Suramin As a Chemo- and Radio-Sensitizer

SURAMIN AS A CHEMO- AND RADIO-SENSITIZER:

PRECLINICAL TRANSLATIONAL STUDIES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Yan Xin, M.S.

*****

The Ohio State University 2006

Dissertation Committee:

Dr. M. Guillaume Wientjes, Adviser Approved by

Dr. Jessie L.-S. Au ______Adviser Dr. Duxin Sun Graduate Program in Pharmacy

ABSTRACT

Previous studies in our laboratory showed that low-dose suramin, an inhibitor of fibroblast growth factor action, enhances sensitivity of various human tumors in preclinical and clinical studies to chemotherapy. Chemosensitization required apoptosis, and increased the extent and duration of the induction of apoptosis. The primary focus of this dissertation research was to explore, in preclinical studies, therapeutic approaches for therapy enhancement based on this mechanism.

A phase III clinical trial in superficial bladder cancer, which emanated from our laboratory, showed that optimizing mitomycin C delivery nearly doubled the recurrence- free survival of treated patients to 40%. Tumor sensitization with suramin might yield further therapeutic improvements, and was investigated in in vitro and in vivo studies. Studies in Chapter 2 demonstrated enhanced antitumor activity of mitomycin C, administered at subtherapeutic and therapeutic regimens.

Various preclinical and clinical studies determined that suramin sensitized tumor tissue at low but not at high concentrations, presumably due to additional pharmacologic effects at elevated concentrations. Studies to overcome this limitation, especially in tumors containing high fibroblast growth factor concentrations, used pentosan polysulfate, another nonspecific FGF inhibitor. This agent was also a chemosensitizer, but combined use with suramin did not increase the overall efficacy (Chapter 3).

Radiotherapy depends on induction of apoptosis for its anticancer effect, as is the case for many forms of chemotherapy, and led to our evaluation of suramin as radiosensitizer. Results in Chapter 4 and Chapter 5 showed that low-dose suramin sensitized the radiation response of both radiosensitive (prostate PC-3) and relatively radioresistant (pharynx FaDu, pancreatic Hs 766T) xenograft tumors, thereby further extending the clinical application of suramin to modulate radiotherapy. More importantly, the radiosensitizaiton effect of suramin had tumor type specific dose dependence, with a narrow sensitiziting window in FaDu and a wider range in Hs 766T (pancreatic) xenograft tumors (Chapter 5), suggesting the necessity of drug target-specified dosing regimen for suramin. To better understand the biphasic effect of suramin in combination with radiation, pharamacodynamic studies at a microscopic level were conducted

(Chapter 6). The results indicated that low-dose but not high-dose suramin enhanced radiation-induced apoptosis, inhibiting radiation-induced upregulation of phospho-ERK and survivin, two survival signals involved in radioresistance.

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DEDICATION

Dedicated to my dear parents, my brothers, sisters-in-law, and nephews

ACKNOWLEDGMENT

I wish to express my sincere appreciation to my advisor, Dr. M. Guillaume

Wientjes, who has made this dissertation come true. His scientific enthusiasm, integrity and dedication have been inspiring me throughout my graduate study. His patience and encouragement helped me through the frustrated moments. He is a great mentor and friend.

My deep appreciation also goes to Dr. Jessie L-S. Au, for all her invaluable contribution to my overall scientific training and growth, as well as her kind friendship in daily life. Being a successful scientist, a wife, and a mother, she is definitely an amazing role model for me.

Special thanks go to Dr. Yong Wei for his invaluable scientific suggestions, discussions, and input into my thesis work. His enthusiasm and dedication to science have greatly inspired me. I appreciate his encouragement and friendship.

A special recognition is extended to our collaborators, Dr. John C. Grecula and Dr.

Nilendu Gupta, in the College of Medicine and Public Health. It is their dedication that made our cooperation productive.

Everyone in the lab has played a special role in my training. I feel really grateful to have such an enjoyable environment to finish the most important training in my career.

I want to thank each of them for the sweet memorable days. In particular, my sincere

gratitude goes to Dr. Yuebo Gan and Dr. SaeHeum Song, who walked me through the

starting stage in this laboratory. I would also like to recognize the following individuals

for their direct experimental contribution to my thesis: Dr. Ling (Lucy) Chen for the

collaboration in phospho-ERK and survivin staining and discussion in pathology, Dr.

Greg Lyness for the coopperation of patient tissue culture, Jianning Yang for the help

with PK modeling, Tong Shen for assisting me with the suramin concentration analysis,

Dr. Ling Chen and Adrianne Lovelace for the help of tissue sectioning. In addition, I

want to express my gratitude to the following labmates for their friendship and help: Dr.

Xiao (Shelley) Hu, Dr. Jie (Jack) Wang, Dr. Ze Lu, Bei Yu, Dan Lu, Jing Li, Jia Ji, Dr.

Leijun Hu, Dr. Colin Walsh, Dr. Danny Chen, Dr. Liang Zhao, Dr. Mingjie Liu, and Dr.

Zancong Shen. My appreciation also goes to Kathy Brooks, Sharon Palko, and Emily

Noble for their patient help during my study.

My sincere thanks also extend to my dear roommate, Xiaohui (Tracey) Wei, for her friendship and encouragements during the last few years. Thanks to all my other friends for their support during the whole training.

Last but not the least, my deepest appreciation goes to my parents, my brothers and sisters-in-law. Without their endless love and support, I would not be able to go through all the way here.

VITA

September 12, 1975 ...... Born – Hubei, P.R. China

July, 1998 ...... ….…...... B.S., Pharmacy School of Pharmacy Beijing Medical University

July, 2001 ...... ….…... M.S., Biochemical Engineerin Tsinghua University

September, 2001 – present .....………...... …...... Graduate Research Associate College of Pharmacy The Ohio State University

PUBLICATIONS Papers Xin Y., Lyness G., Chen D., Song S., Wientjes M.G., and Au J. L.-S., Low dose suramin as a chemosensitizer of bladder cancer to mitomycin C. J Urol. 2005 Jul;174(1):322-7.

Xin Y., Li Q., and Cao Z.. Investigation of glutamine metabolism in hybridoma cell C50. Chinese Journal of Biotechnology, Vol. 17(4), 2001.

Presentations Xin. Y., Grecula J.C., Gupta N., Wei Y., Chen L., Au J. L.-S., and Wientjes M.G., Pharmacodynamics of the chemosensitizer suramin in radiotherapy. Proceedings of the American Association for Cancer Research Annual Meeting 97, 2006. Xin Y., Chen D., Song S., Lyness G., Wientjes M.G., and Au J. L.-S., Low-dose suramin enhances antitumor activity of mitomycin C in bladder tumors. Proceedings of the American Association for Cancer Research Annual Meeting 95, 2004.

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FIELDS OF STUDY

Major field: Pharmacy

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TABLE OF CONTENTS

Page

ABSTRACT...... ii

ACKNOWLEDGMENT...... v

VITA...... vii

LIST OF TABLES...... xv

LIST OF FIGURES ...... xvii

Chapter 1 ...... 1

BACKGROUND INFORMATION ...... 1

1.1 Introduction...... 1

1.2 Chemoresistance and modulation strategies ...... 1

1.2.1 Chemoresistance ...... 1

1.2.2 Growth factor signaling and resistance to cancer chemotherapy...... 4

1.2.3 Other targeted therapies to modulate chemoresistance...... 8

1.3 Radioresistance and modulation strategies ...... 10

1.3.1 Overview...... 10

1.3.2 Combination of conventional cytotoxic agents with radiation ...... 11

1.3.3 Modulating radiotherapy with molecular targeted agents ...... 13

1.4 Modulation of hypoxia in chemo- and radiotherapy...... 17

1.5 Protection of normal tissue in cancer therapy...... 18

1.6 Suramin...... 19

1.7 Overview of dissertation...... 22

Chapter 2 ...... 25

LOW-DOSE SURAMIN AS A CHEMOSENSITIZER OF BLADDER CANCER TO

MITOMYCIN C ...... 25

2.1 Introduction...... 25

2.2 Materials and methods ...... 27

2.2.1 Cell and tumor cultures...... 27

2.2.2 Establishment of RT4 xenograft tumors...... 27

2.2.3 Drug activity evaluation in tumor histocultures...... 28

2.2.4 In vivo drug activity evaluation: animal treatment protocols...... 29

2.2.5 Statistical analysis...... 31

2.3 Results...... 31

2.3.1 Effects of suramin on MMC activity in histocultures of RT4 tumors ...... 31

2.3.2 Effect of suramin on MMC activity in histocultures of human tumors.... 32

2.3.3 Enhancement of in vivo MMC activity by suramin in RT4 xenograft

tumors: tumor size changes...... 33

2.3.4 Enhancement of in vivo MMC activity by suramin in RT4 xenograft

tumors: tumor histological data ...... 34

2.4 Discussion...... 34

2.5 Acknowledgements...... 37

Chapter 3 ...... 47

COMBINATION OF NON-SPECIFIC bFGF INHIBITOR AND PACLITAXEL IN

TREATMENT OF PANCREATIC XENOGRAFT TUMORS ...... 47

3.1 Introduction...... 47

3.2 Materials and methods ...... 52

3.2.1 Chemicals and reagents...... 52

3.2.2 Cell culture...... 52

3.2.3 Animal and drug treatment protocols ...... 53

3.2.4 Drug activity evaluation...... 54

3.2.5 Statistical analysis...... 56

3.3 Results...... 56

3.3.1 Enhancement of paclitaxel activity by PPS: short-term tumor size change .

...... 56

3.3.2 Enhancement of paclitaxel activity by PPS: overall survival ...... 57

3.3.3 Histological evaluation: proliferation and apoptosis ...... 58

3.3.4 Effects of drug treatment on animal body weights ...... 59

3.4 Discussion...... 59

Chapter 4 ...... 73

RADIOSENSITIZATION EFFECT OF SURAMIN IN PROSTATE AND PHARYNX

XENOGRAFT TUMORS...... 73

4.1 Introduction...... 73

4.2 Materials and methods ...... 76

4.2.1 Cell cultures...... 76

4.2.2 Establishment of xenograft tumors ...... 77

4.2.3 Animal treatment protocol: in vivo drug activity evaluation...... 77

4.2.3.1 PC-3 tumors ...... 77

4.2.3.2 FaDu tumors...... 80

4.2.4 Statistical Analysis...... 81

4.3 Results...... 81

4.3.1 Radiosensitization effect of low-dose suramin in PC-3 tumors...... 81

4.3.2 Dose-dependent radiosensitization effect of suramin in FaDu tumors..... 83

4.3.3 Toxicity of the treatments ...... 84

4.3 Discussion...... 85

Chapter 5 ...... 94

RADIOSENSITIZATION EFFECT OF SURAMIN IN PHARYNX AND

PANCREATIC XENOGRAFT TUMORS: DOSE DEPENDENCE AND

PHARMACOKINETICS...... 94

5.1 Introduction...... 94 xii

5.2 Materials and methods ...... 97

5.2.1 Chemicals and Reagents ...... 97

5.2.2 Apparatus ...... 97

5.2.3 Animal protocol: in vivo drug activity evaluation...... 97

5.2.4 Pharmacokinetic (PK) study of suramin...... 98

5.2.4.1 Sampling ...... 98

5.4.2.2 Plasma and tumor tissue extraction...... 99

5.2.4.3 HPLC analysis...... 100

5.2.4.4 Pharmacokinetic analysis...... 101

5.2.4.5 Pharmacokinetic/Pharmacodynamic (PK/PD) correlation ...... 101

5.2.5 Statistical Analysis...... 101

5.3 Results...... 102

5.3.1 Dose-dependent radiosensitization effect of suramin in FaDu tumors... 102

5.3.2 Dose-dependent radiosensitization effect of suramin in Hs 766T tumors....

...... 102

5.3.3 Tumor type specific dose dependence of suramin as radiosensitizer ..... 104

5.3.4 Suramin PK in plasma and tumor ...... 104

5.3.5 Pharmacokinetic/pharmacodynamic (PK/PD) correlation in FaDu tumors..

...... 106

5.4 Discussion...... 108

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Chapter 6 ...... 121

CELLULAR/MOLECULAR PHARMACODYNAMICS OF SURAMIN IN

COMBINATION WITH IONIZING RADIATION ...... 121

6.1 Introduction...... 121

6.2 Materials and methods ...... 125

6.2.1 Chemicals and reagents...... 125

6.2.2 Cell cultures and xenograft tumor establishments...... 125

6.2.3 Animal treatment protocol ...... 126

6.2.4 Histological evaluation...... 127

6.2.5 Statistical analysis...... 130

6.3 Results...... 130

6.3.1 Antiproliferation effect (BrdU labeling)...... 130

6.3.2 Apoptosis (M30 staining) ...... 131

6.3.3 bFGF induction by irradiation ...... 132

6.3.4 Phospho-ERK ...... 132

6.3.5 Survivin...... 133

6.3.6 Correlation analysis...... 133

6.4 Discussion...... 134

Chapter 7 ...... 150

PERSPECTIVES AND CONCLUSION...... 150

REFERENCES ...... 154 xiv

LIST OF TABLES

Table 2.1 Enhancement of in vitro MMC activity by low-dose suramin ...... 38

Table 2.2 Human patient tumor characteristics and effects of suramin on MMC activity39

Table 2.3 Effect of low-dose suramin on in vivo MMC activity on tumor size and body weight...... 40

Table 2.4 Enhancement of in vivo antitumor effect of MMC by suramin: histological evaluation...... 41

Table 3.1 Short-term effect of PPS/suramin on in vivo antitumor activity of paclitaxel: tumor size and body weight ...... 64

Table 3.2 Long-term effect of PPS/suramin on in vivo antitumor activity of paclitaxel: growth rate and survival...... 65

Table 3.3 Enhancement of antitumor effect of paclitaxel by PPS/Suramin (histological evaluation)...... 66

Table 4.1 Radiosensitization effect of suramin in PC-3 tumors ...... 88

Table 4.2 Dose-dependent radiosensitization effect of suramin in FaDu tumors...... 89

Table 4.3 Loss of radiosensitization in FaDu tumors with split-radiation dose: repeated doses of suramin ...... 90

Table 5.1 Dose-dependent radiosensitization effect of suramin in FaDu tumors...... 112

Table 5.2 Dose-dependent radiosensitization effect of suramin in Hs 766T tumors...... 113

Table 5.3 Tumor type specific dose dependence of suramin as a radiosensitizer ...... 114

Table 5.4 Plasma and tumor pharmacokinetic data of suramin...... 115

Table 5.5 Pharmacokinetic/pharmacodynamic (PK/PD) correlation in FaDu tumors ... 116

Table 6.1 Pharmacodynamics of suramin in combination with irradiation in FaDu tumors ...... 139

Table 6.2 Enhancement of antitumor effect of irradiation by suramin in PC-3 tumors (histological evaluation)...... 140

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LIST OF FIGURES

Figure 2.1 Enhancement of MMC activity in tumor histocultures by suramin: inhibition of BrdU incorporation in tumor cells...... 42

Figure 2.2 Enhancement of MMC-induced apoptosis in tumor histocultures by suramin: induction of apoptosis...... 43

Figure 2.3 Enhancement of in vivo antitumor activity of MMC by low-dose suramin: tumor size reduction...... 44

Figure 2.4 Enhancement of in vivo antiproliferation effect of MMC by low-dose suramin ...... 45

Figure 2.5 Effect of low-dose suramin on the apoptotic effect of MMC in vivo...... 46

Figure 3.1 Effects of paclitaxel, PPS, and suramin on tumor growth in pancreatic tumors ...... 67

Figure 3.2 Overall survival of mice treated with paclitaxel, PPS, and suramin (Kaplan- Meier plot) ...... 68

Figure 3.3 Tumor regrowth following treatment with paclitaxel, PPS, and suramin...... 69

Figure 3.4 In vivo antiproliferative activity of paclitaxel/PPS/suramin...... 70

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Figure 3.5 Enhancement of in vivo apoptotic activity of paclitaxel by PPS/suramin: TUNEL staining...... 71

Figure 3.6 Enhancement of in vivo apoptotic activity of paclitaxel by PPS/suramin: M30 staining...... 72

Figure 4.1 Suramin enhanced radiation response in PC-3 tumors...... 91

Figure 4.2 Dose-dependent radiosensitization effect of suramin in FaDu tumors ...... 92

Figure 4.3 Loss of radiosensitization effect in FaDu tumors with split-dose irradiation: repeated doses of suramin...... 93

Figure 5.1 Dose-dependent radiosensitization effect of suramin in FaDu tumors ...... 117

Figure 5.2 Dose-dependent radiosensitization effect of suramin in Hs 766T tumors: tumor growth curves...... 118

Figure 5.3 The five-compartment linear model used to fit the plasma and tumor concentration simultaneously...... 119

Figure 5.4 Measured and the best predictions of suramin concentrations in plasma and tumor...... 120

Figure 6.1 Antiproliferation effect of irradiation/suramin in FaDu tumors...... 141

Figure 6.2 Antiproliferation effect of irradiation/suramin in PC-3 tumors ...... 142

Figure 6.3 Apoptotic effect of radiation/suramin in FaDu tumors ...... 143

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Figure 6.4 Apoptotic effect of irradiation/suramin in PC-3 tumors...... 144

Figure 6.5 bFGF induction by irradiation in FaDu tumors...... 145

Figure 6.6 Effect of radiation/suramin on phospho-ERK level in FaDu tumor...... 146

Figure 6.7 Effect of radiation/suramin on survivin expression in FaDu tumors ...... 147

Figure 6.8 Changes of survivin expression in PC-3 tumors after irradiation ...... 148

Figure 6.9 Results of correlation analysis...... 149

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CHAPTER 1

BACKGROUND INFORMATION

1.1 Introduction

Cancer is a major public health problem in the United States and other developed countries. Currently, one in four deaths in the United States is due to cancer, accounting for the second leading cause of death besides heart disease (1). Chemotherapy, radiotherapy and surgery are the major three treatment modalities for cancer. Resistance to chemotherapy and radiotherapy remains a major problem in cancer therapy.

The work in this dissertation focuses on the investigation of developing suramin as a chemo- and radiosensitizer.

1.2 Chemoresistance and modulation strategies

1.2.1 Chemoresistance

Chemoresistance is a main obstacle to successful treatment. There are two general categories of mechanisms for chemoresistance: host factors and specific genetic or

epigenetic alterations in the cancer cells (2; 3). Host factors include: (a) poor absorption, rapid metabolism or excretion of drugs, which results in low serum drug levels; (b) poor tolerance to drug effect, thus resulting in dose reduction below optimal levels; (c) impaired drug delivery, as could occur with biological agents of high molecular weight and low tissue penetration (e.g. monoclonal antibodies and immunotoxins) (4); (d) alterations in the host-tumor environment that affect response of the tumor (5).

In the past 40 years, various mechanisms by which cancer cells in culture become resistant to anticancer drugs have been explored in experimental models generated by in vitro selection with cytotoxic agents. Some of these mechanisms only convey resistance to a small number of related drugs. For example, loss of a cell surface receptor or transporter for a drug, change in drug specific metabolism, or alteration of the specific target of a drug, as occurred for antifolates such as methotrexate (6). In such cases, combination of multiple drugs entering cells with different mechanisms and with different cellular targets will achieve effective chemotherapy and high cure rates (2).

However, very often, cells might also show cross-resistance to other structurally and mechanistically unrelated drugs after selection for resistance to a single drug, a phenomenon that is known as multidrug resistance (MDR) (7). Multidrug resistance can result from limited accumulation of drugs within cells by limiting uptake, enhancing efflux, or affecting membrane lipids such as ceramide (8).

Accumulated experimental data indicate that the major mechanism of multidrug resistance in cultured cancer cells was the expression of P-glycoprotein (P-gp) or the

multidrug transporter (9; 10). As one of 48 known ATP-binding cassette (ABC) transporters in the human, P-gp is an efflux pump encoded by the human MDR1 gene

(11). P-gp can detect and bind a large variety of hydrophobic natural-product drugs, such as doxorubicin, vinblastin, and taxol. Binding of these drugs leads to activation of one of the ATP-binding domains, and the hydrolysis of ATP causes a major conformational change of P-gp, which ultimately results in the release the drug into the extracellular space (12). Other ABC members such as MRP1 (multidrug resistance associated protein

1), MXR, BCRP (breast cancer resistance protein), or LRP (lung resistance-related protein) may also associate with cancer drug resistance (3).

Although there are lines of evidence that P-gp and other transporter proteins are involved in clinical drug resistance (13-15), modulation of MDR by competitive inhibition of drug binding to P-gp or MRP1, or by changing the conformation of transport proteins, have not yielded any clinical benefits to improve chemotherapy for most solid tumors (16). The effect of MDR modulation has been limited by multiple and redundant cellular mechanisms of resistance, pharmacokinetic interactions with chemotherapy and undesired toxicities (17).

With the increasing knowledge in molecular processes and signaling pathways in cancer development and drug response, more interests have been attracted to cellular chemoresistance mechanisms in clinical settings.

1.2.2 Growth factor signaling and resistance to cancer chemotherapy

Neoplastic transformation is frequently associated with concomitant enhancement

of growth factor signaling, which conveys growth and survival advantages to transformed

cells. The enhanced growth factor signaling mainly result from increased expression of

growth factors and their receptors, and activating mutations of growth factor receptors

(18).

Strong evidence supports an important role of growth factors and their signaling pathways in chemosensitivity and –resistance. Growth factor signaling triggers chemoresistance probably by multiple mechanisms, including apoptosis suppression (19) and altered expression of various genes that affect cytotoxic drug potency, such as P-gp

(20). Since apoptosis is the major mode of cell death for a majority of chemotherapeutic drugs, its suppression by growth factor signaling could be a main mechanism of multidrug resistance. For instance, vascular endothelial growth factor (VEGF) was shown to induce survivin expression synergistically with basic fibroblast growth factor (bFGF) via

signaling through the PI3K/Akt pathway and protect endothelial cells against drug-

induced apoptosis (21). Other inhibitors of apoptosis, such as members of the Bcl-2 and

inhibitor of apoptosis protein (IAP) families, are also regulated by ERK and Akt

mediated NF-κB activation (19). For example, Bcl-2, Bcl-XL, and Mcl-1 are upregulated

by MEK/ERK signaling, thus preventing apoptosis in human pancreatic cancer cells (22).

In addition to the proliferative and anti-apoptotic effects of growth factor signaling, the

downstream target gene MDR1 also contributes to chemoresistance (23). Acid and basic

FGF have been shown to induce epigenetic chemoresistance to tumor cells of metastatic rat prostate MAT-LyLu tumor (24).

Therefore, novel drugs have been designed to target growth factor signaling and

enhance chemosensitivity of tumor cells. These include antibodies directly interfering

growth factor-receptor interactions, or tyrosine kinase inhibitors inhibiting signaling

activity of activated growth factor receptors (reviewed by Dai, et al. (23)).

EGFR signaling The oncogenic potential of epidermal growth factor receptor

(EGFR) signaling has been well characterized. Receptors of the EGFR family include

EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4. Activation of EFGR signaling has been reported to promote tumorigenesis, including angiogenesis, cell survival, invasion, and metastasis. Two strategies have been developed to interfere the EGFR signaling: monoclonal antibodies blocking ligand binding and small molecular-weight tyrosine kinase inhibitors.

In preclincal studies, anti-EGFR monoclonal antibodies (e.g. IMC-C225

(cetuximab)), synergistically inhibits the growth of a variety of human xenografts in mice when combined with various anticancer drugs, such as CPT-11 and topotecan, doxorubicin, cisplatin, and paclitaxel. In clinical trials, the combination of IMC-C225 and cisplatin yielded superior objective response rates compared to either agent alone.

However, no increase in survival times was achieved in one phase III clinical trial (25).

The relatively selective tyrosine kinase inhibitor of EGFR, ZD1839 (IressaR), was reported to significantly increase cytotoxic drug potency in vitro and in vivo; but no

improvement in patient survival was achieved when combined with conventional

chemotherapeutic drugs. Since ZD1839 given alone has shown significant clinical

benefits in a subset of patients, it was approved in May 2003 for treatment of non-small cell lung carcinomas in late stage (26).

Another small molecule tyrosine kinase inhibitor of HER1/EGFR, Tarceva

(erlotinib), demonstrated increased survival in advanced non-small cell lung cancer

(NSCLC) and received regular approval as monotherapy for the treatment of patients

with locally advanced or metastatic NSCLC after failure of at least one prior

chemotherapy regimen in November 2004. Tarceva has also shown improved overall

survival when added to gemcitabin as initial treatment in pancreatic cancer in a phase III

trial (1-year survival increased from 19% to 23%). It was approved in November 2005 as

first-line treatment in combination with gemcitabine for locally advanced, inoperable or

metastatic pancreatic cancer (www.osip.com).

ErbB2 has also been associated with resistance to chemotherapy in breast cancer

cells (20). Anti-ErbB2 antibody HerceptinR (trastuzumab) was reported to have

chemosensitizing effects with different chemotherapeutic agents in both preclinical and

clinical settings (reviewed in (23)). It is the only FDA-approved therapeutic for HER2

protein overexpressing metastatic breast cancer. It is approved for first-line use in

combination with paclitaxel, and as a single agent for those who have received one or

more chemotherapy regimens (www.herceptin.com).

VEGF Although vascular endothelial growth factor (VEGF) has attracted

much attention due to its angiogenic effect, its role in chemoresistance needs further

investigation. Anti-VEGF monoclonal antibody bevacizumab in combination with

fluorouracil and leucovorin has been proven to be an active regimen as first-line

treatment for metastatic colorectal cancer (27). In phase II trials, bevacizumab enhances

the activity of gemcitabine in advanced pancreatic cancer patients (28) and the efficacy of

doxorubicin for patients with metastatic soft-tissue sarcomas (29). In addition,

simultaneous targeting VEGF and EGF signaling pathways, i.e., combined use of

bevacizumab and erlotinib, has shown encouraging antitumor activity and safety in a phase I/II trial for patients with recurrent NSCLC, supporting further development of this combination for patients with advanced NSCLC and other solid tumors (30). Substantial activity has been reported in initial clinical trials with VEGF-targeting agents (reviewed

in (31)).

Fibroblast growth factors (FGFs) Au and collaborators have found that elevated levels of acidic (aFGF) and basic (bFGF) fibroblast growth factors induce broad- spectrum resistance to chemotherapeutic drugs (24). Specific (bFGF antibody) and nonspecific (suramin) inhibitors enhance the antitumor activity of several chemotherapeutic agents in vitro and in vivo (32-34) (please see the section of suramin for more details). Several other groups have also found bFGF-mediated chemoresistance.

For example, bFGF has been reported to prevent etoposide-induced apoptosis in H-510

SCLC cells, by inducing translational regulation of Bcl-XL and Bcl-2 via a MEK-

dependent pathway (35) and transfection of bFGF in bladder cancer cells induces higher

resistance to cisplatin, probably through protection of cisplatin-induced apoptosis (36).

Other growth factors The activation of insulin-like growth factor 1 (IGF-1) signaling through its receptor (IGF-1R) may cause chemoresistance due to its anti- apoptotic function in solid tumors (37). Inhibitors of platelet-derived growth factor

(PDGF) receptor tyrosine kinase activity have been used in clinical trials such as SU101

and Gleevec, but more studies are needed to determine the role of PDGF signaling in chemoresistance. Brain-derived neurotropic factor (BDNF) signaling and other signaling

proteins interacting with growth factors such as integrins and cytokines may also

contribute to chemoresistance (reviewed in (23)).

Since chemosensitivity involves multiple growth factors and pathways that

contribute to overall response, the interaction between multiple growth factors and their

combined role in chemoresistance should be understood to predict a patient’s response

and to identify novel drug targets and regimens (23).

1.2.3 Other targeted therapies to modulate chemoresistance

In the last 10-15 years, the emphasis in search for new anticancer drugs has

shifted from the development of effective cytotoxic agents to the identification of small

molecules targeted to particular, (usually) protein targets (38). Targeted therapies

selectively restoring defective programs such as apoptosis or cellular senescence are

expected to improve the tumoricidal efficacy of conventional anticancer therapies. For

example, compounds that restore wild-type function in p53-mutant cells and antisense

oligonucleotides that degrade bcl2 transcripts are under investigation (reviewed in (39)).

Cyclooxygenase-2 (COX-2) is the enzyme that normally synthesizes

prostaglandins during an inflammatory response. It is considered a reasonable "target" for

cancer therapy because it is found in many epithelial tumors and its expression was

associated with poor prognosis (e.g. (40)), and is involved in many processes that

promote cancer progression and chemotherapy resistance (41). Combining chemotherapy

with selective Cox-2 inhibitors has shown very promising results in both preclinical and

clinical studies for different cancers. For example, docetaxel in combination with

cyclooxygenase-2 inhibitor celecoxib seems to prolong time to disease progression

compared to docetaxel alone for advanced NSCLC progressing after platinum-based

chemotherapy in a multicenter phase II trial (42).

Therapeutic targeting of ras via the inhibition of the covalent attachment of the farnesyl-isoprenoid group to the ras proteins has been studied. The results of preclinical studies of farnesyltransferase inhibitors (FTIs) have been promising, but clinical studies have not successfully demonstrated their usefulness in common cancers (43).

Downstream to ras, the deregulation of the PI3K/Akt pathway contributes to both malignancy progression and treatment resistance. Chemical inhibitors of Akt have a potential use as suppressors of tumor growth. Examples are wortmannin, LY294002, rapamycin, and rapamycin derivatives and preclinical studies have shown promising data

(43). Targeting of the NF-κB pathway is also under investigation (43).

1.3 Radioresistance and modulation strategies

1.3.1 Overview

Radiation therapy has been the choice of treatment for locally or regionally

advanced, unresectable cancers, either with curative intent or with palliative intent, both

of which contribute much to cancer management.

Local tumor control after curative radiotherapy relies on the successful killing of all clonogenic cells capable of tumor regrowth. This is influenced by two types of resistance factors, i.e., factors acting at cellular level, such as intrinsic cellular radiosensitivity, the repair of irradiation-induced DNA damage, redistribution of tumor cells into more resistant cell cycles; and mircoenvironmental resistance factors at the three-dimensional level of the stromal tissues and tumors including tumor hypoxia and its reoxygenation as well as accelerated tumor cell repopulation (44).

Two approaches have been widely studied to improve the local response of radiation without increasing normal tissue toxicities. One is the physical approach of localizing irradiation at more accurate doses to spare normal tissue as much as possible.

Examples are intensity-modulated radiation therapy, brachytherapy, and the use of proton beams. Despite the great advances in radiation technology and methodology, local failure rates of primary tumors following radiotherapy remain high for many solid tumors; thus, the enhancement of radiotherapy effects is necessary and another approach, the biological approach, has been intensively investigated. This includes the alteration of fractionation

scheduling of radiation to achieve the best response or the combination of chemical or

biological agents with radiation. The latter will be further discussed in the following.

1.3.2 Combination of conventional cytotoxic agents with radiation

Recently, the concurrent use of chemotherapy (CT) and radiotherapy (RT) has

become a standard treatment for many types of cancer. Combination of chemotherapy

and radiotherapy is based on two ideas, spatial cooperation and enhancement of radiation

effects (45). Adding systemic drugs to radiation exposes both the tumor lesions within the

irradiated field and micrometastases outside the irradiated area to the cytotoxic agent. In addition, chemotherapeutic drugs may increase the efficacy of ionizing radiation at killing tumor cell clonogens (46). Many chemotherapeutic agents interact with radiation

and enhance the cytotoxic or antitumor effect of radiation through a number of

mechanisms. These include an increase in initial radiation damage, inhibition of cellular

repair, cell cycle redistribution, counteracting hypoxia, and overcoming accelerated

repopulation (reviewed in (45; 46)).

The most closely investigated conventional chemotherapeutic agents combined

with radiotherapy include platinum analogues, nucleotide analogues, and taxanes.

Platinum analogs are believed to enhance cell killing after radiation through

several mechanisms, including enhanced formation of toxic platinum intermediates in the

presence of radiation-induced free radicals, inhibition of DNA repair, cell-cycle arrest,

and increased cellular platinum uptake by radiation (reviewed in (47)). Platinum analogs,

and specifically cisplatin, carboplatin, and oxaliplatin, are combined with radiation in the

treatment of a variety of solid tumors. Cisplatin/carboplatin-based chemoradiotherapy has

become the best first-line treatment for patients with unresectable locally advanced non

small cell lung cancer, small cell lung cancer (48), and locally-advanced cervical cancer

(49). Clinical trials confirming an overall improved outcome of radiation delivered

concurrently with cisplatin/carboplatin are also observed in trials involving cancers of head and neck (50), or muscle-invasive bladder cancer (51).

The rationale for exploring nucleoside analogues as potential radiosensitizer is

their ability to inhibit DNA replication and repair. The most commonly studied two drugs

are 5-fluorouracil and gemcitabine. Fluorouracil-based chemoradiotherapy is the standard

treatment for locally advanced pancreatic cancer (52) and rectal cancer after surgery (53).

Gemcitabine-based chemoradiotherapy is widely tested in clinical setting now and has

shown some promising results in both pancreatic cancer (54) and NSCLC (48), but this

approach remains investigational and no clear dose or schedule of chemotherapy or radiation has been accepted as standard (55). There seem to be multifactorial mechanisms

by which nucleotide analogs enhance tumor radioresponse, although not fully explained

yet, including inhibition of repair of nonlethal radiation damage, depletion of

radioresistant S-phase cells by inducing apoptosis, reoxygenation of hypoxic tumor cells,

and reducing tumor cell repopulation (reviewed in (46; 47)).

Taxanes such as paclitaxel and docetaxel inhibit tubulin depolymerization and

promote microtubule assembly and stability, leading to cell cycle arrest in the

radiosensitive G2 and M phase. Both paclitaxel and docetaxel have been effective in

enhancing tumor radioresponse in extensive preclinical testing, both in vitro and in vivo.

Increasing investigations are conducted for their clinical efficacy when combined with

radiotherapy in various human cancers, such as NSCLC (48), advanced head and neck

squamous cell carcinoma (56), locally advanced pancreatic cancer (54), and uterine cancer (57), either alone or in combination with other chemotherapeutic agents.

Combined irradiation and methotrexate-based chemotherapy is now the standard treatment for malignant lymphomas (58).

Despite the successful application of chemoradiotherapy over the last two decades, the main drawback of chemotherapy lies in the fact that it does not discriminate between normal and tumor cells and could result in severe damage to normal tissues as well. For example, the use of gemcitabine that sensitizes both normal and tumor tissue as a radiosensitizer has resulted in major toxicity due to the sensitization of normal tissues

(59). Therefore, tumor specific targeting agents are needed to potentially minimize the overlapping toxicity with radiation on normal tissue.

1.3.3 Modulating radiotherapy with molecular targeted agents

Increasing evidence suggests that the dysregulated molecular processes and signaling pathways in cancer cells may be responsible for tumor resistance to cytotoxic therapies, including radiotherapy and chemotherapy. Hence, strategies counteracting molecular processes involved in tumor radioresistance or chemoresistance are rapidly emerging for improving the efficacy of cancer therapy. Among these pathways or processes are epidermal growth factor receptor (EGFR), cyclooxygenase-2 (COX-2)

enzyme, mutated ras, and various proangiogenic substances. Interfering with these

processes in combination with radiotherapy and chemotherapy provides theoretical benefit because agents that interfere with these processes preferentially or selectively

affect malignant cells, toxicity to normal tissues is either minimal or absent (46).

EGFR inhibitors The epidermal growth factor receptor (EGFR) is a rational

target for cancer therapy due to its overexpression in a wide range of solid tumors and its

implication in the control of cell survival, proliferation, metastasis and angiogenesis (60).

EGFR overexpression has been found to be associated with more aggressive tumor behavior, adverse patient survival, and poor tumor response to conventional therapy (61).

Recent publications suggest that EGFR activates anti-apoptotic signaling pathways and

increases the radiation resistance of tumor cells (62-64). Two types of EGFR inhibitors

have been widely studied, anti-EGFR antibody (e.g. C225) or tyrosine kinase inhibitors

(e.g. gefitinib or Iressa). Inhibition of EGFR has proven to enhance radiation response in

preclincal studies and clinical trials are awaited. For instance, C225 has safely exerted

pronounced radiosensitization effect in vivo and phase III trials are on the way (46).

Gefitinib could potentially improve radiation response in NSCLC, head-and-neck cancers,

and other solid tumors, considering its effect in enhancing radiation-induced growth

inhibition in xenograft tumors and its efficacy with favorable tolerability as monotherapy

for patients with NSCLC or head-and-neck carcinomas in randomized clinical trials (65).

COX-2 inhibitors Cyclooxygenase-2 (COX-2) is overexpressed in many

human carcinomas, thus a potential target for radiotherapy to spare normal tissues. COX-

2 inhibitors, such as SC-236, celecoxib, rofecoxib, and NS-398, were shown to increase the radiosensitivity of tumor cells in preclinical studies but did not gain too much benefit in combination with radiotherapy in clinical trials. The underlying mechanisms for radiosensitization are still poorly understood, and may include inhibition of sublethal radiation damage, elimination of prostaglandins (PGs) as radioprotective molecules, inhibition of tumor angiogenesis, and removal of PG-mediated immunosuppression (46;

66).

Angiogenesis Antiangiogenic agents have been found to enhance tumor growth delay and tissue oxygenation during radiotherapy (67). In preclinical studies, two groups of antiangiogenic agents have been investigated to augment the radiation response of tumors: drugs inhibiting activation of angiogenesis (e.g. inhibitors of vascular endothelial growth factor (VEGF)) and inhibitors of endothelial cell function. The tyrosine kinase receptor of VEGF, VEGFR, is overexpressed in many cancers and its expression can be induced by exposure to ionizing radiation. Inhibitors of the VEGFR tyrosine kinase domain, as well as monocloncal antibodies against VEGF (e.g., bevacizumab) and its receptor, have been developed. When combined with radiotherapy, these inhibitors have shown radiosensitizing activity in different cancer models and some of them have entered phase I/II clinical trials. Inhibitors of endothelial cell function such as angiostatin, TN-

470 also entered phase I evaluation in combination with radiotherapy (reviewed in (44)).

Mutated ras The RAS protein-mediated pathway, which is important for signal transduction initiated by growth factors, has also been implicated in tumor

radioresistance. A critical step of the activation of RAS proteins is mediated by farnesyl transferase (FTase), which allows for RAS protein docking into the plasma membrane.

Farnesyltransferase inhibitors (FTIs) have been shown to radiosensitize cells both in vitro and in vivo. Early clinical studies are currently underway to test farnesyltransferase inhibition combined with fractionated radiotherapy of head and neck cancers (reviewed in (68). Downstream to RAS, the RAF-MEK-ERK and phosphatidylinositol-3 kinase

(PI3K)-Akt/PKB are two separate cascades putatively linked to tumor radioresistance.

Inhibitors of PI3K signaling such as LY294002 and wortmannin significantly enhanced response to radiotherapy in different cancer cells carrying mutated RAS and activated

PI3K. But few studies have reported the in vivo antitumor activity of these two PI3K inhibitors and no human studies have been performed due to their presumed toxicity in humans (44).

Others The NF-κB-signaling and ATM/p53-pathways are important sensors of irradiation-induced DNA damage. Blockade of NF-κB by proteasome inhibitor PS-341 has been reported to enhance radioresponse in vitro but has not undergone clinical evaluation (44). Several targeting strategies focus on p53 via transfection with p53 containing retroviruses or adenoviruses and have shown promising result, however, the low in vivo transfection of gene therapy limits the potential of this strategy (59).

Other strategies involve induction of cellular senescence (e.g., retinoids), proapoptotic cytokines (e.g., interferon), apoptotic effectors and regulators (e.g.,

ceramide, cytochrome c), modulation of cell cycle (e.g., CDK inhibitor UCN-01 and flavopiridol) (reviewed in (44)), and inhibition of protein kinase C activity (69).

1.4 Modulation of hypoxia in chemo- and radiotherapy

Hypoxia is considered to be a major cure-limiting factor in radiotherapy by virtue of increasing radiation resistance, associating with poor prognosis, and poor response to local control (68). Hypoxia also has negative influence on tumor cell response to most chemotherapy because drugs do not reach hypoxic regions easily and tumor cells there do not proliferate well, which makes them resistant to drugs. Although tumor hypoxia could be improved by chemotherapy-induced reoxygenation, a hypoxic region can be targeted through the use of drugs that only become cytotoxic in a hypoxic environment, the so- called hypoxic cytotoxins (45), or the use of hypoxic cell radiosensitizers such as nitroimidazoles.

The first drug introduced into the clinic purely as a hypoxic cytotoxin is tirapazamine (TPZ). The mechanism for the preferential toxicity of TPZ to hypoxic cells is well understood. Catalyzed by intracellular reductases, TPZ forms a highly reactive radical that will abstract hydrogen from macromolecules, including DNA, producing both single- and double-stand breaks, thus leading to chromosome breaks and cell killing (70).

Under aerobic conditions, the TPZ radical is back-oxidized to the nontoxic parent by the oxygen (71). Treatment regimens combining TPZ with conventional chemotherapy or radiotherapy have undergone extensive preclinical and clinical investigations and have

shown favorable results. For example, TPZ with cisplatin and vinorelbine in patients with

advanced non-small-cell lung cancer might be more active than the cisplatin/vinorelbine

combination. This triplet is currently being evaluated in a phase III study (72). The

combination of TPZ, cisplatin, and radiation has shown promising efficacy in patients

with locally advanced head and neck cancer in a randomized phase II trial and is now being evaluated in a large phase III trial (73).

Overall results from clinical studies suggest that modification of tumor hypoxia yields treatment benefits for head and neck tumors as well as bladder tumors. However, no significant improvement was found for uterine cervix, lung, esophagus, or central nervous system (45).

1.5 Protection of normal tissue in cancer therapy

Another way to improve cancer therapy is to protect normal tissues from cytotoxic effects of chemotherapy and radiotherapy, thus allowing for safer and more effective administration of anticancer therapy. A successful example is amifostine.

Developed as a radiation protector, amifostine also has exhibited activity as a chemoprotector, preventing radiation- and chemotherapy-induced cellular injury through free-radical scavenging, hydrogen donation, and inhibition of DNA damage (74).

Amifostine is favorably metabolized in normal cells than in tumor cells, hence, it protects normal tissues from toxicity induced by chemo- or radiotherapy without reducing the antitumor efficacy (74; 75). The protective effect of amifostine against radiation damage

and against the myelotoxic, nephrotoxic and neurotoxic effects of chemotherapeutic

agents such as alkylating agents and platinum compounds has been confirmed in

extensive preclinical studies. In some cases, amifostine even potentiated the antitumour

effects of these agents (75). Numerous clinical trials have confirmed that amifostine

protects a broad range of normal tissues from the toxic effects (e.g. nephrotoxicity,

neurotoxicity and neutropenia) of chemotherapy (e.g alkylating drugs, taxanes) and radiotherapy without attenuating tumor response in various tumor types (e.g. (76-79)).

1.6 Suramin

Suramin, a polysulfonated naphthylurea, is an old drug that has been used for the treatment of parasitic diseases such as African trypanosomiasis and onchocerciasis since the 1920’s (80). Since the late 1970’s, suramin was tested as an antiviral agent due to its potency to inhibit the reverse transcriptases of several retroviruses including human immunodeficiency virus (81-83). Unfortunately, the National Cancer Institute-sponsored multi-institutional trials failed to yield clinical efficacy (84).

Suramin has shown preclinical activity against prostate and other solid tumors, either as single agent or in combination with other chemotherapeutics (85). It has also been evaluated clinically since the early 1980’s. At least 33 trials have been published. In all these trials, suramin was used as a cytotoxic agent, given at its maximum tolerated steady state concentrations of 100-200 µM. Suramin has also been tested in breast cancer patients as an antiangiogenic agent, again requiring the maintenance of high

concentrations, i.e., above 140 µM (86). At these concentrations, suramin shows only

modest activity in patients but associated with significant toxicities including

polyneuropathy (87), adrenal insufficiency (88; 89), coagulopathy (90), and renal

insufficiency (91) at plasma concentration above 200 µM. Furthermore, suramin- containing combination therapy did not show a benefit over monotherapy. This has led to recommendations, by multiple investigators, against its clinical use (e.g., (92)).

Au and collaborators recently reported that elevated levels of acid and basic fibroblast growth factor (aFGF and bFGF) in solid and metastatic tumors confers broad- spectrum resistance to chemotherapy drugs with diverse structures and mechanisms of

action, and that nontoxic, low concentrations/doses of suramin reverse the FGF-induced

chemoresistance in vitro and in vivo in mice with well established lung metastases (24;

32). Since then, the chemosensitization effect of low-dose suramin was further

demonstrated in more common solid tumors including prostate (34; 93), breast (94), lung

(95), bladder (96), pancreatic (97), colorectal (33), and renal cell carcinoma (98). Inspired

by the promising preclinical data, several clinical trials testing low-dose suramin as a

chemosensitizer have been launched, including the finished phase I/II trials in non-small

cell lung cancer (NSCLC), phase I trial in renal cell carcinoma, and phase I trial in tumor-

bearing pet dogs. Another phase I/II trial in breast cancer is ongoing and a randomized

two-arm phase II trial in prostate cancer has been proposed. The results from the clinical

trials have been very encouraging. In the completed NSCLC trial, where suramin in

combination with standard treatment was used as first-line treatment, median TTP (7.2

months vs. 4.4 months), response rate (43.5% vs. 28.0%), and 1-year survival (47.9% vs.

37.7%) were all improved compared to historical data for the standard treatment regimen

(unpublished data). When suramin combined with taxanes was evaluated as a second-line

treatment in lung cancer and breast cancer, 11 of the 22 responders had previously progressed on prior taxanes, suggesting that suramin really reversed the chemoresistance

to taxanes (unpublished data).

The precise mechanism underlying the impressive chemosensitization effect of

low-dose suramin is still under investigation. Suramin has many pharmacological

activities other than bFGF inhibition that could be responsible for the established

chemosensitization effect, including inhibition of binding of other growth factors and

cytokines to their receptors. The targets that are inhibited by <50 µM suramin are: FGFs

(99-102), reverse transcriptase (103), PKC (104), RNA polymerase (105), and

transforming growth factor $ (106), with respective IC50 of 15-50, 1, 30, 35, and 1 µM.

At concentrations >50 µM suramin also inhibits IL-2 (107), insulin growth factor-1 (108),

tumor necrosis factor " (109), and topoisomerase II (110) with respective IC50 values of

700, 200, 650 and 100 µM. Suramin also inhibits DNA polymerase " (111), glycosaminoglycan metabolism (112), platelet-derived growth factor (113), epidermal growth factor (114; 115), and vascular endothelial growth factor (116).

1.7 Overview of dissertation

The rest of this dissertation is divided into six chapters. Each chapter begins with a brief introduction of background information and the purpose of the study, followed by a description of the methods and materials used. Experimental findings are summarized in the results section, followed by more detailed discussion regarding the significance and implications of the findings in the discussion section. Tables and figures are attached at the end of each chapter, while references are included at the end of the dissertation.

Chapter 2 evaluates the effects of low-dose suramin on the activity of mitomycin

C (MMC) in bladder cancer, using both RT4 xenograft tumors and bladder cancer patient tumors. Low-dose suramin enhanced the in vitro and in vivo antitumor activity of MMC at both subtherapeutic and therapeutic treatment regimens without enhancing the host toxicity, thus providing the proof-of-concept for evaluating low-dose suramin as a chemosensitizer with MMC in bladder cancer. This is the second strategy developed by our laboratory to improve the response of intravesical MMC therapy since the successful phase III trial in which optimized MMC delivery nearly doubled the recurrence-free survival in patients with superficial bladder cancer (117).

Chapter 3 further evaluates whether another potent nonspecific FGF inhibitor pentosan polysulfate (PPS), either alone or in combination with suramin, can enhance the activity of paclitaxel in pancreatic cancer that has high FGF levels. The results showed that PPS significantly enhanced the antitumor activity of paclitaxel in Hs 766T pancreatic xenograft tumors, presumably via an apoptotic pathway. However, the toxicity of the

combination group was serious and limited the potential of further investigation.

Combination of PPS and low-dose suramin failed to achieve better chemosensitization effect.

Chapter 5 further evaluates the dose dependence of suramin in mediating radiosensitization effect and its pharmacokinetic (PK) characteristics, thus providing preclinical translational data for optimized dosing regimen design in clinical setting.

Tumor type specific dose dependent radiosensitization effect of suramin was observed, with a narrow sensitizing window in FaDu tumors and a wider range in Hs 766T tumors.

PK study suggested no dose dependence or tumor dependence in suramin PK, thus the tumor type specificity in dose dependence of suramin resulted from the biological difference between FaDu and Hs 766T tumors. The biphasic effect of suramin in FaDu tumors also provided an ideal model for further pharmacodynamic evaluation.

Chapter 6 evaluated several microscopic pharmacodynamic endpoints to understand the radiosensitization effect of low-dose (10 mg/kg) but not high-dose (200

mg/kg) suramin, and possibly identify potential biomarkers. The results showed that low- dose suramin enhanced the apoptotic effect of irradiation without affecting its antiproliferative activity. The radiosensitization effect of low-dose suramin was probably due to its inhibitory effect on irradiation-induced upregulation of phospho-ERK and survivin, with the latter being a long-term effect.

The final chapter summarizes the conclusions and contributions of this dissertation research and discusses future investigations.

CHAPTER 2

LOW-DOSE SURAMIN AS A CHEMOSENSITIZER OF BLADDER CANCER

TO MITOMYCIN C

2.1 Introduction

Superficial bladder cancer is often managed by transurethral surgical resection, followed by intravesical chemotherapy with, e.g., mitomycin C (MMC) and doxorubicin.

Our laboratory has established that the efficacy of these chemotherapies is limited by two factors, i.e., inadequate drug delivery to tumor cells and low chemosensitivity of the more rapidly proliferating tumors (117), and have devised two therapeutic strategies.

The first strategy was to enhance the MMC delivery to tumors. We established mathematical models to determine the effects of physiological and pharmacological parameters on drug delivery, and used model-based computer simulations to identify a new method of MMC administration. This method was predicted to double the recurrence-free survival rate from 20% to 40% (118), and was tested in an NCI-supported phase III trial; the results confirm that maximizing the MMC delivery significantly

prolong the median time to recurrence from 11.8 to 29.1 months and improves the 5-year

recurrence-free fraction from 23% to 42% (117).

The finding of recurrent disease in ~60% patients in spite of maximal drug

delivery has led to the development of the second strategy of enhancing tumor sensitivity

to MMC. We recently identified an epigenetic, broad-spectrum mechanism of anticancer

drug resistance, caused by acidic and basic fibroblast growth factors (aFGF and bFGF)

expressed in solid tumors. We further showed that FGF inhibitors, including monoclonal

antibodies and suramin, completely reverse the FGF-induced resistance and enhance the

activity of chemotherapy in cultured human tumor cells and xenograft tumor-bearing animals. The suramin concentration and dose required for chemosensitization was nontoxic and subtherapeutic (24; 32; 34). These encouraging preclinical results have led to four phase I/II trials using low-dose suramin to enhance the antitumor activity of standard chemotherapies in advanced lung (two trials), breast, and kidney cancer patients.

The results of the first phase II trial in chemotherapy-naïve nonsmall cell lung cancer

patients treated with paclitaxel plus carboplatin suggest therapeutic benefits by adding

nontoxic doses of suramin (119; 120).

The present study was to evaluate whether low-dose suramin enhances MMC

activity in bladder cancer, under in vitro (histocultures of human RT4 bladder xenograft tumors and bladder cancer patient tumors) and in vivo (mice bearing subcutaneous RT4 tumors) conditions. RT4 cells are derived from a low-grade superficial bladder carcinoma

and express FGF receptors (121). Further, as our previous studies indicate inadequate

drug delivery as a major cause of treatment failure, we also addressed the question

26 whether suramin enhances MMC activity when tumors are presented with subtherapeutic

drug exposure. This was accomplished by evaluating the effects of suramin on two MMC

treatment schedules, a subtherapeutic schedule that retarded tumor growth but did not

produce tumor regression and a therapeutic schedule that produced tumor regression.

2.2 Materials and methods

2.2.1 Cell and tumor cultures

Patient tumors were obtained via the Tissue Procurement Shared Resourse of the

Comprehensive Cancer Center, The Ohio State University and The Cleveland Clinic.

Because chemosensitization experiments required a relatively large tumor volume that

was not readily available from surgical specimens of superficial bladder tumors (~ 200

mg), we also used specimens from cystectomy containing high-stage and muscle-

invading tumors. Human RT4 cells (American Type Culture Collection, Rockville, MD) and xenograft histocultures were maintained in McCoy 5A, and patient tumor histocultures in Minimum Essential Medium. All culture media were supplemented with

9% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 90 µg/ml

gentamicin, and 90 µg/ml cefatoxime.

2.2.2 Establishment of RT4 xenograft tumors

Female athymic nude mice (5-6 weeks) were purchased from the National Cancer

Institute (Bethesda, MD). Subconfluent RT4 cells were harvested and implanted

27 subcutaneously into both flanks of mice (3 × 106 cells per site). After 3-4 weeks, animals

bearing tumors >3 mm in diameter were used for experiments.

2.2.3 Drug activity evaluation in tumor histocultures

Drug effect was measured as inhibition of bromodeoxyuridine (BrdU)

incorporation (122). Briefly, tumor fragments (1mm3) were cultured on collagen gel, and

treated with MMC (Bristol-Myer Squibb Co., Wallingford, CT) for 2 hr, with or without

20 µM (or 28.6 µg/ml) suramin (Sigma, St. Louis, MO). The 2 hr treatment is the same as in intravesical therapy. Tumors were then incubated with fresh media containing 40 µM

BrdU for 48 hr, and then processed for BrdU immunostaining using LSAB (Dako,

Carpinteria, CA) and DAB (BioGenex, San Ramon, CA) kits. The fraction of BrdU- labeled cells was determined by microscopic examination. In the same sections, apoptotic cells were identified by condensed nuclei and membrane blebbing (123). Typically, 5-12 tumor pieces and a minimum of 70 cells per piece were counted per concentration.

The drug concentration-effect data were analyzed using the following equation and nonlinear regression with PROC NLIN (SAS; Cary, NC).

C n E = (E − R ) • (1 − ) + R o e K n + C n e

Where E is the BrdU labeling index (LI) of drug-treated tissues, Eo is the LI of untreated

controls, Re is the residual fraction, C is the drug concentration, K is the drug

concentration at one-half (Eo-Re), and n is a curve shape parameter.

28 2.2.4 In vivo drug activity evaluation: animal treatment protocols

MMC (0.6mg/ml) and suramin (2 mg/ml) stock solutions were prepared in physiological saline. RT4 tumor-bearing mice were randomized according to initial tumor sizes and body weights (see Results), and received physiological saline, MMC (3 mg/kg/dose), suramin (10 mg/kg/dose), or MMC/suramin combination. We studied two

MMC schedules, i.e., subtherapeutic (3 intravenous treatments, every 4 days) and therapeutic (6 treatments, twice weekly). Partly due to the difficulty to give repeated injections via tail vein and partly because of the rapid and extensive absorption from the peritoneal cavity (124), the therapeutic treatments were given intraperitoneally.

Drug treatment effects were measured using several pharmacodynamic endpoints.

The first endpoint was tumor size measurement and was applied to animals in both subtherapeutic and therapeutic MMC groups. The widths and lengths of tumors were measured using calipers. Tumor volume was calculated as 50% of (width)2 × (length).

The second set of pharmacodynamic endpoints was drug-induced changes in proliferation index and apoptotic cell fraction in tumors. This was studied in a subset of animals treated with the subtherapeutic schedule. The animals treated with the therapeutic schedule, due to the small size of post-treatment residual tumors, were not studied. For this purpose, an animal was euthanized 3 days after the final treatment and tumors were harvested for histological evaluation. Tumors were excised, weighed and fixed in 10% phosphate-buffered neutral formalin and embedded in paraffin. Five µm histologic sections were prepared for immunostaining. The apoptotice cells were identified by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) method

29 (Apoptag, Intergen, NY) and the fraction of apoptotic cells was obtained by counting the

number of TUNEL-labeled tumor cells and total tumor cells in 4-5 randomly selected

microscopic fields at 400x magnification, as described elsewhere (125). On average, we

counted 1160 ± 280 and 1300 ± 180 cells per tumor in the control and suramin groups,

and 768 ± 125 and 682 ± 186 cells per tumor (mean ± SD) in the MMC and combination

groups, respectively.

The proliferation index was determined by staining for Ki67 nuclear antigen

(Ki67 Antigen Kit, Novocastra Lab Ltd, UK), which is expressed throughout the cell

cycle except in the resting G0 cells. Briefly, tissue sections of paraffin-embedded tumors

were deparaffinized, rehydrated, and boiled in 10 mM sodium citrate buffer (pH 6) for 15

min for antigen retrieval. After washing, quenching of endogenous peroxidase activity

(1% hydrogen peroxide for 10 min), and blocking, the sections were incubated with anti-

Ki67 monoclonal antibody at room temperature for 2 hr, followed by incubation with biotinylated secondary antibody (diluted 1:500) for 30 min and then streptavidin- peroxidase complex for 30 min. Positive staining was developed using diaminobenzidine as the chromogen substrate, and sections were counterstained with hematoxylin, dehydrated and mounted using Permount®. Human tonsil sections were used as positive controls. For negative controls, we replaced the primary antibody with the blocking reagent. The proliferation index (or, Ki67 labeling index) was calculated as (number of

Ki67-stained cells) divided by (total cell number) in five microscopic fields at 400×

magnification. On average, we counted 1325 ± 210 and 1436 ± 130 cells per tumor in the

30 control and suramin groups, and 854 ± 112 and 877 ± 124 cells per tumor (mean ± SD) in

the MMC and combination groups, respectively.

2.2.5 Statistical analysis

The effects of suramin on MMC activity in tumor histocultures were analyzed

using paired Student’s t-test. For in vivo studies, differences in tumor growth were assessed by ANOVA for repeated measures and differences in other parameters were

assessed by Tukey test after ANOVA.

2.3 Results

2.3.1 Effects of suramin on MMC activity in histocultures of RT4 tumors

MMC reduced the BrdU labeling index in a concentration-dependent manner and

showed an IC50 of 2.45 µg/ml. Suramin (20 µM) had no activity, as indicated by the similar labeling index between suramin and control groups, but significantly enhanced the

MMC activity, such that the IC50 and IC90 of MMC were reduced by about 2-3 folds

(Table 2.1).

MMC induced apoptosis in a concentration-dependent manner (Figure 2.2A),

whereas suramin had no effect. The MMC/suramin combination showed a trend of higher

apoptosis at higher MMC concentrations (at and above 1 µg/ml), but the differences

between MMC and MMC/suramin combination groups were not statistically significant

except at one MMC concentration (3 µg/ml).

31 2.3.2 Effect of suramin on MMC activity in histocultures of human tumors

In human tumors, MMC reduced BrdU incorporation in a concentration-dependent

manner; the IC50 ranged from 1.4 to 15.9 µg/ml with an average value of 7.9 µg/ml

(Figure 2.1B, Table 2.2). These values are consistent with our earlier studies, in which the

IC50 ranged from 0.17 to 18.1 µg/ml with an average value of 6.0 µg/ml (122; 126).

Suramin showed an IC50 of about 12 mM (Figure 2.1C). Similar to the findings in RT4 tumors, 20 µM suramin did not show activity as single agent but significantly reduced the

IC50 and IC90 of MMC in all 11 tumors, by 2.5-fold.

Figure 2.2B shows the drug-induced apoptosis. Suramin alone at 20 µM produced

a higher apoptotic index compared to untreated control, but the difference was not

statistically significant. On the other hand, MMC induced significantly higher apoptosis

beginning at 0.333 µg/ml (p<0.05 compared to control). Interestingly, the apoptotic

fraction decreased as MMC concentration increased such that the apoptotic fractions at the

two highest concentrations were not significantly different from the control. The cause for

the latter observation is unclear, but may be a result of concentration-dependent kinetics of

apoptosis. For example, an earlier onset and completion of apoptosis at higher drug concentrations (therefore disappearance of apoptotic cells prior to the time of tumor harvesting) would explain the lower apoptotic index at those concentrations. In general,

MMC/suramin combination showed a trend of higher apoptosis at all MMC

concentrations compared to single agent MMC, but the differences between MMC and

MMC/suramin combination groups were not statistically significant except at one MMC

concentration (6.67 µg/ml).

32 2.3.3 Enhancement of in vivo MMC activity by suramin in RT4 xenograft tumors: tumor size changes

The results are shown in Figure 2.3 and Table 2.3. The control group and the three

drug-treated groups showed similar initial body weight. The tumor size distribution in

animals treated with the subtherapeutic schedule was such that we were not able to

achieve comparable initial tumor sizes in all groups. Hence, we placed the animals with

larger tumors in the treatment groups, in order to minimize the potential bias due to

favorable outcome associated with smaller tumor size. Note the comparable initial tumor

sizes of the single agent MMC and MMC/suramin combination groups.

At the end of the 30-day experiment, the tumor volume in the control group was

~500% of the initial volume. Low-dose suramin had no antitumor effect, consistent with

the previous results in other tumor-bearing mouse models (24; 32; 34).

The subtherapeutic MMC schedule significantly retarded tumor growth, but did

not cause tumor regression. In contrast, the therapeutic MMC schedule yielded significant

tumor regression (27% reduction). Addition of suramin significantly enhanced the MMC

activity; the respective tumor sizes were 40% and 63% of the sizes after subtherapeutic

and therapeutic treatments of MMC alone (p<0.01).

Animals in the subtherapeutic MMC schedule group showed tumor regrowth

starting within 4 days after the final treatment (i.e., day 16) such that the tumor size

increased by 83% on day 30. The addition of low-dose suramin delayed the onset of tumor

regrowth to 14 days after treatment (i.e., day 26) and decreased the regrowth rate as

indicated by the more shallow tumor growth curve.

33 The maximum body weight loss was ~2% for the subtherapeutic MMC schedule

and 18% for the therapeutic schedule. Single agent suramin did not cause body weight loss

and adding suramin to MMC did not enhance the body weight loss for either MMC

schedule.

2.3.4 Enhancement of in vivo MMC activity by suramin in RT4 xenograft tumors:

tumor histological data

Table 2.4, Figure 2.4 and 2.5 showed the drug effects on a cellular level. The

control group showed a ~35% Ki67 labeling index and ~1% apoptosis. Suramin had no

effects. MMC significantly reduced the Ki67 labeling index by about one-third to 24%

and increased the apoptotic fraction in the residual tumors by ~2.6-fold to 4%. Addition of suramin further significantly reduced the Ki67 labeling index by 2.5-fold to 10% and further enhanced the apoptotic fraction by 1.8-fold to 7%.

2.4 Discussion

The present study demonstrated that nontoxic, low-dose suramin significantly enhanced the activity of MMC in 3-dimensional histocultures of human bladder tumors and RT4 xenograft tumors. The chemosensitization effect of suramin in human tumors was found in both low-stage, superficial tumors and in high-stage, muscle-invading and metastatic tumors. Similarly in mice bearing RT4 tumors, low-dose suramin had no antitumor effects, but significantly enhanced the activity of subtherapeutic or therapeutic

34 MMC treatments, on both macroscopic level (i.e., tumor growth retardation and

regression) and cellular level (i.e., drug-induced antiproliferative and apoptotic effects).

The therapeutic benefits were accomplished without enhancing host toxicity. These

results are consistent with our earlier findings that low-dose suramin enhanced the in vitro and/or in vivo antitumor activity of other chemotherapeutic agents, i.e., paclitaxel, doxorubicin, and 5-fluorouracil, in primary and metastatic tumors in animals, and with our phase II data in non small cell lung cancer patients (24; 32; 34; 119; 120).

Treatment of bladder cancer with an FGF inhibitor is appealing in concept since there are multiple lines of evidence to indicate the involvement of bFGF in the development of bladder cancer and/or prognosis in patients. bFGF affects various cellular functions including protease production, angiogenesis, tumor cell migration and invasion.

The gene encoding FGF receptor-3 is the most commonly mutated gene in transitional cell carcinoma (127), and all bladder cancer cell lines expressed bFGF receptors (121). In patients, higher expressions of bFGF are found in precursor lesions of muscle-invading tumors compared to superficial papillary tumors (128), and are associated with higher risk of developing metastasis (129). Compared to healthy individuals, the urinary aFGF and bFGF levels in bladder cancer patients are up to 100-fold higher (130). In addition, bFGF-transfected bladder cancer cells show a three- to four-fold higher resistance to cisplatin compared to bFGF-negative parental cells (131).

Suramin has undergone extensive clinical evaluation in various solid tumors, either as single agent or in combination with other chemotherapeutics, since the early

1980’s. In all these trials, the therapeutic plasma concentrations of suramin were between

35 100 and 200 µM, which had significant toxicities and only modest activity. This has led

to recommendations, by multiple investigators, against its clinical use (e.g., (92)). The

major difference between the previous clinical studies with suramin and our ongoing studies is the intended use of suramin and, accordingly, the selection of the

dose/concentration. In the current study, suramin is used to reverse bFGF-induced

resistance, an effect requiring only 10-20 µM concentration, which has no cytotoxicity in

cultured tumor cells nor in animals or patients. Another important consideration is the

unintended effects at higher drug concentration. For example, suramin at concentrations

above 50 µM arrests cells in the G1 phase (e.g., (132)), which may diminish the activity

of other agents that exert their action in the later cell cycle phases. An example is the

antagonism between suramin at >50 µM concentration and radiation which is most

effective in the G2/M phase (133).

In summary, results of the present study support using nontoxic, low-dose suramin to improve the MMC efficacy during intravesical therapy of superficial bladder cancer. In order to translate these preclinical data to develop clinical protocols to test the concept of using MMC/suramin combination, we are conducting studies in dogs to identify the intravesical suramin dose to deliver the target extracellular concentrations (i.e., 10-50 :M) that produce chemosensitization to bladder tumor cells. Furthermore, we are conducting computer simulations using our previously described mathematical models that depict the relationship between drug delivery/exposure, drug efficacy and treatment outcome (118), to address the question whether the chemosensitization effect of suramin is materially important and may improve the treatment outcome.

36 2.5 Acknowledgements

This work was performed in collaboration with Drs. Greg Lyness, Danny Chen, and SaeHeum Song, and has been published in Journal of Urology, Vol. 174, 322-327,

July 2005.

This work was supported in part by MERIT grant (R37CA49816) from the

National Cancer Institute, DHHS. We thank the Tissue Procurement Shared Resource of the Comprehensive Cancer Center, The Ohio State University and The Cleveland Clinic, for providing surgical tumor specimens from bladder cancer patients. The OSU CCC

Tumor Procurement Service was supported partly by grant P30 CA16059, National

Cancer Institute, Bethesda, M.D.

37 No Suramin + 20 µM Suramin

Control LI MMC IC50 MMCIC90 Control LI MMC IC50 MMC IC90 (%) (µg/ml) (µg/ml) (%) (µg/ml) (µg/ml) Mean±SD 57.6±5.0 2.45±0.45 22.1±2.66 59.8±6.0 0.73±0.37 10.4±1.31 Range 53.4–63.1 2.13–2.96 19.2–24.5 54.1–66.1 0.51–0.16 9.45–11.9 Median 56.3 2.26 22.5 59.2 0.53 9.87

Table 2.1 Enhancement of in vitro MMC activity by low-dose suramin

Histocultures of RT4 xenograft tumors were treated with MMC, with or without suramin

(20 µM). IC50 and IC90 are the concentrations of MMC required for 50 and 90%

inhibition of BrdU incorporation. Single agent suramin had no effects. Differences

between the IC50 and IC90 values for single agent MMC and MMC/suramin combinations

are significant (p<0.05). Data were obtained from three experiments, with three replicates per experiment.

38 and and 50 denotes that the M). Differences µ

are significant (p<0.005). IC incorporation. MX MMC/suramin combinations red for 50 and 90% inhibition of BrdU tumors were treated with MMC, with or without suramin (20 (20 suramin MMC, with or without with treated were tumors values of MMC alone and asis cannot be assessed. 90 and IC 50 Human patient tumor characteristics and effects of suramin on MMC activity of effects and characteristics tumor Human patient are the concentrations of MMC requi

90 Histocultures of human bladder IC between the IC presence of distant metast Table 2.2

39 t treatments, every 4 days.

nk regions of immunodeficien SD ± r size and body weight the right and left fla utic schedule: 3 intravenous Mean > 3 mm, animals received physiological saline, 3 mg/kg MMC, physiological saline, 3 > 3 mm, animals received MMC activity on tumo MMC activity anted subcutaneously into in vivo treatments twice weekly intraperitoneal hedule: 6 Effect of low-dose suramin on

Table 2.3 Human bladder RT4 tumor cells were impl weeks or when tumors reached a diameter mice. After 3 mg/kg suramin, or a combination of both drugs. Subtherape 10 Therapeutic sc

40 Total Cell Viable Cell Ki67-Positive cells TUNEL-Positive cells Density Density Density % Density % Group (Cells/Field) (Cells/Field) Control 265 ± 42 261 ± 24 92 ± 17 34.7±1.1 3.8 ± 0.5 1.4±0.3 Suramin 287 ± 26 291 ± 7 97 ± 8 34.0±1.7 4.0 ± 0.6 1.4±0.2 a a a a a a MMC 171 ± 22 168 ± 16 41 ± 8 24.1±3.5 6.5 ± 1.3 3.7±0.6 a a a, b a, b a, b a, b Combination 175 ± 25 166 ± 31 18 ± 9 9.8±3.3 11.5 ± 2.8 6.6±1.5 a. p<0.05 compared to control by Tukey test after ANOVA b. p<0.05 compared to MMC by Tukey test after ANOVA

Table 2.4 Enhancement of in vivo antitumor effect of MMC by suramin: histological evaluation

Animals were treated with the subtherapeutic MMC schedule, with or without low-dose suramin, as described in Table 2.3. Tumors were removed and processed for histological and immunohistochemical staining for Ki67 and TUNEL staining. For each tumor, cell density, Ki67 labeling index and apoptotic index were determined using five randomly selected microscopic fields at 400× magnification. Mean ± S.D. Six tumors per group. Differences among the four groups are significant by ANOVA (p < 0.01).

Figure 2.1 Enhancement of MMC activity in tumor histocultures by suramin: inhibition of BrdU incorporation in tumor cells

Tumor histocultures were prepared and treated with drugs. Drug effect was measured as reduction of BrdU labeling index. Results of representative experiments are shown. (A) RT4 xenograft tumors treated with MMC, with (open circle) or without (closed circle) 20 µM suramin (three experiments with three replicates per experiment). (B) A representative human tumor treated with MMC, with (open circle) or without (closed circle) 20 µM suramin. (C) A human tumor treated with single agent suramin. Three replicates per data point. Note the mM suramin concentration in Panel C.

A. RT4 B. Patient 40

20 *

10 * Apoptotic Index (%) Index Apoptotic 0 00.10.3131030 0 0.067 0.333 0.667 3.33 6.67 33.3 66.7 MMC, µg/ml

Figure 2.2 Enhancement of MMC-induced apoptosis in tumor histocultures by suramin: induction of apoptosis

RT4 (A) and human (B) tumor histocultures were prepared and treated with MMC, with (white bar) or without (black bar) suramin, as described in Figure 2.1. * indicates statistically significant differences between the effects of single agent MMC and MMC/suramin combination (p<0.02, paired Student’s t-test). Note the different MMC concentrations used for RT4 and patient tumors.

Figure 2.3 Enhancement of in vivo antitumor activity of MMC by low-dose suramin:

tumor size reduction

Animals with well-established, subcutaneously implanted RT4 tumors were treated with physiological saline (control, closed circle), 10 mg/kg suramin (open circle), 3 mg/kg MMC (closed triangle), and MMC plus suramin (open triangle). Changes in tumor size were expressed as fraction of initial tumor size. (A) Subtherapeutic schedule (3 intravenous treatments, every 4 days). (B) Therapeutic schedule (6 intraperitoneal treatments, twice weekly). Arrows indicate treatments. Mean plus 1 S.D. Differences between MMC/suramin combination and all other groups are statistically significant (p < 0.01, ANOVA for repeated measures).

Figure 2.4 Enhancement of in vivo antiproliferation effect of MMC by low-dose suramin

Animals were treated as described in Figure 2.3. Tumors were harvested 3 days after the last treatment from animals treated with the subtherapeutic schedule. Paraffin embedded tumor specimens were cut into 5 µm section and processed for BrdU immunohistochemical staining. Dark color indicates positive staining. Micrographs were taken under 400x magnification for tumors treated with physiological saline, suramin (10 mg/kg), MMC (3 mg/kg) and combination.

Figure 2.5 Effect of low-dose suramin on the apoptotic effect of MMC in vivo

Animals were treated as described in Figure 2.3. Tumors were harvested 3 days after the last treatment from animals treated with the subtherapeutic schedule. Paraffin embedded tumor specimens were cut into 5 µm section and apoptosis was detected by TUNEL staining (arrows). Micrographs were taken under 400x magnification for tumors treated with physiological saline, suramin (10 mg/kg), MMC (3 mg/kg) and combination.

46 CHAPTER 3

COMBINATION OF NON-SPECIFIC bFGF INHIBITOR AND PACLITAXEL IN

TREATMENT OF PANCREATIC XENOGRAFT TUMORS

3.1 Introduction

Pancreatic cancer remains the fourth leading cause of cancer death in the United

States. Its lethality is demonstrated by the fact that the estimated annual incidence in

2005 (32180) almost equals the annual deaths (31800) (1). The overall 1- and 5- year survival rates are 23% and 4%, respectively, and median survival is only three to six months (134). Several factors underlie the poor prognosis of pancreatic cancer, including late presentation, aggressive local invasion, early and extensive metastasis, as well as poor response to conventional chemoradiotherapy strategies (135). Advances in the management of this disease are urgently needed.

Surgical resection remains the only chance of cure. However, only 15-20% of patients have resectable pancreatic cancer and actual 5-year survival rates in this subgroup remains as low as 20% due to the propensity of the disease to relapse (136).

Treatment for patients with locally unresectable disease involves radiation therapy and

47 systemic chemotherapy (137) or chemotherapy alone (138). About 40-50% of

patients present with metastatic pancreatic cancer, and are treated with systemic chemotherapy. Gemicitabine has replaced fluorouracil (5-Fu) as the standard care for metastatic pancreatic cancer since 1996, based on a pivotal phase III trial by Burris et al., who reported a significant improvement in survival among patients treated with gemcitabine (1-year survival of 18% with gemcitabine compared to 2% with 5-FU, P

= .003) (139). However, the treatment outcome is still poor. Attempts to improve the efficacy of single-agent gemcitabine through optimal administration of this prodrug have been unsuccessful so far. Other ongoing clinical investigations are designed either to compare the new agent (e.g. exatecan (140)) directly against gemcitabine or to combine the new agents with gemcitabine, including antimetabolites, topoisomerase I inhibitors, platinums, taxanes, as well as molecularly targeted agents such as VEGF-A antibody bevacizumab, EGFR antibody cetuximab, small molecule TKI anti-EGFR agents gefitinib (Iressa) and erlotinib (Tarceva) (reviewed in (141)). Some of the trials have indicated that the new combinations may have better treatment outcome, but further studies are needed to assess the value of these treatments. In November 2005, FDA approved Tarceva (erlotinib) in combination with gemcitabine chemotherapy as first-line

treatment in locally advanced, inoperable or metastatic pancreatic cancer based on its

significant improvement in overall survival in a phase III trial.

We recently reported an epigenetic, broad-spectrum mechanism of anticancer drug resistance caused by acidic and basic fibroblast growth factors (aFGF and bFGF) expressed in solid tumors (24). Although the basis for the chemoresistance of pancreatic

48 cancer has not been elucidated, we hypothesize that it is, at least in part, due to the high

expression of fibroblast growth factors (FGFs) in pancreatic cancer. Considering the

implication of FGFs in the tumorigenesis of a variety of human tumors, FGFs may also

play an important role in the pathogenesis and prognosis of pancreatic cancer (142; 143).

Expression of aFGF and bFGF is low in normal pancreas (143; 144), but in pancreatic

adenocarcinoma aFGF and bFGF were found to be overexpressed and localized to the

malignant ductal cells, and advanced clinical tumor stage is correlated with the presence

of either aFGF or bFGF (143; 145; 146). Shorter survival has been noted in patients with

bFGF-positive tumors but not in patients with aFGF-positive tumors. Increased aFGF and

bFGF were also observed in chronic pancreatitis (144; 145). bFGF produced by human

pancreatic carcinoma was found to induce ductal and stromal hyperplasia (147).

Overexpressed aFGF and bFGF in pancreatic adenocarcinoma have also been

accompanied by the overexpression of FGF receptor (FGFR) (142; 148). bFGF may also

be implicated in human pancreatic cancer cell growth and invasion to basement membrane (149; 150). A number of other FGFs are also overexpressed in pancreatic cancer, and may have roles in tumorigenesis (151). For example, FGF-5 was proposed to participate in autocrine and paracrine pathways promoting pancreatic cancer cell growth in vivo (152). These observations suggest that FGFs provide growth advantages for pancreatic tumors, and interfering with FGF signaling may lead to improved treatments for pancreatic cancer.

Our laboratory further found that FGF inhibitors, including monoclonal antibody and suramin, reversed the FGF-induced resistance and enhance the activity of

49 chemotherapy on different tumors, both in vitro and in vivo. The suramin concentration

and dose required for chemosensitization was nontoxic and subtherapeutic (24; 32-34;

96). More specifically, suramin also sensitized the efficacy of paclitaxel and gemcitabine in pancreatic Hs 766T xenograft tumors. However, the best dose of suramin was higher than in other tumor types (30 and 50 mg/kg vs. 10 mg/kg) (97). This observation is in line with our hypothesis because of the high bFGF level in pancreatic tumors (153). In addition, suramin showed biphasic effect in combination with paclitaxel, with sensitization effect at low dose (<50 µM in culture medium or plasma) whereas antagonism at high dose (>50 µM in culture medium or plasma) in PC-3 tumors (93).

Similar phenomenon was also observed in RCC human tumors treated with 5-FU (154).

Therefore, two questions need to be answered when developing treatment regimens for tumors with high bFGF expression such as pancreatic tumors (153). First, can we use a more selective and potent bFGF inhibitor to achieve better chemosensitization effect?

Second, will combination of suramin with other bFGF inhibitors that share different targeting site from suramin, reach greater chemosensitization effect not achievable by suramin alone?

Pentosan polysulfate (PPS), a semi-synthetic sulfated heparinoid polysaccharide, serves as a good candidate because it can selectively and potently inhibit aFGF and bFGF at a nontoxic concentration. In adrenal cancer cell line SW13, whose anchorage- independent growth depends on the presence of FGF, PPS inhibited aFGF and bFGF- induced growth and binding to their receptors at an IC50 of 1-3 µg/ml, compared to that of

100 µg/ml for suramin (155). PPS has been used in the United States for interstitial

50 cystitis (156) and in Europe as an anticoagulant (157). It is obtained from extracts of beechwood shavings and consists of a mixture of polymers with molecular weights ranging from 1800 to 9000 daltons (mean 4700 daltons), containing ~1.8 sulphate groups per monosaccharide unit (158). The interaction between FGF2 and heparan sulfate proteoglycans is essential for receptor-mediated signal transduction. Recent studies have demonstrated that the interaction of FGF2 with heparin-derived oligosaccharides results both FGF2 and FGFR dimerization, subsequently leading to dimerization and activation of the tyrosine kinase receptor (159). As a heparinoid, PPS is capable of abrogating the effects of FGFs and other heparin-binding growth factors in vitro and in vivo (160; 161), presumably by binding to FGF and preventing them from reaching their receptors on tumor or stromal cells (161; 162); or by occupying the heparin binding site on FGFRs

(163).

Paclitaxel has minimal activity in pancreatic cancer (164-166), which is possibly due to the FGF-related tumor resistance. Using 96 tumors obtained from patients with bladder, breast, head and neck, ovarian or prostate cancer, our laboratory has shown that bFGF level, but not other proteins known to contribute to resistance (i.e., mdr1 p- glycoprotein, p53 and Bcl-2), is the best indicator of paclitaxel resistance. Overcoming this resistance may result in good antitumor activity, justifying preclinical investigation.

The purpose of this study was to evaluate whether a more potent nonspecific inhibitor PPS, either alone or in combination with suramin, can enhance the activity of paclitaxel in Hs 766T pancreatic tumors, thereby further prove our hypothesis regarding

51 FGF-induced chemoresistance in pancreatic cancer as well as develop a more effective treatment strategy.

3.2 Materials and methods

3.2.1 Chemicals and reagents

Paclitaxel, suramin, cremophor EL, and proteinase K were obtained from Sigma

(St. Louis, MO); pentosan polysulphate from the National Cancer Institute (Bethesda,

MD); cell culture supplies from Invitrogen (Carlsbad, CA); Matrigel™ basement

membrane matrix from BD Biosciences (Bedford, MA); Ki67 Antigen Kit from

Novocastra Laboratories Ltd. (United Kingdom); M30 CytoDEATH antibody from

Roche (Germany); ApopTag® Peroxidase In Situ Apoptosis Detection Kit from Intergen

Company (Purchase, NY) and Chemicon International (Temecula, CA); LSAB kit from

Dako (Carpinteria, CA); and liquid DAB kit from BioGenex (San Ramon, CA).

3.2.2 Cell culture

Human Hs 766T cells (a gift from Dr. Byoungwoo Ryu, Johns Hopkins Medical

Institute, Baltimore, MD) were maintained as monolayer cultures at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM)

supplemented with 9% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential

amino acids, 90 µg/ml gentamicin, and 90 µg/ml cefatoxime.

52 3.2.3 Animal and drug treatment protocols

Female athymic nude mice (5-6 weeks old) were purchased from the National

Cancer Institute (Bethesda, MD). Mice had free access to sterilized rodent food and

water. Sub-confluent Hs 766T cells were suspended in a 1:1 solution of Matrigel™ and

sterile physiological saline and subcutaneously injected into both flanks of mice (2 × 106 cells per site in 200 µl solution). After ~2 weeks, mice were randomized into control and treatment groups according to initial tumor volume and body weight.

PPS (4 mg/ml) and suramin (1 mg/ml) stock solution was prepared in physiological saline and filtered using sterilized 0.2 µm pore size syringe filters.

Paclitaxel was dissolved at a concentration of 20 mg/mL in a 1:1 solution of Cremophor

EL and ethanol and diluted with sterile physiological saline right before use. In the short- term effect study, mice received vehicle (0.5:0.5:9 solution of Cremophor EL, ethanol and physiological saline), 20 mg/kg PPS, 20 mg/kg paclitaxel (T20), or a combination of both drugs with (TPS) or without the addition of 10 mg/kg suramin (TPS). Treatment was conducted twice weekly for three weeks. Mice were anesthetized with Avertin three days following administration of the final doses. A second group of mice were used for the survival study and received the same treatment but included two more groups, 20 mg/kg paclitaxel in combination with 5 mg/kg PPS (TP5) or 10 mg/kg PPS (TP10). In both studies, suramin was injected intraperitoneally, and paclitaxel and control vehicle were administered through tail vein injection. PPS was dosed intraperitoneally for convenience in the short-term study, and changed to tail vein injection in the survival study due to serious body weight loss.

53 3.2.4 Drug activity evaluation

Standard macroscopic pharmacodynamic end points, i.e., changes in tumor

volume and survival, and microscopic end points, i.e., changes in proliferation and

apoptosis in post-treatment tumors, were used to evaluate the drug activity. Body weights

were monitored before the administration of each dose and used as an indicator of drug

toxicity.

Tumor size measurement was applied to animals in both short-term and survival studies. The widths and lengths of tumors were measured using calipers and tumor volume was calculated as 50% of (width)2 × (length). For the survival study, mice were

euthanized when moribund (i.e., tumor size reached ten times of the initial value or when tumor ulceration affected health as required by our animal care and use protocol). Group mean tumor volumes at time points subsequent to animal removal were calculated using the final volumes of the euthanized animals. Median survival times (MST) were calculated from the day of cell implantation to animal death. The tumor growth rate was calculated during the treatment, and the regrowth rate after the termination of treatment was also calculated because most mice had no tumor regression following drug treatment.

Average tumor regrowth rate was calculated as the tumor size difference, between the onset of tumor regrowth and the final measurement, divided by the number of days over which the tumor size measurements were made. For mice that did not have tumor regression, regrowth rate was calculated as the tumor size increase from day 21 to the day of sacrifice.

54 Histological evaluation was conducted on samples from the short-term effect

study but not the survival study, due to the big size and necrosis of tumors in the latter.

Mice were anesthetized with Avertin three days following administration of the final dose,

and tumors were excised, fixed in 10% phosphate buffered formalin, dehydrated, and

embedded in paraffin wax. Five µm histologic sections were prepared for

immunostaining. Ki67 staining was performed following the protocol described in

Chapter 2. The apoptotic indices were identified by both the terminal deoxynucleotidyl

transferase biotin-dUTP nick end labeling (TUNEL) method (123) and monoclonal

antibody M30 CytoDEATH (M30), which is specific for neo-epitope in cytokeratin 18

that becomes available at an early caspase cleavage step during apoptosis (167). TUNEL

staining protocol followed the protocol described in Chapter 2. For M30 staining,

rehydrated tissue sections were incubated in methanol containing 3% H2O2 for 10 min

before antigen retrieval, which was conducted by microwaving the samples in preheated

citric sodium buffer (1 mM, pH 6.0) at sub-boiling temperature for 15 min. After cooling

down for 5 min, the sections were washed, and blocked with incubation buffer for 10 min,

followed by incubation with primary antibody (1:250 dilution) at 37°C for 1 hr. The

incubation buffer without antibody was used for negative controls. Then the sections were stained using LSAB and DAB kits, and counter-stained with hematoxylin, dehydrated, and mounted.

The tissue after staining was scanned at low magnification (100 ×) using a Zeiss

Axiovert 35 microscope (Carl Zeiss, Thornwood, NY) to find the most active area of positive staining, the so-called “hot-spot” area, and manual cell counting was done in 4-5

55 fields of the “hot-spot” area at 400× magnification (168). The proliferation index and

fractions of apoptotic cells were calculated as the (number of Ki 67-positive, TUNEL- or

M30-stained cells) divided by (total cell number) in each tumor section, respectively. At

least 800 cells were counted per tumor.

3.2.5 Statistical analysis

Statistical significance of the differences between treatment groups was assessed

using the following tests. Differences in tumor growth were analyzed by ANOVA for

repeated measures. Survival probabilities were estimated with the Kaplan-Meier method.

The log-rank test was used for comparison of survival between groups. All other comparisons were performed by Tukey test after ANOVA. Differences were considered

significant when p<0.05.

3.3 Results

3.3.1 Enhancement of paclitaxel activity by PPS: short-term tumor size change

The results were shown in Table 3.1 and Figure 3.1. All the experimental groups

showed comparable initial tumor sizes. PPS alone did not exhibit significant antitumor

activity, indicated by the similar tumor growth profile as the control group. Tumor

growth was retarded by single-agent paclitaxel, with an average tumor volume ~4-fold of initial value on day 21 (compared to ~11-fold in control and PPS alone groups, p<0.001).

The addition of PPS to paclitaxel therapy significantly enhanced the activity of paclitaxel,

56 reducing the average tumor volume by ~64% on day 21 (p<0.003). Yet the inclusion of a

third drug, low-dose suramin, did not cause any further benefit in terms of tumor size. In

contrast, the three-drug combination was even worse than the two-drug combination up

to two weeks from the first treatment, although the difference was not statistically

significant. In summary, the tumor response was still poor and there was no universal

tumor regression in either paclitaxel or the two combination groups.

3.3.2 Enhancement of paclitaxel activity by PPS: overall survival

Overall survival was shown in Table 3.2 and Figure 3.2. MST was 32 days for

both control and PPS alone groups. Single-agent paclitaxel significantly prolonged the

MST to 42 days (p<0.03 compared to control and single-agent PPS), which was further

extended to 55, 60, and 70 days by the addition of 5, 10, and 20 mg/kg PPS to paclitaxel

treatment (TP5, TP10, TP20), respectively. But statistical difference was only reached at the two higher PPS doses (p<0.03 for TP10 and TP20, and p=0.15 for TP5, compared to paclitaxel alone). Again, the three-drug combination was not different from the two-drug

combination, with a MST also at 70 days, reaching marginal statistical significance

compared to paclitaxel alone (p<0.063), probably due to the smaller sample size (n=3).

Tumor regrowth curves for mice in survival study were shown in Figure 3.3 and

growth rates were listed in Table 3.2. Consistent with the tumor size data, PPS showed a

growth rate comparable to control groups. During treatment, pacliatxel significantly

slowed down the tumor growth (17.5 ± 14.4 %/day for paclitaxel versus 52.3 ± 11.3

%/day for control, p<0.001), although there is no tumor regression. The combination of

PPS and paclitaxel further decreased the growth rate in a dose-dependent manner, with

57 the maximal effect at the dose of 20 mg/kg which reduced growth rate to 4.6 ± 3.5 %/day

(p<0.03 compared to paclitaxel alone). After the end of treatment, the tumor regrowth

rate increased more than 3 folds in both single-agent paclitaxel and combination groups,

compared with that during treatment. The addition of PPS had a trend to slow down the

tumor regrowth, but it’s not significantly different from paclitaxel alone due to the large

variation in all groups. In the combination groups, there seemed to be three phases of the

tumor growth curve. After a short phase of rapid growth during the first four treatments,

the tumor growth was retarded, showing a plateau of tumor size until five weeks. After

that, the tumor regrowth resumed and exhibited a similar growth rate as in control, indicated by the almost paralleled growth curves.

The three-drug combination (TPS) had comparable effect on tumor regrowth as

TP20 (Figure 3.3B), either during treatment (4.6 ± 3.5 %/day for TP20 vs. 2.3 ± 5.7

%/day for TPS), or after treatment (26.8 ± 14.0 %/day for TP20 vs. 24.3 ± 21.8 %/day for

TPS). These data provided additional evidence that the slightly worse efficacy of TPS

group in the short-term effect study was probably only due to the big variation.

3.3.3 Histological evaluation: proliferation and apoptosis

As shown in Table 3.3 and Figures 3.4, single-agent PPS had no effect on tumor

cell proliferation compared to untreated controls, as indicated by a similar proliferation

index around 85%. Single-agent paclitaxel significantly decreased the proliferation index

and proliferating cell density by more than 15% (p<0.05 compared to control and single-

agent PPS), but the addition of PPS did not significantly further decrease the proliferation

index or proliferating cell density, with or without the presence of low-dose suramin. 58 TUNEL and M30 CytoDEATH monoclonal antibody staining gave similar results

regarding apoptosis (Table 3.3, Figure 3.5, and 3.6). Again, PPS alone did not induce

apoptosis, reflected by an apoptotic index close to the untreated controls (<2%). Single-

agent paclitaxel increased the apoptotic index by more than 3-fold (p<0.05 compared to

control and single-agent PPS), which was further enhanced by an additional 40% by the

combination of PPS and paclitaxel (p<0.05 compared to single-agent paclitaxel). The

inclusion of the third drug, low-dose suramin, failed to further increase the paclitaxel-

induced apoptosis.

3.3.4 Effects of drug treatment on animal body weights

Initial body weights were similar for all groups as shown in Tables 3.1 and Table

3.2. Both single-agent PPS and paclitaxel treatment were generally well tolerated at the

doses administered, with body weight loss less than 5% in average. However, once PPS was combined with paclitaxel, serious toxicity occurred, accompanied by a body weight loss more than 15%, no matter low-dose suramin was co-administered or not.

3.4 Discussion

The goal of this study was to evaluate the effect of PPS on in vivo antitumor activity of paclitaxel in human pancreatic xenograft tumors. This study was motivated by the finding that low-dose suramin, a nonspecific FGF inhibitor, enhanced the antitumor activity of different chemotherapeutic agents in various xenograft tumors (32-34; 96),

59 including pancreatic tumors (97), and results of Phase I/II clinical trials suggesting therapeutic benefits by adding low-dose suramin to paclitaxel and carboplatin in NSCLC

(119; 120). The enhancing activity of suramin was observed at low, but not at high dose.

As suramin is known to have a myriad of pharmacological activities, it is quite likely that suramin at high doses causes undesirable actions that counteract the low-dose activity.

PPS has greater specificity as FGF inhibitor (161) and may therefore lack the U-shaped dose-activity relationship.

Results of the present study indicated that subtherapeutic, nontoxic doses of PPS significantly enhanced the antitumor activity of paclitaxel in Hs 766T xenograft tumors.

This result is consistent with previous studies which showed sensitization effect of low- dose suramin to both Gemzar® and paclitaxel without enhancing host toxicity (97). These findings further prove our hypothesis regarding FGF-induced chemoresistance in addition to studies applying bFGF antibody and/or suramin in other tumor models in our laboratory. Although both PPS and suramin sensitized the tumor response to paclitaxel and enhanced paclitaxel-induced tumor cell apoptosis, the two FGF inhibitors had some differences. The most important one is that low-dose suramin did not increase host toxicity in combination with paclitaxel while PPS did, which certainly limited the potential of applying PPS as a chemosensitizer. Besides, PPS has little impact on the antiproliferation effect of paclitaxel whereas low-dose suramin significantly reduces the proliferation index in combination with paclitaxel (97). The reason of the toxicity induced by the combination of paclitaxel and PPS was not clear, since both agents were well tolerated when administered individually and even higher doses of PPS

60 (intraperitoneal dose at 25 mg/kg/day, 6 days per week) could be tolerated by the animals

for up to 3 months without any apparent toxic effects (160).

In addition to FGF antagonism, both suramin and PPS have multiple other targets.

Suramin inhibits protein kinase C isoforms, reverse transcriptase, mitocondrial oxidative

enzymes, and the binding of other growth factors to their respective receptors, including

platelet derived growth factor, epidermal growth factor, vascular endothelial growth

factor, transforming growth factor $, and insulin-like growth factor 1 (119). PPS also

binds to many other heparin-binding growth factors, such as Kaposi’s sarcoma-derived

fibroblast growth factor, and extracellular growth factor (EGF) associated tyrosine kinase

in lysed tumor cells (155; 160; 169). At low concentration (1-10 µM), PPS competitively

inhibits PKC β1 catalyzed phosphorylation of EGFR substrate (104). PPS also prevents

the interaction of HIV-1 with target cells (170). Therefore, combining these two inhibitors with paclitaxel would theoretically generate greater chemosensitization effect than applying one single inhibitor PPS. However, the result failed to show any difference.

One possible reason is that PPS already caused maximal sensitization effect; thus the inclusion of suramin would have no additional benefit. This is reasonable considering the higher potency and selectivity of PPS at inhibiting bFGF (155). The redundancy of PPS and suramin effect in the current study is also indicated by the distinct pharmacokinetic profiles of these two inhibitors. PPS is eliminated with a half-life of 2-4 hours whereas suramin has a long half-life of ~60 hours in mice (171). To design a better combination strategy, further studies are needed to elucidate the mechanisms of the in vivo chemosensitization effect of PPS.

61 Single-agent PPS did not show antitumor activity in our study. This is not

surprising. Although PPS was widely studied as an anticancer agent in 1990’s because of its antiangiogenetic effect by blocking FGFs, the promising preclinical data in tumor growth inhibition (160; 161; 172) did not lead to clinical efficacy (169; 173-175). The main reason was that tumors were treated at very early stage in preclinical studies, which was not true in clinical setting. PPS effectively inhibited tumor growth when the treatment was initiated 1 day after tumor cell inoculation, but had no effect on tumors beyond a certain size (161). In contrast, all the treatments started about 2 weeks after tumor cell implantation with tumor size around 5-9 mm in diameter in our study. Further more, in preclinical studies testing the antitumor activity of PPS, both higher dose of PPS and more frequent dosing schedules were used, i.e., daily intraperitoneal dose at 25 mg/kg for six days per week (160; 161) or daily oral dose of 150 or 1500 mg/kg (172).

Targeting growth factors to modulate chemotherapy is an appealing approach.

Numerous other studies have used inhibitors of growth factors to enhance the effects of chemotherapy in pancreatic tumors and have shown very promising results. Clinical trials are underway testing both radiation and chemotherapy in combination with growth factor-targeted agents, such as EGFR antibody ZD1839 and HER2 antibody cetuximab

(55), VEGF-A antibody bevacizumab, EGFR antibody cetuximab, small molecule TKI anti-EGFR agents gefitinib (Iressa) and erlotinib (Tarceva) (Reviewed in (141)). Tarceva targets the EGFR/HER1 pathway and is the first drug in a phase III trial to have shown a significant improvement in overall survival when added to gemcitabine chemotherapy as

62 initial treatment for pancreatic cancer, thus providing a successful example in the

modulation of chemotherapy targeting at growth factors.

In conclusion, PPS enhanced the antitumor effect of paclitaxel, presumably via an

apoptotic mechanism, but the serious toxicity of the combination limited the potential for

further investigation. Yet as a proof-of-concept study, results of the present study further proved our hypothesis that chemosensitization effect could be achieved by inhibiting bFGF, the epigenetic inducer of broad-spectrum chemoresistance. The combination of two bFGF inhibitors, PPS and low-dose suramin, failed to achieve better

chemosensitization effect, probably due to the redundancy.

of ept for

d twice weekly for three weeks. litaxel (T20), or a combination l: tumor size and body weight l: tumor ven intraperitoneally. n=6 mice per group exc n=6 ven intraperitoneally. rol), 20 mg/kg PPS, 20 mg/kg pac rol), 20 mg/kg g suramin. Treatment was g suramin. conducte antitumor activity of paclitaxe activity antitumor in vivo vein injection. Suramin and PPS were gi

Short-term effect of PPS/suramin on Hs 766T tumor-bearing mice received treatments of vehicle (cont Table 3.1 Vehicle and paclitaxel were given via tail control (n=5). both drugs with (TPS) or without (TP20) the addition of 10 mg/k the addition (TP20) (TPS) or without drugs with both

# rent ken as

r three weeks. Suramin was een groups were compared by owth rate and survival a combination of three drugs (20 mg/kg a combination of 20 mg/kg paclitaxel and diffe a combination of 20 mg/kg p<0.05 compared to control and single-agent PPS. † ± S.D. Survival times betw S.D. Survival times ± activity of paclitaxel: gr was conducted twice weekly fo ered via tail vein injection. Median survival time (MST) was ta

d as TP5, TP10, and TP20, respectively), or vehicle, 20 mg/kg PPS, 20 mg/kg paclitaxel (T20), 20 mg/kg vehicle, 20 mg/kg PPS, p<0.063 compared to paclitaxel alone.

* Long-term effect of PPS/suramin on in vivo antitumor Kaplan Meier analysis. Other comparisons used Tukey test after ANOVA. time from tumor implantation until moribund. n=3-4 mice per group. Mean n=3-4 time from tumor implantation until moribund. injected intraperitoneally and all the other drugs were administ paclitaxel, 20 mg/kg PPS, and 10 mg/kg suramin, TPS). Treatment and 10 mg/kg paclitaxel, 20 mg/kg PPS, Table 3.2 Animals received injections of p<0.05 and 20 mg/kg, designate doses of PPS (5, 10, and

65 Ki67-positive cells TUNEL-positive M30-positive cells Group ells Cells/field % Cells/field % Cells/field % Control 236 ± 22 85.0 ± 2.0 4.7 ± 0.9 1.5 ± 0.3 2.7 ± 0.5 0.8 ± 0.1 PPS 241 ± 42 85.5 ± 3.8 5.3 ± 0.7 1.7 ± 0.3 3.8 ± 1.3 1.2 ± 0.4 T20 198 ± 24a 70.5 ± 5.0a 13.1 ± 2.1a 5.3 ± 1.0a 21.2 ± 3.7a 6.3 ± 0.7a TP20 151 ± 56a 63.5 ± 7.3a 20.7 ± 2.6a, b 8.2 ± 1.1a, b 26.2 ± 3.3a, b 9.1 ± 1.5a,b TPS 183 ± 16a 67.4 ± 7.4a 21.8 ± 2.0a, b 8.7 ± 1.3a, b 27.3 ± 4.1a, b 9.1 ± 1.2a,b a. p<0.05 compared to control by Tukey test after ANOVA b. p<0.05 compared to paclitaxel by Tukey test after ANOVA

Table 3.3 Enhancement of antitumor effect of paclitaxel by PPS/Suramin (histological

evaluation)

Animals received doses of paclitaxel alone (T20), or paclitaxel in combination with PPS (TP20), as described in Table 3.1. Tumors were removed three days after the last treatment and processed for histological and immunohistochemical staining for Ki67, M30 and TUNEL. Cell density, Ki67 labeling index and apoptotic index were determined using five “hot-spot” microscopic fields at 400× magnification in each tumor. Three to six tumors per group. Mean ± S.D. Differences among the four groups are significant by ANOVA (p < 0.001).

66 1000

500 †

†# †# Tumor size (% of initial) Tumor size (% 100

0 7 14 21

Time, day

Figure 3.1 Effects of paclitaxel, PPS, and suramin on tumor growth in pancreatic tumors

Mice with well-established, subcutaneously implanted Hs 766T tumors were treated with vehicle (closed circles), 20 mg/kg PPS (open circles), 20 mg/kg paclitaxel (closed triangles), a combination of both drugs with (TPS, closed squares) or without suramin (TP20, open triangles), twice weekly for three weeks. Vehicle and paclitaxel were given via tail vein injection. Suramin and PPS were injected intraperitoneally. Arrows indicate administration of doses. n=5-6 mice per group except for control group. Mean + standard deviation. † p<0.0001 compared to control and single-agent PPS and # p<0.02 compared to single-agent paclitaxel, by ANOVA for repeated measurements.

100

† †* 25 † Overall survival, % †# †#

0 0 142842567084

Time, day

Figure 3.2 Overall survival of mice treated with paclitaxel, PPS, and suramin (Kaplan-

Meier plot)

Mice with well-established, subcutaneous Hs 766T tumors were treated with vehicle (control, closed circles), 20 mg/kg PPS (PPS20, open circles), 20 mg/kg paclitaxel (T20, closed triangles), a combination of 20 mg/kg paclitaxel and different doses of PPS (5 mg/kg, TP5, open triangles; 10 mg/kg, TP10, closed squares; and 20 mg/kg, TP20, open squares), or a combination of three drugs (20 mg/kg paclitaxel, 20 mg/kg PPS, and 10 mg/kg suramin, TPS, closed diamonds), twice weekly for three weeks. Suramin was given intraperitoneally and all the other drugs were given via tail vein injection. Mice survival was observed until moribund. Arrows indicate administration of doses. n=3-4 mice/group. Survival times between groups were compared by log-rank test. † p<0.05 compared to control and single-agent PPS. # p<0.05 compared to single-agent paclitaxel. * p<0.063 compared to single-agent paclitaxel.

1000 A

500

of initial) Tumor size (% 100

0 1428425670 Time, day

1000 B

500

Tumor size (% of initial) Tumor size (% 100

0 1428425670 Time, day

Figure 3.3 Tumor regrowth following treatment with paclitaxel, PPS, and suramin.

Mice with well-established, subcutaneous Hs 766T tumors were treated as described in Figure 3.2. Tumor size and body weight were monitored for 7 weeks. Panel A, dose- dependent chemosensitizing effect of PPS. Panel B, no additional benefit with the addition of 10mg/kg suramin.

Figure 3.4 In vivo antiproliferative activity of paclitaxel/PPS/suramin

Mice received treatments as described in Figure 3.1. Three days following administration of the final dose, tumors were harvested and fixed in formalin. Histological sections were stained for Ki67, a marker of tumor cell proliferation. The proliferation index was calculated as (number of Ki67- stained cells) divided by (total cell number) in 4-5 “hot- spot” microscopic fields at 400× magnifications in each tumor. Three to six tumors per group. Mean + standard deviation. † p<0.05 compared to control and single-agent PPS.

Figure 3.5 Enhancement of in vivo apoptotic activity of paclitaxel by PPS/suramin: TUNEL staining Tumor samples were obtained as described in Figure 3.4. Histological sections were stained for apoptosis using TUNEL. The apoptosis index was calculated as (number of TUNEL- positive cells) divided by (total cell number) in 4-5 “hot-spot” microscopic fields at 400× magnifications in each tumor. Three to six tumors per group. Mean + standard deviation. † p<0.05 compared to control and single-agent PPS. # p<0.05 compared to paclitaxel alone.

Figure 3.6 Enhancement of in vivo apoptotic activity of paclitaxel by PPS/suramin: M30 staining Tumor samples were obtained as described in Figure 3.4. Histological sections were stained for apoptosis using M30 CytoDEATH antibody. The apoptosis index was calculated as (number of M30- stained cells) divided by (total cell number) in 4-5 “hot- spot” microscopic fields at 400× magnifications in each tumor. Three to six tumors per group. Mean + standard deviation. † p<0.05 compared to control and single-agent PPS. # p<0.05 compared to paclitaxel alone.

72 CHAPTER 4

RADIOSENSITIZATION EFFECT OF SURAMIN IN PROSTATE AND

PHARYNX XENOGRAFT TUMORS

4.1 Introduction

Radiation therapy has been the treatment choice for locally or regionally advanced, unresectable cancers, either as curative therapy or as palliative therapy. Two approaches have been widely studied to improve the local response of radiation without increasing normal tissue toxicities. One is the physical approach of localizing radiation more precisely at the tumor tissue while sparing normal tissue as much as possible.

Examples are intensity-modulated radiation therapy, brachytherapy, and the use of proton beams. The other is aiming at improving the antitumor effect of radiation by modifying fractionation schedule and by combining radiation with chemical or biological agents.

Combination of chemotherapy and radiotherapy is based on two ideas, spatial cooperation and enhancement of radiation effects (45). Adding systemic drugs to radiation exposes both the tumor lesions within the irradiated field and micrometastases outside the irradiated area to the cytotoxic agent. In addition, chemotherapeutic drugs may increase the efficacy of ionizing radiation at killing tumor cell clonogens (46). The 73 most closely investigated conventional chemotherapeutic agents in combination with ionizing radiation include platinum analogues, nucleotide analogues, and taxanes

(reviewed in (46; 47)). Cisplatin/carboplatin-based chemoradiotherapy has become the standard treatment for patients with unresectable locally advanced non small cell lung cancer, small cell lung cancer (48), and locally-advanced cervical cancer (49). Clinical trials have confirmed an overall improved outcome of radiation delivered concurrently with cisplatin/carboplatin in head and neck cancer (50), and muscle-invasive bladder cancer (51). Fluorouracil-based chemoradiotherapy is the standard treatment for locally advanced pancreatic cancer (54) and rectal cancer after surgery (53).

Despite the successful application of chemoradiotherapy over the last two decades, the main drawback lies in the fact that all chemotherapeutic agents enhance radiation damage to normal tissue as well. For example, the use of gemcitabine as a radiosensitizer has been limited because of the major toxicity resulted from the sensitization of normal tissues (59).

Consequently, agents targeting specifically at the tumor tissue may offer a theoretical advantage over chemotherapy, because overlapping toxicity on normal tissue is potentially minimized. This concept attracted more attention with the increasing evidence showing that the dysregulated molecular processes and signaling pathways in cancer cells may be responsible for tumor resistance to both radiotherapy and chemotherapy. Thus strategies counteracting molecular processes responsible for tumor radioresistance or chemoresistance constitute a rapidly emerging treatment paradigm for improving the efficacy of chemoradiotherapy. Targeting agents under investigation now

74 include inhibitors of the EGFR pathway, tyrosine-kinase inhibitors, protein-kinase

inhibitors, cyclooxygenase-2 (COX-2) inhibitors, and angiogenesis inhibitors (reviewed

in (44; 46; 66; 68).

As stated in earlier chapters, our laboratory recently identified an epigenetic,

broad-spectrum mechanism of anticancer drug resistance, caused by acidic and basic

fibroblast growth factors (aFGF and bFGF) expressed in solid tumors. In the literature,

studies on the role of FGFs in radiation mainly focused on their proangiogenice effect thus protecting endothelial cells against radiation damage (e.g. (176)). Several other reports demonstrated the implication of bFGF-mediated signal transduction pathway in the cellular resistance to ionizing radiation. For example, Cohen-Jonathan and colleagues found that hela cells transfected with 24 kDa bFGF had significantly higher radioresistance than parent cells (177). They further proved that the 24 kDa FGF-2- induced radioresistance was controlled by Rho pathways (178). Recently, the

RAS/MAPK/BAD pathway was found to participate in the bFGF-induced effect on survival of HUVECs exposed to radiation, suggesting that RAS/ MAPK pathway in tumor vascular endothelium could be a potential therapeutic target to enhance the efficacy of ionizing radiation (179; 180).

We also found that FGF inhibitors, including monoclonal antibodies and suramin, reversed the FGF-induced resistance and enhanced the activity of chemotherapy in cultured human tumor cells and xenograft tumor-bearing animals. The suramin concentration and dose required for chemosensitization was nontoxic and subtherapeutic.

One of the major mechanisms of chemosensitization effect by low-dose suramin involves

75 the enhancement of apoptosis. It has been hypothesized that one of the most important

determinants of cancer cell resistance towards chemotherapy or radiation is a generalized

resistance to apoptosis (181).

Based on the above information, we hypothesized that low-dose suramin can

sensitize the radiation response of solid tumors. To test this hypothesis, we used prostate

PC-3 tumor and pharynx FaDu subcutaneous xenograft tumors in the current study.

Previous studies in our laboratory have shown that suramin sensitizes the effect of apoptosis-inducing therapies (32; 34; 93-96). Among the tumor types studied, PC-3 tumor shows the most significant enhancement. Therefore, any existing radiosensitization effect of suramin will be apparent in PC-3 tumor model. FaDu cells are known to be less sensitive to radiation-induced cell death, and were selected to evaluate sensitization in a more resistant tumor model. An additional reason to study prostate and head and neck cancer models is that radiotherapy is a major treatment modality for both tumor types.

4.2 Materials and methods

4.2.1 Cell cultures

Cell culture supplies were obtained from Invitrogen (Carlsbad, CA). Human PC-3

and FaDu cells (American Type Culture Collection, Rockville, MD) were maintained in

RPMI and MEM, respectively. All culture media were supplemented with 9% fetal

bovine serum, 0.1 mM non-essential amino acids, 1% antimitotic-antimycotic (100×).

76 4.2.2 Establishment of xenograft tumors

Male Balb/c nu/nu mice (5-6 weeks) and female athymic nu/nu mice (5-6 weeks)

were purchased from the National Cancer Institute (Bethesda, MD). Mice had free access

to sterilized rodent food and water. Subconfluent PC-3 cells were harvested and

implanted subcutaneously into the right hind limb of Balb/c nu/nu mice (3 × 106 cells per

site in 100 µl sterile physiological saline). About six weeks later, animals bearing tumors

between 5-9 mm in diameter were used for experiments. Similarly, subconfluent FaDu

cells were harvested and implanted subcutaneously into the right hind limb of athymic

nu/nu mice (2 × 106 cells per site in 100 µl sterile physiological saline). After ~2 weeks,

animals bearing tumors between 5-9 mm in diameter were used for experiments.

4.2.3 Animal treatment protocol: in vivo drug activity evaluation

4.2.3.1 PC-3 tumors

Suramin (5 mg/ml) stock solution was prepared in physiological saline, filtered

using syringe filters with pore size of 0.2 µm, and diluted to 1 mg/ml before administration. PC-3 tumor-bearing mice were treated with physiological saline only,

ionizing radiation only, or suramin pretreatment followed by ionizing radiation. Both

physiological saline and suramin were given intraperitoneally twice weekly for three

weeks. Two suramin pretreatment schedules were used, i.e., four days (2 doses in total)

or one day (one dose) before the initial radiation. No suramin alone control group was

included because multiple previous studies showed an absence of antitumor effect for this

treatment (32; 34; 93; 94; 96; 97).

77 Ionizing radiation was conducted as described in the following text. All the mice

were anaesthetized with Avertin (240 mg/kg) right before each irradiation treatment. The

tumor-implanted right hind limbs of animals were arranged in a circular fashion and

secured to 5 cm of solid water with hypoallergenic tape. One centimeter of tissue

equivalent bolus (superflab) was placed over the hind limbs to increase the surface dose.

The torsos of all animals were shielded with a custom block consisting of 5 half-value

layers of Cerrobend (decreasing the transmission of the radiation to < 5%). The radiation

was then delivered with a linear accelerator (Siemens Medical Systems, Concord, CA) at

a dose rate of 250 monitor units per minute. Three daily doses of 500 cGy were delivered

and six million volt X-rays (6 MV) prescribed to D-Max was utilized.

We measured changes in tumor volume (50% of width2 ×length) using calipers.

Body weights were monitored as an indicator of drug toxicity. Both tumor size and body

weight were measured twice weekly until one week after the last treatment and the

measurement frequency decreased to once a week afterwards. PC-3 tumor-bearing mice

were observed for survival and mice were euthanized when moribund (i.e., tumor size

reached 5 folds of initial value, when tumor ulceration affected health as required by our

animal care and use protocol, or when significant body weight loss occurred). The

experiment was terminated at 112 days after the last irradiation.

Tumor growth rates during different phases of the experiment were also analyzed.

Overall tumor growth rate was calculated as the tumor size difference from the beginning of the experiment to the time of mouse removal divided by the duration of the observation. The average tumor size reduction rate was calculated as the difference of

78 initial tumor volume and the value on day 39, i.e., when maximal tumor reduction was

reached in irradiation alone group, divided by the time. The average tumor regrowth rate

was calculated as the tumor volume difference, between the onset of tumor regrowth and

the final measurement, divided by the number of days over which the measurements were

made. Some animals were euthanized during the experiment. Group mean tumor volumes

at time subsequent to animal removal were calculated using the final tumor volume of the

euthanized animals.

Response analysis using clinically relevant pharmacodynamic end points were

performed based on Response Evaluation Criteria in Solid Tumors (RECIST) (182). Mice

showing no evidence of tumors for two consecutive measurements were termed complete

response (CR), and euthanized if CR lasted more than 30 days. Partial response (PR) was

achieved when a tumor volume of less than 50% of pretreatment size was maintained for

at least two consecutive measurements. If the tumor volume increased by >50% relative

to the pretreatment size, it was considered a progressive disease (PD). Tumors were classified as stable disease (SD) if there was neither sufficient increase to qualify for PD

nor sufficient shrinkage to qualify for PR. The onset of response was calculated as the date of the first measurement when the tumor volume change qualified for PR or CR. The duration of response was counted from the onset of PR or CR until the first date the response was lost, i.e., the tumor size bigger than 50% relative to the pretreatment size for the loss of PR and tumor recurrence for the loss of CR. Progression-free survival was calculated as the duration from the initiation of treatment to the first time point tumor volume increased >50% compared to the lowest tumor volume.

79 4.2.3.2 FaDu tumors

Suramin stock solution (5 mg/ml) was prepared in sterile physiological saline, and diluted to 0.5, 1, and 3 mg/ml with physiological saline for the doses of 5, 10, and 30 mg/ml. FaDu tumor-bearing mice were randomized to different treatment groups according to initial tumor size and body weight. Two treatment regimens of suramin were studied, single dose and repeated dose.

In the first study, we evaluated the dose dependence of suramin in combination with split-dose irradiation. Mice received one intraperitoneal dose of physiological saline alone, irradiation alone, or irradiation combined with sruamin pretreatment at different doses (5, 10, or 30 mg/kg, single dose) one day prior to irradiation. Irradiation was conducted the same way as described in PC-3 tumors. The reason for choosing one-day pretreatment of suramin was because this regimen resulted in significant radiosensitization effect in PC-3 tumors.

In the second study, we further evaluated the radiosensitization effect of repeated doses of suramin by targeting plasma concentration of 10-30 µM, the concentration that would induce chemosensitization effect. Mice received physiological saline alone, three daily injections of suramin (10 mg/kg/day) alone, ionizing radiation alone, or a combination of irradiation and suramin, with suramin administered eight hours before each irradiation. Ionizing radiation was conducted the same way as in PC-3 tumors except for that six million electron volt (6 MeV) (not X-rays) prescribed to D-Max was utilized.

80 Tumor size and body weights were monitored. Survival was also observed for the

study using repeated suramin dose. Mice were removed when moribund (i.e. tumor size

reached 2 cm in diameter or when tumor ulceration affected health as required by our

animal care and use protocol). The calculation of tumor growth rates and the response

analysis were performed the same way as described for PC-3 tumors. Progression-free

survival and overall survival were calculated for survival study.

4.2.4 Statistical Analysis

Statistical significance of the differences between treatment groups was assessed

using the following tests. Differences in tumor growth were analyzed by ANOVA for

repeated measures. Survival probabilities were estimated with the Kaplan-Meier method.

The log-rank test was used for comparison of survival between groups. Tumor responses were compared by Fisher’s exact test. All other comparisons for more than two groups

were performed by Tukey test after ANOVA, and comparisons of two groups by

Student’s t-test. Differences were considered significant when p<0.05.

4.3 Results

4.3.1 Radiosensitization effect of low-dose suramin in PC-3 tumors

Two pretreatment schedules of suramin, i.e., one-day (one dose) or four-day

pretreatment (totally two doses), were used to mimic the situation in chemotherapy. Since

these two pretreatment regimens showed almost equivalent radiosensitization effect, mice

81 in these two groups were grouped together. All the tumor sizes were then normalized to

their values at one day before the first irradiation. The results were shown in Table 4.1

and Figure 4.1A.

PC-3 s.c. tumors were very sensitive to the radiation treatment, with a lowest

tumor volume of ~42% relative to initial value at five weeks after the last irradiation,

which was further decreased by ~7 folds to ~6% with the addition of low-dose suramin.

The final tumor volume was also >3-fold lower in the combination group at the end of

observation (~56% in combination vs. ~194 % in irradiation alone group, p≤0.01).

Consistent results were obtained by comparing tumor growth rates. The addition of low- dose suramin to irradiation dramatically enhanced the tumor reduction rate by >50%, slowed down the overall growth rate by ~2 fold and decreased the regrowth rate by >90%

(p<0.05 compared to irradiation alone). Further more, the combination treatment delayed the onset of tumor regrowth for two weeks (day 53 in combination versus day 39 in irradiation alone group).

Response analysis of PC-3 xenograft tumors using clinical RECIST criteria gave similar results (Table 4.1). Irradiation effectively caused tumor regression, achieving three PRs and three SDs out of six animals (p<0.01 compared to control). The addition of low-dose suramin significantly enhanced the radiation responses, with three PRs and four

CRs out of seven animals (p<0.05 compared to irradiation alone). More importantly, the

CRs in combination group were actually “cure” because no tumor recurrence occurred, with a median duration of 81 days. It should be noted that in the combination group, the onset of PR was earlier (day 18 in combination group vs. day 32 in irradiation group) and

82 the duration was longer (53 days for combination group versus 42 days for irradiation

alone). The addition of low-dose suramin also significantly prolonged the median TTP

from 85 days in irradiation alone group to >116 days (Figure 4.1B).

4.3.2 Dose-dependent radiosensitization effect of suramin in FaDu tumors

Single-dose suramin showed a dose-dependent radiosensitization effect in FaDu

tumors (Table 4.2 and Figure 4.2). Irradiation alone only retarded tumor growth, shown

by the lowest tumor volume at ~1.5-fold of initial value, which was significantly

decreased to ~42%, and ~74% by the pretreatment of suramin at 5, and 10 mg/kg,

respectively. The final tumor volume at the end of observation showed the same trend,

which reached ~3.2 fold of initial value in the irradiation alone group and was decreased

to ~74%, and ~1.5 fold with the addition of 5, and 10 mg/kg suramin, respectively.

Interestingly, the sensitization effect of suramin was lost at 30 mg/kg, suggested by the similar final and lowest tumor size compared to irradiation alone, and the almost overlapping tumor growth curve.

Comparison of tumor growth rates gave similar results. Pretreatment of both 5 and 10 mg/kg suramin decreased the overall tumor growth rate and increased the tumor reduction rate compared to irradiation alone, with statistical significance achieved only at

5 mg/kg suramin. There was no difference between irradation and combination groups in tumor regrowth rate, indicating suramin did not inhibit the regrowth of FaDu tumors after irradiation.

Tumor response analyses using RECIST criteria also demonstrated the radiosensitization effect of low-dose suramin. All combination groups showed higher

83 overall tumor response compared to irradiation alone, with statistical difference and

marginal difference achieved at 5 mg/kg (p<0.05) and 10 mg/kg suramin (p<0.07),

respectively.

The loss of radiosensitization effect of suramin at higher dose was further confirmed with three repeated daily doses of suramin at 10 mg/kg (Table 4.3). Irradiation

alone and combination groups were almost equivalent in short-term antitumor activity,

indicated by overlapping tumor growth curves (Figure 4.3A), comparable tumor

reduction rates and lowest tumor volumes. Combination group had a trend to shorten the

median TTP (31 days in combination versus 56 days in irradiation alone group) and MST

(91 days in combination versus 107 days in irradiation alone group), although no

statistical significance (Figure 4.3B and C). The overall tumor response was also

comparable between irradiation alone and combination group using RECIST criteria.

Very interestingly, one mouse reached PR with the treatment of single-agent suramin.

4.3.3 Toxicity of the treatments

The radiation dose used was generally well tolerated in both PC-3 (Table 4.1) and

FaDu tumors (Table 4.2, Table 4.3), with body weight loss less than 10%. The

combination of suramin with ionizing radiation did not show additional toxicity. For PC-

3 tumor-bearing mice, both irradiation alone and combination group had one mouse

showing body weight loss more than 10% two months after irradiation, and was thus

removed before the end of experiment. There was also one mouse had edema in the

control group, probably due to infection.

84 4.3 Discussion

Two sets of pharmacodynamic end points were used to evaluate the radiosensitization effect of suramin in the current study, the conventional tumor volume changes and growth rates, as well as clinically relevant response analysis (PR, CR, SD,

PD, and median TTP). Results showed that subtherapeutic, nontoxic doses of suramin significantly improved the radiation response of both radiosensitive PC-3 prostate and relatively radioresistant FaDu pharynx xenograft tumors without increasing the host toxicity. Therefore, suramin could be widely used as a radiosensitizer in solid tumors, in addition to its broad-spectrum chemosensitization effect established in our laboratory in various tumors in vitro, in vivo (24; 32-34; 96), and in clinical evaluations (119; 120).

Suramin had been combined with radiation in both preclincal and clinical studies by other groups, but the suramin dose or concentrations used were cytotoxic (183-185).

Only one report suggested an effective concentration lower than 100 µM (186). In the current study, suramin is used to reverse bFGF-induced resistance, an effect requiring only less than 50 µM concentration, which has no cytotoxicity in cultured tumor cells, animals or patients.

The radiosensitization effect of nontoxic suramin could be applied to radiation therapy in several ways. First, it can be used to enhance radiation response of primary tumors and thereby improve the local control, because one major reason of local recurrence tumors after radiation therapy is the inherent biologic resistance of tumor cell clones to radiation (187). Second, it can be used to lower the radiation dose yet still achieve the same response, thus decrease the toxicities related to radiation treatment. This

85 is important in clinical situation for patient to achieve a better quality of life. For example,

comprehensive head and neck radiation therapy is associated with a wide spectrum of normal tissue toxicities (188). Third, suramin might also sensitize recurrent tumor cells to subsequent radiotherapy, which is highly likely, as our laboratory already reported that

nontoxic suramin enhance the tumor response to chemotherapy in chemotherapy-

pretreated non-small cell lung xenograft tumors (95) and breast xenograft tumors (94).

The intrinsic radiosensitivity of PC-3 tumors is higher than FaDu tumors, as

indicated by better tumor response after the same dose of ionizing radiation. This could

be well explained by the literature report. A major determinant of biologic effect of

radiation is the response of the target tissue to radiation, quantified in a value called the

alpha/beta ratio (α/β ratio). The α/β ratio for prostate cancer has been demonstrated to be

substantially lower than values commonly attributed to tumor tissue (189; 190). The

reason for this is believed to be the slow growth kinetics and low proportion of

proliferating cells in prostate cancer (189). The slow growth kinetics has been seen in the

current study, with an average growth rate of ~40%/day in FaDu tumors versus ~8%/day

in PC-3 tumors, and the low proliferation of PC-3 tumors will be further shown in

Chapter 6. Please note that the radiation response of FaDu tumors were better in the

experiment using repeated suramin doses (Table 4.3), but the mice received different

irradiation source (electron beads instead of high-energy X-ray, please see methods and

materials) in that experiment because of technique reason, thus it is not fair to compare to

PC-3 tumors.

86 Radiotherapy induces both acute and late toxicities. Serious body weight loss

(>10%) of the two PC-3 tumor bearing mice did not occur until two months after irradiation. It was probably due to the late toxicity of ionizing radiation. However, the one mouse with edema in the control group indicated that it might also be caused by carelessness in animal care. Other than those two animals, localized high-energy X-ray was generally well tolerated in both PC-3 and FaDu tumors at the dose used; more importantly, low-dose suramin did not add toxicity to irradiation. Since the radiation response in FaDu xenograft tumors was poor, higher tolerable irradiation dose could be applied for further investigation, and longer observation might be needed to exclude radiation-induced late toxicity for future translation into the clinical setting.

In conclusion, nontoxic doses of suramin enhances the radiation response in both prostate and pharynx xenograft tumors. Due to the enhancement of antitumor response and the lack of toxicity, suramin is an efficient enhancer of radiation response for both

PC-3 tumors and FaDu tumors. Therefore, the results of this study support the use of combining suramin and radiation for the radiotherapy of prostate and head and neck cancer, and possibly other tumors. Suramin showed a biphasic radiosensitization effect in

FaDu tumors, which will be further investigated in the next two chapters.

87 Group Control IR Sur + IR Total # of mice 4 6 7 Body Weight Pre-treatment, g 23.1 ± 1.0 21.8 ± 1.3 22.3 ± 1.8 Post-treatment a, g 23.7 ± 3.0 21.2 ± 2.1 22.1 ± 2.1 Post-treatment a, % 102 ± 8.7 99.6 ± 5.7 102 ± 8.7 Tumor Volume, mm3 Initial 212 ± 67 185 ± 59 226 ± 41 Final a, % of initial 492 ± 346 194 ± 172 56± 122 Lowest, % of initial -- 42 ± 19 5.8 ± 6.3 Growth Rate (%/day) Overall 7.6 ± 3.0 0.96 ± 1.5 † -0.86± 0.29 † # Reduction -- -2.1 ± 0.68 -3.3 ± 0.27 # Regrowth -- 2.4 ± 2.1 0.14 ± 0.34 # (RECIST) Tumor Response † † # CR b, # (%) of Mice 0 0 4 (57) Onset, days c -- -- 36 Duration, days c -- -- 81 PR, # (%) of Mice 0 3 (50) 3 (43) Onset, days c -- 32 18 Duration, days c -- 42 53 SD, # (%) of Mice 0 3 (50) 0 Duration, days c -- 67 -- PD, # (%) of Mice 4 (100) 0 0 PFS, days c 13 85 † >116 † # a. The end of observation b. CR is actually “cure” here, no tumor recurrence at the end of observation c. Median value; PFS: progression-free survival

Table 4.1 Radiosensitization effect of suramin in PC-3 tumors PC-3 tumor-bearing mice received intraperitoneal injection of physiological saline, localized ionizing radiation (500 cGy per day for three consecutive days), or irradiation combined with suramin. The treatment of physiological saline or suramin was given intraperitoneally twice weekly for three weeks. Tumor size and body weight were monitored for 112 days since the last irradiation. Difference in tumor response was analyzed by Fisher’s exact test. Survival differences were compared by log-rank test. Differences in growth rate were analyzed by Tukey test after ANOVA for three groups and Student’s t-test for two groups. † p<0.01 compared to control. # p<0.05 compared to irradiation alone. 88 Group Control IR S5 + IR S10 + IR S30 + IR

Total # of Mice 6 5 4 5 4 Body Weight Initial, g 21.4 ± 1.4 23.6 ± 1.9 22.6 ± 2.2 23.4 ± 0.5 23.6 ± 2.0 Final, % of initial 108 ± 4.3 107 ± 2.7 109 ± 2.5 105 ± 1.2 107 ± 2.3 Tumor Volume, Initial, mm3 116 ± 70 126 ± 95 115 ± 61 112 ± 48 111 ± 32⋅ Final a, fold of initial 9.9 ± 3.8 3.2 ± 1.4 0.74 ± 1.2 1.5 ± 1.3 3.3 ± 2.1 Lowest, fold of initial -- 1.5 ± 0.21 0.42 ± 0.34 0.74 ± 0.62 1.1 ± 0.36 (RECIST) Tumor Response † # †* † PR, # (%) of mice 0 0 2 (50) 1 (20) 1 (25) CR, # (%) of mice 0 1 (20) 1 (25) 1 (20) 0 SD, # (%) of mice 0 0 1 (25) 2 (40) 2 (50) PD, # (%) of mice 6 (100) 4 (80) 0 1 (20) 1 (25) Growth Rate, %/day Overall 42.4 ± 18.2 10.3 ± 6.5† -0.75 ± 4.9†# 2.3 ± 6.3† 10.8± 10.1† Reduction -- 1.2 ± 2.2 -4.2 ± 1.9# -2.2 ± 5.1 0.54 ± 3.5 Regrowth -- 15.1 ± 11.6 5.0 ± 6.8 9.1 ± 8.1 24.9 ± 16.7 a. The end of observation.

Table 4.2 Dose-dependent radiosensitization effect of suramin in FaDu tumors

FaDu tumor-bearing mice received intraperitoneal injection of physiological saline, localized split-dose irradiation (500 cGy per day for three consecutive days, IR), or radiation with suramin pretreatment at different doses (5, 10, and 30 mg/kg, designated as S5+IR, S10+IR, and S30+IR, respectively). The first dose of physiological saline or suramin was administered one day prior to the first irradiation. Differences in tumor response were analyzed by Fisher’s exact test. Differences in growth rates were compared by Tukey test after ANOVA for more than two groups and Student’s t-test for two groups. † p<0.05 compared to control. # p<0.05 compared to irradiation alone.

89 Group Control Sur IR Sur + IR Total # of Mice 4 4 5 5 Tumor Volume, Initial, mm3 240 ± 130 236 ± 108 254 ± 119 247 ± 80.9 Lowest, % of initial -- -- 58.3 ± 78.0 61.0 ± 46.2 Tumor Response * * CR, # (%) of mice 0 0 1 (20) 0 PR, # (%) of mice 0 1 (25) 2 (40) 2 (40) SD, # (%) of mice 0 0 1 (20) 3 (60) PD, # (%) of mice 4 (100) 3 (75) 1 (20) 0 Median PFS, days 7 8.5 56† 31† MST, days 49 51 107† 93† Growth Rate, %/day Overall 35.5 ± 16.7 34.4 ± 22.3 7.3 ± 10.8† 15.8 ± 5.2 Reduction -- -- -3.0 ± 2.1 -3.0 ± 2.3 Regrowth -- -- 13.2 ± 10.1 21.4 ± 5.0 PFS: progression-free survival. MST: median survival time.

Table 4.3 Loss of radiosensitization in FaDu tumors with split-radiation dose: repeated doses of suramin

Mice with well-established FaDu tumors were treated intraperitoneally with physiological saline, localized ionizing radiation (three daily doses of 500 cGy per day), with or without three daily doses of suramin (10 mg/kg per day for three conscutive days, starting 8 hrs before each irradiation). Differences of survival between groups were compared by log-rank test. Differences in growth rates were analyzed by Tukey test after ANOVA. † p<0.03 compared to control. * p<0.05 compared to control by Fisher’s Exact test.

90 A 1000

†

100 † #

10 Tumor size, % of initial of initial % size, Tumor

0 20406080100120

B 100

†# 50

25 † Progression-free survival,% 0 0 306090120 Time, day

Figure 4.1 Suramin enhanced radiation response in PC-3 tumors Mice received physiological saline (closed circles in A, solid line in B), ionizing radiation (open circles in A, dotted line in B), or suramin pretreatment before ionizing radiation (closed triangles in A, dashed line in B). Suramin was given intraperitoneally twice weekly for three weeks. Tumor size was normalized to one day before irradiation for all the groups. n=4-7 mice per group. Panel A, tumor growth curve. Mean + standard deviation. † p<0.001 compared to control and # p≤0.01 compared to radiation alone by ANOVA for repeated measurements. Panel B, Progression-free survival. † p<0.04 compared to control and # p<0.03 compared to radiation alone by log-rank test. 91

1000

500 † †

†# 100

% of initial tumor size of initial % † #

0 7 14 21 Time, day

Figure 4.2 Dose-dependent radiosensitization effect of suramin in FaDu tumors Mice were treated with physiological saline (closed circles), ionizing radiation (open circles), or suramin pretreatment (5 mg/kg, closed triangles; 10 mg/kg, open triangles; or 30 mg/kg, closed squares) one day prior to irradiation. The upward arrow indicates the treatment of suramin, and the downward arrows indicate the conduction of irradiation. Mean + SD. † p≤0.01 compared to control and # p<0.05 compared to radiation alone by ANOVA for repeated measurements.

92 A

1000

500

† † 100 % of initial tumor size initial of %

0 7 14 21 28

100 B 100 C

75 75

50 50 †

25 † 25 Overall survival, % † † Progression-free survival, % 0 0 0 285684112 0 28 56 84 112 140 Time, day

Figure 4.3 Loss of radiosensitization effect in FaDu tumors with split-dose irradiation: repeated doses of suramin Mice were treated physiological saline (closed circles in A, solid line in B and C), three daily doses of suramin (open circles in A, dotted line in B and C), irradiation (500 cGy per day for three days, closed triangles in A, dash-dot-dot line in B and C), or combination of irradiation and suramin, with suramin treatment at 8 hrs before each irradiation (open triangles in A, dash line in B and C). Panel A, Growth curve. Mean + SD, † p<0.002 compared to control and suramin alone. Panel B, Progression-free survival. Panel C, Overall survival. † p<0.02 compared to control and suramin alone in panel B. † p<0.05 compared to control and single-agent suramin in panel C.

93 CHAPTER 5

RADIOSENSITIZATION EFFECT OF SURAMIN IN PHARYNX AND

PANCREATIC XENOGRAFT TUMORS: DOSE DEPENDENCE AND

PHARMACOKINETICS

5.1 Introduction

As discussed in Chapter 4, suramin had biphasic effects in FaDu tumors when combined with split-dose irradiation, that is, suramin at low dose (5 or 10 mg/kg, single dose) was able to enhance the radiation response, but failed to achieve sensitization effect at a higher single dose of 30 mg/kg or three daily doses of 10 mg/kg/day. Hence, FaDu tumor is a good model to further study the differential effect of suramin in combination with irradiation, which will ultimately help to optimize suramin application. On the other hand, irradiation is a one-time insult thus single-dose irradiation serves as an ideal model for time-course study for mechanistic evaluation of drug action in xenograft tumors.

Since studies in Chapter 4 were performed with split-dose irradiation, and the sensitization effect of suramin might differ from single-dose to split-dose irradiation, the current study was designed to find out the most effective sensitizing dose and the highly antagonistic dose of suramin in combination with single-dose irradiation. These two 94 doses will be used for microscopic pharmacodynamic evaluation in the next

chapter.Since our final goal is to translate the preclinical findings into clinical application,

we also want to know if there is tumor type specificity in the dose dependence of suramin

in mediating radiosensitization effect. Therefore, another tumor model, Hs 766T

xenograft pancreatic tumor that has high FGF expression, was also included in the current study. This study was motivated by the previous finding in our laboratory that pancreatic tumors needed higher suramin dose than other tumor types (e.g. PC-3 tumor) to achieve the best chemosensitization effect. When combined with paclitaxel in pancreatic Hs 766T tumors, 30 and 50 mg/kg suramin enhanced the Hs 766T tumor response to a larger extent than 10 mg/kg, in terms of tumor volume change, progression-free survival and overall survival (97). Thus, we tested whether higher doses of suramin could also result in a greater extent of radiosensitizing effect in pancreatic tumors.

Another reason for testing the radiosensitization effect of suramin in pancreatic cancer is because of the severe lethality of this malignant cancer and its high resistance to radiotherapy. Radiotherapy combined with conventional chemotherapy has been widely used as neoadjuvant or adjuvant treatment in resectable pancreatic cancer. Specifically, radiotherapy combined with 5-fluorouracil (5-FU) treatment is now the standard treatment for locally advanced pancreatic cancer, based on the result of an early GITSG trial, in which the combination of fluorouracil with radiation almost doubled survival duration from 5.3 months to 9.3 to 9.7 months (191). However, eventually all patients will develop disease progression and death (192; 193). Therefore, developing a more active local-regional therapy for pancreatic cancer is necessary. Newer approaches to

enhance radiation response include the combination of conventional cytotoxic agents

such as gemcitabine and paclitaxel. Despite its high potency as a radiosensitizer, full-dose

gemcitabine combined with full-dose radiation has severe toxicity in clinical application and difficulty exists in identifying an “optimal” dose and schedule for gemcitabine in

combination with radiotherapy. Recently, several phase I and phase II studies have

evaluated the feasibility and efficacy of chemoradiotherapy with taxanes for locally

advanced pancreatic cancer (clinical trials for gemcitabine and paclitaxel in combination

with radiotherapy were recently reviewed (54; 194)). The main drawback of applying

conventional cytotoxic agents in radiotherapy lies in the fact that all chemotherapeutic

agents enhance radiation damage to normal tissue as well. Hence, interventions with specific molecular targets may be applied to improve tumor control. Several novel approaches, including the combination of cetuximab, bevacizumab, and other growth- factor and cell-signal inhibitors with radiation are being studied (141). Suramin enhanced radiation response in both FaDu and PC-3 tumors without increasing the host toxicity

(see Chapter 4), thus might be an ideal candidate to improve the radiation response in pancreatic cancer.

In addition, a pharmacokinetic study of suramin was performed in FaDu tumors to further understand the dose dependence of suramin in mediating radiosenstization effect in different tumors. A comparison of suramin PK data in different tumor types was also performed to identify if there is any tumor type specific PK characteristics of suramin.

5.2 Materials and methods

5.2.1 Chemicals and Reagents

Cell culture supplies were purchased from Invitrogen (Carlsbad, CA). Suramin,

trypan blue, tetrabutylammonium bromide (TBAB), and tetratutylammonium

hydrogensulfate (TBAHS) were purchased from sigma (St. Louis, MO). Methanol and

acetonitrile were purchased from Fisher Scientific Company (Fair Lawn, NJ).

Suramin stock solution (20 mg/kg) was prepared by dissolving suramin in

physiological saline at 20 mg/ml and filtered using sterile syringe filters with pore size of

0.2 µm. Stock solution was diluted to 3, 1, and 0.5 mg/ml with sterile physiological saline

for dosing.

5.2.2 Apparatus

HPLC analysis was performed on an Agilent 1100 system, consisted of a G1311A

quaternary pump, G1379A vacuum degasser, G1314A variable wavelength detector,

G1313A autosampler, and G1316A thermostatted column compartment (Agilent Tech.,

Palo Alto, CA). Tumor was homogenized with Brinkmann Polytron Homogenizer

(Brinkmann Instrument, Inc., Westbury, NY).

5.2.3 Animal protocol: in vivo drug activity evaluation

FaDu tumors FaDu tumor establishment was performed as described in Chapter

4. The mice received a single intraperitoneal injection of physiological saline or different

doses of suramin (5, 10, 30, or 200 mg/kg), with or without localized high-energy X-ray

at a single dose of 1080 cGy, which was biologically equivalent to 3 daily doses of 500 cGy/day, on tumors one day afterwards.

Hs 766T tumors Hs 766T tumor-bearing mice were randomized into different treatment groups as described in Chapter 2. The control group only received one injection of physiologic saline. Five groups of mice received a single dose of localized high-energy X-ray at 1080 cGy on tumors, with or without one-day pretreatment of suramin at different doses, i.e., 5, 10, 30, or 50 mg/kg. Three of the mice receiving 10 mg/kg suramin before irradiation received a second suramin dose (10 mg/kg) 3 days after irradiation. Another group of mice received a 50% higher irradiation dose (1620 cGy, single dose) without suramin treatment. The effect of single-agent suramin was not studied here because it had no antitumor activity in the same tumor model from other people’s study in our laboratory (97).

The radiation was conducted as described in Chapter 4 for both FaDu and Hs

766T tumors. Tumor size and body weight were monitored and tumor response analysis was performed the same way as described in Chapter 4.

5.2.4 Pharmacokinetic (PK) study of suramin

5.2.4.1 Sampling

FaDu tumor-bearing mice were treated with intraperitnoneal injection of suramin

at 10 mg/kg. At 0, 0.5, 1, 3, 6, 12, 24, 48, 96, 168, 336, 504, and 672 hrs after drug

treatment, mice (n=3 per time point) were anaesthetized with Avertin (240 mg/kg). Blood

samples were collected maximally by cardiac puncture, placed into heparinized tubes on

ice, and centrifuged at 1000 g for 10 minutes to collect the plasma. Tumors were quickly

removed and stored in eppendorf tubes at –70 °C until analysis.

Plasma and tumor samples at 24 hrs and/or 504 hrs after the administration of

different suramin doses (5, 30, or 200 mg/kg, and three daily doses at 10 mg/kg/day) were obtained and the concentrations of suramin were analyzed. Comparison of the

predicted values and experimental data at corresponding time points was used to evaluate

the linearity of PK with dose.

5.4.2.2 Plasma and tumor tissue extraction

Suramin extraction was performed following the standard protocol in our

laboratory. Briefly, 100 µl plasma was mixed with 100 µl of 0.5 M tetrabutylammonium bromide (TBAB, pH 8.0) and 10 µl internal standard (250 µg/ml trypan blue) and vortexed for 30 seconds. Then 300 µl acetonitrile was added and the samples were vortexed for another 30 seconds, followed by storage at 4 °C for more than 2 hours. The stored samples were centrifuged at 1000 g for 5 min and 50 µl of the supernatant were injected into the HPLC sampling tubes. The extraction ratio was shown to be >90% (171).

Suramin tumor extraction was performed following the method established in our laboratory. In brief, tumor samples were weighed and placed in 15 ml glass tubes. 10 µl internal standard (250 µg/ml trypan blue), 0.4 ml TBAB solution (pH 8.0) and 2 ml acetonitrile were added into each tube. Tumor was homogenized and a second tube containing the same volume of TBAB solution and acetonitrile was used to collect the remaining tissue and solution on the probe of the homogenizer. The first tube was then centrifuged at 2600 g for 15 minutes and the supernatant was taken. The solution from 99

the second tube was used to resuspend the tissue pellet during 1 min vortexing. After

centrifugation of the second tube, the supernatant was combined with that from the first one and evaporated to dryness. The processed samples were then stored at -20°C until

HPLC analysis, when the processed samples were reconstituted with 300 µl mobile phase and centrifuged at 1,000 g for 5 minutes. The supernatant was collected and 50 µl of

them was injected into the HPLC sampling tubes. This method yields an extraction rate

of >90% (195).

5.2.4.3 HPLC analysis

Suramin was analyzed using the standard protocol in our laboratory. The

stationary phase consisted of an analytical column (C18, 3 µm, 4.6 x 8.3 mm, Perkin

Elmer Instruments, Norwalk, CT) and a guard column (Nova-pak C18 guard column

insert, Waters Associates, Milford, MA). The mobile phase consisted of two solvents.

Solvent A was a mixture of 200 ml of methanol, and 800 ml of 0.2 M phosphate buffer

(pH 6.5) containing 6.25 mM of tetrabutylammonium hydrogensulfate. Solvent B was

100% methanol. The two solvents were mixed with a linear gradient condition: 0 min: A

= 80%, B = 20%; 0.1-17 min: A = 46.4%, B = 53.6%; 17-22 min: A = 80%, B = 20%.

The flow rate was kept at 1 ml/min. The detection limit of suramin was 0.5 µg/ml of plasma and 1.5 to 2 µg/g of tissue (93). Retention times of suramin and trypan blue were

11 min and 10 min, respectively.

100

5.2.4.4 Pharmacokinetic analysis

Plasma and tumor suramin concentration-time profiles were simultaneously fitted

by a five-compartment linear model using ADAPTII. Goodness-of-fit was analyzed by the Akaike and SCHWARZ Information Criteria. AUC was estimated using trapezoidal

method.

5.2.4.5 Pharmacokinetic/Pharmacodynamic (PK/PD) correlation

Radiation response was evaluated comparing the final tumor volume (day 21) to

the value of irradiation alone group for all the radiation studies done in FaDu tumors.

Suramin concentrations in plasma and tumor at the time of irradiation were simulated

using the parameters estimated by ADAPTII-based fitting constants for a 5-compartment

model. Dose dependence of suramin in FaDu and Hs 766T tumors was also compared.

5.2.5 Statistical Analysis

Statistical significance of the differences between treatment groups was assessed

using the following tests. Differences in tumor growth were analyzed by ANOVA for

repeated measures. Tumor responses were compared by Fisher’s exact test. Dose- normalized suramin concentrations in different tumors were compared by ANOVA.

Linear correlation between measured and predicted suramin concentratios was analyzed

by Pearson correlation. Differences were considered significant when p<0.05.

101

5.3 Results

5.3.1 Dose-dependent radiosensitization effect of suramin in FaDu tumors

As shown in Table 5.1 and Figure 5.1, single-agent suramin did not show any antitumor effect with a single intraperitoneal dose of up to 200 mg/kg. This is consistent with our data in other tumor types (Chapter 2, and (32; 34; 93; 97)). Single-dose irradiation (1080 cGy) only delayed tumor growth, achieving ~2.6 fold of initial tumor volume 20 days after irradiation, which was decreased to ~1.5 fold with the addition of

10 mg/kg suramin (p<0.03 compared to irradiation alone). The sensitizing effect of suramin was lost at either lower dose (5 mg/kg) or higher dose (30 and 200 mg/kg).

Tumor response analysis showed similar result (Table 5.1). Suramin alone had no impact on tumor growth, showing 100% PD at all the three doses, similar to control. All the PRs were achieved in the combination groups, three PRs with the pretreatment of 10 mg/kg suramin, and one PR at other suramin doses. The overall response rate (PR plus

SD) was higher in all the combination groups except for 200 mg/kg suramin, but the statistical significance was not reached compared to irradiation alone.

5.3.2 Dose-dependent radiosensitization effect of suramin in Hs 766T tumors

Dose-dependent radiosensitization effect was also observed in Hs 766T xenograft

tumors (Table 5.2 and Figure 5.2).

Hs 766T tumors grew faster than FaDu tumors (control tumor ~13-fold of initial

volume on day 14 versus ~9.8-fold on day 18 in FaDu tumors) and thus had worse

radiation response. After a same dose of irradiation (1080 cGy), Hs 766T tumors

achieved ~4.4-fold of initial volume on day 21, compared to ~2.6-fold in FaDu tumors. 102

Suramin at 5 mg/kg did not sensitize the radiation response significantly, although the final tumor volume was 25% lower than that of irradiation alone group. The pretreatment of suramin at all the other three doses (10, 30, and 50 mg/kg) significantly improved the radiation response. The tumor volume on day 21 was 3.2, 2.5, and 2.8 fold of initial value

(compared to ~4.4 fold in irradiation alone) for 10, 30, and 50 mg/kg suramin pretreatment, respectively.

Similar results were obtained by the response analysis. The overall response was so poor that the best response was SD. All the SDs were achieved in the combination groups, but only the 50 mg/kg group reached statistical difference compared to control or irradiation alone.

Taken together, higher suramin dose at 30 or 50 mg/kg had a trend of achieving greater sensitization effect than 10 mg/kg, but there was no significant difference among these three doses. Further more, the effect of combining 30 or 50 mg/kg suramin with irradiation at 1080 cGy was comparable to that of irradiation at a 50% higher dose (1620 cGy, IR2), indicated by the overlapping tumor growth curve, final tumor volume, median

TTP as well as response rate (Table 5.2 and Figure 5.2).

Interestingly, a second dose of 10 mg/kg suramin at three days after irradiation in the 10 mg/kg suramin pretreatment group (2×S10+IR1 in Table 5.2 and Figure 5.2) caused additional sensitization effect. The tumor volume and response were comparable to that of the 30 and 50 mg/kg suramin combination group, suggesting that suramin apparently acted as a sensitizer not only at the time of radiation insult, but also at later times.

103

5.3.3 Tumor type specific dose dependence of suramin as radiosensitizer

Dose dependence of suramin as a radiosensitizer in FaDu and Hs 766T tumors

were compared in Table 5.3. Radiation response was evaluated by normalizing the final

tumor volume on day 21 in combination groups to the average volume in irradiation

alone group. In FaDu tumors, sensitization effect of suramin was achieved only at 10

mg/kg and lost at 30 or 200 mg/kg. In contrast, suramin sensitized the radiation response

of Hs 766T tumors from 10 to 50 mg/kg, with the trend of higher dose associated with

greater sensitization effect.

5.3.4 Suramin PK in plasma and tumor

Suramin concentrations in plasma and tumor after an intraperitoneal dose of 10

mg/kg were listed in Table 5.4A. Suramin was rapidly absorbed after intraperitoneal

injection, reaching peak plasma concentration of 35.5 µg/ml at 1 hr after administration.

The tumor uptake was rather fast, with tumor concentration close to the plateau value as early as 3 hrs. There seemed to be an up-and-down pattern of suramin concentration in tumor, which was observed before in our laboratory (93). Suramin was retained in tumor

for a relatively long time, with a half-life of 289 hrs (Table 5.4B), which was longer than

the half-life in plasma of 67 hrs.

Both the plasma and tumor concentration-time profiles of suramin were

simultaneously fitted by ADAPT II using a five-compartment linear model (Figure 5.3)

modified from a population PK-PD linear model (93). The corresponding differential

equations are shown as below:

dy1(t)/dt = ka × y5(t) + k31 × y3(t) + k21 × y2(t) - k10 × y1(t) - k12 × y1(t) - k13 × y1(t) 104

dy2(t)/dt = - k21 × y2(t) + k12 × y1(t)

dy3(t)/dt = - (k31 + k34 ) × y3(t) + k13 × y1(t) + k43 × y4(t)

dy4(t)/dt = k34 × y3(t)- k43 × y4(t)

dy5(t)/dt = - ka × y5(t)

y5(0) = 0 mg/kg, y1(0) = y2(0) = y3(0) = y4(0) = 0 mg/kg

Here, yi(t) represents the drug amount in the ith compartment. Tumor was split

into two intrinsic compartments, a fast-distribution compartment and a slow-distribution

compartment. By assumption, these two compartments shared the same volume of

distribution (V3 = V4), and the tumor suramin concentration was calculated as (y3(t) + y4(t))/ V3. The plasma concentration was calculated as y1(t)/V1.

The estimated values of model parameters were listed in Table 5.4A. Both plasma

and tumor suramin concentrations were well fitted (Figure 5.4). The predicted and

measured suramin concentrations were linearly related (R2= 0.91, p<0.0001 for plasma

and R2= 0.78, p<0.001 for tumor).

The linearity of suramin PK with dose was further evaluated by comparing the

predicted and measured suramin concentrations in plasma and tumor at 24 hr and/or 504

hr after different doses. As shown in Figure 5.4, suramin concentrations were well

predicted in both plasma and tumor after either a single dose (5, 30, or 200 mg/kg) or repeated doses (10 mg/kg/day for three consecutive days). The predicted and measured suramin concentrations were linearly related (R2= 0.83, p<0.01 for plasma and R2= 0.96, p<0.0001 for tumor), suggesting that the chosen model was very efficient to predict suramin concentrations in plasma and tumor after different doses. Therefore, dose-

105

dependent PK was unlikely between 5 and 200 mg/kg at macroscopic level in FaDu tumors.

To further identify the tumor type specificity of suramin PK, data obtained in different tumor types at different suramin doses were compared. Since suramin was given by intraperitoneal injection in FaDu tumors but via intravenous route in HT-29 and Hs

766T tumors, we compared suramin concentration in tumor at 24 hrs (i.e., the time when suramin reached the peak concentration in tumors), half-life in tumor, as well as tumor accumulation (AUCtumor/AUCplasma). As shown in Table 5.4B, suramin concentrations at

24 hrs were comparable if normalized to the dose of 10 mg/kg and tumor accumulation was also similar in different tumors. Although the half-life of suramin in tumor seemed longer (289 hrs vs. 239 hrs), no statistical difference was found if comparing suramin concentration in FaDu and HT-29 tumors at each individual time point used to calculated half-life. Therefore, the type specific PK of suramin was highly unlikely.

5.3.5 Pharmacokinetic/pharmacodynamic (PK/PD) correlation in FaDu tumors

To better understand the dose-dependent radiosensitization effect of suramin in

FaDu tumors, an attempt to correlate PK and PD was made. Suramin concentrations in plasma and tumor at the time of irradiation were simulated using the parameters estimated by ADAPTII-based fitting constants. Radiation response was evaluated by normalizing the final tumor volume on day 21 in combination groups to the average volume in irradiation alone group. The results were listed in Table 5.5. For single-dose irradiation, suramin concentration at the time of irradiation was the same as C1st for split- dose irradiation at corresponding suramin dose and the response was listed in Table 5.3.

106

At a single dose of 10 mg/kg which reached steady tumor concentration around

10 µg/g at each irradiation, suramin sensitized radiation response in combination with

either single-dose or split-dose irradiation. Very interestingly, a lower dose at 5 mg/kg

achieved even greater sensitizing effect if combined with split-dose irradiation. Since the

plasma concentration after this dose declined from 4.5 to 1.6 µg/ml from the first

irradiation to the last one, while the tumor concentration stayed around ~5 µg/g during all

the irradiation, it suggested that the tumor concentration might govern the sensitization

effect. However, this tumor concentration did not improve the tumor response in

combination with single-dose irradiation. Thus there might be some biological changes

after the first irradiation, resulting in the request of lower suramin exposure. Following

this idea, the antagonistic effect of a single higher dose (30 mg/kg), which generated

tumor concentration around 30 µg/g during the course of irradiation, could be better explained. One-day pretreatment of suramin at 30 mg/kg before single-dose irradiation

achieved an average of 80% tumor volume compared to irradiation alone, yet there was

no difference in average tumor volume (103%) in combination with split-dose irradiation.

Three repeated doses of 10 mg/kg also reached tumor concentrations higher than

necessary at the time of irradiation, consistent with its failure to sensitizing radiation response.

107

5.4 Discussion

The tumor type specific dose dependence of suramin as a radiosensitizer may be due to two possible reasons: tumor specificity in its PK characteristics or in biological targets. PK study in the current study indicates that tumor-dependent suramin PK is highly unlikely at least at macroscopic level and further PK study at microscopic level is warranted to elucidate more information. It is very possible that suramin has different biological targets for sensitizing or antagonistic effect in specific tumors. Suramin is a multiple-target agent. Besides inhibition of the binding of growth factors to their respective receptors such as FGFs, PDGF, EGF, TGFβ, and IGF-1, suramin also inhibits protein kinase C (PKC) isoforms, reverse transcriptase, and mitochondrial oxidative enzymes (119). The expression of these targets may differ in different tumors. For example, the major target of suramin that we hypothesize to induce chemo- and radioresistance, FGF, has about six-fold higher expression in Hs 766T cells than in FaDu cells (unpublished data). This might at least partly explain the wide sensitizing dose range of suramin in Hs 766T tumors. In addition, it has been established that suramin has biphasic effect on PKC isoforms, activating non-active PKCs in low concentration range while inhibiting active PKCs in higher concentration range (IC50 of 30-50 µM) (196; 197).

Preliminary data by Dr. Yong Wei shows that specific PKC inhibitor antagonizes cisplatin effect in FaDu cells, suggesting PKC isoforms as one of the targets leading to the loss of radiosensitization effect at higher suramin dose in FaDu tumors. The differential expression of PKC isoforms might therefore contribute to the tumor specificity in the dose dependence of suramin. 108

On the other hand, the distinct differences in the dose dependence of suramin

between FaDu and Hs 766T tumors suggest that these two tumors could serve as good in

vivo models to identify key targets responsible for the differential effect of suramin

through side-by-side comparison. Higher dose of suramin might be studied in Hs 766T

tumors to test whether antagonistic effect could be achieved. More importantly, the tumor

type specific dose dependence of suramin also indicates that the conventional concept in

the relationship between drug exposure and effect might be reconsidered for a special

drug like suramin. A third component, drug target, may also need to be included.

The PK/PD correlation analysis raises more questions for future investigations.

Studies in the present study only suggest that tumor concentration of suramin might be

the factor governing the sensitization effect if considering the concentration at time of

irradiation as the most important. The 5-compartment linear model well predicts suramin

concentrations in plasma and tumor for single dose and repeated doses, thus providing a

good tool for future studies to differentiate the suramin concentration range (sensitizing

or antagonistic) in plasma and tumor by adjusting the dosing regimen. This is important because it will help to design efficient preclinical studies to identify the differential molecular targets of suramin, as well as guiding the dosing regimen optimization in translating preclinical results into clinical application.

In addition, lower tumor concentration of suramin was required in FaDu tumors to achieve sensitization effect in combination with split-dose than with single-dose irradiation. This could be due to the biological changes after the first irradiation, lowering the request of suramin for the subsequent irradiations. Since radiation has the unique

109

characteristics as a single time point of insult, time course study of microscopic

pharmacodynamic endpoints is needed in FaDu tumors. Another possibility might be

radiation-induced change of microscopic disposition and subsequent pharmacological

activity of suramin since it is a multiple-target agent. Advancements in mechanistic study

and microscopic disposition of suramin might clarify the discrepancy.

The observation that higher suramin doses (30 or 50 mg/kg) tend to achieve greater radiosensitization effect in Hs 766T tumors is consistent with our finding in chemotherapy in the same tumor model (97), suggesting the possible similarity between chemosensitization and radiosensitization effect of suramin. Hence, the mechanistic study performed using this radiation model will also help to explain the chemosensitization effect of suramin. On the other hand, combination of 30 or 50 mg/kg suramin with irradiation results in tumor responses comparable to a 50% higher dose of irradiation alone. Therefore, suramin can be applied as a radiosensitizer to reduce the radiation dose and subsequent toxicity while still achieving the same response. This is important because the dose-limiting toxicity of radiotherapy is a serious problem in clinical setting.

Further more, the second dose of suramin after irradiation further improves the sensitization effect is very interesting. Future studies are needed to explore the exact mechanism underlying this additional benefit.

In summary, results of the present study support that suramin sensitizes the radiation response in a tumor type-specific dose-dependent manner, with a narrow window in FaDu tumors but a wider range in Hs 766T tumors, which may be mainly due to the biological differences of specific tumors. The pharmacokinetic model is efficient to

110

predict suramin concentrations in plasma and tumor after either single dose or repeated doses, thus providing a good tool for further PK/PD studies.

111

SD. * p<0.05 by Fisher’s ± saline, different doses of suramin (5, 10,

ng radiation (1080 cGy, single dose). Mean ffect of suramin in FaDu tumors intraperitoneal injection of physiological hout localized ionizi Dose-dependent radiosensitization e FaDu tumor-bearing mice received a single 30, or 200 mg/kg), with wit exact test. Table 5.1 112

Body Weight Tumor Volume Tumor Response Group (# of mice) Initial, Day 21, fold SD Initial, g Day 21, % mm3 of initial # (%) Control (5) 21.4 ± 2.4 107 ± 8.2 a 160 ± 66 12.9 ± 7.8 a -- IR1 (5) 21.4 ± 1.3 107 ± 9.4 157 ± 53 4.8 ± 0.43 -- S5+IR1 (6) 20.9 ± 1.5 109 ± 5.6 128 ± 32 4.4 ± 2.5 1 (17) S10+IR1 (6) 22.6 ± 1.5 113 ± 10.2 155 ± 74 3.2 ± 1.1 2 (33) S30+IR1 (5) 21.0 ± 2.6 109 ± 9.2 128 ± 42⋅ 2.5 ± 1.2 3 (60) S50+IR1 (5) 21.4 ± 1.9 113 ± 5.4 176 ± 59 2.8 ± 0.47 5 (100) # 2×S10+IR1(3) 21.5 ± 1.8 111 ± 2.0 195 ± 67 2.3 ± 0.95 2 (67) IR2 (6) 21.3 ± 1.9 105 ± 4.3 162 ± 101 2.3 ± 1.2 3 (50) a. Body weight or tumor volume at day 14.

Table 5.2 Dose-dependent radiosensitization effect of suramin in Hs 766T tumors

Immunodeficient mice bearing well-established Hs 766T subcutaneous tumors received intraperitoneal injection of physiological saline, different doses of suramin (5, 10, 30, and 50 mg/kg), with or without localized ionizing radiation (1080 cGy, single dose, IR1) on tumors. Physiological saline or suramin was administered one day prior to irradiation. Three of the mice receiving 10 mg/kg suramin before irradiation were treated with a second dose of suramin (10 mg/kg) three days after irradiation (2×S10+IR1). Another group of mice received a higher radiation dose (1620 cGy, single dose, IR2). All tumor responses were SD or PD, with SD defined as <50% increase in tumor volume for at least 7 days. # p<0.05 compared to control or IR1 by Fisher’s exact test.

113

Suramin Tumor volume, % of IR p-value (animal #) FaDu Hs 766T FaDu Hs 766T (mg/kg)

5 92.5 ± 48.7 91.1 ±5 1.2 0.50 (n=9) 0.85 (n=6) 10 58.3 ± 34.7 66.1 ± 23.6 0.03 (n=13) 0.03 (n=6) 30 82.4 ± 40.3 51.9 ± 25.5 0.38 (n=8) 0.01 (n=6) 50 -- 57.4 ± 9.7 -- 0.01 (n=5) 200 105 ± 55.7 -- 0.50 (n=7) --

Table 5.3 Tumor type specific dose dependence of suramin as a radiosensitizer

Radiation response was evaluated by comparing the tumor volume at day 21 to the tumor volume of irradiation alone group. p-value was obtained by ANOVA for repeated

measures.

114

Time, hr Plasma conc. Tumor conc. Model parameters Estimate (µg/ml) (µg/g) -1 0.5 26.7 ± 1.8 3.3 ± 1.3 k10 0.03463 (hr ) -1 1 35.5 ± 4.7 5.3 ± 1.0 k12 25.79 (hr ) -1 3 22.4 ± 1.9 7.8 ± 1.4 k21 23.28 (hr ) -1 6 19.3 ± 3.0 7.0 ± 1.4 k34 0.08337 (hr ) -1 12 12.8 ± 0.6 8.2 ± 0.3 k43 0.008244 (hr ) -1 24 7.7 ± 1.2 8.6 ± 1.4 θ8 (k13=TW*θ8*k12) 0.1582 (g ) -1 48 5.7 ± 0.8 7.7 ± 2.0 θ9 (k31=TW*θ9*k21) 0.1678 (g ) 96 3.0 ± 0.5 9.5 ± 3.3 V1 0.1565 (ml/g) 168 2.0 ± 0.1 9.4 ± 0.1 V3 0.5244 (ml/g) -1 336 -- 4.1 ± 1.3 ka 6.897 (hr ) 504 -- 3.6 ± 1.5 672 -- 3.0 ± 0.7 AIC 95.36 SCHWARZ 106.71 R2 WSSR SSR Plasma 0.91 15.6 182 Tumor 0.78 10.9 23.4

A. IP suramin at 10 mg/kg in FaDu tumors FaDu tumor-bearing mice received one intraperitoneal injection of suramin at 10 mg/kg. Plasma and tumor samples were taken at predetermined time points and analyzed by HPLC. 3 animals per time point. Suramin concentrations in plasma and tumor were fitted by ADAPT II using a five-compartment linear model described in Figure 5.4. AUC was calculated by the trapezoidal method. TW= tumor weight. WSSR: weighted sum of squared residuals. SSR: sum of squared residuals.

Dose Tumor Normalized C 24 hr T1/2 (hr) AUCtumor/AUCplasma (mg/kg) type tumor p-value plasma tumor 0-96 hr 0-168 hr 10 FaDu 8.6 ± 1.4 67 289 1.10 1.61 10 HT-29 10.9 ± 3.9 0.232 60 239 # -- 1.70 30 Hs 766T 7.0 ± 1.4 79.7 -- 0.88 -- 200 Hs 766T 8.1 ± 2.2 43.6 -- 1.18 --

B. Comparison of suramin pharmacokinetics in different tumor types Suramin was given through intraperitoneal injection in FaDu-tumor bearing mice and intravenous injection in HT-29 and Hs 766T tumor-bearing mice. Tumor concentrations at 24 hr (C 24 hr) in different tumors were normalized to the dose of 10 mg/kg and compared by ANOVA. # p>0.37 if comparing suramin concentrations in FaDu and HT-29 tumors at each individual time points used to calculate T1/2.

Table 5.4 Plasma and tumor pharmacokinetic data of suramin

115

Suramin Plasma Tumor Tumor vol., p-value

(mg/kg) C1st C2nd C3rd C1st C2nd C3rd % of IR (animal #)

5 4.5 2.4 1.6 4.9 5.5 5.5 23.4 ± 36.7 <0.01(n=4) 10 9.0 4.8 3.2 9.9 10.911.0 46.7 ± 42.6 0.04 (n=5) 30 27.1 14.3 9.7 29.7 32.832.9 103 ± 67.0 0.72 (n=4) 3 × 10 # 15.6 22.7 26.8 7.7 16.7 29.2 102 ± 72.2 0.70 (n=5)

Table 5.5 Pharmacokinetic/pharmacodynamic (PK/PD) correlation in FaDu tumors

Suramin concentrations in plasma and tumor at the time of irradiation (C1st, C2nd, and C3rd for the first, second, and third irradiation with split-dose irradiation, respectively) were simulated using the parameters estimated by ADAPT II-based fitting constants for a 5- compartment model. Radiation response was evaluated by comparing the final tumor volume (day 21) to the value of irradiation alone group. p-value was obtained by ANOVA for repeated measures. Suramin was given 8 hrs before each irradiation for three consecutive days. # Suramin was administered as single dose one day before IR in all the other groups for FaDu tumors.

116

Control S5 S10 S30 S200 IR 1000 S5+IR S10+IR S30+IR S200+IR 500

† †

of initial tumor size % †# 100

0 7 14 21 Time, day

Figure 5.1 Dose-dependent radiosensitization effect of suramin in FaDu tumors Mice were treated with a single dose of physiological saline, or suramin (5, 10, 30, or 200 mg/kg), with or without ionizing radiation (1080 cGy, single dose) the second day. Mean + standard deviation. † p<0.001 compared to control and single-agent suramin and # p<0.03 compared to ionizing radiation by ANOVA for repeated measurements.

117

A Control (n=5) IR1 (1080cGy, n=5) S5+IR1 (n=6) S10+IR1 (n=6) 1000 S30+IR1 (n=5) S50+IR1 (n=5)

500 † † †# †# †# % of tumor initial size % 100

0 7 14 21

B Control (n=5) IR1 (1080cGy, n=5) IR2 (1620cGy, n=6) S30+IR1 (n=5) 1000 2*S10+IR1 (n=3)

500 †

†# †# †# % of initial tumor size % 100

0 7 14 21 Time, day

Figure 5.2 Dose-dependent radiosensitization effect of suramin in Hs 766T tumors: tumor growth curves Tumor-bearing mice were treated with a single dose of physiological saline, ionizing radiation (IR1, 1080 cGy, single dose) with or without one-day pretreatment of suramin (5, 10, 30, or 200 mg/kg). Additional comparison groups received an additional dose of suramin after irradiation (2*S10+IR1) or a single dose of higher irradiation (IR2, 1620 cGy). Sensitization studies to compare a series of increasing suramin doses were presented in panel A, while additional comparison groups were presented in panel B. Mean + standard deviation. † p<0.001 compared to control. # p≤0.03 compared to radiation alone in panel A. # p≤0.01 compared to radiation alone in panel B.

118

IP Dose K10

Peritoneal Ka Central K12 Peripheral (5) (1), V1 (2) K21

TumorWT*Theta8*K12 = K13 K31 = TumorWT*Theta9*K21

Tumor/Shallow (3) K43 K34 Tumor/Deep (4)

Figure 5.3 The five-compartment linear model used to fit the plasma and tumor concentration simultaneously

Compartment 1 represents the central compartment; compartment 2 the peripheral compartment; compartment 3 & 4 the fast & slow distribution intrinsic tumor compartments; compartment 5 the peritoneal compartment. The default drug transfer rate from compartment i to compartment j will be labeled Kij. Observation compartments represent the compartments with experimental measurements of drug concentration. Tumor weight will use the average of all tumor weights.

119

100 10 mg/kg

0.1 0 168 336 504 672

100 100 5 mg/kg 30 mg/kg g/g) µ 10 10 g/ml or µ 1 1

0.1 0.1 Suramin ( 0 168 336 504 0 168 336 504

1000 100 200 mg/kg 3×10 mg/kg

100 10

10 1

1 0.1 0 168 336 504 0 168 336 504

Time, hr

Figure 5.4 Measured and the best predictions of suramin concentrations in plasma and tumor FaDu-tumor bearing mice received intraperitoneal doses of suramin. Suramin concentrations in plasma and tumor after a single dose of 10 mg/kg were simultaneously fitted by a five-compartment linear model (Figure 5.3) using ADAPT II. Suramin concentrations in plasma and tumor after other doses (single dose at 5, 30, or 200 mg/kg; three daily doses at 10 mg/kg/day) were simulated using the estimated model parameters and compared to the actual values 24 hrs and/or 504 hrs after dosing. Solid and dotted lines are the best-fit prediction for plasma and tumors, respectively. Closed and open circles are measured suramin concentrations in plasma and tumor, respectively. 120

CHAPTER 6

CELLULAR/MOLECULAR PHARMACODYNAMICS OF SURAMIN IN

COMBINATION WITH IONIZING RADIATION

6.1 Introduction

Suramin has a biphasic effect in FaDu tumors when combined with irradiation, with sensitizing effect at low dose but antagonistic effect at high dose. To better understand the underlying mechanisms of this unconventional effect, pharmacodynamic studies at a microscopic level are necessary. On the other hand, radiation has the unique characteristics as a single time point of insult, thus serving as an ideal model for mechanistic time-course study in xenograft tumors. The current chapter evaluated the radiation effect, with or without the pretreatment of low-dose and high-dose suramin at both cellular and molecular level. Two common cellular pharmacodynamic endpoints, i.e., cell proliferation and apoptosis, were investigated. At molecular level, phospho-ERK and survivin were studied. It has been reported that bFGF could induce the upregulation of inhibitor-of-apoptosis proteins (IAPs) XIAP and IAP-1 through ERK pathway, which resulted in blocking of etoposide-induced apoptosis in small cell lung cancer cells (198).

Similarly, preliminary data in our laboratory suggested that ERK and 121 survivin, another member of IAPs family, but not anti-apoptotic proteins Bcl-2 and Bcl-

XL, were involved in the chemosensitization effect of low-dose suramin. Since our final

goal is to translate the preclinical findings into clinical application, studies at molecular

level may help us to identify useful biomarker in the future.

After exposure to irradiation, cell death may occur by one or more of the following mechanisms: immediate or delayed apoptosis, mitotic-linked death (i.e., mitotic catastrophe), and terminal growth arrests (i.e., senescence) associated with

necrosis (44). It is proposed that impairment of apoptotic cell death might be the generalized mechanism for cancer cell resistance towards chemotherapy or radiation (181;

199; 200). Based on the accumulated knowledge regarding apoptosis induction and regulation, strategies to modulate apoptosis in combination with radiotherapy have been widely explored and several promising approaches have entered clinical trials (reviewed in (66)).

Survivin is the smallest mammalian member of the inhibitor of apoptosis (IAP)

gene family with a molecular weight of 16.5 kDa (201). Survivin has a dual function at both cell division regulation and apoptosis suppression (202). Accumulated experimental

evidence supports the anti-apoptotic role of survivin (reviewed in (203)), although the precise mechanism is still incompletely understood (204). Survivin has differential expression in tumor cells and it has been proven to be associated with disease progression, which makes survivin a particularly attractive target in cancer therapy (205). Overall experimental results obtained in different studies indicate survivin as a cellular factor potentially involved in the chemo-resistant and radio-resistant phenotypes of human

122 tumor (205). Preliminary results in our laboratory also suggest the association of survivin

with the chemosensitization effect of low-dose suramin in HT-29 and PC-3 tumors.

In particular, the role of survivin in determining radio-sensitivity profiles of tumor

cells has been investigated in various cancer cells. For instance, survivin plays a critical

role in mediating radiation resistance in human primary glioblastoma multiforme cells

(206). In pancreatic cancer cells, survivin has been shown as both a constitutive and an

inducible radio-resistance factor (207). Similar results have been found in colorectal

cancer, where survivin expression acts as a marker of radiation resistance in vitro, and

more importantly, a predictive factor for radiation response and local control in the

clinical setting (208; 209). More direct evidence of survivin in radioresistance come from

the findings that inhibition of survivin expression through different molecular strategies increases the radiosensitivity of different cancer cells (210-212). All these data suggest survivin as a good molecular target to evaluate the radiosensitization effect of suramin.

The extracellular signal-regulated kinase (ERK) cascade is the first mitogen- activated protein kinase (MAPK) cascade elucidated (213). Interacting with the JNK, p38MAPK, ERK5, and probably other MAPK cascades, ERK cascade forms a complex network of interacting proteins, which governs most stimulated physiological processes

(214). Two ERK genes are known, named ERK1 and ERK2, encoding two main proteins p44 and p42, respectively (215). Because of the high degree of similarity, ERK1 and

ERK2 are usually considered to be functionally redundant, although their substrate specificity differs (213). The ERKs are phosphorylated and activated by a variety of

extracellular stimuli, including growth factors, hormones, and neurotransmitters, and

123 thereby regulate cellular processes such as proliferation, differentiation, and oncogenic

transformation (216).

Although there are few conflicting reports suggesting that activation of

Raf/MEK/ERK is essential for drug-induced cell death (217), the majority of literature

supports the pro-survival role of classical MAPK signaling with regards to the

development of chemotherapeutic drug resistance. Preliminary results in our laboratory

indicate that ERK cascade may be involved in the chemosensitization effect of low-dose suramin in HT-29 and PC-3 tumors (unpublished data). How ERK is involved in radioresistance is not fully understood yet. Ionizing radiation triggers several mitogenic intracellular signaling cascades including the Raf-MEK-ERK kinase cascade. Activated

MEK and ERK may thus promote cell proliferation and also prevent cell death induced by cytotoxic stimuli. Experimentally, it has been observed that Raf, MEK, and ERK can be activated by irradiation in some tumor cell lines (e.g. (218; 219)), suggesting that irradiation-induced activation of this MAPK signaling pathway may enhance the survival of irradiated cells. It has been reported that MEK/ERK-mediated signals selectively inhibite irradiation-induced loss of mitochondrial membrane potential and subsequent cell death in lymphocytic leukemia cells (220). The MEK/ERK pathway has been identified to act upstream of NF kappa B1 (p50) homodimer activity and Bcl-2 expression in a murine B-cell lymphoma cell line, and MEK inhibition restores radiation- induced apoptosis (221). On the other hand, there is conflicting result demonstrating that

MEK and ERK do not affect the clonogenic survival of irradiated UM-SCC6 cells (218).

124 Taken together, cell proliferation, apoptosis, bFGF, ERK activity (phospho-ERK) and survivin at predetermined time points were evaluated in FaDu tumors to explore the

cellular/molecular mechanism underlying the biphasic effect of suramin in combination

with radiation. In addition, cell proliferation and apoptosis were evaluated in PC-3 tumors

at two weeks after irradiation to elucidate the molecular changes. Survivin was also

detected in PC-3 tumors at 112 days after irradiation (study in Chapter 4) to see the long-

term effect.

6.2 Materials and methods

6.2.1 Chemicals and reagents

Cell culture supplies were obtained from Invitrogen (Carlsbad, CA); Suramin and

5-Bromo-2’-deoxyuridine (BrdU) and monoclonal anti-human bFGF mAB from sigma

(St. Louis, MO); Mouse anti-BrdU antibody from DakoCytomation (Denmark); M30

CytoDEATH antibody from Roche (Germany); phospho-p44/42 MAPK (ERK1/2) rabbit

mAB (#4376) from cell signaling (Danvers, MA); Survivin antibody from Labvision

(United Kingdom); LSAB kit from Dako (Carpinteria, CA); and liquid DAB kit from

BioGenex (San Ramon, CA); and ABC kits from (Vectastain, ABC kits).

6.2.2 Cell cultures and xenograft tumor establishments

PC-3 and FaDu cell cultures and xenograft tumor establishments were performed

as described in Chapter 3.

125 6.2.3 Animal treatment protocol

FaDu tumors FaDu tumor-bearing mice were randomized according to initial

tumor size and body weight, and received one intraperitoneal dose of physiological saline

or suramin (10 mg/kg for low dose, or 200 mg/kg for high dose), followed by a single

dose of high-energy X-ray (1080 cGy) as described in Chapter 4 on the second day.

Tumor samples were harvested before irradiation (controls), or 3 hr, 1 day, 3 days, and 2

weeks after irradiation. Tumor cell proliferation was determined by in vivo BrdU labeling.

Mice received tail vein injection of BrdU (100 mg/kg) 1 hour before euthanization.

Tumors were fixed in 10% formalin, embedded in paraffin, and then cut into 4 µm-thick sections using a microtome.

PC-3 tumors To evaluate the short-term effect of radiation and suramin at cellular level in PC-3 tumors, tumor-bearing mice received one intraperitoneal injection of physiological saline or suramin (10 mg/kg), with or without receiving localized ionizing radiation (1080 cGy, single dose) on tumors the second day. Physiological saline or suramin treatment was continued twice weekly for three more times until the mice were sacrificed at 14 days after irradiation and tumors were harvested for histological evaluation. In vivo BrdU labeling was performed the same way as in FaDu tumors for detection of tumor cell proliferation. Tumor samples were fixed in 10% formalin, embedded in paraffin, and then cut into 4 µm-thick sections by microtome.

126 6.2.4 Histological evaluation

BrdU, M30, survivin and phospho-ERK1/2 staining was performed on all the

FaDu tumor samples. For PC-3 tumors, BrdU and M30 staining were done in short-term samples, while survivin was detected in long-term samples from the study in Chapter 4.

6.2.4.1 BrdU staining

Briefly, the sections were deparaffinized, hydrated, and boiled for 7-8 minutes in citric buffer (10 mM, pH=6.0), in a microwave oven. After cooling down for another 15 min, the sections were incubated with 1% BSA for 25 min before incubation with anti-

BrdU antibody (1:250 dilution in 0.5% BSA) for 90 min at room temperature. The staining was continued using LSAB and liquid DAB kits, and counter-stained with hematoxylin.

6.2.4.2 M30 staining

The apoptotic cells were identified by monoclonal antibody M30 CytoDEATH

(M30), following the staining protocol described in Chapter 3.

The tissue after both BrdU and M30 staining was scanned at low magnification

(100 ×) using a Zeiss Axiovert 35 microscope (Carl Zeiss, Thornwood, NY) to find the most active area of positive staining, the so-called “hot-spot” area, and manual cell counting was done in 4-5 fields of the “hot-spot” area at 400× magnification (168). The proliferation index and fractions of apoptotic cells were calculated as (the number of

BrdU- or M30-positive cells) divided by (total cell number) in each tumor section, respectively. At least 800 cells were counted per tumor.

127 6.2.4.3 bFGF staining

Briefly, after de-waxing and rehydration sequentially in xylene, ethanol and water,

tissue sections were boiled for 7-8 minutes in citric buffer (10 mM, pH=6.0), in a

microwave oven, and cooled down for another 15 min before incubation with 3% H2O2 for 10 min. The tissue sections were then incubated with mouse anti-human bFGF antibody (1:100 dilution in PBS containing 5% bovine serum albumin) for 2 hrs in a humidified chamber at room temperature, followed by incubation with the linker solution

(DAKO kit) for 40 min, and then with peroxidase-conjugated streptavidin solution for 40 minutes (DAKO kit). After washing twice with PBS, tissue sections were incubated for 5 min with diaminobenzidine and counterstained with hematoxylin. Negative control specimens were included by replacing the primary antibody with blocking solution.

6.2.4.4 Phospho-ERK staining

In brief, 4 µm-thick tumor sections were rehydrated through a xylene and alcohol series, and incubated with 3% hydrogen peroxide in ddH2O for 10 min to block

endogenous peroxidase activity. The sections were then heated in 10 mM citrate buffer

(pH 6.0) until boiling in a microwave oven and kept at boiling temperature for 2-3 min,

followed by another 10 min at sub-boiling temperature. After cooling down for 5 min,

and washing two times with ddH2O and one time with TBST (Tris-buffered saline

containing 0.1% Tween 20), the sections were incubated with 5% goat serum diluted in

TBST for 1 hr to block nonspecific binding (all incubations were carried out at room

temperature), followed by incubation of the primary phospho-ERK mAB (1:300 dilution

in blocking solution) overnight at 4 °C. After three times washing in TBST, sections were 128 incubated with a 1:1000 dilution of biotinylated secondary antibody for 1 hr, followed by

30-min incubation with streptavadin/biotin–horseradish peroxidase complex in blocking solution. Liquid DAB was applied for five minutes to develop the brown color. Sections were counterstained in hematoxylin before dehydration using a sequential alcohol and

xylene series. Negative control specimens were included by replacing the primary antibody with blocking solution (222).

6.2.4.5 Suvivin staining

Staining protocol of survivin was similar to that of phospho-ERK, except for three changes. First, 1 mM EDTA (ethylenediaminetetraacetate disodium dihydrate, pH 8.0) solution replaced the 10 mM citrate buffer (pH 6.0) for antigen retrieval. Second, the primary antibody changed to survivin antibody and the dilution ratio was 1:500. Last, the

DAB development time decreased to 3 min.

For tissue samples stained for bFGF, phospho-ERK or survivin, images were

captured in 4-5 randomly selected microscopic fields at 400× magnification and

quantified with a custom macro written in Optimas® (version 6.51, Media Cybernetics,

Silver Spring, MD) developed by Dr. Colin T. Walsh (153). The macro opened each image in turn and applied a user-defined threshold to select the brown bFGF, survivin or phospho-ERK from blue hematoxylin counterstain, then extracted the sum of the optical densities for each image and exported the data to an excel spreadsheet. The intensity of bFGF, phospho-ERK or survivin staining was normalized to the average value in control groups.

129 6.2.5 Statistical analysis

Statistical significance of the differences between different groups was assessed

by Tukey test after ANOVA. Pearson correlation analysis was conducted to test the

association between the levels of the investigated pharmacodynamic endpoints. When

time was significantly correlated with the interested endpoint or there was interaction

between time and the endpoint, correlation analysis was performed at each individual time point; otherwise, data from different time points were grouped together for analysis.

Differences were considered significant when p<0.05.

6.3 Results

Quantification results for all the endpoints were summarized in Table 6.1 and

Table 6.2 for FaDu and PC-3 xenograft tumors, respectively. Representative staining

pictures and plots were shown in the corresponding figures.

6.3.1 Antiproliferation effect (BrdU labeling)

Single-agent suramin did not show any effect on cell proliferation in either FaDu

(Table 6.1, Figure 6.1) or PC-3 (Table 6.2, Figure 6.2) tumors at the doses used. When

suramin was given at a single dose of 10 or 200 mg/kg, the proliferation indices right

before irradiation (control for S10+IR and S200+IR in Table 6.1), were comparable to

control of IR alone group in FaDu tumors. Similarly, four biweekly doses of suramin at

10 mg/kg also did not show any antiproliferation effect in PC-3 tumors. 130 Irradiation effectively decreased the cell proliferation in FaDu tumors as early as

three hours after irradiation, and reached the maximal effect (>30% reduction) after 3

days. Cells started to recover at two weeks after irradiation, although the proliferation

index was still significantly lower than control (24.2 ± 1.0 % vs. 29.4 ± 0.7%, p<0.05).

Neither low-dose (10 mg/kg) nor high-dose (200 mg/kg) suramin affected the

antiproliferation effect of irradiation, as indicated by comparable proliferation indices

among the three groups at the same time point after irradiation.

PC-3 tumors were more sensitive to the antiproliferation effect of irradiation. One

single dose of irradiation at 1080 cGy almost completely blocked the tumor cell

proliferation, dramatically decreasing the BrdU labeling index from ~14% in control and

suramin alone group to 0.7% in the two irradiation groups (with or without the combination of suramin) at two weeks after irradiation.

6.3.2 Apoptosis (M30 staining)

A single dose of irradiation at 1080 cGy induced time-dependent apoptosis in

FaDu tumors (Table 6.1, Figure 6.3). Apoptosis happened as early as three hours after

irradiation and reached a peak at 3 days after irradiation, with an apoptosis index ~6-fold

of the control level (7.3 ± 1.3% vs. 1.3 ± 0.1%). The apoptosis decreased after two weeks,

yet it was still significantly higher than the control value. Pretreatment with low-dose

suramin (10 mg/kg) did not affect the baseline level of apoptosis compared to the control

of irradiation alone group. But it significantly enhanced the irradiation-induced apoptosis,

reaching higher apoptosis index at every time point after irradiation. However, the

enhancement of irradiation-induced apoptosis by suramin was lost at high dose (200

131 mg/kg), indicated by the comparable apoptosis index to irradiation alone group at every time point after irradiation.

Consistent results were obtained in PC-3 tumors (Table 6.2, Figure 6.4). Suramin alone showed an apoptosis index comparable to control (~1%). Irradiation significantly increased the apoptosis to >4 folds compared to control and suramin alone group, which was further enhanced by 70% with the addition of low-dose suramin.

6.3.3 bFGF induction by irradiation

As shown in Table 6.1 and Figure 6.5, irradiation tended to induce bFGF expression, although only marginal statistical significance was achieved at 1 day and 3 days after irradiation, suggesting that bFGF was involved in the irradiation response.

6.3.4 Phospho-ERK

Irradiation also affected the ERK activation in FaDu tumors (Table 6.1, Figure

6.6). It increased the phospho-ERK level at early time points after irradiation, which remained up to three days and declined back to baseline level after two weeks. However, the statistical significance was only reached at one day after irradiation. Low-dose suramin did not change the phospho-ERK expression in control tumors, but partially reversed the irradiation-induced upregulation of phospho-ERK. The difference between irradiation alone and low-dose suramin plus irradiation group was significant at both three hours and one day after irradiation, but not at the other time points. In contrast, combination of high-dose suramin and irradiation showed a similar trend of phospho-

ERK expression compared to irradiation alone but statistical difference was not reached in comparison to control. 132 6.3.5 Survivin

Irradiation significantly increased the survivin expression (> 2 fold compared to

control, Table 6.1, and Figure 6.7) in FaDu tumors as early as three hours after irradiation,

and this upregulation maintained until the end of the experiment, i.e., two weeks after

irradiation. Single-agent suramin had no impact on survivin expression, either at low dose

(10 mg/kg) or high dose (200 mg/kg). The addition of low-dose suramin to irradiation

partially reversed the irradiation-induced upregulation of survivin, from as early as 3 hrs until 2 weeks later (p<0.05 compared to irradiation alone at the same time point except for one day (p<0.10)).

Irradiation-induced upregulation of survivin and its reversal by low-dose sruamin was a long-term effect. As shown in Figure 6.8, survivin expression was significantly increased by ionizing radiation (12.7±2.9 fold compared to control group at 74 day) until

112 days after treatment and the upregulation was partially inhibited by the combination of low-dose suramin (>40% reduction, p<0.05 compared to irradiation alone).

The inhibitory effect on irradiation-induced survivin upregulation of suramin was lost at high dose (200 mg/kg), as indicated by the similar survivin level compared to that of irradiation alone group at the same time points.

6.3.6 Correlation analysis

Pearson correlation analysis showed that there was no significant correlation between bFGF and survivin, phospho-ERK, or apoptosis. Neither was there any association between phospho-ERK level and apoptosis.

133 The expression level of survivin and phospho-ERK showed significant positive correlation from 3 hrs to 3 days after irradiation (Figure 6.9), but the significance was lost if including the levels at two week. Negative, correlation between survivin level and apoptosis reached statistical significance at 2 weeks after irradiation, and marginal significance at 3 hrs and 1 day after irradiation, but not at the peak time of apoptosis, i.e.,

3 days after irradiation.

6.4 Discussion

The mechanism of radiosensitization effect by low-dose suramin was presumably via modulation of apoptotic pathways in both FaDu and PC-3 tumors, as supported by several lines of evidence. First, the antiproliferation effect of irradiation was not affected by either low-dose or high-dose suramin. It could be argued that the cell proliferation was almost completely blocked by irradiation alone in PC-3 tumors that there was hardly any room for suramin to improve. However, considering the observation that suramin had no additional antiproliferation effect in FaDu tumors, which had a much higher proliferation index in irradiation alone group, it is quite clear that low-dose suramin sensitized the radiation response through mechanisms other than antiproliferation. Second, apoptosis index was significantly increased by the combination of low-dose suramin. Third, irradiation-induced upregulation of survivin, an apoptosis inhibitor, could be partially inhibited by the addition of low-dose suramin. Last but not the least, the loss of

134 radiosensitization effect of suramin at high dose (200 mg/kg) in FaDu tumors was associated with the loss of apoptosis enhancement and failure to inhibit the irradiation- induced survivin upregulation.

A generalized resistance to apoptosis has been hypothesized as one of the most important determinants of cancer cell resistance towards chemotherapy or radiation (199;

200). Accumulated results have clearly demonstrated that active modulation of apoptotic and survival pathways may substantially enhance radiation response, although the significance of apoptosis and apoptosis-associated gene products for radiation responses in tumors awaits further elucidation. For example, an antibody against EGFR

(C225/cetuximab) has safely exerted pronounced radiosensitization effect in vivo and phase III trials are on the way (46). The effects were based on a modulation of JAK/Stat signaling as well as Bcl-2 and Bax level. Synthetic phospholipid analogues inhibiting

JNK activation, PKC inhibitors, NF-κB inhibitors, and COX-2 inhibitors are also actively

investigated in preclinical and clinical studies and have shown some promising results

(reviewed in (66)).

The partial inhibitory effect of suramin on irradiation-induced survivin

upregulation was consistent with its ability to increase the irradiation-induced apoptosis.

As the smallest member of the inhibitor of apoptosis (IAP) gene family (201), survivin

has been shown to suppress apoptosis by accumulated experimental evidence (reviewed

in (203)), thus potentially involved in cellular resistance to both chemo- and radio-

therapy of human tumors (205). However, the precise mechanism is still incompletely

understood. Currently, several theories have been proposed, including direct suppression

135 of caspase-3 or caspase-9 by survivin, or indirect inhibition of caspase via intermediate

proteins. Example for the latter is that survivin may bind to Smac/DIABLO and prevent

this proapoptotic protein from blocking IAP proteins (204). Correlation analysis failed to

show a tight association between survivin level and apoptosis, suggesting that survivin was not the only factor regulating apoptosis and some other survival pathways might have also contributed.

Irradiation-induced upregulation of survivin in both FaDu and PC-3 tumors is consistent with literature reports (207). The interesting point is that the upregulation could remain up to 16 weeks after irradiation. This might explain the radioresistance of recurrent tumors in clinical settings, considering the potential role of survivin in radioresistance. Hence, the inhibition of irradiation-induced survivin upregulation by

low-dose suramin, which happened early and remained long, has more significance in

translational research. Low-dose suramin can thus be applied to enhance both the

intrinsic tumor cell sensitivity to radiation and the radiosensitivity of recurrent tumors,

which is more difficult to treat in patients. Further studies are still warranted to identify if

survivin is the only or the key target, and possible biomarker for the radiosensitization effect of low-dose suramin by applying available molecular antagonists of survivin such

as antisense and siRNAs.

The link between the evaluated pharmacodynamic endpoints awaits further

investigation. It has been reported that bFGF released by squamous cell carcinoma cells

after irradiation increases resistance to subsequent irradiation (223). Irradiation-induced

upregulation of bFGF was also observed in the current study but it was not correlated

136 with phospho-ERK, survivin, or apoptosis. Further more, phospho-ERK and survivin

were correlated at early time (3 hrs to 3 days) but not long time (2 weeks) after irradiation,

suggesting the co-regulation of these two molecules to some extent but also the

involvement of some different cellular processes. In addition, survivin is a relatively

short-lived protein (t1/2=30 min), yet the irradiation-induced upregulation of survivin

could last as long as 16 weeks, therefore, some upstream signaling pathways must have

been involved. Taken together, future studies are needed to elucidate the exact link

between bFGF signaling, ERK cascade and survivin regulation after irradiation, thus

identify how low-dose suramin exerted the radiosensitization effect. Molecular strategies

such as antisense and siRNAs could be applied.

Interestingly, PC-3 tumor cells had much lower baseline level of proliferation

index compared to FaDu tumors (13.8 ± 1.9% versus 29.4 ± 0.7%). This is consistent

with the finding that PC-3 tumors were more sensitive to ionizing radiation than FaDu

tumors (Chapter 4) and further proves the reason proposed, i.e., slow growth kinetics and

low proportion of proliferating cells in prostate cancer (189).

The observation that low-dose suramin did not affect the antiproliferation effect

of ionizing radiation was not consistent from previous studies where low-dose suramin

enhanced antiproliferation effect of chemotherapeutic agents such as mitomycin C

(Chapter 1) and paclitaxel (97). The confliction lies in the different methodology. Cell proliferation was evaluated with Ki67 staining in the chemotherapy studies, while in vivo

BrdU labeling was used in the current study. In vivo BrdU labeling detects the

proliferating cells more accurately and specifically, because only actively cycling cells

137 can uptake BrdU and be detected in BrdU staining. Yet Ki67 nuclear antigen is expressed throughout the cell cycle except in the resting G0 cells and thus may overestimate the cell proliferation.

In summary, results of the current study support that low-dose suramin sensitized radiation response of FaDu and PC-3 tumors via enhancement of irradiation-induced apoptosis, thereby providing a piece of direct evidence that radiosensitization effect could be achieved through apoptosis modulation. At a molecular level, low-dose suramin partially inhibited irradiation-induced ERK activation and subsequent survivin upregulation. The loss of radiosensitization effect of suramin at high dose was probably due to the failure to inhibit irradiation-induced ERK activation, survivin upregulation, and thus the apoptosis enhancement.

138 Control 3hr 1 day 3 day 2 wk Proliferation index, % IR 29.4 ± 0.7 24.3 ± 4.0 21.5 ± 0.3† 17.9 ± 1.6† 24.2 ± 1.0† S10+IR 29.7 ± 3.3 23.3 ± 2.9 20.3 ± 1.9† 17.9 ± 1.8† 23.1 ± 0.9† S200+IR 28.1 ± 1.6 24.2 ± 1.1 -- 20.0 ± 2.5† 23.8 ± 3.0 Apoptosis index, % IR 1.3 ± 0.1 2.7 ± 0.8 4.3 ± 0.8† 7.3 ± 1.3† 3.3 ± 1.0† S10+IR 1.3 ± 0.1 4.5 ± 0.6†# 6.5 ± 1.4†# 11.3 ± 1.7†# 4.1 ± 0.7† S200+IR 1.6 ± 0.4 2.4 ± 0.8 -- 8.1 ± 0.9† 2.7 ± 0.3 bFGF, % of control IR 100 ± 31.9 96.2 ± 35.9 157 ± 47.3 166 ± 45.9* 104 ± 37.9 Phospho-ERK, % of control IR 100 ± 76.8 265 ± 206 614 ± 311† 219 ± 119 44.2 ± 78.2 S10+IR 92.3 ± 71.1 23.1 ± 8.2# 234 ± 3.8†# 86.2 ± 132 196 ± 140 S200+IR 230 ± 270 124 ± 93.5 -- 501 ± 535 96.3 ± 36.0 Survivin, % of control IR 100 ± 12.9 208 ± 52.4† 249 ± 73.6† 149 ± 21.3 231 ± 45.9† S10+IR 98.8 ± 26.5 85.7 ± 50.8# 137 ± 99.8 48.1 ± 71.5# 126 ± 25.6# S200+IR 96.6 ± 7.0 210 ± 93.5† -- 189 ± 60.3† 246 ± 53.0†

Table 6.1 Pharmacodynamics of suramin in combination with irradiation in FaDu tumors FaDu tumor-bearing mice received radiation (IR, 1080 cGy, single dose), with one-day pretreatment of normal saline or suramin (single dose, 10 or 200 mg/kg). Tumor samples were harvested at predetermined time points with one hour in vivo BrdU labeling. Controls for all three groups were harvested right before irradiation. Apoptotic cells were detected by M30 CytoDEATH antibody. Proliferation index and apoptosis index were calculated as BrdU- or M30- positive cells divided by total cells in 4-5 “hot-spot” microscopic fields, respectively. bFGF, survivin and phospho-ERK were detected by immunohistochemistry and quantified using Optimas® in 4-5 randomly selected microscopic fields. n=3-5 mice per point. Differences between groups were assessed by Tukey test after ANOVA. † p<0.05 compared to control and # p<0.05 compared to IR alone at the same time point. * p<0.05 compared to control by Student’s t-test.

139 Total cell density BrdU-positive cells M30-positive cells Group cells/field Cells/field % Cells/field % Control 249 ± 6 31 ± 6 13.8 ± 1.9 2.1 ± 1.0 0.8 ± 0.4 Suramin 267 ± 12 36 ± 3 14.4 ± 0.5 3.0 ± 0.5 1.1 ± 0.1 IR 212 ± 10 # 2.3 ± 1.5 # 0.7 ± 0.9 # 10.6 ± 3.5 # 4.9 ± 1.4 # ∗ ∗ Sur + IR 207 ± 10 # 1.6 ± 0.8 # 0.6 ± 0.6 # 18.5 ± 2.4 #, 8.6 ± 0.8 #,

Table 6.2 Enhancement of antitumor effect of irradiation by suramin in PC-3 tumors

(histological evaluation)

PC3 tumor-bearing mice received intraperitoneal injection of physiological saline, suramin (10 mg/kg), localized ionizing radiation (1080 cGy), or radiation with one-day pretreatment of suramin (10 mg/kg). Three more doses of physiological saline or suramin were given after irradiation on a twice-weekly schedule. Two weeks following irradiation, tumors were labeled with BrdU (tail vein injection at 100 mg/kg, labeling for 1 hr), harvested and fixed in formalin. Histological sections were stained for BrdU and M30. For each tumor, BrdU labeling index and apoptosis index were calculated as (number of BrDu- or M30- stained cells) divided by (total cell number) in 4-5 “hot-spot” microscopic fields at 400× magnifications. Four to five tumors per group. Mean ± S.D. For all comparisons, differences among the four groups are significant by ANOVA (p < 0.001). # p<0.05 compared to control/suramin by Tukey test after ANOVA. ∗ p<0.05 compared to IR by Tukey test after ANOVA.

140

40 IR S10+IR S200+IR

† † † † † 20 † †

10 BrdU labeling index, % index, BrdU labeling

0 Control0 hr 3 hr 1 d 3 d 2 w k Tim e after IR

Figure 6.1 Antiproliferation effect of irradiation/suramin in FaDu tumors Upper panel, representative pictures of BrdU staining. Lower panel, time course of BrdU labeling index. N=3-4 tumors per point. Mean+SD. † p<0.05 compared to control (samples harvested right before IR) by ANOVA.

141

5 BrdU labeling index, % index, labeling BrdU

0 C o n tro l S u r IR S u r + IR

Figure 6.2 Antiproliferation effect of irradiation/suramin in PC-3 tumors

Tumor samples were harvested as described in Table 6.2. Histological sections were stain2d for BrDu and the BrdU labeling index was calculated as (number of BrDu- stained cells) divided by (total cell number) in 4-5 “hot-spot” microscopic fields at 400× magnifications in each tumor. Four to five tumors per group. Mean+SD. † p<0.0001 compared to control and single-agent suramin.

142

15 IR S10+IR †# S200+IR

10 † † †#

†# † † 5 † Apoptosis index, %

0 0 hr 3 hr 1 d 3 d 2 w k Tim e after IR

Figure 6.3 Apoptotic effect of radiation/suramin in FaDu tumors

Upper panel, representative pictures of M30 staining. Lower panel, time course of apoptosis index. N=3-4 tumors per point. Mean+SD. †p<0.05 compared to control and # p<0.05 compared to IR alone at the same time point by Tukey test after ANOVA. 143

4 Apoptotic index, %

0 C o n tro l S u ra m in IR S u r + IR

Figure 6.4 Apoptotic effect of irradiation/suramin in PC-3 tumors

Tumor samples were obtained as described in Table 6.2. Histological tumor sections were stained for apoptosis using M30 CytoDEATH antibody. The apoptosis index was calculated as (number of M30- stained cells) divided by (total cell number) in 4-5 “hot- spot” microscopic fields at 400× magnifications in each tumor. Four to five tumors per group. Mean+SD. † p<0.001 compared to control and single-agent suramin and # p<0.05 compared to irradiation alone by Tukey test after ANOVA.

144

250 † #

200

150

100

ofcontrol bFGF, % 50

0 Control 3 hr 1 d 3 d 2 wk Time after IR

Figure 6.5 bFGF induction by irradiation in FaDu tumors

Upper panel, representative pictures of bFGF staining. Lower panel, time course of bFGF expression. n=4-5 tumors per point. †p<0.05 and # p<0.07 compared to control by Student’s t-test.

145

800 IR S10+IR S200+IR 600

400 †#

200 #

of control % phospho-ERK, 0 Control0 hr 3 hr 1 d 3 d 2 w T im e after IR

Figure 6.6 Effect of radiation/suramin on phospho-ERK level in FaDu tumor

Upper panel, representative pictures of phospho-ERK staining. Lower panel, time course of phospho-ERK level. n=3-4 tumors per point. Mean+SD. † p<0.05 compared to control and # p<0.05 compared to IR alone at the same time point by Tukey test after ANOVA.

146

400 IR S10+IR S200+IR † † † 300 † † † *

200 # # # 100 Survivin, % of control of % Survivin,

0 Control0 hr 3 hr 1 d 3 d 2 w k Tim e after IR

Figure 6.7 Effect of radiation/suramin on survivin expression in FaDu tumors

Upper panel, representative pictures of survivin staining. Lower panel, time course of survivin expression. n=3-4 tumors per point. Mean+SD. †p<0.05 compared to control and # p<0.05 compared to IR alone at the same time point by Tukey test after ANOVA.

147

Control IR Sur+IR

10000 † †# 1000

100 of control % Survivin,

Control IR Sur+IR

Figure 6.8 Changes of survivin expression in PC-3 tumors after irradiation

PC-3 tumor bearing mice received intraperitoneal injection of physiological saline (control), ionizing radiation (IR), or radiation in combination with radiation (Sur+IR) Control tumors were harvested at 74 days while tumors in IR and Sur+IR groups were harvested at 112 days after the last irradiation treatment (for details, please see the long- term effect study in Chapter 4). Histological sections were stained for suvivin and quantified with Optimas® in 4-5 randomly selected microscopic fields at 400× magnifications in each tumor. Three to six tumors per group. Mean + SD. † p<0.001 compared to control and # p<0.05 compared to ionizing radiation by Tukey test after ANOVA.

148

400 IR_3hr, 1day, 3 day p = 0.018

300

200

100 Survivin, % of control % Survivin,

0 500 1000 1500 phospho-ERK, % of control

10 IR_3 hr IR_1 day p = 0.069 p = 0.068

e 0 100 200 300 400 0 100 200 300 400

s 15

o IR_3 day IR_2 wk

p p = 0.34 p = 0.028

A 10

0 100 200 300 400 0 100 200 300 400

Survivin, % of control

Figure 6.9 Results of correlation analysis

Upper panel: Expression level of survivin versus phospho-ERK for individual tumors at 3 hrs, 1 day, and 3 days after irradiation. Lower panels: Apoptosis index versus expression level of survivin for individual tumors at different time points after irradiation. p-value was obtained by Pearson correlation analysis. 149 CHAPTER 7

PERSPECTIVES AND CONCLUSION

Resistance to conventional cytotoxic treatments including chemotherapy and radiotherapy remains a major obstacle to successful management of cancer. The work presented in this dissertation has focused on the preclinical translational studies of developing suramin as chemo- and radiosensitizer, with the final goal to maximize the clinical application of suramin.

Intravesical adjuvant MMC treatment could be significantly improved by maximizing MMC delivery, increasing recurrence-free survival from 23% to 42%. The remaining ~60% patients with recurrence in spite of maximal drug delivery urges the development of a second strategy, i.e., enhancing the chemosensitivity of tumor cells.

Low-dose suramin therefore could be evaluated in bladder cancer to further improve the tumor response after maximal drug delivery. Low-dose suramin sensitizing the subtherapeutic treatment regimen (i.e., only stable disease was achieved) of MMC is also very interesting. It suggests that suramin can also improve the treatment outcome in case no therapeutic efficacy (partial or complete response) was achieved, which is very common in cancer patients.

150 Although PPS, another nonspecific FGF inhibitor with high potency, caused serious toxicity in combination with paclitaxel, it significantly improved the antitumor activity of paclitaxel in pancreatic tumors (Chapter 3). Considering the high FGF in pancreatic tumors, other more specific and potent but nontoxic FGF inhibitors might be studied to develop more effective treatment for this deadly disease.

The finding that low-dose suramin sensitized the radiation response of different tumor types (PC-3, FaDu, and Hs 766T) is very important. This further extends the potential application of suramin in improving cancer treatment, i.e., low-dose suramin could be applied as both a chemosensitizer and a radiosensitizer, and highly likely in a wide range of solid tumors.

The finding that the radiosensitization effect of suramin had tumor type specific dose dependence (Chapter 4 and 5) suggests the necessity to optimize suramin dose regimens according to tumor type when combined with radiotherapy, more likely, chemotherapy too. Suramin showed a narrow sensitizing window in FaDu but a wider range, or maybe no antagonism at all, in Hs 766T tumors. Therefore, these two tumors can serve as ideal in vivo models to study the differential biological targets of suramin.

Since our final goal is to translate the preclinical results into clinical application, future investigations to identify the exact biological targets of suramin responsible for sensitization or antagonism are warranted, so that dosing regimen could be adjusted accordingly.

The observation that there was no significant dose dependence or tumor type specificity in suramin PK simplified the future study of developing the most effective

151 combinatory therapy with suramin. Suramin concentrations in plasma and tumors after

different dosing regimens could be simulated using the PK model. Thus, further studies

could be conducted to differentiate the suramin concentrations (sensitizing or antagonistic)

in plasma and tumors by adjusting dosing schedules of suramin and identify which one is

the real governing factor in mediating radiosensitization effect. The differentiation of

plasma and tumor concentration is important because it will help to design more efficient

preclinical studies to identify the differential molecular targets of suramin, as well as guiding the dosing regimen optimization in the translation of preclinical results.

With the advancement in studying the microscopic disposition of suramin and

identifying important suramin targets for sensitization effect, further efforts of differentiating shallow and deep compartment of tumors could be considered. Success of future PK/PD correlation study also relies on the identification of key suramin targets that will provide more sensitive and accurate pharmacodynamic end points than tumor volume, thus finally help to optimize the treatment regimen of suramin in different cancers.

Irradiation-induced upregulation of survivin and its consistent inhibition by low- dose suramin suggests survivin as a potential biomarker to predict suramin sensitization effect. Therefore, further investigations could be designed to identify if survivin is the only or key target for the sensitization effect of suramin. More importantly, survivin upregulation remained up to 16 weeks after irradiation treatment. This might be one of the reasons for the resistance of recurrence tumors. The inhibitory effect of low-dose suramin on irradiation-induced survivin upregulation also remained long, suggesting its

152 potential role in preventing radioresistance for recurrent tumors. The effect of suramin in

modulating radiation response of recurrent tumors could be further evaluated in the future.

In summary, low-dose suramin sensitizes tumor response to the two major two modalities of cancer therapy, chemotherapy and radiotherapy, thus could be widely applied in the management of solid tumors. The tumor type specific dose dependence of suramin in mediating radiosensitization effect indicate that optimized dosing regimens of suramin should be considered in treating cancers with different biological targets. ERK activation and survivin are found to be associated with the radiosensitization effect of suramin. Future PK/PD studies are warranted to identify the exact molecular targets of suramin and ultimately optimize the clinical application of suramin to achieve maximal clinical benefit, either as a chemosensitizer or a radiosensitizer.

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