ca^"^ in the apoptotic process induced by Sophoraflavanone G. Apart from induction of apoptosis as a strategy of leukemia therapy, differentiation therapy has become another novel and targeted approach to leukemia treatment (Leung et al., 2005). Normally, hematopoietic stem cells (HSC) are programmed to differentiate into restricted cell lineage pathways of blood cell development through regulation of certain external growth factors that keep balancing cell growth, differentiation and apoptosis in the bone marrow (Tsifsoglou et al., 2003). In leukemia cells, early hematopoietic progenitors fail to differentiate, leading to an accumulation of immature blast cells in the bone marrow and in peripheral blood (Tsifsoglou et al., 2003; Leung et al, 2005). With the treatment of different biological or chemical inducing agents, differentiation therapy induces terminal differentiation of neoplastic cells with reduced malignancy (Leung et al., 2005). Previous studies demonstrated that the human promyelocytic leukemia HL-60 cell line was able to be induced to differentiate along the monocytic and granulocytic lineages by various inducers, such as vitamin D3 and ATRA. Therefore, in the present study, the HL-60 cell line was chosen as an in vitro model for examining the differentiation-inducing effect of Sophoraflavanone G. Our preliminary results indicate that Sophoraflavanone G is unable to trigger terminal differentiation of HL-60 cells under the prescribed experimental conditions, as judged by morphological analysis on cytospin preparations of Sophoraflavanone G-treated HL-60 cells and functional assessment of differentiation by the nitroblue tetrazolium (NBT) reduction assay. Morphological studies showed that -146 - Chapter 4 Apoptosis- and Differentiation-inducing Effects Sophoraflavanone G did not increase the vacuolation and ratio of cytoplasm to nucleus in the HL-60 cells even after 72 hours of treatment. This morphological finding suggests that the treated leukemia cells fail to differentiate into the more mature monocytic or granulocytic lineage cells. In addition, when HL-60 cells undergo mature differentiation along the granulocytic or monocytic linages, the conversion of oxygen to superoxide anions will markedly increase upon exposure to phorbol myristate acetate (PMA), owing to induction of respiratory burst activity (Collins, 1987)，and the production of superoxide anions can be monitored by Nitroblue tetrazolium (NBT). In our study, it was observed that, Sophoraflavanone G failed to enhance the NBT-reducing activity of HL-60 cells under the prescribed experimental conditions. It has been reported that with lower concentrations and longer treatment periods, some members of the flavonoids family, such as genistein, quercetin，and apigenin, can trigger differentiation in HL-60 cells (Takahashi et al., 1998; Kawaii et al, 1999). Therefore, further studies of the possible differentiation-inducing effect of Sophoraflavanone G should be performed by using lower concentrations of the compound and longer incubation periods so as to clarify whether Sophoraflavanone G can trigger leukemic cell differentiation. In conclusion, our results in Chapter Four suggest that Sophoraflavanone G can trigger apoptosis mainly through the intrinsic pathway in the human promyelocytic leukemia HL-60 cells. Ca^^ and ROS may also act as an apoptotic initiator and/or mediator in the Sophoraflavanone G-induced apoptosis in the HL-60 cells. On the other hand, induction of myeloid leukemic cell differentiation is unlikely to be responsible for the observed anti-proliferative effect of Sophoraflavanone G on the HL-60 cells. -147 - Chapter Five Conclusions and Future Perspectives Chapter 5 Conclusions and Future Perspectives Sophoraflavanone G, also known as 5,7,2',4'-tetrahydroxy-8-lavandulyl- flavanone, was firstly reported by Shirataki and his colleagues in 1988. This compound belongs to the lavandulylflavonoids family which is found to be enriched in Sophorae Radix {Kushen). In nature, members of this family mainly function as phytoalexins which protect the host plants from attack by parasites such as insect pests, fungi, bacteria and nematodes (Rao, 1990). Laboratory studies have provided growing evidence that certain members of this lavandulylflavonoids family exert pleiotropic bioactivities including anti-inflammatory, antioxidant, anti-tumor and tyrosinase-inhibitory activities (Ko et al., 2000; Chi et al, 2001; Son et al, 2003; Jung et al, 2008). Among all these bioactivities, the anti-tumor activities have received much attention. Previous studies on bioassay-guided isolation of bioactive compounds from Kushen against human hepatocellular carcinoma showed that Sophoraflavanone G exhibited the most potent anti-proliferative effect on human hepatoma HepG2 cells with the highest yield among the six bioactive flavonoid compounds purified from Kushen (Tsang, 2006). Apart from human hepatoma HepG2 cells, Sophoraflavanone G was also reported to have potent in vitro growth-inhibitory effects on a variety of human tumor cell lines such as human skin melanoma SK-MEL-2 cells, human colorectal carcinoma HCT-15 cells, human lung carcinoma A549 cells, human ovarian cancer SK-OV-3 cells, and human acute leukemia HL-60 cells (Ryu et al, 1997; Ko et al, 2000). Nevertheless, the modulatory effects of Sophoraflavanone G on human myeloid leukemia cells and its possible anti-leukemic mechanisms are still unclear. Sophoraflavanone G was successfully purified from Sophora flavescens by our previous labmate Ms. Tsang Kit Man (Tsang, 2006). In the beginning of this study, further characterization of this purified compound was carried out by Mass -148- Chapter 5 Conclusions and Future Perspectives Spectrometry (MS), Nuclear Magnetic Resonance (^H-NMR and ^^C-NMR) and High Performance Liquid Chromatography (HPLC). By direct comparison of our spectral data (MS, ^H-NMR, and ^^C-NMR) with the published values (Shirataki et al, 1988), the isolated compound, with an estimated purity of 93% as measured by HPLC, was identified as Sophoraflavanone G, i.e. 5,7,2',4'-tetrahydroxy-8-1 avanduly 1 -flavanone (chemical formula: C25H28O6). The modulatory effects of Sophoraflavanone G on human myeloid leukemia cells and the possible underlying anti-leukemic mechanisms were the major focus of the present study. As shown in Chapter Three, Sophoraflavanone G could inhibit the proliferation of human acute promyelocytic leukemia HL-60 cells in a dose- and time-dependent manner. The observed growth-inhibitory effect of Sophoraflavanone G was not tumor cell line specific, as similar anti-proliferative effect of Sophoraflavanone G was also demonstrated in other human myeloid leukemia cell lines, including EoL-1, K-562 and NB-4. On the other hand, our results also indicated that Sophoraflavanone G exhibited minimal, if any, in vitro cytotoxicity on human peripheral leukocytes and murine bone marrow cells at its growth-inhibitory concentrations for the leukemia HL-60 cells. Moreover, the observed anti-proliferative activity of Sophoraflavanone G was most likely to be caused by its cytostatic rather than direct cytotoxic effect on HL-60 cells as suggested by the trypan blue exclusion assay. In order to elucidate the mechanisms by which Sophoraflavanone G could inhibit the in vitro proliferation of HL-60 cells, the effects of Sophoraflavanone G on the cell cycle progression, the induction of apoptosis and differentiation in the HL-60 cells were investigated. Our findings revealed that Sophoraflavanone G could arrest the cell cycle progression at the GQ/GI phase. Cell cycle is tightly regulated by a network of cell cycle regulatory protein complexes, which include cyclins and -149- Chapter 5 Conclusions and Future Perspectives cydin-dependent kinases (CDK). The main cyclin/CDK complexes responsible for Gi/S checkpoint are cyclin A/CDK-1 or -2，cyclin D/CDK-4 or -6, and cyclin E/CDK-2 (Quereda et al, 2007). Using the technique of Western blotting analysis, Sophoraflavanone G was found to decrease the expression level of CDK-6, which may affect the assembly of the cyclin D/CDk-6 complex and thus results in the Gi arrest. Furthermore, the activities of CDK are also regulated by various cyclin-dependent kinase inhibitors (CKI), which are grouped into two families: the INK4 inhibitors (pl5腿朴，pis�脳 a,pigiNMc ^^ pi9腿4d) ^^ the Cip/Kip inhibitors and pST^^'^^) (Quereda et al.’ 2007). Therefore, more detailed molecular studies on other cyclin/CDK complexes and their corresponding CDK inhibitors should be carried out so as to provide deeper insights on the underlying mechanisms of cell cycle arrest induced by Sophoraflavanone G. Since Sophoraflavanone G has shown to exhibit anti-proliferative effect and modulate the cell cycle profile in the HL-60 cells in vitro, therefore, the suppressive effect of Sophoraflavanone G on the tumorigenicity of the HL-60 cells in BALB/c nude mice was also examined. The results show that pretreatment of HL-60 cells with Sophoraflavanone G in vitro could significantly inhibit the in vivo growth of leukemia cells in nude mice. Nonetheless, more studies should be performed to evaluate the in vivo potency of Sophoraflavanone G on prevention of tumor formation and/or inhibition of established tumor growth in leukemia-bearing mice. Moreover, development of multidrug resistance (MDR) remains to be a major problem encountered in the chemotherapeutic therapy against cancer (Stavrovskaya, 2000; Ullah, 2008). The applications of natural products as potential anti-MDR molecules have received growing attention since many of these are expected to have fewer side effects and may possibly represent a new generation of MDR modulators -150- Chapter 5 Conclusions and Future Perspectives (Ullah, 2008). In order to mimic the situation of MDR in vitro, the anti-leukemic effect of Sophoraflavanone G on HL-60/MX2 cells, a well-characterized multidrug resistant leukemia cell model (Harker et al., 1991)，was also investigated in Chapter Three. Interestingly, Sophoraflavanone G was found to inhibit the growth of the HL-60/MX2 cells in a dose- and time-dependent manner, as well as triggering cell cycle arrest at the GQ/GI phase, which are similar to its parental cell line HL-60 cells. In addition, since the resistance of HL-60/MX2 cells is caused by reduced expression of DNA topoisomerase II (3 isoform (Harker et al, 1991)，the susceptibility of HL-60/MX2 to Sophoraflavanone G suggests that topoisomerase II may not be the cellular target for the action of Sophoraflavanone G. However, whether Sophoraflavanone G could be a potential MDR modulator still requires further studies and pinpointing the cellular target of Sophoraflavanone G in the HL-60/MX2 cells is an intriguing aspect that is worthy of further investigation. The induction of cell cycle arrest could be one of the possible mechanisms responsible for the observed anti-proliferative activity of Sophoraflavanone G on human myeloid leukemia cells. Apart from proliferating independently of growth signals, tumor cells are also characterized as being capable of avoiding apoptosis which leads to cancer cell death. Consequently, induction of apoptosis in tumor cells has become an attractive strategy and further leads to the recent success stories in cancer therapy (Roderick and Cook, 2008). In this regard, a series of bioassays which measure different aspects of apoptosis were carried out to investigate the apoptosis-inducing activity of Sophoraflavanone G and the possible underlying action mechanisms. Our results clearly demonstrated that Sophoraflavanone G was able to induce apoptosis in the HL-60 cells mainly through mitochondria-mediated death pathway (i.e. intrinsic pathway) in a dose-response maimer, as evidenced by (1) -151 - Chapter 5 Conclusions and Future Perspectives induction of DNA fragmentation; (2) phosphatidylserine (PS) externalization; (3) activation of caspase-9; (4) mitochondrial membrane depolarization; and (5) induction of imbalance in the expression of Bcl-2 family members between the anti-apoptotic and pro-apoptotic proteins. Based on the results so far obtained, a hypothetical model can be proposed to illustrate how Sophoraflavanone G may exert its apoptotic activity on the HL-60 cells. As shown in Figure 5.1, Sophoraflavanone G can activate the apoptosis intrinsic pathway by up-regulating the pro-apoptotic Bcl-2 family members Bax and Bak proteins, and down-regulating the anti-apoptotic member Bcl-2 protein. Leaning to the apoptosis-promoting signal, the imbalance between the pro-apoptotic and the anti-apoptotic Bcl-2 family of proteins further induces the formation of mitochondrial membrane permeability transition pore (FTP). The formation of PTP leads to the mitochondrial membrane depolarization which triggers the release of cytochrome c from the mitochondrial intermembrane space into the cytoplasm. Nevertheless, whether Sophoraflavanone G can induce the release of cytochrome c awaits further investigation. Cytochrome c binds to an adaptor protein Apaf-1 and this complex further recruits procaspase-9 to form an apoptosome. The formation of apoptosome triggers autocatalytic activation of procaspase-9 to caspase-9 and then further activates the downstream "executioner" caspase-3. Subsequently, activated caspase-3 signals the downstream apoptotic pathways by cleaving a number of critical proteins and ultimately leads to the final stages of cell death. Moreover, Ca^"^ may also play a role as an apoptotic initiator and/or mediator in the Sophoraflavanone G-induced apoptosis. On the other hand, high concentration of Sophoraflavanone G can activate the extrinsic pathway "initiator" caspase-8 and enhance the level of intracellular ROS. However, the effect of Sophoraflavanone G -152- Chapter 5 Conclusions and Future Perspectives on upstream signaling molecules of caspase-8, such as the death receptor Fas and adaptor molecule FADD，should be investigated so as to delineate the role of Sophoraflavanone G in the activation of the extrinsic apoptotic pathway. Apart from induction of apoptosis, stimulation of differentiation has become another novel and targeted strategy for leukemia therapy. With the availability of a wide variety of differentiation-inducing agents, differentiation therapy will lead to the terminal differentiation of neoplastic cells resulting in reduced malignancy (Leung et al., 2005). Our preliminary results demonstrated that Sophoraflavanone G is unable to trigger terminal differentiation of HL-60 cells under the prescribed experimental conditions, as judged by morphological analysis on cytospin preparations of Sophoraflavanone G-treated HL-60 cells and functional assessment of differentiation by the nitroblue tetrazolium (NBT) reduction assay. Further confirmation of our results should be carried out by studying the expression of differentiation antigens on the surface of Sophoraflavanone G-treated HL-60 cells using immunophenotyping and flow cytometry. In conclusion, our results show that Sophoraflavanone G can inhibit the growth of human myeloid leukemia HL-60 cells in vitro, possibly through triggering cell cycle arrest at the GQ/GI phase and by inducing mitochondria-mediated apoptosis. Therefore, Sophoraflavanone G, as a naturally occurring compound, might have the potential to be exploited as a therapeutic agent against certain forms of human leukemia. However, it is a long way to go and many problems need to be solved in the therapeutic development of Sophoraflavanone G as an effective anti-leukemic agent. It is obvious that more in-depth investigations are required to reveal the molecular mechanisms and signaling pathways by which Sophoraflavanone G can modulate the proliferation and apoptosis of human myeloid leukemia cells. -153 - Chapter 5 Conclusions and Future Perspectives Additionally, more in vivo evidences should be provided on the safety and efficacy of using Sophoraflavanone G as an anti-leukemic agent in animal models, which is essential before Sophoraflavanone G can be used for the clinical trials in leukemia therapy. -154- Chapter 5 Conclusions and Future Perspectives Sophoraflavanone G \ Extrinsic Intrinsic pathway ^^ pathway Other Stress Death ligands Death receptors (TNF Rl, Fas, Cell UT U Trail Rl, Trail R2) membrane “1/\ 丄 — \r � U ( ( Mitochondria ) ” ^ \ BCI-2 ~1 ^ Bax/Bak Oxidative Stress \ Cytochrome c ROS \ \ i C^Caspase^^ 各 Cytoplasmic & nuclear death substrates wmm Fig. 5.1 Proposed apoptotic pathways in the human promyelocytic leukemia HL-60 cells induced by Sophoraflavanone G. -155- References References American Cancer Society. 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