• unlimited access with print and download
    $ 37 00
  • read full document, no print or download, expires after 72 hours
    $ 4 99
More info
Unlimited access including download and printing, plus availability for reading and annotating in your in your Udini library.
  • Access to this article in your Udini library for 72 hours from purchase.
  • The article will not be available for download or print.
  • Upgrade to the full version of this document at a reduced price.
  • Your trial access payment is credited when purchasing the full version.
Buy
Continue searching

Investigating Molecular Targets of Phosphaplatins: A Class of Novel Non-DNA-Binding Platinum Anticancer Agents in the Treatment of Ovarian Cancer

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Pooja M Majmudar
Abstract:
Platinum drugs have been the cornerstone in treating human ovarian cancer, as a result of major advances in the field of synthetic and analytical chemistry. Drugs such as cisplatin and carboplatin that are routinely used for chemotherapy, form intra- and inter-strand platinum-DNA cross links, ultimately leading to cancer cell death. But unfortunately in many cases this platinum-DNA association is also responsible for treatment resistance, leading to patient death. It has been extensively documented that inhibition of DNA repair pathways and a greater understanding of cellular reactions upon platinum drug treatment, are crucial in diminishing toxicity and improving the effectiveness of such drugs. Pyrodach-2, belonging to a class of novel platinum compounds called phosphaplatins, demonstrated enhanced cytotoxicity against ovarian cancer cells and no DNA-binding in a recently published study (Bose et. al., 2009). These observations inspired the following study, which aims at determining the anti-cancer properties of two other pyrophosphate complexes, namely pyrodach-4 and RRD4. Platinum compounds have been known to channel their effects through multiple pathways. In the first part of this study, the signaling mechanism(s) of pyrodach-4, which induced ovarian cancer cell death upon treatment, were investigated. Cell-death and apoptosis associated genes along with their products make good targets, which can be exploited in the development of drugs to treat human diseases. Using various assays such as gene expression microarray and qRT-PCR, levels of various such genes were analyzed. Interestingly pyrodach-4 utilized genes belonging to both the extrinsic and intrinsic apoptotic cell death machinery, namely Bax, Fas, PTEN and PUMA to induce maximal cisplatin-sensitive and -resistant ovarian cancer cell death at 24 hours post treatment. Further, anti-apoptotic genes such as Bcl-2 decreased in treated cells. In contrast, protein expression results for Fas and Bax proteins did not exactly mimic the results from the gene expression analyses, suggesting that post-transcriptional modifications might be at play. Additionally, findings from investigating RRD4 cytotoxicity, suggested that both the cisplatin-sensitive and resistant cell lines were sensitive to 24 hour treatment with RRD4. Combined results from the gene expression, protein expression and ELISA analyses proved that RRD4-treated cells may be differentially regulated than those treated with pyrodach-4, hinting a faster uptake process, and a distinct mechanism of action of RRD4, in comparison to pyrodach-4. Effects of platinum drugs on tumor vasculature have become the subject of increased study. In part two of this study, treatment of HUVECs with pyrodach-4 and RRD4 caused intense cell death, adding anti-angiogenesis to the repertoire of phosphaplatin anti-cancer functions. Finally, in accordance with the anti-cancer activities seen in vitro , pyrodach-4 toxicity and efficacy studies were performed, as the third part of the study. Results from the toxicity and efficacy studies demonstrated that phosphaplatins were well tolerated and performed much better in comparison to cisplatin- and carboplatin-treated mice. Collectively, data presented in this study reveal the probable pathways triggered upon pyrodach-4 and RRD4 treatments, making phosphaplatins a class of novel targeted compounds, which can be used to treat human ovarian cancer.

TABLE OF CONTENTS Page Abstract .............................................................................................................................. 3 Dedication .......................................................................................................................... 6

Acknowledgements ........................................................................................................... 7

List of Tables ................................................................................................................... 11

List of Figures .................................................................................................................. 12 List of Abbreviations…………………………………………………………………...14

Chapter 1: Review of Literature ................................................................................... 16

1.1 History of Cancer Treatment .......................................................................... 16

1.2 Ovarian Cancer ............................................................................................... 21

1.3 Current Therapies for Ovarian Cancer ............................................................ 25

1.4 Phosphaplatins (Pyrodach-2 and -4) ............................................................... 32

1.5 p53 Tumor Suppressor Gene .......................................................................... 35

1.6 Cellular Signaling in Cellular Stress Responses & Cell Death: ..................... 42

1.7 Death Receptor Signaling In Chemotherapy .................................................. 58

1.8 PI3K/AKT Cell Survival Pathway in Cancer ................................................. 64

1.9 Anti-Angiogenesis .......................................................................................... 69

1.10 Significance of Study and Specific Aims ..................................................... 72

Chapter 2: Materials & Methods .................................................................................. 75

2.1 Cell Lines and Platinum compounds .............................................................. 75

2.2 Phase Contrast Microscopy............................................................................. 78

2.3 RT 2 Profiler™ Apoptosis PCR Array ............................................................. 78

2.4 Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR or quantitative Real-Time PCR) ................................................................................ 80

2.5 Immunofluorescence ....................................................................................... 85

2.6 Cell Death Detection ELISA .......................................................................... 86

2.7 In vivo Toxicity Study ..................................................................................... 88

10

2.8 In vivo Efficacy Study- Subcutaneous Xenograft model of Human Ovarian Cancer ................................................................................................................... 91

Chapter 3: Results......................................................................................................... 100

3.1 Clonogenic Assay (Colony Forming Assay) & Cell viability analyses ........ 100

3.2 Phase Contrast Microscopy........................................................................... 102

3.3 RT 2 Profiler™ Apoptosis PCR Array ........................................................... 105

3.4 RNA Expression Analyses using qRT-PCR ................................................. 109

3.5 Protein Expression Analysis by Immunofluorescence ................................. 133

3.6 Cell Death Detection ELISA ........................................................................ 142

3.7 Preclinical Animal Studies ............................................................................ 148

Chapter 4: Discussion ................................................................................................... 161

4.1 Summary ....................................................................................................... 180

Chapter 5: Conclusion & Future studies .................................................................... 183

References……………………………………………………… ……………………..187

11

LIST OF TABLES Page Table 1: Survival statistics based on the number of surviving cancer patients ................ 22

Table 2: Platinum compounds used in the in vitro studies ............................................... 78

Table 3: Inventoried Taqman® Gene Expression Assays ................................................ 81

Table 4: Treatment conditions for RNA isolation and subsequent Real-time PCR ......... 82

Table 5: Cell treatments conditions for the Cell Death Detection ELISA ........................ 88

Table 6: Treatment groups for the in vivo cytoxicity Study ............................................. 91

Table 7: Treatment groups for the in vivo efficacy assay ................................................. 95

Table 8: Sensitivity of A2780 and A2780/C30 to Pyrodach-4 and RRD4 at 24 hour exposure time .................................................................................................................. 102

Table 9: Selected apoptotic gene families screened using the RT 2 Profiler PCR Array 106

Table 10: Genes analyzed by qRT-PCR ......................................................................... 110

Table 11: Summary of in vitro treatments done using A2780 and A2780/C30 cell line 111

Table 12: Relative fold changes in gene expression levels in A2780 cell line ............... 113

Table 13: Comparing relative fold changes in gene expression in the A2780 cell line of pyrodach-4 treatment with RRD4 treatment of A2780 cells after 24 hours ................... 116

Table 14: Comparing change in gene expression levels between pyrodach-4 only and co- treatment of pyrodach-4 with PFT-α after 24 hour treatment ......................................... 118

Table 15: Comparing change in gene expression levels between RRD4 only and co- treatment of RRD4 with p53 inhibitor PFT-α after 24 hour treatment ........................... 119

Table 16: Time course study with A2780/C30 cells ....................................................... 127

Table 17: A2780/C30 cells after pyrodach-4 and RRD4 treatment ................................ 130

Table 18: Mean %T/C values for A2780 xenografts in four treatment groups .............. 155

Table 19: Gross log cell kill index .................................................................................. 157

12

LIST OF FIGURES Page Figure 1: Steps involved in the drug development process. ............................................. 18

Figure 2: Cellular uptake and mechanism of action and inactivation of cisplatin ........... 28

Figure 3: Input signals and responses of an active p53 protein ........................................ 38

Figure 4: Overview of apoptotic fate of the cell under stress conditions ......................... 44

Figure 5: Classic Extrinsic Cell Death Pathway ............................................................... 49

Figure 6: Intrinsic (Mitochondrial) cell death pathway & the ceramide pathway ........... 51

Figure 7: Morphological features of an apoptotic and necrotic cell ................................ 55

Figure 8: Schematic representation of forms of death induced by cell death receptors ... 56

Figure 9: Type I and II cell signaling via the Fas death receptor ..................................... 63

Figure 10: The phosphatidylinositol 3-kinase pathway .................................................... 66

Figure 11: PTEN and PI3K signaling ............................................................................... 68

Figure 12: Pathological angiogenic process ..................................................................... 71

Figure 13: Pyrodach-4 Toxicity Study ............................................................................. 93

Figure 14: Pyrodach-4 Efficacy Study ............................................................................. 97

Figure 15: Phase contrast images of live A2780 cells .................................................... 103

Figure 16: Phase contrast images of live A2780/C30 cells ............................................ 104

Figure 17: Fold changes in the expression of selected genes ......................................... 106

Figure 18: Relative fold changes in gene expression in the A2780 cell line ................. 115

Figure 20: Relative fold changes in gene expression, upon treatment with D4 and RRD4 in the presence and absence of PFT-α ............................................................................ 124

13

Figure 21: Time-course study with A2780/C30 cells ..................................................... 129 Figure 22: Expression patterns of gene after 24 hour treatment with D4 and RRD4 .... 132 Figure 23: A2780 cells: Cellular localization of Fas receptor after 1 hour treatment with IC 50 values of pyrodach-4 and cisplatin in A2780 ovarian cancer cells ......................... 134

Figure 24: Cellular localization of (A) BAX (B) E-cadherin (C) Fas (D) PUMA (E) p53 in A2780 cells ................................................................................................................. 137

Figure 25: A2780/C30 cells: Cellular localization of Fas receptor after 1 hour treatment with IC 50 values of pyrodach-4 and cisplatin in A2780/C30 ovarian cancer cells. ....... 138

Figure 26: Cellular localization of (A) BAX (B) E-cadherin (C) Fas (D) PUMA (E) p53 in A2780/C30 cells ......................................................................................................... 141

Figure 27: Pyrodach-4 treatment for 24 hours, induced apoptosis in A2780 ovarian cancer cells, with and without the presence of p53 inhibitor PFT-α (15 μM) and PI3 kinase inhibitor LY294002 (30 μM) ............................................................................... 143

Figure 28: Pyrodach-4 treatment for 24 hours, induced apoptosis in A2780/C30 ovarian cancer cells, with and without the presence of p53 inhibitor PFT-α (15 μM) and PI3 kinase inhibitor LY294002 (30 μM) ............................................................................... 144

Figure 29: Time-course study with HUVEC (human umbilical vein endothelial cells) 148

Figure 30: Percent weight loss/gain in animals .............................................................. 150 Figure 31: Anti-tumor activity in human epithelial ovarian cancer xenograft A2780 ... 153

Figure 32: Percent Tumor Regression ............................................................................ 156 Figure 33: Percent Tumor Growth Inhibition (%TGI) ................................................... 157 Figure 34: Net and Gross Cell Kill Index ....................................................................... 159 Figure 35: The stepwise approach to the development of a drug ................................... 162 Figure 36: Potential mechanisms of phosphaplatin action ............................................. 178

14

List of Abbreviations

A2780 Human ovarian cancer cell line A2780/C30 Cisplatin resistant ovarian cancer cell line ACS American Cancer Society AKT/PKB Serine-threonine specific protein kinase, protein kinase B BAD Bcl-2-associated death promoter protein BRCA1, BRCA2 Breast cancer type 1 and 2 susceptibility protein Cisplatin (CP) Cis-diamminedichloridoplatinum(II) Carboplatin (CR) Diammine(1,1-dicarboxylatocyclobutane)platinum(II)) CD Circular dichromism spectroscopy cIAP D4 Cellular inhibitors of apoptosis Pyrodach-4 DD Death Domain DNA Deoxyribonucleic acid DISC complex Death inducing signal complex EDTA Ethylenediaminetetraacetic acid FAS CD95, death receptor from TNF receptor superfamily FBS Fetal Bovine Serum FIGO International Federation of Gynecology and Obstetrics HBSS Hank's balanced salt solution IC 50

Inhibitory dose to achieve 50% cell death IKK IκB kinase, phosphorylates IκBα protein to activate NF-κB

15

MDM2 Murine double minute oncogene μl Microliter μM Micromolar NER Nuclear excision repair NMR Nuclear magnetic resonance PI3K Phosphoinositide 3-kinase PIK3CA p110α catalytic subunit of PI3K PIP2 Phosphatidylinositol (4, 5) diphosphate PIP3 Phosphatidylinositol (3, 4, 5)-trisphosphate P/S Penicillin/ Streptomycin Pt Platinum PTEN Phosphatase and tensin homolog deleted on chromosome 10 Oxaliplatin Trans-1,2-cyclohexanediamine)oxalatoplatinum(II) RNA Ribonucleic acid RPMI Roswell Park Memorial Institute medium RTK Receptor tyrosine kinase TNFα Tumor necrosis factor α TNFR Tumor necrosis factor family of ligands and receptors TRAIL TNF-related apoptosis-inducing ligand Ub Ubiquitinated VEGF Vascular endothelial growth factor VEGFR-2 Vascular endothelial growth factor

16

CHAPTER 1: REVIEW OF LITERATURE Despite intense efforts in the diagnosis and treatment of various cancers, ovarian cancer still remains a highly lethal malignancy. A recent study from the Duke University Medical Center, reported modest effectiveness of the current screening techniques in predicting and preventing fatalities from ovarian cancer (Havrilesky et al., 2010). Such studies highlight the importance of investing efforts and resources in preventing, along with treatment of the disease. Platinum-based drugs, though widely used in cancer treatment, ultimately become clinically ineffective and yield no response from the growing tumors. Phosphaplatins are a class of novel platinum-based compounds which have shown tremendous potential in studies in recent years. Considering the dearth of effective screening and treatment options, it is imperative to design targeted therapies with minimal toxicities. The objectives of the following research endeavour are to outline the mechanistic details of the novel platinum-based compounds called phosphaplatins.

1.1 History of Cancer Treatment

One in eight deaths worldwide is cancer-related, with over 12 million cases being diagnosed annually. Cancer chemotherapy which involves the systemic use of drugs is an important and aggressive treatment option, with over a hundred drugs currently in use (Goldblatt & Lee, 2010). The word ‘Chemotherapy’ was first coined by the German chemist Paul Ehrlich in the 1900s, in connection with treatment of syphilis. Subsequently, the use of animals (rabbits) to test different compounds marked the birth of using chemicals to treat diseases (DeVita & Chu, 2008). During WWII in 1943, an

17

accidental air raid destroyed seventeen allied ships, including one containing mustard bombs (being stored as weapons for chemical warfare). Soldiers exposed to the gas experienced bone marrow atrophy and disappearance of lymphoid tissue, which was previously reported with sulfur mustard gas during World War I. These events provided the impetus to use nitrogen mustard gas in the treatment of lymphomas (cancer of the lymph nodes) (DeVita & Chu, 2008; Muggia, 2009). The discovery of Penicillin in 1928, medical studies related to nitrogen mustard by the U.S governmental agencies, screening of chemicals at Yale University using animal models of lymphomas in 1942, and such similar events, set the stage for the creation of the National Institutes of Health (NIH) and the National Cancer Institute (NCI) (Muggia, 2009) which today is a part of NIH, and the U.S. Department of Health and Human Services. The NCI as a Federal Government agency, coordinates cancer research, investigates its causes, prevention, detection and treatment, by using the outcomes of various clinical trials and research projects (www.cancer.gov ). A few decades later, the 1960s and 1970s saw an upswing in pharmaceutical drug development, which primarily focused on designing semi-synthetic drugs, especially chemical analogs antibiotics. Similarly analogs of plant based extracts were also intensively studied and synthesized. These set of occurrences laid the ground for biochemists and pharmacologists to synthesize and screen therapeutic agents. These agents were used in experimental settings to treat various pathologies including cancer, out of which the most promising candidates were chosen by the Food and Drug Administration (FDA) for their use in clinical treatments.

18

Figure 1: Steps involved in the drug development process. New targets for cytotoxicity have been possible for the most part due to our increasing knowledge of cancer. Interestingly, the steps leading to the development of a drug have not changed much over the years. The use of microarray techniques have improved the potential to investigate a number of potential drug candidates simultaneously, while advancements in in vitro and in vivo testing have helped recognize many genes as probable targets for anti-cancer drugs. Identify needs in specific areas of cancer therapeutics Use medicinal chemistry to make/ improve drugs Establish libraries of structurally and chemically stable compounds Screen compounds with biopharmaceutical properties Conduct in vitro screening tests to identify potential gene targets Carry out pathway analysis, gene knock-out studies to study the impact of drug Estimate efficacy of compound(s) using animal models of diseases Establish safety margins and maximum tolerated doses in patients Obtain regulatory approval on the compound(s)

19

Pt NH 3 NH 3 Cl Cl Cisplatin Platinum Therapy and Cisplatin invention Anticancer drugs are a select group of cytotoxic agents, designed to kill cancer cells by various modes. Platinum drugs are the most widely used and studied anticancer agents available to the clinician. Over the years platinum chemistry has been at the forefront of cancer treatment regimens and achievements in this field have continued to expand. Specifically the treatment of ovarian cancer is closely linked to the therapeutic

Figure 2: Cisplatin (cis-diammine-dichloro-platinum)

developments in platinum compounds. Many platinum-based anti-cancer drugs have been developed and screened, but only a handful are being used in the clinics today. Of these cisplatin and carboplatin and more recently oxaliplatin are being used worldwide. The heavy metal derivative cis-diammine-dichloro-platinum (CDDP) or cisplatin (Platinol®- Bristol Myers Squibb), was introduced into clinical investigations by the NCI in 1971 and approved by the FDA in 1978 for use in the treatment of testicular, ovarian and head and neck cancers by Bristol-Myers Squibb (Muggia, 2009). Since then, cisplatin is known as one of the most potent broad spectrum anti-tumor agents, used in treating many solid tumors.

20

Cisplatin is a neutral, highly polar inorganic compound. Normally administered as a prodrug, cisplatin gets into the blood circulation very quickly upon intraperitoneal administration. Once administered, intact cisplatin is passively transported from the blood plasma into the cell across the plasma membrane. Due to the low chloride content within the cytoplasm, the inactive prodrug undergoes a series of spontaneous aquation and hydrolysis reactions, which involve the substitution of the chloro-groups with (OH)- groups, to get converted into its active form. The mono-aquated form of cisplatin is its most active form, which mainly reacts with the nitrogen bases (G-C rich regions) in the DNA, and not the sugar phosphate backbone (Lippert, 1999). In addition, due to the high propensity of sulfur- platinum binding, active cisplatin also interacts with various sulfur- containing nucleophiles such as glutathione, methionine, various proteins and metalothionine (Siddik, 2003). Cisplatin has shown remarkable clinical activity and helped in increasing the survival rates in ovarian cancer patients (Siddik, 2003). It also has remained an important treatment option for cancers of the head and neck, bladder, oesophagus and lung cancer (Lebwohl & Canetta, 1998). But the use of cisplatin in the treatment of ovarian cancer is plagued with several drawbacks, and new approaches aimed at optimizing the treatment options for cancer patients are constantly being explored. Phosphaplatins, a new class of Pt-based compounds (described in detail below), have been shown to be not only biologically active, but less toxic in comparison to the industry standard cisplatin.

21

1.2 Ovarian Cancer

Ovarian cancer is the cancer of the ovaries and can be classified into three types, namely epithelial, germline and stromal ovarian cancer. Most ovarian cancers are epithelial in origin and are among the most lethal gynecological malignancies, and account for the fifth most frequent cause of death in women. According to the National Cancer Institute it is estimated that in the current year (2010), approximately 21,880 new cases of ovarian cancer will be diagnosed, and 13,850 will succumb to the disease. Despite the medical breakthroughs in the field of cancer research, ovarian cancer accounts for nearly 3% of all the cancers afflicting women in the United States and ranks second among gynecological cancers, following cancer of the uterine corpus (American Cancer Society, 2010). The incidence rates of ovarian cancer have been on a decline since the 1990s, and between the years 2001-2006, the decline` rate was 2.1% per year (American Cancer Society, 2010; National Cancer Institute (NCI), 2009), but this promising statistic is dampened by the lack of early signs and symptoms and inaccurate screening and detection tests. This makes cancer of the ovaries, the number one cause of gynecological-related deaths in the United States. Most of the cases diagnosed are in the advanced stages, with very little room for optimal treatment options. If diagnosed early the survival rate for the patient is 94%, but only 15% of the cases are known to be detected early (American Cancer Society, 2010). The International Federation of Gynecology and Obstetrics (FIGO) has categorized the stages of ovarian cancer, depending on the progression levels and malignancy. Stage I, when the disease is restricted to the ovaries, until the most advanced

22

stage IV, when the one or both ovaries are involved, with the presence of lung or liver metastases.

Table 1: Survival statistics based on the number of surviving cancer patients.

Numbers are compared to healthy individuals, for the years 1999-2006. (National Cancer Institute (NCI)

The Surveillance Epidemiology and End Results (SEER) database of the National Cancer Institute (NCI), a widely accepted index highlighting the improvements in therapies, compares the survival of cancer patients with age and sex-matched controls. The survival statistics from SEER underscore the fact that the survival rates decline sharply as the disease progresses from a local, to a distant metastatic type, as shown in Table 1. Unfortunately, most cases are diagnosed when the disease has progressed to stage III and IV and has reached the peritoneal cavity and other organs, causing the Stage at Diagnosis Stage Distribution (%) 5- year Relative Survival (%) Localized (confined to primary site) 15 93.5 Regional (spread to regional lymphnodes) 17 73.4 Distant (cancer has metastasized) 62 27.6 Unknown (unstaged) 7 27.2

23

survival rates to drop sharply, between 1 and 5 years post diagnosis (FIGO committee on gynecologic oncology, 2009).

Ovarian Cancer Risk Factors and Etiology The likelihood of ovarian cancer increases with advancing age, and is common among women from industrialized nations. Age-based incidences increase from 0.26 per 100,000 women between the ages 5-9 and to 58.3 per 100,000 women between the ages of 80-84 (National Cancer Institute - NCI). 90% of the cases diagnosed with epithelial ovarian cancer are sporadic with no apparent cause, family history or genetic influence. According to the American Cancer Society, the lifetime risk of a woman getting ovarian cancer is 1 in 71 and the chances increase with other environmental promoting factors such as diet, use of fertility drugs, alcohol, smoking, and use of talcum powder (American Cancer Society). Other minor predisposing factors explaining the epidemiology of ovarian cancer are (i) incessant ovulation, which causes the disruption of the germinal epithelium leading to activation of cellular repair processes and with this the likelihood of somatic gene deletions and/or mutations aiding tumor initiation or progression, (Cannistra & McGuire, 2007), (ii) hormone replacement therapy and/or increase in the stimulatory signals from the pituitary gonadotropins leading to the formation of ovarian cysts that have the potential to undergo a malignant change, and (iii) diet high in saturated animal fats. In contrast conditions such as pregnancy (a reason for anovulation), multiparity, lactation, and use of oral contraceptives, reduce the risk of ovarian cancer.

24

Further each pregnancy reduces the risk by 13-19% (Sueblinvong & Carney, 2009), and incidence rates are higher in white women in comparison to black, Hispanic and women from other ethnic groups (National Program of Cancer Registries - NPCR). In addition to the above mentioned factors, a family history of breast, colon, ovarian and prostate cancer is the strongest risk factor for the development of ovarian cancer. Hereditary predisposition to ovarian cancer accounts for the remaining 10% of all epithelial ovarian cancers, resulting from genetic susceptibility to autosomal dominant mutations within high-penetrance genes such as the Breast and Ovarian Cancer gene (BRCA 1 and 2) or due to mismatch-repair gene mutations, or a strong family of ovarian cancer (Landen, Birrer, & Sood, 2008).

Clinical Screening for Ovarian Cancer Women over the age of 50 have a higher chance of developing ovarian cancer and present with vague initial symptoms such as abdominal/pelvic pain, nonspecific bloating, gastrointestinal symptoms, and urinary urgency (Clarke-Pearson, 2009). This inconspicuous nature of the symptoms often cause a delay in disease detection by the clinician, unless a significant amount of suspicion is raised due to increased frequency and severity of the symptoms (Willmott & Fruehauf, 2010). Also unlike other cancers afflicting women such as breast, cervical and colon cancers, there are no clinically identifiable pre-malignant phases or obvious precursor lesions associated with ovarian cancer. In addition, most of the screening is restricted to post-menopausal women, limiting detection options for pre-menopausal women. Since a diagnosis of ovarian

25

cancer is generally followed by surgical excision of the ovaries, a sensitive and specific test is important to prevent false positives. Transvaginal ultrasonography imaging is superior in detecting physical changes in size, structure and morphological characteristics of the ovaries, in comparison to transabdominal ultrasonography. But the positive predictive value of imaging techniques are very low and interpretations of the results equally difficult. Hence peripheral blood analysis of tumor markers such as cancer antigen 125 (CA-125) are routinely done in conjunction with imaging techniques, since 1983 (Clarke-Pearson, 2009) (Rosen, Sevelda, Klein, Spona, & Beck, 1990).

1.3 Current Therapies for Ovarian Cancer

Ovarian Cancer Treatment Women with a high risk of ovarian cancer, such as those with a family history of BRCA 1 or 2 mutations are advised to undergo formal screening as well as genetic counseling. Prophylactic oophorectomy (removal of ovaries) is generally lifesaving in such high risk patients as the chances of developing ovarian cancer is as high as 50% (Sueblinvong & Carney, 2009). Standard therapy in the early stages (FIGO stage I-II) generally consists of cytoreduction measures. In addition the following procedures are conducted depending on the histological grade and stage of the disease; surgical excision of ovaries (bilateral-oophorectomy), removal of the fallopian tubes along with the affected ovary or ovaries (salpingo-oophorectomy), total abdominal hysterectomy, removal of lymph nodes and tumor debulking (Han, Lin, & Wakabayashi, 2009). But due to lack of adequate screening tools, the detection is only possible unfortunately at much

26

advanced stages, at which time surgery alone is insufficient. Residual tumors less than 1 cm in diameter post-debulking are treated by radiation and systemic cytotoxic chemotherapy, using platinum-based drugs such as cisplatin and carboplatin (Cannistra & McGuire, 2007). Taxanes introduced in the 1990s, are also used in adjuvant chemotherapy in combination with platinum drugs, as a standard option of care for early stage epithelial ovarian cancer (EOC). Second-line treatment options include the use of drugs that target critical components of growth or survival pathways such as anthracyclines (antibiotics namely doxorubicin, epirubicin), gemcitabine, and etoposide. These are generally used in patients with recurrent and metastatic cancer, after the initial cisplatin and taxane-based treatments have failed (Eifel, 2006). But despite optimal cytoreduction surgeries and most effective chemotherapies in advanced stage patients, over 70-80% of them experience a recurrence in cancer and ultimately succumb to the disease (Han et al., 2009).

Full document contains 201 pages
Abstract: Platinum drugs have been the cornerstone in treating human ovarian cancer, as a result of major advances in the field of synthetic and analytical chemistry. Drugs such as cisplatin and carboplatin that are routinely used for chemotherapy, form intra- and inter-strand platinum-DNA cross links, ultimately leading to cancer cell death. But unfortunately in many cases this platinum-DNA association is also responsible for treatment resistance, leading to patient death. It has been extensively documented that inhibition of DNA repair pathways and a greater understanding of cellular reactions upon platinum drug treatment, are crucial in diminishing toxicity and improving the effectiveness of such drugs. Pyrodach-2, belonging to a class of novel platinum compounds called phosphaplatins, demonstrated enhanced cytotoxicity against ovarian cancer cells and no DNA-binding in a recently published study (Bose et. al., 2009). These observations inspired the following study, which aims at determining the anti-cancer properties of two other pyrophosphate complexes, namely pyrodach-4 and RRD4. Platinum compounds have been known to channel their effects through multiple pathways. In the first part of this study, the signaling mechanism(s) of pyrodach-4, which induced ovarian cancer cell death upon treatment, were investigated. Cell-death and apoptosis associated genes along with their products make good targets, which can be exploited in the development of drugs to treat human diseases. Using various assays such as gene expression microarray and qRT-PCR, levels of various such genes were analyzed. Interestingly pyrodach-4 utilized genes belonging to both the extrinsic and intrinsic apoptotic cell death machinery, namely Bax, Fas, PTEN and PUMA to induce maximal cisplatin-sensitive and -resistant ovarian cancer cell death at 24 hours post treatment. Further, anti-apoptotic genes such as Bcl-2 decreased in treated cells. In contrast, protein expression results for Fas and Bax proteins did not exactly mimic the results from the gene expression analyses, suggesting that post-transcriptional modifications might be at play. Additionally, findings from investigating RRD4 cytotoxicity, suggested that both the cisplatin-sensitive and resistant cell lines were sensitive to 24 hour treatment with RRD4. Combined results from the gene expression, protein expression and ELISA analyses proved that RRD4-treated cells may be differentially regulated than those treated with pyrodach-4, hinting a faster uptake process, and a distinct mechanism of action of RRD4, in comparison to pyrodach-4. Effects of platinum drugs on tumor vasculature have become the subject of increased study. In part two of this study, treatment of HUVECs with pyrodach-4 and RRD4 caused intense cell death, adding anti-angiogenesis to the repertoire of phosphaplatin anti-cancer functions. Finally, in accordance with the anti-cancer activities seen in vitro , pyrodach-4 toxicity and efficacy studies were performed, as the third part of the study. Results from the toxicity and efficacy studies demonstrated that phosphaplatins were well tolerated and performed much better in comparison to cisplatin- and carboplatin-treated mice. Collectively, data presented in this study reveal the probable pathways triggered upon pyrodach-4 and RRD4 treatments, making phosphaplatins a class of novel targeted compounds, which can be used to treat human ovarian cancer.