This thesis has been approved by The Honors Tutorial College and the Department of Biological Sciences __________________________ Dr. Rathindra Bose Vice President for Research and Creative Activity Dean of the Graduate College Thesis Advisor __________________________ Dr. Soichi Tanda Honors Tutorial College, Director of Studies Biological Sciences __________________________ Jeremy Webster Dean, Honors Tutorial College PROTEIN TARGETS OF TWO NOVEL ANTICANCER AGENTS __________________________________ A Thesis Presented to The Honors Tutorial College Ohio University __________________________________ In Partial Fulfillment of the Requirements for Graduation from The Honors Tutorial College with the degree of Bachelor of Science in Biological Sciences __________________________________ by Nyssa R. Adams June 2011 i TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................v ABSTRACT...............................................................................................vi INTRODUCTION......................................................................................1 I. OVARIAN CANCER .............................................................................1 A. A Deadly Malignancy: Statistics and Survival Rates...................1 B. The “Silent” Killer......................................................................2 C. Treatment of Ovarian Cancer......................................................3 D. Recurrence and Resistance .........................................................4 II. PLATINUM CHEMOTHERAPY..........................................................5 A. Platinum Drugs: A Brief History ................................................5 B. Current Uses of Platinum Drugs .................................................6 C. The Mechanism of Action of Platinum Drugs .............................6 1. DNA-Binding Activity ....................................................6 2. Alternative Mechanisms ..................................................10 D. Unresolved Problems .................................................................11 1. Physical Problems: Solubility and Stability......................12 2. Toxicity ...........................................................................14 3. Acquired Resistance ........................................................17 ii E. The Continuing Search ...............................................................20 III. RRD2 AND RRD4 ...............................................................................20 A. The Discovery of Two Novel Platinum Analogs.........................20 B. The Unique Properties of RRD2 and RRD4................................23 IV. INVESTIGATING THE MECHANISM OF ACTION OF RRD2 AND RRD4..............................................................................................26 A. Intrinsic and Extrinsic Apoptotic Pathways ................................27 1. The Intrinsic Apoptotic Pathway......................................28 2. The Extrinsic Apoptotic Pathway.....................................32 B. Growth and Survival Signaling Pathways ...................................35 1. IGF Signaling ..................................................................35 2. Heat Shock Proteins.........................................................38 3. Cell Cycle Regulatory Proteins ........................................38 4. PI3K Signaling ................................................................40 C. Activation of Acid Sphingomyelinase.........................................42 V. EXPERIMENTAL GOALS ...................................................................44 MATERIALS AND METHODS ................................................................46 I. SAMPLE COLLECTION .......................................................................46 A. Cell Culture................................................................................46 B. Drug Synthesis ...........................................................................46 C. Drug Treatment and Sample Collection ......................................47 iii D. Determination of Protein Concentration .....................................48 II. ARRAY OF APOPTOTIC PROTEINS..................................................48 III. SDS-PAGE AND WESTERN BLOT ...................................................52 IV. ASSAY OF ACID SPHINGOMYELINASE ACTIVITY .....................54 RESULTS ..................................................................................................57 I. EFFECTS ON INTRINSIC AND EXTRINSIC PATHWAY PROTEINS .......................................................................................................57 A. RRD2 and RRD4 Activate the Intrinsic Apoptotic Pathway........57 B. RRD2 and RRD4 Favor Caspase Activation ...............................59 C. RRD2 and RRD4 Activate Extrinsic Apoptotic Pathways...........60 II. EFFECTS ON SIGNALING PROTEINS...............................................61 A. RRD2 and RRD4 Modulate IGF Signaling .................................61 B. RRD2 and RRD4 do not Alter Expression of HSPs ....................63 C. RRD2 Inhibits p21 and p27 ........................................................63 D. RRD2 and RRD4 Modulate PI3K Signaling ...............................64 III. RRD2 AND RRD4 ACTIVATE ACID SPHINGOMYELINASE.........70 A. RRD2 and RRD4 Activate Acid Sphingomyelinase Activity in A2780 Cells.........................................................................70 B. Activation of Acid Sphingomyelinase is Diminished in A2780/C30 Cells ....................................................................................71 iv DISCUSSION AND FUTURE DIRECTIONS ...........................................73 I. RRD2 AND RRD4 INDUCE APOPTOSIS THROUGH THE INTRINSIC AND EXTRINSIC APOPTOTIC PATHWAYS..............................73 A. Activation of the Intrinsic Apoptotic Pathway ............................73 B. Activation of the Extrinsic Apoptotic Pathway ...........................74 C. Involvement of Acid Sphingomyelinase .....................................75 D. Other Pro-Apoptotic Effects .......................................................77 II. RRD2 AND RRD4 MODIFY GROWTH AND SURVIVAL SIGNALS .......................................................................................................78 A. RRD2 and RRD4 Modulate IGF Signaling .................................78 B. RRD2 and RRD4 Alter Signaling by the PI3K Pathway .............79 III. RRD2 OR RRD4? A POSSIBLE PREFERENCE BASED ON MECHANISTIC DIFFERENCES..............................................................................81 IV. CLOSING COMMENTS......................................................................82 REFERENCES...........................................................................................83 v ACKNOWLEDGEMENTS I gratefully acknowledge the support of many people in the composition of this thesis. I would like to thank Dr. Rathindra Bose for his support and instruction. I would also like to thank Dr. Shadi Moghaddas; without her guidance, none of the results presented in this thesis would have materialized. I am thankful to all of the women working in the Bose lab, including Dr. Pooja Majmudar, Dr. Anna Qi, Homa Dezvareh, Farina Mahmud, and Jamee Miller. I am grateful to Dr. Soichi Tanda for his guidance and encouragement. I would also like to thank Jan Hodson for her continuing support and advice. I would like to thank Dean Webster and the staff of the Honors Tutorial College for their support throughout my undergraduate education. Finally, I thank my friends and family for all they have done for me. vi ABSTRACT This thesis investigates the mechanism of action of two novel anticancer drugs, RRD2 and RRD4. RRD2 and RRD4 are platinum analogs that show promise in the treatment of ovarian cancer . Little is known about the mechanism by which these two drugs induce apoptosis in cancerous cells. This thesis examines the effects of RRD2 and RRD4 on the expression and activity of various proteins in an effort to understand the changes that result in drug-induced apoptosis. Alterations in the expression of intrinsic and extrinsic apoptotic pathway proteins, insulin-like growth factor signaling (IGF) proteins, heat shock proteins, cell cycle regulatory proteins, and phosphatidylinositol-3 kinase (PI3K) signaling proteins are investigated. The effects of RRD2 and RRD4 treatment on acid sphingomyelinase activity are also elucidated. Experimental results suggest that RRD2 and RRD4 induce apoptosis via the intrinsic and extrinsic apoptotic pathways. Results also demonstrate pro-apoptotic alterations in the expression of cell cycle regulatory proteins and modifications of the IGF and PI3K signaling pathways. Further, RRD2 and RRD4 cause robust activation of the acid sphingomyelinase enzyme. Several differences in the activities of RRD2 and RRD4 are apparent, suggesting a possible advantage of one drug over the other. 1 INTRODUCTION I. OVARIAN CANCER A. A Deadly Malignancy: Statistics and Survival Rates Ovarian cancer is the deadliest gynecologic cancer in the United States, claiming over 14,000 lives annually . While significant advances have been made in the management of many cancers, the diagnosis and treatment of ovarian cancer has not changed greatly in several decades . This lack of progress is evidenced by the low survival rate associated with ovarian cancer. The overall five-year survival rate for ovarian cancer is a dismal 44%, meaning only 44% of women diagnosed with ovarian cancer will survive the first five years following their diagnosis . The remaining 56% of women diagnosed with ovarian cancer will succumb to the disease within this five- year period. The survival rate for ovarian cancer is starkly low when compared with the survival rates of other cancers. Breast cancer, for instance, has a five-year survival rate of 89% . The five-year survival rate for males diagnosed with gonadal cancer (testicular cancer) is 95% . Strikingly, although the five-year survival rate for ovarian cancer has improved slightly over the past 20 years, the overall cure rate for ovarian cancer remains a disappointing 30% . The low survival and cure rates associated with ovarian cancer are partially due to the difficulty of detecting ovarian cancer at an early stage . If ovarian cancer is detected at a localized stage, when it is confined to one or both ovaries, the five-year survival rate is 92% . However, only 15% of all ovarian cancers are detected at this 2 early stage . The majority of ovarian cancers, 63%, are not detected until distant metastasis has occurred . At this stage, cancerous tumors have metastasized, or spread, to other organs in the abdominal cavity or beyond. In particular, the position of the ovaries facilitates widespread metastasis within the peritoneal cavity . Ovarian cancers that are not diagnosed until this later stage are more difficult to cure and have a five- year survival rate of only 27% . Unfortunately, the delayed detection of most ovarian cancers prevents successful treatment for a majority of patients. B. The “Silent” Killer The drastic difference in survival rate depending on the stage of ovarian cancer at diagnosis underlines the importance of early detection. Regrettably, the generalized symptoms associated with early-stage ovarian cancer and the lack of an effective screening strategy are significant factors contributing to the difficulty of detecting ovarian cancer at an early stage. Early detection of ovarian cancers remains difficult because the symptoms of early-stage ovarian cancer are nonspecific . Although ovarian cancer has been called a “silent killer,” this label is a misnomer . Over 80% of women with ovarian cancer experience symptoms, even in the early stages of the disease . However, these early warning signs are generalized and can easily be mistaken as symptoms of other, more common diseases . The symptoms most frequently reported by women with ovarian cancer include abdominal bloating and pain, fatigue, indigestion, urinary frequency and incontinence, pelvic pain, and constipation . When women present with these vague symptoms, they are often misdiagnosed and their symptoms are 3 mistakenly attributed to more common diseases such as irritable bowel, stress, gastritis, or depression . Although early-stage ovarian cancer produces observable symptoms, they cannot be relied upon to make a diagnosis. Because the symptoms of early-stage ovarian cancer are not specific enough for efficient diagnosis, an effective screening method is highly desirable. Unfortunately, none of the screening methods currently available have been proven effective . At present, screening of the general population for ovarian cancer is not recommended by any medical society . Screening recommendations are not consistent and are limited to women with a family history of ovarian cancer or a known genetic predisposition to cancer, such as a mutation in the BRCA1 or BRCA2 genes . When combined with the lack of specific symptoms in the early stages of ovarian cancer, the absence of an effective screening system precludes early detection of ovarian cancer in the majority of cases. C. Treatment of Ovarian Cancer Because most ovarian cancers are not detected until they are of a late stage in which metastasis has occurred, they must be treated with an aggressive regimen that includes multidrug chemotherapy. The first goal of treatment is to remove most of the cancerous tissue . This is achieved through surgical debulking, in which any visible tumors and parts of the female reproductive tract, including the ovaries, fallopian tubes, and uterus, are surgically removed . Even with optimal debulking of all tumors and affected tissues, some cancerous cells remain in the patient. If left untreated, these residual cells will proliferate, resulting in recurrence of the cancer. Therefore, 4 targeting residual cancer cells with radiation or chemotherapy is a crucial component of treatment. Radiation therapy is rarely used in the treatment of ovarian cancer because administering a dose of radiation to the entire abdominal cavity is difficult and dangerous . Instead, ovarian cancer is treated with multidrug chemotherapy, which typically includes a taxane and a platinum drug . Currently, the standard treatment is a combination of the taxane, paclitaxel, and the platinum drug, carboplatin . Cisplatin, another platinum drug, and docetaxel, an alternative taxane, are also used commonly in the treatment of ovarian cancer . The combination of surgical debulking with a taxane-platinum drug cocktail has been the first line of defense against ovarian cancer since the early 1990’s . D. Recurrence and Resistance Recurrence is a major obstacle in the treatment of ovarian cancer and is responsible for the low overall cure rate of patients with ovarian cancer . Most ovarian tumors display sensitivity to initial chemotherapy, with over 70% of patients responding well to a taxane-platinum drug combination . However, recurrence is a prolific problem in patients with ovarian cancer . The majority of patients with advanced ovarian cancer will experience a recurrence and eventually succumb to the disease . In some cases, relapse occurs many months after initial treatment . Most of these long-term recurrent cancers remain platinum sensitive, allowing for the possibility of successful second-line treatment . If, however, recurrence occurs within six months of initial therapy, the tumor will likely be resistant to treatment . Few options exist for the treatment of ovarian cancers that have developed resistance 5 to chemotherapy. Currently, the best strategy for the treatment of chemoresistant ovarian cancer is simply a high-dose chemotherapy regimen, which can be extremely toxic and may not provide a successful cure . II. PLATINUM CHEMOTHERAPY A. Platinum Drugs: A Brief History Platinum compounds comprise a fundamental class of chemotherapy drugs and are used in the treatment of a variety of cancers . The first platinum drug discovered, cis-diamminedichloroplatinum(II), commonly known as cisplatin, was found to have anticancer activity in the late 1960’s . After extensive clinical investigation, cisplatin was approved by the Food and Drug Administration in 1979 for the treatment of ovarian, testicular, and head and neck cancers . Since then, thousands of platinum compounds have been screened for antitumor activity . This research has led to the discovery of analogous platinum drugs, including carboplatin (cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)), which was introduced in 1983 and is used extensively in the treatment of ovarian cancer . Oxaliplatin ((trans-L-diaminocyclohexane)oxalatoplatinum(II)) is another important member of the platinum drug family, although it is not used as commonly in ovarian cancer chemotherapy . Structurally, cisplatin, carboplatin, and oxaliplatin are highly similar (Figure 1). They each consist of a central platinum atom and differ in the arrangement and identity of surrounding functional groups . Together, cisplatin, 6 carboplatin, and oxaliplatin are some of the most important chemotherapeutic drugs available to treat cancer . Cisplatin Carboplatin Oxaliplatin Figure 1. Structures of the platinum drugs, cisplatin, carboplatin, and oxaliplatin. B. Current Uses of Platinum Drugs Platinum drugs are commonly prescribed in the treatment of solid tumors, such as ovarian, testicular, non-small cell lung, and head and neck cancers . Platinum drugs are also considered in the first-line treatment of tumors of epithelial origin, including esophageal, gastric, bladder, and pancreatic cancers . Metastatic cancers, such as melanoma, mesothelioma, malignant glioma, leiomyosarcoma, and prostate cancer, can be treated with platinum drugs as well . Platinum drugs are frequently used in combination with other chemotherapy drugs and, in some cases, they are combined with radiation therapy . C. The Mechanism of Action of Platinum Drugs 1. DNA-Binding Activity Traditionally, the cytotoxicity of platinum drugs has been attributed to their ability to bind to DNA . Platinum drugs are transported into the cell via heavy metal transport proteins, such as the copper transporter, CTR1 . Within the cell, 7 cisplatin and carboplatin undergo chemical changes that result in their activation . Due to the low intracellular concentration of chloride ions, cisplatin exchanges one of its chloride ligands for a water molecule . This monoaquated form of cisplatin is highly reactive and capable of binding to DNA . Monoaquated cisplatin forms DNA adducts by binding to nucleophilic sites on purine bases in the DNA molecule . These platinum-DNA adducts can be either mono- or bifunctional (Figure 2). Bifunctional adducts, or crosslinks, may be intrastrand, involving only one strand of DNA, or interstrand, involving both strands of DNA . Intrastrand crosslinks are the most common adducts formed by cisplatin . However, bifunctional adducts between cisplatin, DNA, and nuclear proteins are also possible . Carboplatin undergoes a similar aquation reaction within the cell, in which its carboxylate ligand is exchanged for a water molecule . Once aquated, carboplatin forms DNA adducts identical to those of cisplatin . Oxaliplatin also forms DNA crosslinks, although the structure of oxaliplatin-DNA adducts differs from that of cisplatin- and carboplatin-DNA adducts . 8 Figure 2. Platinum drugs can form a variety of DNA adducts. 1: Monofunctional platinum-DNA adduct. 2: Intrastrand platinum-DNA adduct. 3: Interstrand platinum-DNA adduct. 4: Bifunctional protein-platinum-DNA adduct. The DNA adducts formed by platinum drugs cause damage to the DNA, which inhibits the growth of cancerous cells by interfering with transcription and DNA replication. The crosslinks formed by the binding of platinum drugs cause extensive physical damage to the DNA molecule . Intrastrand crosslinks can cause the DNA helix to bend and unwind . Interstrand crosslinks, while rarer than intrastrand crosslinks, result in more extensive bending and unwinding of the DNA helix . This physical damage to the DNA molecule interferes with the process of transcription . First, platinum-DNA adducts present a physical block to the process of transcription . Additionally, the formation of platinum-DNA adducts inhibits the transcription of specific genes . In particular, platinum-DNA adducts interfere with the transcription of ribosomal RNA (rRNA) . UBF, a transcription factor that enhances rRNA expression, binds to platinum-DNA adducts with high affinity . 9 This binding competes with the normal activity of UBF, resulting in reduced transcription of rRNA . As a result, the rapidly growing cancer cell cannot produce the proteins needed for continued proliferation and survival . The damage resulting from DNA crosslinks also inhibits the growth of cancerous cells by preventing DNA replication . The DNA replication machinery is physically blocked by platinum- DNA adducts. When DNA replication stops, the cancerous cell cannot divide and tumor growth is inhibited. By preventing the processes of DNA transcription and replication, the physical distortions created by platinum-DNA adducts effectively inhibit cell growth. The DNA damage caused by platinum drugs goes beyond growth inhibition and includes induction of apoptosis, or programmed death. The mechanism by which platinum drugs induce apoptosis has not been fully elucidated and probably involves several signaling pathways . Many nuclear proteins recognize and bind to platinum- DNA adducts . Over 20 different proteins demonstrate binding activity to cisplatin-DNA intrastrand crosslinks . One such protein, DNA mismatch repair (MMR) protein, recognizes platinum-DNA adducts . Normally, the MMR protein proceeds in repairing the DNA molecule following recognition of a damaged area. However, the MMR protein cannot repair platinum-DNA adducts and instead activates an apoptotic signaling cascade after it fails to repair the damage . Other nuclear proteins that may be involved in the DNA-dependent induction of apoptosis include high mobility group (HMG) proteins, nucleotide excision repair (NER) proteins, and the transcription factor, TATA-binding protein (TBP) . Once these proteins 10 recognize platinum-DNA adducts, they can trigger apoptosis by activating signaling pathways that may include p53 and the Bcl-2 family . Although the exact pathway remains unknown, the induction of apoptosis by platinum-DNA adducts is an essential component of the cytotoxicity of platinum drugs. 2. Alternative Mechanisms In recent years, the centrality of DNA-binding as the primary mediator of the cytotoxicities of platinum drugs has been questioned. The broad spectrum of cytotoxic effects elicited by platinum drugs cannot be explained by the DNA-binding model alone . Non-DNA targets may play crucial roles in the anticancer activities of platinum drugs . Platinum drugs undoubtedly induce a wide range of cellular changes because they are capable of interacting with sulfur-containing proteins and RNA in addition to DNA . Cisplatin, carboplatin, and oxaliplatin can react with various sulfur-containing proteins via covalent linkages . Notably, platinum drugs interact with proteins that are members of the signaling pathways that regulate cell proliferation and survival . Signaling pathways are rational targets for anticancer drugs because they are frequently dysregulated in cancer cells, permitting aberrant growth and uninhibited proliferation. For example, cisplatin modifies expression of proteins in the MAPK, PI3K, and Src kinase signaling pathways . Platinum drugs can also interact with tumor suppressor proteins to enhance their activity . Cisplatin treatment enhances the effects of the tumor suppressor, p53, by increasing its stability, in addition to 11 activating p53 via the formation of platinum-DNA adducts . These and other protein targets are likely mediators of the anticancer effects of platinum drugs. The cytotoxicity of platinum drugs may also result from interactions with the plasma membrane and cytoskeleton. Platinum drugs can bind to and modify phospholipids and other molecules located at the plasma membrane . These interactions can occur before the drug enters the cell, providing a possible route for the rapid changes induced by platinum drugs . Interactions between platinum drugs and the cytoskeleton may represent another mechanism of action. For example, cisplatin induces dramatic changes in the morphology of breast cancer cells via rapid restructuring of the actin cytoskeleton . Platinum drugs probably exhibit anticancer activity through several distinct mechanisms that include direct interactions with cellular proteins, the plasma membrane, and the cytoskeleton. Although platinum drugs have been used to treat cancer patients for over 30 years, their mechanism of action has yet to be fully elucidated . D. Unresolved Problems Since the discovery of cisplatin over 40 years ago, researchers have tested thousands of analogous platinum compounds in hopes of overcoming the limitations associated with cisplatin . The principal problems associated with cisplatin are limited solubility and stability, toxicity to healthy tissues, and acquired drug resistance. Unfortunately, while carboplatin and oxaliplatin have overcome some of 12 the obstacles associated with administration and toxicity, the problem of acquired resistance remains unresolved. 1. Physical Problems: Solubility and Stability A key objective to be addressed in the search for platinum analogs is the improvement of drug solubility and stability . Cisplatin is poorly soluble in water, which causes difficulties in administering the drug in the clinic. Prior to administration, cisplatin must undergo forced hydration and diuresis, a time- consuming and inefficient process for healthcare personnel . Once dissolved in aqueous solution, cisplatin is extremely unstable. The drug’s chloride ligands quickly undergo an aquation reaction, generating monoaquated cisplatin, a highly reactive product that will readily bind to a wide range of biomolecules . This reaction occurs while cisplatin circulates in blood plasma, enabling toxic reactions with healthy tissues throughout the body. Besides causing undesirable toxicity, these side reactions consume the active drug before it reaches its target tissue, the cancerous tumor . Overcoming the poor solubility and stability associated with cisplatin will allow for administration of platinum chemotherapy that can be dispensed efficiently to target cancerous tissue. Carboplatin and oxaliplatin demonstrate improvements in both solubility and stability compared to cisplatin . Carboplatin is readily soluble in water thanks to the polarity of its carboxylate ligand . The polar oxalate ligand of oxaliplatin creates a similar improvement in solubility . Carboplatin and oxaliplatin exhibit greater stability because they undergo slower aquation reactions when they are kept at high 13 concentrations . This eases the administration process because carboplatin and oxaliplatin can be purchased as ready-to-use infusion solutions that have shelf-lives of several years . However, the increased stability of carboplatin and oxaliplatin is not maintained once the drug is administered . The stability of carboplatin and oxaliplatin results from self-association of drug molecules, an interaction that only occurs in highly concentrated solutions . Once the drugs are diluted to an appropriate concentration for chemotherapy, self-association no longer occurs and stability deteriorates . Resultantly, carboplatin and oxaliplatin still cause systemic side reactions that increase toxicity and decrease drug concentration in cancerous tissue. Although carboplatin and oxaliplatin significantly enhance solubility and stability, the search for targeted drug delivery continues. To avoid consumption of cisplatin, carboplatin, and oxaliplatin by intravenous side reactions, intraperitoneal drug delivery is often used in place of intravenous drug delivery. Intraperitoneal delivery is the direct administration of platinum drugs to the peritoneal cavity through a catheter. This approach shows promise in the treatment of advanced ovarian cancer, which has often metastasized throughout the peritoneal cavity . Intraperitoneal delivery allows platinum drugs to reach ovarian tumors more quickly and at higher concentrations, reducing systemic side reactions and enhancing drug activity . Clinical studies have shown a statistically significant improvement in the survival rate of ovarian cancer patients receiving intraperitoneal chemotherapy over intravenous chemotherapy . However, intraperitoneal administration has not replaced intravenous administration in the standard treatment of ovarian cancer 14 because it is also associated with a considerable increase in drug toxicity . In addition, intraperitoneal drug delivery is more likely to cause treatment complications such as catheter occlusion, infection, bowel perforation, and fistula formation . To reduce toxicity and the technical complexity of administration, platinum chemotherapy is frequently administered intravenously despite the inferior efficacy of this delivery route . 2. Toxicity Historically, platinum drugs have been plagued by noxious toxicity profiles. While platinum drugs kill cancerous cells quite effectively, they can also damage healthy tissues . The DNA-binding activity of platinum drugs causes toxic effects in proliferative tissues, including the skin, hair follicles, small intestine, and bone marrow . Side reactions between platinum drugs and proteins cause toxicities in other healthy tissues, such as the kidneys and peripheral nerves. These toxic side effects pose a great threat to patients and limit the maximum dosages of cisplatin, carboplatin, and oxaliplatin. Cisplatin is the most toxic platinum drug currently in use . During the 1970’s, the toxicity of cisplatin was a critical concern that delayed approval of the drug by the Food and Drug Administration . Cisplatin treatment causes a broad spectrum of unpleasant side effects, including nausea and vomiting, which can occur as early as one hour post-treatment . The toxic effects of cisplatin are thought to result from the drug’s protein-binding activity . The instability of cisplatin allows aquation reactions to occur in the blood plasma, generating highly reactive species that react 15 readily with proteins and other biomoleucles . Cisplatin has a high affinity for proteins containing thiol groups and binds indiscriminately to a number of proteins, including albumin, transferrin, and globulins . Reactions that bind cisplatin to plasma proteins cause progressive accumulation of the drug in blood plasma over the course of chemotherapy . Cisplatin also accumulates in the liver, although no adverse effects of this accretion have been documented . Additional toxicity can occur when cisplatin induces apoptosis in healthy cells . In particular, cisplatin stimulates apoptosis in the cells lining the gastrointestinal tract . Cisplatin also exhibits toxicity toward the peripheral nervous system and the inner ear . Most importantly, cisplatin accumulates in the kidneys, causing severe kidney damage, or nephrotoxicity . The nephrotoxicity that results from cisplatin treatment is so great that it limits the maximum dose that can be administered during chemotherapy . Larger doses of cisplatin can result in acute renal failure and patient death . For this reason, nephrotoxicity is considered the dose-limiting toxicity of cisplatin. An important goal in the search for platinum analogs is the development of an effective drug with a reduced toxicity profile . To date, the discovery of carboplatin has been the most important advancement in the development of less toxic platinum drugs. Carboplatin is as effective as cisplatin but does not cause many of the toxicities associated with cisplatin . Carboplatin is better tolerated by patients, which allows for an improved quality of life during chemotherapy . For instance, carboplatin causes less nausea and vomiting than cisplatin . The reduced toxicity of carboplatin results from the slower rate of hydrolysis of carboplatin . This reduces the affinity of 16 carboplatin for proteins, preventing many of the side reactions responsible for the toxicity of cisplatin . Importantly, carboplatin administration is not restricted by the dose-limiting nephrotoxicity of cisplatin . Additionally, carboplatin shows reduced neurotoxicity when compared to cisplatin . However, carboplatin treatment is not without side effects. Carboplatin causes thrombycytopenia and exhibits a dose- limiting myelosuppression . Nonetheless, because carboplatin is as effective as cisplatin but produces fewer side effects, it is typically given preference over cisplatin . Oxaliplatin also shows less toxicity than cisplatin, though to a lesser extent than carboplatin. Oxaliplatin does not exhibit the reduced protein-binding activity of carboplatin . Even so, oxaliplatin does not accumulate in blood plasma, although this effect may be due to accumulation of the drug in red blood cells via covalent linkages with hemoglobin . Like carboplatin, oxaliplatin is not toxic to the kidneys . However, oxaliplatin does produce a dose-limiting neurotoxicity, but this effect is reversible . Intraperitoneal administration of platinum drugs is associated with increased toxicity . In particular, neurotoxicity and local intolerance are exacerbated when platinum drugs are administered intraperitoneally . Local discomfort, including abdominal bloating, pain, and distension, is a common side effect of intraperitoneal therapy . The toxicity associated with intraperitoneal chemotherapy is so great that many patients discontinue treatment before completion . As a result of the toxicity 17 associated with intraperitoneal administration, the use of this route remains controversial and is not usually considered in first-line therapy . 3. Acquired Resistance The greatest problem associated with the use of platinum drugs in chemotherapy is the development of acquired resistance . Over the course of treatment, tumors that are initially sensitive to platinum chemotherapy can become resistant. This phenomenon, called acquired resistance, is much more problematic than intrinsic resistance, in which a tumor is not sensitive to chemotherapy from the outset. Platinum drugs are especially prone to generating resistance in cancerous cells. Once a tumor has become resistant, platinum drugs are not effective treatment options. While the response rate to cisplatin is 50% in the first-line treatment of advanced tumors, it is only 15% in second- and third-line treatments because acquired resistance is so widespread . Acquired resistance to platinum chemotherapy is especially problematic in the treatment of ovarian cancer . The persistence of even a few cancerous cells after initial chemotherapy can lead to growth of a dangerous tumor that is resistant to further chemotherapy . Most patients diagnosed with advanced ovarian cancer will eventually succumb to resistant secondary tumors . To cure these patients, a drug that remains effective against tumors that have developed resistance to extant platinum drugs must be found. Because platinum drugs exert their cytotoxic effects through a variety of cellular pathways, cancerous cells can develop resistance in diverse ways . One possible route of platinum resistance is the alteration of membrane transport . This 18 can include reduced uptake of platinum drugs at the plasma membrane or increased efflux of platinum drugs that have entered the cell . Additionally, cancerous cells can acquire platinum resistance by activating metal detoxification pathways . Enhanced expression of glutathione and metallothionine, for instance, allows the cell to deactivate cisplatin, forming inactive product compounds that are unable to exert cytotoxic effects . Cells can also develop resistance to platinum drugs by improving the repair of platinum-DNA adducts or finding ways to continue DNA replication through damaged areas . Treatment with cisplatin induces a transient elevation in the expression of DNA repair proteins . If these proteins successfully repair platinum-DNA adducts, apoptosis can be avoided . Translesion synthesis, the ability to continue DNA replication past a damaged region, is also associated with acquired resistance to platinum drugs . Apoptotic pathways may be directly involved in the generation of platinum resistance . Activation of anti-apoptotic pathways can cause platinum resistance . Alternatively, the deactivation of pro- apoptotic pathways can contribute to platinum resistance . For instance, resistance to cisplatin can be induced by blocking activation of caspases . Cell lines that are deficient in MMR proteins, which play an important role in the recognition of platinum-DNA adducts and subsequent activation of apoptosis, are also resistant to platinum drugs . Similarly, cells that are p53-deficient are platinum-resistant . Loss of certain death receptors can also cause platinum resistance in several cell lines . Other genes that have been implicated in the acquisition of platinum resistance include Bcl-2, AKT, HMG proteins, and DNA polymerases . Changes at the level of the 19 plasma membrane may also generate platinum resistance . Altered membrane fluidity contributes to cisplatin resistance, as cell lines that exhibit decreased membrane fluidity are resistant to cisplatin treatment . The development of one or more of these cellular mechanisms of resistance can render platinum drugs ineffective in further chemotherapy. Unfortunately, the platinum analogs screened to date have not adequately addressed the problem of resistance. The search for new platinum drugs has focused on compounds that lack cross-resistance to cisplatin . Many analogs have been designed to interact with DNA in a manner that is distinct from extant drugs. Unfortunately, many of these DNA-binding analogs remain ineffective in tumors that have developed resistance to other platinum drugs. Carboplatin, arguably the most important platinum analog developed, is cross-resistant with cisplatin . This means that tumors that are resistant to cisplatin will not respond to carboplatin treatment and vice versa. Oxaliplatin is only partially cross-resistant with cisplatin and carboplatin . This lack of cross-resistance probably results from the unique DNA adducts formed by oxaliplatin . Oxaliplatin-DNA adducts inhibit DNA replication more effectively than the DNA adducts produced by cisplatin and carboplatin . As a result, oxaliplatin is effective in some tumors, such as colon cancer, that do not respond to cisplatin or carboplatin . However, oxaliplatin does show cross resistance with cisplatin and carboplatin in platinum-resistant ovarian cancer . Thus, the search for platinum analogs that truly evade acquired resistance continues. 20 E. The Continuing Search Forty years of research into platinum drugs has not yielded an ideal drug for the treatment of ovarian cancer. Research continues in hopes of developing a platinum drug that is soluble and stable, nontoxic to healthy tissues, and effective against resistant tumors. Two platinum drugs stemming from this research are RRD2 and RRD4, developed by Dr. Rathindra Bose . These novel drugs overcome all of the inadequacies associated with extant platinum drugs. RRD2 and RRD4 are currently undergoing preclinical testing with the hope that they will someday be used in the treatment of patients with ovarian cancer. III. RRD2 AND RRD4 A. The Discovery of Two Novel Platinum Analogs RRD2 and RRD4 were discovered by Dr. Rathindra Bose in an effort to develop platinum analogs that overcome the shortcomings of extant platinum drugs . These novel drugs are structurally similar to other platinum drugs. However, they display marked improvements in solubility and stability, decreased toxicity, and efficacy against platinum-resistant cancer cells. RRD2 and RRD4 induce apoptosis in cancerous cells via a unique mechanism that has yet to be elucidated. 21 Oxaliplatin RRD2 RRD4 Figure 3. Structures of the platinum drugs, oxaliplatin, RRD2, and RRD4. Structurally, RRD2 and RRD4 are quite similar to oxaliplatin (Figure 3). These three drugs consist of a central platinum atom complexed with a cyclohexanediammine ligand and a bifunctional oxygen-containing ligand. Oxalate is the oxygen-containing ligand of oxaliplatin and pyrophosphate is the equivalent ligand of RRD2 and RRD4 . RRD2 and RRD4 differ in the oxidation state of their central platinum atom. RRD2 incorporates a platinum(II) atom while RRD4 incorporates a platinum(IV) atom that is bound to two hydroxyl groups in addition to its cyclohexanediammine and pyrophosphate ligands. RRD2 and RRD4 are specific optical isomers of the compounds, pyrodach-2 and pyrodach-4, respectively. Both pyrodach-2 and pyrodach-4 contain two stereocenters, which can exist in either the R,R or S,S conformations. Therefore, three distinct formulations of each drug exist: the racemic mixture of R,R and S,S isomers, and the purified isomers, R,R or S,S. Because optical isomers frequently display 22 differences in biological activity, all three isomeric formulations have been studied. In vitro tests demonstrate that, of these formulations, the purified R,R optical isomers are most effective against cancer cells (S. Moghaddas, pers. comm.). The efficacies of RRD2 and RRD4 have been examined in both in vitro and in vivo experiments (S. Moghaddas, pers. comm.). In vitro efficacy tests demonstrate the ability a compound to inhibit proliferation of cells that are cultured outside of a living system. The efficacies of RRD2 and RRD4 were examined in vitro by determining half maximal inhibitory concentration (IC50) values via clonogenic assay. IC50 values are a common measure of efficacy and indicate the drug concentration that inhibits proliferation of cancerous cells by 50%. Clonogenic assays and IC50 value calculations establish that racemic mixtures of pyrodach-2 and pyrodach-4 are as effective as cisplatin and carboplatin against the A2780 human ovarian cancer cell line . It should be noted that the A2780 cell line is considered sensitive to cisplatin and carboplatin treatment. Purified RRD2 and RRD4, which are more potent than the racemic stereoisomer mixtures, are more effective against A2780 cells than cisplatin and carboplatin (S. Moghaddas, pers. comm.). The in vivo efficacies of RRD2 and RRD4 have also been established. In vivo efficacy tests demonstrate the ability of a compound to inhibit tumor growth in a living system. Murine xenograft models were used to characterize the efficacies of RRD2 and RRD4 in vivo. In these studies, mice were inoculated with A2780 human ovarian cancer cells. Once tumors had formed, drug treatment was started and efficacy was determined by monitoring tumor regression. These xenograft models show that 23 RRD2 and RRD4 are as effective as cisplatin and carboplatin against the A2780 cell line in vivo (S. Moghaddas, pers. comm.). The results of in vitro and in vivo efficacy studies illustrate that RRD2 and RRD4 are as effective as extant platinum drugs. However, the most exciting properties of RRD2 and RRD4 are their differences from currently available drugs. These unique properties allow RRD2 and RRD4 to overcome the inadequacies associated with extant platinum drugs. B. The Unique Properties of RRD2 and RRD4 While RRD2 and RRD4 are structurally comparable to cisplatin, carboplatin, and oxaliplatin, these novel drugs demonstrate unique properties that represent improvements over extant platinum drugs. RRD2 and RRD4 dissolve readily in water and chemical studies show that these drugs are remarkably stable in solution . Further, RRD2 and RRD4 exhibit dramatic reductions in toxicity (S. Moghaddas, pers. comm.). Most importantly, RRD2 and RRD4 retain their efficacies against cancer cells that are resistant to other platinum drugs (S. Moghaddas, pers. comm.). The lack of cross-resistance between these drugs and extant platinum drugs is due to a novel mechanism of action, which does not involve DNA-binding. RRD2 and RRD4 exhibit significant improvements in solubility and stability over extant platinum drugs . Polar cyclohexanediammine and pyrophosphate ligands allow RRD2 and RRD4 to dissolve readily in water. Once dissolved, RRD2 and RRD4 are remarkably stable . Nuclear magnetic resonance studies show that RRD2 and RRD4 do not decompose, even after six days in aqueous solution . These 24 characteristics suggest that RRD2 and RRD4 can overcome the problems associated with the clinical administration of cisplatin and carboplatin. The stability of RRD2 and RRD4 eliminates the need for hospital personnel to suspend the drugs in solution immediately prior to the administration of chemotherapy. Further, the absence of aqueous decomposition may permit RRD2 and RRD4 to exhibit full efficacy when administered intravenously. In vivo toxicity studies, conducted in mice, demonstrate that RRD2 and RRD4 are less toxic than cisplatin and carboplatin (S. Moghaddas, pers. comm.). Previous studies demonstrated the reduced toxicities of pyrodach-2 and pyrodach-4 compared to cisplatin and carboplatin . In these experiments, mice treated with cisplatin and carboplatin exhibited standard signs of toxicity, including loss of appetite, decreased activity levels, weight loss, and loss of coat luster . Cisplatin-induced weight loss was so severe that some of the cisplatin-treated mice were humanely sacrificed before completion of the toxicity study . The pyrodach-2- and pyrodach-4-treated mice did not display loss of appetite or decreased activity levels . These mice experienced some weight loss and loss of coat luster, but these changes reversed in the week following drug treatment . These results demonstrated the improved tolerance of pyrodach-2 and pyrodach-4 over cisplatin and carboplatin . Similar results were obtained from a recent study of the in vivo toxicities of RRD2 and RRD4 (S. Moghaddas, pers. comm.). Notably, both RRD2 and RRD4 are effective against ovarian cancer cells that are resistant to extant platinum drugs (S. Moghaddas, pers. comm.). The efficacies of 25 RRD2 and RRD4 against platinum-resistant cells were examined in the A2780/C30 cell line. The A2780/C30 cell line was cultured by scientists at the Fox Chase Cancer Center to exhibit resistance to cisplatin at concentrations up to 30 μM. By comparison, the IC50 value for cisplatin in the A2780 cell line is seven μM. As expected, the A2780/C30 cell line demonstrates cross-resistance to carboplatin. Clonogenic assays demonstrate that pyrodach-2 and pyrodach-4 maintain their efficacies in the A2780/C30 ovarian cancer cell line . These experiments show that the IC50 value of cisplatin increases 15-fold in the A2780/C30 cell line . In contrast, the IC50 value of pyrodach-2 increases 2.5-fold in the A2780/C30 cell line . The IC50 of pyrodach-4 shows a slight decrease in the A2780/C30 cell line . RRD2 and RRD4 also maintain their efficacies in the A2780/C30 cell line (S. Moghaddas, pers. comm.). Because these drugs retain their efficacies against resistant cells, RRD2 and RRD4 may provide a cure for the thousands of ovarian cancer patients who succumb to platinum-resistant tumors each year. The efficacies of RRD2 and RRD4 against platinum-resistant cells result from their unique mechanism of action . Remarkably, pyrodach-2 and pyrodach-4 do not bind to DNA . Pyrodach-2 and pyrodach-4 cannot be detected bound to DNA, even after 24-hour treatment . Chemical studies demonstrate similar results, showing that pyrodach-2 and pyrodach-4 do not bind to DNA after seven-day incubation with DNA molecules . This represents a crucial difference that distinguishes pyrodach-2 and pyrodach-4 from all extant platinum drugs . Because RRD2 and RRD4 exhibit cytotoxicity through a unique mechanism, they should easily overcome acquired 26 resistance to other platinum drugs . The absence of DNA-binding activity demonstrates that the cytotoxic effects of RRD2 and RRD4 are mediated by unique mechanisms, which are the subject of current study. IV. INVESTIGATING THE MECHANISM OF ACTION OF RRD2 AND RRD4 Elucidating the novel mechanism of action of RRD2 and RRD4 is a priority in the study of these two drugs. Clonogenic assays indicate that RRD2 and RRD4 inhibit the proliferation of cancerous cells, but cannot demonstrate whether this effect is due to inhibition of the cell cycle or induction of cell death. However, microscopy and cell death ELISA experiments verify that RRD4 and RRD2 cause apoptosis in A2780 and A2780/C30 ovarian cancer cells (S. Moghaddas, pers. comm.). Induction of apoptosis occurs rapidly during treatment with RRD2 and RRD4 (S. Moghaddas, pers. comm.). One-hour RRD2 and RRD4 treatment is sufficient to induce apoptosis in A2780 and A2780/C30 ovarian cancer cells (S. Moghaddas, pers. comm.). After six-hour treatment, the rate of apoptosis is greatly enhanced, with further increases in the rate of apoptosis occurring with increasing treatment length . RRD2 and RRD4 could stimulate apoptosis via numerous pathways by causing changes at multiple cellular levels. Because RRD2 and RRD4 do not bind to DNA, the apoptotic pathways activated by these novel drugs must be unique from the apoptotic pathways that are activated in response to the DNA damage that is induced by other platinum drugs. Although RRD2 and RRD4 do not bind to DNA, they cause important changes in gene expression. These changes can occur at the levels of gene 27 transcription and mRNA translation. Previous studies examined these effects by characterizing changes in gene expression following pyrodach-4 and RRD4 treatments . While documentation of these changes is important, almost all cellular functions are carried out by proteins rather than mRNA molecules. Therefore, examining the effects of RRD2 and RRD4 treatment on protein expression and activity is of vital importance. Undoubtedly, a multitude of proteins are involved in the cytotoxicities of RRD2 and RRD4. These drugs probably modify the expression and activity of proteins involved in cell growth, survival, and apoptosis. RRD2 and RRD4 could modulate any of the numerous proteins involved in the intrinsic and extrinsic apoptotic pathways. Signaling proteins that regulate cell cycle progression, growth, and survival are also likely targets of RRD2 and RRD4. Finally, the apoptotic effects of RRD2 and RRD4 may be mediated by a pathway that involves activation of the enzyme, acid sphingomyelinase, at the plasma membrane. A. Intrinsic and Extrinsic Apoptotic Pathways Induction of apoptosis can be mediated by multiple cellular pathways in response to various stimuli. Intracellular signaling molecules can induce apoptosis in response to cellular damage or stress . This route, called the intrinsic apoptotic pathway, mediates the apoptotic effects of many anticancer treatments, including platinum drugs and radiation therapy . Extracellular signaling molecules can also trigger apoptosis, via the extrinsic apoptotic pathway . Apoptotic pathways are frequently dysregulated in cancerous cells . As a result, malignant cells proliferate 28 uncontrollably despite the presence of apoptotic signals . Many anticancer agents induce apoptosis of malignant cells by resensitizing them to apoptotic signals . Sensitization of ovarian cancer cells to apoptotic signals may play a role in the cytotoxicities of RRD2 and RRD4. RRD2 and RRD4 probably induce apoptosis through both the intrinsic and extrinsic apoptotic pathways. Involvement of both pathways would provide flexibility in the induction of apoptosis, allowing RRD2 and RRD4 to retain efficacy in cancer cells that have defects in one of the apoptotic pathways. 1. The Intrinsic Apoptotic Pathway RRD2 and RRD4 may induce apoptosis through the intrinsic apoptotic pathway. The intrinsic apoptotic pathway triggers cell death by releasing death- inducing molecules from the mitochondria . Mitochondria are organelles that produce energy through the process of cellular respiration. Additionally, they play an important role in the induction of apoptosis . Mitochondrial structure is central to their role as regulators of apoptosis. Mitochondria are surrounded by a double membrane that encloses an intermembrane space. Apoptosis-inducing molecules, including cytochrome C, second mitochondria-derived activator of caspases (SMAC), and high temperature requirement serine protease A (HtrA), are contained within the intermembrane space . Within the intermembrane space, these proteins are inactive. When they are released into the cytosol, however, they initiate a proteolytic cascade that culminates in apoptosis . 29 The release of cytochrome C, SMAC, and HtrA from the intermembrane space is regulated by proteins of the Bcl-2 family . Pro-apoptotic members of the Bcl-2 family, which include Bad, Bax, Bid, and Bim, increase the permeability of the outer mitochondrial membrane and promote the release of cytochrome C, SMAC, and HtrA . Bad and Bax self-associate to form a pore complex, called the mitochondrial apoptosis-induced complex (MAC) . Once associated, MACs are inserted in the outer mitochondrial membrane . Incorporation of these pore complexes permeabilizes the outer mitochondrial membrane, facilitating the release of cytochrome C, SMAC, and HtrA . Bid and Bim augment this apoptotic process, although they do not associate into MACs . The exact roles of Bid and Bim remain unknown and may involve activation of Bad and Bax . An alternative role for Bid and Bim may be the inhibition of anti-apoptotic Bcl-2 proteins . The association and membrane insertion of MACs is regulated by anti-apoptotic members of the Bcl-2 family, such as Bcl-w and Bcl-2 . Bcl-w and Bcl-2 are expressed at the outer mitochondrial membrane, where they interfere with the formation and membrane insertion of MACs . Once the outer mitochondrial membrane has been permeabilized by MACs, an apoptotic cascade is initiated in the cytosol . Cytochrome c diffuses from the intermembrane space into the cytosol, where it associates with protease-activating factor-1 (APAF-1) and procaspase-9 to form a complex, called the apoptosome . The apoptosome is a proteolytic oligomer that cleaves proscaspase-9 to generate the active protease, caspase-9 . Caspase-9 propagates the apoptotic cascade by cleaving 30 procaspase-3 into the active protease, caspase-3. Caspase-3 is an executioner caspase that cleaves structural proteins and causes apoptosis. The apoptotic caspase cascade can be abrogated by cytosolic proteins, aptly named inhibitors of apoptosis (IAPs) . The IAP family includes the proteins cIAP-2, livin, survivin, and XIAP . IAPs bind caspases, preventing their activation and halting the apoptotic cascade . SMAC and HtrA are released from the mitochondrial intermembrane space to combat the inhibitory effects of IAPs . SMAC and HtrA are proteases that bind to and cleave IAPs . These proteins antagonize IAP function and prevent deactivation of the apoptotic caspase cascade . 31 Figure 4. The intrinsic apoptotic pathway. 1: Bax and Bad associate to form a MAC. 2: The MAC is inserted in the outer mitochondrial membrane. Bid and Bim promote the association and insertion of MACs. Bcl-2 and Bcl-w inhibit the association and insertion of MACs. 3: MAC insertion permeabilizes the outer mitochondrial membrane, allowing release of cytochrome C (cytoC) into the cytosol. 4: Cytochrome C and APAF-1 associate to form the apoptosome. 5: Active caspase-9 is released from the apoptosome. 6: Caspase-9 cleaves procaspase-3 to produce active caspase-3. Caspase-3 induces apoptosis. IAPS inhibit activation of caspase-9 and -3. RRD2 and RRD4 could activate the intrinsic apoptotic pathway by modulating any of these proteins. For instance, RRD2 and RRD4 could enhance expression of the pro-apoptotic Bcl-2 proteins, Bad, Bax, Bid, or Bim. RRD2 and RRD4 might also inhibit expression of the anti-apoptotic Bcl-2 proteins, Bcl-w and Bcl-2. Caspases and the proteins that modify their activity may also be affected by RRD2 and RRD4 32 treatment. RRD2 and RRD4 could amplify intrinsic apoptotic signals by enhancing expression of caspases-9 and -3. In parallel, RRD2 and RRD4 may inhibit IAPs or activate IAP antagonists to improve caspase activation. Clearly, numerous mechanisms have the potential to mediate RRD2- and RRD4-induced apoptosis through the intrinsic apoptotic pathway. 2. The Extrinsic Apoptotic Pathway Alternatively, RRD2 and RRD4 could activate apoptosis through the extrinsic apoptotic pathway. The extrinsic apoptotic pathway involves transduction of apoptotic signals from extracellular ligands by transmembrane receptors, commonly referred to as death receptors . Numerous ligand/receptor pairs can activate the extrinsic apoptotic pathway . While the identities of these signaling molecules and receptors vary, all of the death receptor pathways instigate an apoptotic caspase cascade that resembles the cascade initiated by the intrinsic apoptotic pathway . For the sake of explanation, the FasL ligand/Fas receptor pair will be used to describe the events that occur during activation of the extrinsic apoptotic pathway. Fas is a transmembrane death receptor that activates the extrinsic apoptotic pathway in response to extracellular signals . The extracellular domain of the Fas receptor interacts with the ligand, FasL . The intracellular portion of the Fas receptor is a death domain that binds the adaptor, Fas-associated death domain-containing protein (FADD) . Once bound to the Fas receptor, FADD associates with procaspase-8 . This complex of Fas receptors, FADD, and procaspase-8 is called the death-inducing signaling complex (DISC) . Within the DISC, procaspase-8 is 33 cleaved to produce the active protease, caspase-8 . Caspase-8 plays a role parallel to that of caspase-9 in the intrinsic apoptotic pathway . Caspase-8 is released from the DISC and diffuses through the cytosol, where it cleaves procaspase-3 into the active executioner caspase, caspase-3 . As in the intrinsic apoptotic pathway, caspase-3 degrades cellular proteins and triggers apoptosis . Activation of the extrinsic apoptotic pathway is regulated by conformational changes in the Fas receptor . The Fas receptor can exist in open or closed conformations . Under normal conditions, the Fas receptor is in the closed conformation, with its death domain occluded . This conformation prevents the binding of FADD and cannot induce DISC formation. The Fas receptor exposes its death domain only when it is in the open conformation . However, the open conformation is unstable unless Fas receptors are clustered in a tetramer arrangement . Association of Fas tetramers occurs when Fas receptors bind FasL . In the absence of FasL, Fas receptors are clustered at the plasma membrane in trimers . The binding of FasL causes coclustering of Fas trimers, which facilitates the formation of Fas tetramers . Once tetramers form, Fas receptors can stably adopt the open conformation, exposing their death domains to bind FADD and inducing association of the DISC . 34 Figure 5. The extrinsic apoptotic pathway. The FasL ligand/Fas receptor are illustrated as an example. 1: Binding of FasL to Fas causes aggregation of Fas receptor trimers. This facilitates formation of Fas receptor tetramers. 2: When associated in tetramers, Fas receptors recruit FADD to form the DISC. 3: Caspase-8 is released from the DISC. 4: Caspase-8 cleaves procaspase-3 to produce active caspase-3. Caspase-3 triggers apoptosis. The Fas receptor and its ligand, FasL, are representative members of the tumor necrosis factor (TNF) superfamily . The TNF superfamily includes various signaling molecules and death receptors that can induce the extrinsic apoptotic pathway . Notable ligand/receptor pairs within this superfamily include CD40L/CD40, TNF/TNFR, and TRAIL/TRAILR . Each of these signaling pairs induces apoptosis through the extrinsic apoptotic pathway in a manner similar to FasL/Fas. All of these ligand/receptor pairs cause DISC formation and induce 35 apoptosis via a caspase cascade that involves activation of caspases-8 and -3. In all of these death receptor pathways, DISC formation is regulated by receptor association in response to ligand binding. RRD2 and RRD4 may modulate protein expression in a way that enhances activation of the extrinsic apoptotic pathway. Immunofluorescent protein detection and gene expression experiments suggest that the apoptotic effects of RRD2 and RRD4 are mediated by the extrinsic death receptor pathway . Examining the expression of various extrinsic apoptotic pathway proteins will show whether or not these changes in gene expression translate into changes in protein expression. B. Growth and Survival Signaling Pathways In addition to modulating apoptotic pathways, RRD2 and RRD4 may cause changes in the signaling pathways that regulate cell growth and survival. RRD2 and RRD4 may regulate the signaling activity of insulin-like growth factors. Heat shock proteins and cell cycle regulatory proteins may also act as mediators of the cytotoxicities of RRD2 and RRD4. Finally, RRD2 and RRD4 may exert their toxic effects by modifying activity of the PI3K signaling pathway. 1. IGF Signaling RRD2 and RRD4 may abrogate growth and survival signals by altering the insulin-like growth factor (IGF) pathway. IGFs are crucial signaling molecules that regulate the processes of growth, metabolism, differentiation, and survival . IGFs induce metabolic effects similar to those of insulin and are best known for promoting 36 the growth of bone and skeletal muscle . Recently, novel functions of IGF signaling in apoptosis and oncogenesis have been discovered . Two isoforms of IGF, IGF-1 and IGF-2, are expressed in humans . IGF molecules bind to IGF receptors that are expressed at the plasma membrane of target cells. Humans express two IGF receptors, IGFR-1 and IGFR-2 . Both IGF-1 and IGF- 2 bind to and activate IGFR-1, but only IGF-2 can activate IGFR-2 . IGF signaling is further modulated by six IGF-binding proteins, IGFBPs-1 through -6 . These binding proteins associate with IGF-1 and IGF-2 as they circulate in blood plasma . Some of the IGF binding proteins, namely IGFBP-1 and IGFBP-3, antagonize IGF signaling by sequestering IGF from IGF receptors . Other IGF binding proteins, including IGFBP-2 and IGFBP-5, enhance IGF signaling via an unknown mechanism . 37 Figure 6. IGF signaling pathway. IGFs activate IGF receptor (IGFR). This interaction can be controlled by activating IGF binding proteins (IGFBPs) and inhibitory IGF binding proteins. Active IGF receptors stimulate growth and survival. Importantly, IGF signaling has been implicated in the development of various cancers . IGF signaling enhances tumor growth, protects cells from apoptosis, and promotes tumor vascularization . Inhibition of IGF signaling by RRD2 and RRD4 could inhibit cell growth and sensitize cancerous cells to apoptosis. RRD2 and RRD4 might modulate IGF signaling at several levels to generate an inhibitory effect. Decreased expression of IGF-1 and IGF-2, reduced expression of IGF receptors, and/or altered expression of IGF binding proteins could all result in the inhibition of IGF signaling . 38 2. Heat Shock Proteins Heat shock proteins (HSPs) assist in the folding and transport of cellular proteins and are crucial regulators of apoptosis . Many different heat shock proteins, including HSP27, HSP60, and HSP70, are expressed in cells . The function of HSPs in apoptotic pathways is complex, as some HSPs enhance apoptotic signals while other HSPs provide protection from apoptosis . Most HSPs play a protective role by preventing apoptosis following exposure to stressful stimuli . HSPs are considered oncogenes, which means that overexpression of these proteins can contribute to the development of cancer . The induction of apoptosis by RRD2 and RRD4 might result from reduced expression of HSPs. 3. Cell Cycle Regulatory Proteins Dysregulation of the cell cycle is a common transformation in the process of oncogenesis . Loss of cell cycle regulation permits uncontrolled cell division, enabling the rapid and uninhibited growth of malignant cells . Normally, progression through the cell cycle is regulated by the interaction of cyclin proteins with cyclin- dependent kinases (CDKs) . In the absence of cyclins, cyclin dependent kinases are located in the cytosol, where they remain inactive . When cyclins are present, they associate with cyclin dependent kinases to form heterodimers . These cyclin-CDK complexes are translocated to the nucleus, where they are activated . The kinase portions of active cyclin-CDK complexes phosphorylate protein targets to initiate a cascade that facilitates cell cycle progression . The cell cycle is additionally regulated 39 by cyclin-dependent kinase inhibitors, which halt the cell cycle by preventing activation of cyclin-dependent kinases . p53 is a well-known tumor suppressor that arrests cell cycle progression by activating cyclin-dependent kinase inhibitors in response to DNA damage . p53 also induces apoptosis in response to unrepaired DNA damage . The p53 protein regulates apoptosis by controlling the expression of various apoptotic proteins . p53 halts cell cycle progression by activating the cyclin-dependent kinase inhibitors, p21 and p27 . p21 and p27 mediate arrest of the cell cycle by preventing activation of the cyclin-dependent kinases, CDK1 and CDK2 . Historically, p21 and p27 were thought to be standard tumor suppressor proteins . However, recent studies demonstrate that overexpression of p21 and p27 is oncogenic . Both p21 and p27 promote oncogenesis when they are phosphorylated by the kinase, AKT . Phosphorylation of p21 and p27 alters their cellular distribution and causes them to accumulate in the cytosol . The cytosolic accumulation of p21 and p27 protects cells from apoptosis by inhibiting caspase activation . The cytotoxic effects of RRD2 and RRD4 may be effected by these regulators of the cell cycle. Increased expression of p53 by RRD2 and RRD4 could halt tumor growth and enhance the induction of apoptosis. Interactions with p21 and p27 are more difficult to characterize due to the opposing tumor-suppressive and oncogenic activities of these two proteins. Increased expression of p21 and p27 could enhance p53 signaling, resulting in arrest of the cell cycle and induction of apoptosis. Alternatively, p21 and p27 may act as anti-apoptotic oncogenes in ovarian cancer cells, 40 in which case decreased expression of p21 and p27 would enhance the activation of apoptotic pathways. 4. PI3K Signaling The phosphatidylinositol-3 kinase (PI3K) signaling pathway might also mediate the cytotoxic effects of RRD2 and RRD4. The PI3K pathway controls the processes of cell growth and survival . This signaling pathway can be a cornerstone of oncogenic transformation and is the most frequently overactivated pathway in human cancers . The PI3K pathway is regulated by extracellular growth factors that transmit signals across the plasma membrane via transmembrane receptors . Upon activation by membrane receptors, the PI3K enzyme catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5- triphosphate (PIP3) . PIP3 acts as a second messenger and induces activation of the protein kinase, AKT . AKT is usually present in its dephosphorylated, inactive form and must undergo activation via phosphorylation . PIP3 facilitates phosphorylation of AKT . once activated, AKT phosphorylates a variety of target proteins to propagate PI3K signaling . The protein, phosphatase and tensin homolog (PTEN), provides crucial opposition to PI3K activity by dephosphorylating PIP3 to form PIP2 . PTEN antagonizes PI3K activity and abrogates growth and survival signaling . This inhibitory role makes PTEN a crucial tumor suppressor protein . 41 Figure 7. PI3K signaling pathway. 1: Extracellular growth factors activate receptors at the plasma membrane. 2: Active membrane receptors activate PI3K. 3: PI3K phosphorylates PIP2 to produce the second messenger, PIP3. 4: PIP3 induces activation of AKT via phosphorylation. 5: AKT propagates growth and survival signals by phosphorylating target proteins. 6: PTEN opposes PI3K signaling by dephosphorylating PIP3 to form PIP2. Abrogation of growth and survival signaling through the PI3K pathway could contribute to the cytotoxic effects of RRD2 and RRD4. These drugs could inhibit PI3K signaling through various mechanisms, such as deactivating the PI3K enzyme or enhancing expression of PTEN. Gene expression analysis suggests that RRD2 and RRD4 can increase expression of PTEN, presenting a putative target for blocking growth and survival signals . 42 C. Activation of Acid Sphingomyelinase Activation of acid sphingomyelinase may mediate the induction of apoptosis by RRD2 and RRD4. Acid sphingomyelinase is an enzyme that hydrolyzes the membrane lipid, sphingomyelin, to produce phosphorylcholine and ceramide. Recently, it was discovered that activation of acid sphingomyelinase can induce apoptosis via the extrinsic apoptotic pathway. This mechanism plays a critical role in the induction of apoptosis by several anticancer drugs, including doxorubicin, paclitaxel, and cisplatin . Activation of acid sphingomyelinase causes dramatic changes in the fluidity of the plasma membrane by altering levels of sphingomyelin and ceramide . Sphingomyelin, the substrate of acid sphingomyelinase, is an important constituent of the plasma membrane . Sphingomyelin is concentrated in the extracellular leaflet of the plasma membrane, where it interacts with another membrane lipid, cholesterol . Hydrophobic forces facilitate the interaction of sphingomyelin and cholesterol, causing formation of sphingomyelin- and cholesterol-enriched microdomains . These microdomains, also called lipid rafts, permit the formation of highly specific microenvironments within the plasma membrane . Ceramide molecules undergo a similar aggregation to form ceramide-enriched microdomains within the plasma membrane . Unlike sphingomyelin-containing microdomains, ceramide-containing microdomains can undergo fusion . The fusion of these microdomains creates ceramide-enriched macrodomains that can reach diameters of several micrometers . Activation of acid sphingomyelinase consumes sphingomyelin while generating 43 ceramide, inducing dramatic changes in the microdomain environment of the plasma membrane . The subsequent increase in ceramide levels favors aggregation of large ceramide-enriched macrodomains. These membrane macrodomains can regulate various signaling pathways, including the extrinsic apoptotic pathway . Ceramide-enriched macrodomains create unique microenvironments that serve as signaling platforms . These domains selectively incorporate membrane proteins and enhance signaling pathways by concentrating regulatory proteins in a confined area along with their activators and effectors . Inclusion of the death receptor, Fas, in ceramide-enriched macrodomains induces the extrinsic apoptotic pathway in the absence of the ligand, FasL . Incorporation in the ordered macrodomain environment concentrates Fas receptors, facilitating receptor association and stabilizing supra-molecular receptor complexes . Inclusion in ceramide-enriched macrodomains thus favors the formation of Fas tetramers. Within macrodomains, Fas tetramer formation can occur independently of FasL. Ligand-independent induction of the extrinsic apoptotic pathway might occur when other members of the TNF superfamily associate in ceramide-enriched macrodomains as well . 44 Figure 8. Activation of the extrinsic apoptotic pathway by acid sphingomyelinase. 1: Acid sphingomyelinase (ASMase) cleaves sphingomyelin (SM) to produce ceramide (Cer). 2: Ceramide molecules aggregate to form ceramide-enriched macrodomains (Cer macrodomain). 3: Fas receptor trimers are incorporated in ceramide-enriched macrodomains, where they associate into Fas receptor tetramers. 4: Fas tetramers induce DISC formation and stimulate apoptosis by activating caspase-3. Activation of acid sphingomyelinase may be responsible for the induction of extrinsic death receptor pathways by RRD2 and RRD4 treatment. This mechanism could account for the rapid induction of apoptosis by RRD2 and RRD4 and could augment direct effects on the expression of apoptotic proteins. V. EXPERIMENTAL GOALS The goal of this thesis is to contribute to the elucidation of the mechanism of action of RRD2 and RRD4. Experiments were conducted to determine the effects of RRD2 and RRD4 on protein expression and activity in ovarian cancer cells. Protein expression levels were examined to identify increases or decreases in expression following treatment. The effects of treatment on the enzyme activity of acid 45 sphingomyelinase was also determined. Examining these changes enriches understanding of how RRD2 and RRD4 induce apoptosis of ovarian cancer cells. Additionally, mechanistic studies help to explain differences in the activities of RRD2 and RRD4, allowing for a comparison of these drugs and speculation as to which drug is superior. This is an important topic to address as preclinical testing of RRD2 and RRD4 continues because the clinical trial of one drug is much more feasible than clinical trials of two drugs. 46 MATERIALS AND METHODS I. SAMPLE COLLECTION A. Cell Culture The A2780 and A2780/C30 ovarian cancer cell lines were used for all experiments. Both cell lines were obtained from the Fox Chase Cancer Center in Philadelphia, PA. The A2780 ovarian cancer cell line is sensitive to cisplatin and carboplatin while the A2780/C30 ovarian cancer cell line exhibits acquired resistance to cisplatin and carboplatin. All cells were cultured on 100 mm3 tissue culture plates. Cells were cultured in RPMI 1640 medium, supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.1% human insulin. Culture plates were kept in a humidified incubator at 37°C with a carbon dioxide concentration of 5%. Culture medium was decanted and replaced with fresh medium every one to three days. Cells were grown until they had reached 70- 80% confluency, at which point drug treatment was performed. B. Drug Synthesis RRD2 and RRD4 were synthesized by Dr. Anna Qi as described in the literature . The activity of synthesized drugs was verified by cell viability assay. The same lot of RRD2 and RRD4 was used in all experimental treatments to ensure consistency. Cisplatin was selected for use as a comparative treatment because it is one of the most widely used chemotherapy drugs available . Although carboplatin recently replaced cisplatin as the standard treatment for ovarian cancer, the molecular 47 mechanism of carboplatin is less well characterized, making carboplatin an unfit choice for comparative study. Cisplatin was obtained from Sigma-Aldrich. The same lot was used in all experimental treatments to ensure consistency. C. Drug Treatment and Sample Collection The IC50 value for one-hour treatment was used in all experimental treatment, regardless of duration. This ensured that the only variation within drug treatment groups was treatment duration. Specifically, concentrations of 20 μM RRD2, 45 μM RRD4, and 7 μM cisplatin were used in all experimental treatments. Negative control treatments were included in all experiments. This treatment consisted of normal media without added platinum drugs. All experimental treatments were performed in duplicate or triplicate. Negative control treatments were performed in at least quintuplet. Immediately prior to drug treatment, one mg of the specified drug was dissolved in 22 mL of normal culture medium (RPMI 1640, supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.1% human insulin). This concentrated stock solution was diluted into normal culture medium to achieve the desired drug concentration. At the time of drug treatment, normal culture medium was decanted and plates were washed once with phosphate-buffered saline solution (PBS). Twelve mL of drug-containing medium was added to experimental treatment plates and 12 mL of normal culture medium was added to negative control plates. Plates were incubated at 37°C for the specified time period. At the conclusion of drug treatment, the medium was decanted and plates were washed three times with five mL of PBS to ensure 48 complete removal of the drug-containing medium. Cells were lysed with ice-cold PBS containing protease inhibitors. Cells were scraped off of the culture plates and pelleted by centrifugation. Cell pellets were washed once in PBS containing protease inhibitors. Pelleted cells were resuspended in PBS containing protease inhibitors and stored at -80ºC until use. These pellets were used in sample preparation for apoptosis arrays, Western blots, or acid sphingomyelinase assays. D. Determination of Protein Concentration The total protein concentration of all prepared samples was determined using a Micro BCA Protein Determination Kit, obtained from Thermo Scientific. This kit utilizes a colorimetric assay to quantify protein concentration. Five μL of each prepared sample was diluted to a volume of one mL. One mL of Micro BCA protein detection solution was added to each sample. Samples were incubated at 60°C for 30 minutes. Samples were allowed to cool, then 300 μL of each sample was transferred to a 96-well microplate. Absorbance was measured at 562 nm using a microplate scanner. A standard curve was generated from the absorbance values of samples containing known concentrations of bovine serum albumin (BSA). The standard curve was used to calculate the protein concentration of each experimental sample based on its absorbance. All calculations were performed in Microsoft® Excel. II. ARRAY OF APOPTOTIC PROTEINS Expression of 43 apoptotic proteins was examined using a RayBio® Human Apoptosis Array kit, purchased from RayBiotech, Inc. Arrayed proteins are listed 49 below. Each array membrane included six positive control spots, ten negative control spots, eight blank spots, and two spots for each protein. Four antibody array membranes were purchased, allowing for analysis of two control treatments, one one- hour RRD2 treatment, and one one-hour RRD4 treatment. Protein Number of Antibody Spots Bad 2 Bax 2 Bcl-2 2 Bcl-w 2 Bid 2 Bim 2 Caspase-3 2 Caspase-8 2 CD40 2 CD40L 2 cIAP-2 2 Cytochrome C 2 DR6 2 Fas 2 FasL 2 HSP27 2 HSP60 2 HSP70 2 HtrA 2 IGF-1 2 IGF-2 2 IGFBP-1 2 IGFBP-2 2 50 IGFBP-3 2 IGFBP-4 2 IGFBP-5 2 IGFBP-6 2 IGFR-1 2 Livin 2 p21 2 p27 2 p53 2 SMAC 2 Survivin 2 sTNF-R1 2 sTNF-R2 2 TNF- 2 TNF- 2 TRAILR-1 2 TRAILR-2 2 TRAILR-3 2 TRAILR-4 2 XIAP 2 Positive control 6 Negative control 10 Blank 8 Whole cell lysate was assayed for apoptotic protein expression according to the protocol provided by the manufacturer. Pelleted cells were resuspended in 300 μL of 1X Cell Lysis Buffer. Samples were diluted to 1.8 mL in Blocking Buffer and applied to each antibody array membrane. Membranes were incubated overnight at 4ºC. Samples were washed off with 1X Wash Buffer I followed by 1X Wash Buffer II. 1.5 51 mL of biotin-conjugated secondary antibody mix was added to each membrane and incubated overnight at 4ºC. The secondary antibody mix was washed off with 1X Wash Buffer I followed by 1X Wash Buffer II. Each membrane was incubated in 1.5 mL of HRP-Conjugated Streptavidin for one hour at room temperature. HRP- Conjugated Streptavidin was washed off with 1X Wash Buffer II and membranes were incubated with luminescent Detection Buffers C and D for two minutes. Radiographic films were exposed to each membrane and developed to visualize protein bands. Digital images were created from each radiographic film and protein expression was determined using Adobe® Photoshop® CS5 software. Protein spots were selected from the digital images of the membranes and pixel intensity was quantified using Adobe® Photoshop® CS5 software. An area of constant size was used to select all spots within and across each membrane. Within each membrane, pixel intensity was corrected by subtracting background pixel intensity. Positive control spots were used to normalize protein expression across membranes. Within each membrane, pixel intensity was normalized to the average pixel intensity of the six positive control spots on the membrane. Each membrane contained two spots for each protein, so pixel intensities of each protein were averaged within each control and experimental membrane and standard deviations were determined. Pixel intensities were averaged across the two control membranes. Average pixel intensity of experimental treatments was normalized to average pixel intensity of control treatments to determine fold change. Standard deviations were calculated for control and experimental treatments. Unpaired, two-tailed T-tests were performed to identify 52 significant differences between control and experimental treatments. An of 0.05 was used as a cutoff for significance. All calculations were performed in Microsoft® Excel. It should be noted that only one sample of RRD2 and RRD4 treatment was assayed, so these arrays must be repeated in the future for the results to be considered truly significant. III. SDS-PAGE AND WESTERN BLOT Western blots were conducted to examine the expression of PTEN, dephosphorylated AKT, and phosphorylated AKT (phospho-AKT) proteins following RRD2, RRD4, and cisplatin treatment. Protein expression was measured in A2780 cells following three- and six-hour treatments and in A2780/C30 cells following one-, six-, 12-, and 24-hour treatments. Each treatment was conducted in duplicate and one lane was run per sample. Samples of nuclear and cytoplasmic proteins were used for Western blots. Nuclear and cytoplasmic proteins were extracted from pelleted cells using M-PER® Mammalian Protein Extraction Reagent, obtained from Thermo Scientific. Pelleted cells were resuspended in 100-200 μL of M-PER and centrifuged for 15 minutes. The supernatant was decanted and used for all further assays. The total protein concentration of each sample was determined by Micro BCA kit, as described previously. Protein expression was detected and quantified using SDS-PAGE and Western blot. Fifty μg of protein was used for all A2780 samples and 30 μg of protein was used 53 for all A2780/C30 samples. Protein samples were diluted to a volume of 20 μL in loading buffer containing 5% -mercaptoethanol. Samples were denatured by heating to 80°C for three to five minutes. Samples were then loaded into 12-well Novex® Tris-Glycine Gels, purchased from Invitrogen. SeeBlue® Plus Two, also purchased from Invitrogen, was used as a ladder on each gel. Proteins were separated on the gel by electrophoresis, performed for 90 minutes at 200 V, 120 mA, and 15 W. Proteins were then transferred to a 0.45 μm PVDF membrane. Transfer was carried out for two hours at 100 V, 230 mA, and 10 W. Membranes were washed in Tris-HCl buffered saline solution containing 0.1% Tween (TBST), then blocked overnight in 5% milk, 1% BSA. After washing with TBST, membranes were incubated overnight with the primary antibody, diluted to a concentration of 1:12,500 in 5% milk. The primary antibody was washed off with TBST and membranes were incubated for two hours with HRP-linked secondary antibody, diluted to a concentration of 1:50,000 in 5% milk. Finally, the secondary antibody was washed off with TBST followed by TBS. Membranes were incubated for two minutes in ECL Advance luminescent detection reagent, obtained from GE Healthcare. Radiographic films were exposed to each membrane and developed to visualize protein bands. Band analysis was performed using a Kodak In-Vivo FX imager and corresponding software. The imager was used to produce a digital image of each film. Protein band areas were selected from the digital image and pixel intensity was quantified using Kodak software. An area of constant size was used to select all bands within each film. Within each film, pixel intensity was averaged within each control 54 and experimental treatment. Average pixel intensity of experimental treatments was normalized to average pixel intensity of control treatments to determine fold change. Standard deviations were calculated for each control and experimental treatment. Unpaired, two-tailed T-tests were performed to identify significant differences between control and experimental treatments. An of 0.05 was used as a cutoff for significant. All calculations were performed in Microsoft® Excel. IV. ASSAY OF ACID SPHINGOMYELINASE ACTIVITY Acid sphingomyelinase activity was assayed following 15-minute, 30-minute, and one-hour treatments with RRD2, RRD4, and cisplatin. In the A2780/C30 cell line, enzyme activity was also examined following six-hour treatment. Whole cell lysate was used for the assay of acid sphingomyelinase activity. Cell pellets were suspended in 200 μL of acidic reaction buffer (50 mM sodium citrate, pH 5.0) and lysed by centrifugation. Acid sphingomyelinase activity was measured using an Amplex® Red Sphingomyelinase Assay Kit, obtained from Invitrogen. The kit utilizes a multi- enzyme reaction that generates a fluorescent product to quantify acid sphingomyelinase activity. A two-step assay of acid sphingomyelinase activity was performed according to the instructions provided by the manufacturer. First, the acid sphingomyelinase reaction was carried out in acidic buffer. Each well of a 96-well culture plate was inoculated with 89 μL of acidic reaction buffer (50 mM sodium citrate, pH 5.0) and 10 55 μL of 5 mM sphingomyelin, 2% Triton-X solution. Eleven μL of whole cell lysate was added to each well. Each treatment sample was assayed in two wells. Reactions were incubated at 37°C for 60 minutes. Second, the ceramide-detection reaction was carried out at neutral pH. One hundred μL of base-buffered Amplex® Red detection solution (100 μM Amplex® Red, two U/mL horseradish peroxidase, 0.2 U/mL choline oxidase, eight U/mL alkaline phosphatase, 100 mM Tris-HCl, pH 8.0) was added to each well. Detection reactions were incubated at 37°C for 30 minutes. Fluorescence values were measured with a fluorescence microplate scanner, using an excitation filter of 530 nm with a bandwidth of 25 nm and an emission filter of 590 nm with a bandwidth of 35 nm. Wells containing hydrogen peroxide solution were included as positive detection reaction controls. Fluorescence values were corrected by subtracting the background fluorescence obtained from blank wells containing only acidic reaction buffer, sphingomyelin, and base-buffered Amplex® Red detection solution. Fluorescence values were then normalized to total protein concentration, as determined by Micro BCA assay. Fluorescence values were averaged within all control samples. Absolute fluorescence values were converted to fold change by normalizing to the average fluorescence of control samples. The fold change for each experimental sample was averaged within the two wells in which reactions were conducted. Finally, fold changes were averaged within each experimental treatment and standard deviations were calculated. Unpaired, two-tailed T-tests were performed to identify significant 56 differences between control and experimental treatments. An of 0.05 was used as a cutoff for significance. All calculations were performed in Microsoft® Excel. 57 RESULTS I. EFFECTS ON INTRINSIC AND EXTRINSIC APOPTOTIC PATHWAY PROTEINS The effects of RRD2 and RRD4 treatment on apoptotic pathways were examined using an apoptotic antibody array. This array permitted simultaneous examination of 43 different proteins that are involved in apoptosis. Only four antibody-coated membranes were available, so two control treatments and two experimental treatments were arrayed. These arrays were used to identify changes in the expression of apoptotic proteins that occur during treatment of A2780 ovarian cancer cells with RRD2 and RRD4. Samples were arrayed after one-hour RRD2 and RRD4 treatment to examine early changes in protein expression. Apoptosis is induced within one hour, so it was hypothesized that several pro-apoptotic changes would appear during one-hour treatment with RRD2 and RRD4. A. RRD2 and RRD4 Activate the Intrinsic Apoptotic Pathway Results of apoptotic antibody arrays demonstrate that RRD2 and RRD4 alter protein expression to favor induction of the intrinsic apoptotic pathway (Figure 9). RRD2 and RRD4 induce statistically significant increases in the expression of the pro- apoptotic Bcl-2 protein, Bad. RRD2 also significantly enhances expression of the pro- apoptotic protein, Bax. Additionally, RRD2 appears to amplify Bim expression, but this observation may be the result of experimental error. In addition to enhancing expression of pro-apoptotic members of the Bcl-2 family, RRD2 and RRD4 inhibit 58 expression of anti-apoptotic Bcl-2 proteins. Both RRD2 and RRD4 cause dramatic and statistically significant reductions in the expression of Bcl-2. RRD2 also causes an apparent inhibition of Bcl-w, but this effect is not statistically significant. RRD2 and RRD4 do not produce significant changes in the expression of Bid or cytochrome C (cytoC). Figure 9. Effects of RRD2 and RRD4 on expression of intrinsic apoptotic pathway proteins. A2780 ovarian cancer cells were treated with the indicated drug for one hour. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). 59 B. RRD2 and RRD4 Favor Caspase Activation Figure 10. Effects of RRD2 and RRD4 on expression of caspases, IAPs, and IAP antagonists. A2780 ovarian cancer cells were treated with the indicated drug for one hour. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). Activation of the intrinsic apoptotic pathway by RRD2 and RRD4 is augmented by increased expression of caspases and reduced expression of IAPs (Figure 10). Both RRD2 and RRD4 induce an apparent increase in expression of caspase-3, although this increase is statistically significant only for RRD4. RRD4 also appears to cause an increase in caspase-8 expression, but this increase is not statistically significant. It should be noted that this array examines expression of the active forms of caspase-3 and caspase-8, not the inactive zymogens, procaspase-3 and 60 procaspase-8. Coinciding with caspase activation, RRD2 and RRD4 each demonstrate statistically significant inhibition of an IAP. RRD2 causes a marked decrease in the IAP, livin, while RRD4 causes a significant reduction in survivin expression. Additionally, RRD2 and RRD4 cause trends toward the inhibition of cIAP-2 and XIAP, although these changes are within experimental error. RRD2 and RRD4 do not alter the expression of HtrA and SMAC. C. RRD2 and RRD4 Activate Extrinsic Apoptotic Pathways Figure 11. Effects of RRD2 and RRD4 on expression of extrinsic death receptor pathway proteins. A2780 ovarian cancer cells were treated with the indicated drug for one hour. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). 61 RRD2 and RRD4 induce expression of various signaling molecules and death receptors involved in the extrinsic apoptotic pathway. Both RRD2 and RRD4 cause statistically significant increases in expression of the TNF receptors, sTNF-R1 and sTNF-R2. Additionally, RRD4 induces statistically significant increases in expression of the TRAIL receptors, TRAILR-1 and TRAILR-3. RRD2 and RRD4 do not cause significant changes in expression of the CD40L/CD40 or FasL/Fas ligand/receptor pairs, nor do they significantly alter expression of the ligands, TNF- and TNF- . II. EFFECTS ON SIGNALING PROTEINS The effects of RRD2 and RRD4 on signaling proteins was examined using an apoptotic antibody array and Western blots. The apoptotic antibody array probed expression of IGF, HSP, and cell cycle regulatory proteins. Western blots were used to study expression of PI3K signaling proteins. It was hypothesized that RRD2 and RRD4 treatment would induce changes that abrogate growth and survival and promote apoptosis. Such changes might include downregulation of IGF signaling and HSP expression, enhanced expression of p53, and inhibition of PI3K signaling. A. RRD2 and RRD4 Modulate IGF Signaling RRD2 and RRD4 modify expression of several proteins involved in IGF signaling (Figure 12). Both RRD2 and RRD4 produce an apparent increase in IGF-2 expression, although there is large variation in this effect. Interestingly, RRD2 inhibits expression of five IGF binding proteins. Further, this inhibition is statistically significant for all five IGFBPs. The only IGF binding protein that does not show a 62 significant reduction in response to RRD2 treatment is IGFBP-2. In this case, the lack of statistical significance may due to large variation in the expression of IGFBP-2 in control samples. RRD4 also causes a statistically significant decrease in the expression of two IGF binding proteins, IGFBP-1 and IGFBP-3. Further, RRD2 causes a statistically significant reduction in the expression of the IGF receptor, IGFR-1. Neither RRD2 nor RRD4 produce any change in the expression of IGF-1. Figure 12. Effects of RRD2 and RRD4 on expression of IGF signaling pathway proteins. A2780 ovarian cancer cells were treated with the indicated drug for one hour. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). 63 B. RRD2 and RRD4 do not Alter Expression of HSPs RRD2 and RRD4 do not alter the expression of any of the HSPs arrayed (Figure 13). Figure 13. Effects of RRD2 and RRD4 on expression of HSPs. A2780 ovarian cancer cells were treated with the indicated drug for one hour. Protein expression is expressed as fold change from control treatment. C. RRD2 Inhibits p21 and p27 RRD2, but not RRD4, causes a reduction in the expression of p21 and p27 proteins (Figure 14). RRD2 treatment causes statistically significant downregulation of p21 and p27 expression. RRD4, however, does not affect the expression of p21 or p27. Neither RRD2 nor RRD4 significantly alter p53 expression. 64 Figure 14. Effects of RRD2 and RRD4 on expression of proteins that regulate cell cycle progression. A2780 ovarian cancer cells were treated with the indicated drug for one hour. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). D. RRD2 and RRD4 Modulate PI3K Signaling Results of Western blot experiments demonstrate that RRD2 and RRD4 regulate signal transduction through the PI3K pathway. Both RRD2 and RRD4 induce statistically significant increases in PTEN expression after three- and six-hour treatment of A2780 cells (Figure 15). This increase in PTEN expression coincides with statistically significant increases in levels of inactive AKT following three- and six-hour treatment with RRD2 and RRD4 (Figure 16). However, RRD2 and RRD4 do not alter absolute levels of phospho-AKT, the active form of AKT that participates in PI3K signaling (Figure 17). 65 Figure 15. Effects of cisplatin, RRD2, and RRD4 on expression of PTEN in A2780 cells. A2780 ovarian cancer cells were treated with the indicated drug for the indicated time. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p- value 0.05 as calculated by unpaired, two-tailed T-test). 66 Figure 16. Effects of cisplatin, RRD2, and RRD4 on expression of AKT in A2780 cells. A2780 ovarian cancer cells were treated with the indicated drug for the indicated time. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p- value 0.05 as calculated by unpaired, two-tailed T-test). Figure 17. Effects of cisplatin, RRD2, and RRD4 on expression of phospho-AKT in A2780 cells. A2780 ovarian cancer cells were treated with the indicated drug for the indicated time. Protein expression is expressed as fold change from control treatment. 67 RRD2 and RRD4 also modulate the PI3K pathway in the A2780/C30 cell line. However, enhancement of PTEN expression is only observed following six-hour RRD2 treatment. This may have been due experimental error, as there is large variation in PTEN expression for one-hour RRD2 and RRD4 treatments, as well as for six-hour RRD4 treatment. To investigate whether or not PTEN expression is induced at a later time point, protein expression was also examined after 12- and 24-hour treatments. Results of these experiments demonstrate the PTEN expression is not induced by RRD2 or RRD4 after 12- and 24-hour treatment. Expression of AKT does not correlate with PTEN expression during drug treatment. Although RRD4 does not induce an increase in the expression of PTEN, an increase in expression of inactive AKT is observed. However, elevation of inactive AKT levels is lost after 12 and 24 hours of treatment. As in A2780 cells, neither RRD2 nor RRD4 produce any change in the expression of active phospho-AKT. 68 Figure 18. Effects of cisplatin, RRD2, and RRD4 on expression of PTEN in A2780/C30 cells. A2780/C30 ovarian cancer cells were treated with the indicated drug for the indicated time. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). 69 Figure 19. Effects of cisplatin, RRD2, and RRD4 on expression of AKT in A2780/C30 cells. A2780/C30 ovarian cancer cells were treated with the indicated drug for the indicated time. Protein expression is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). Figure 20. Effects of cisplatin, RRD2, and RRD4 on expression of phospho-AKT in A2780/C30 cells. A2780/C30 ovarian cancer cells were treated with the indicated drug for the indicated time. Protein expression is expressed as fold change from control treatment. 70 III. RRD2 AND RRD4 ACTIVATE ACID SPHINGOMYELINASE Acid sphingomyelinase activity was assayed following treatment with RRD2 and RRD4. Results in the literature show that induction of acid sphingomyelinase activity can occur rapidly following drug treatment, so treatment times of 15 minutes, 30 minutes, and one hour were chosen . Acid sphingomyelinase activity was also assayed following six-hour treatment of A2780/C30 cells. It was hypothesized that RRD2 and RRD4 would rapidly activate the acid sphingomyelinase enzyme. A. RRD2 and RRD4 Activate Acid Sphingomyelinase in A2780 Cells Figure 21. Effects of RRD2 and RRD4 on the activity of acid sphingomyelinase. A2780 ovarian cancer cells were treated with the indicated drug. Acid sphingomyelinase activity was measured after 15 minutes, 30 minutes, and one hour of treatment. Enzyme activity is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). 71 Results of acid sphingomyelinase assays show that RRD2 and RRD4 cause robust activation of the acid sphingomyelinase enzyme in A2780 cells (Figure 21). Both RRD2 and RRD4 induce statistically significant increases in acid sphingomyelinase activity. The effects of cisplatin treatment correspond with those reported in the literature, validating the results of this assay . Like cisplatin, RRD2 and RRD4 cause rapid activation of acid sphingomyelinase. Both RRD2 and RRD4 appear to activate acid sphingomyelinase after only 15 minutes. Remarkably, this increase in activity is statistically significant following 15-minute RRD4 treatment. Activation of the acid sphingomyelinase enzyme is time dependent, increasing with treatment duration. Both RRD2 and RRD4 produce a statistically significant increase in acid sphingomyelinase activity after 30 minutes. Acid sphingomyelinase activity continues to increase after one hour of treatment, although the effects of RRD4 treatment lose significance due to a large variation in enzyme activity. B. Activation of Acid Sphingomyelinase is Diminished in A2780/C30 Cells The results of acid sphingomyelinase assays indicate that sustained activation of acid sphingomyelinase does not occur in A2780/C30 cells (Figure 22). These results are markedly different from the results obtained in A2780 cells. In A2780/C30 cells, RRD2 and RRD4 induce a statistically significant increase in acid sphingomyelinase activity after 15-minute treatment. However, these increases are greatly reduced from the 16- and 43-fold increases in enzyme activity that are observed in A2780 cells after 15-minute treatment. Cisplatin treatment also produces a trend toward activation of acid sphingomyelinase after 15 minutes, but it is not 72 statistically significant. Interestingly, this slight activation of acid sphingomyelinase activity is lost after 15 minutes. Results of 30-minute and one-hour treatments with RRD2, RRD4, and cisplatin do not produce significant alteration of acid sphingomyelinase activity. To exclude the possibility of a slow activation of acid sphingomyelinase, enzyme activity was also assayed after six-hour treatment. Acid sphingomyelinase activity remained unchanged after six-hour treatment with RRD2, RRD4, and cisplatin, demonstrating that acid sphingomyelinase activation is not simply delayed in the A2780/C30 cell line Figure 22. Effects of RRD2 and RRD4 on the activity of acid sphingomyelinase. A2780/C30 ovarian cancer cells were treated with the indicated drug. Acid sphingomyelinase activity was measured after 15 minutes, 30 minutes, one hour, and six hours of treatment. Enzyme activity is expressed as fold change from control treatment. * indicates a statistically significant difference from control treatment (p-value 0.05 as calculated by unpaired, two-tailed T-test). 73 DISCUSSION AND FUTURE DIRECTIONS I. RRD2 AND RRD4 INDUCE APOPTOSIS THROUGH THE INTRINSIC AND EXTRINSIC APOPTOTIC PATHWAYS Results of experiments examining the expression of intrinsic and extrinsic apoptotic pathway proteins indicate that RRD2 and RRD4 can induce apoptosis through activation of both pathways. A. Activation of the Intrinsic Apoptotic Pathway RRD2 and RRD4 activate the intrinsic apoptotic pathway by modifying expression of Bcl-2 proteins, caspases, and IAPs. Both RRD2 and RRD4 strongly inhibit expression of the anti-apoptotic Bcl-2 protein, Bcl-2. Inhibition of Bcl-2 may contribute to the efficacy of RRD2 and RRD4 in platinum-resistant cells. Cisplatin resistance can occur when the anti-apoptotic Bcl-2 proteins, Bcl-2 and Bcl-XL, are overexpressed . Bcl-2-dependent platinum resistance occurs in several cancer cell lines and can be observed in vivo . Analysis of ovarian cancer tumors from human patients shows that tumors expressing Bcl-XL are less likely to respond to platinum chemotherapy than tumors that do not express Bcl-XL . Through inhibition of anti- apoptotic Bcl-2 proteins, RRD2 and RRD4 can resensitize cells to the intrinsic apoptotic pathway. This mechanism should be investigated by examining expression of anti-apoptotic proteins in the platinum-resistant A2780/C30 cell line. The effects of RRD2 and RRD4 on Bcl-XL expression should also be tested. 74 Concurrently, RRD2 and RRD4 enhance expression of the pro-apoptotic Bcl-2 protein, Bad. Additionally, RRD2 amplifies expression of the pro-apoptotic protein, Bax. Together, the downregulation of anti-apoptotic Bcl-2 proteins and stimulation of pro-apoptotic Bcl-2 proteins should result in dramatic activation of the intrinsic apoptotic pathway following RRD2 and RRD4 treatment. The pro-apoptotic effects of RRD2 and RRD4 are augmented by modulation of caspase and IAP expression. RRD4 enhances expression of the executioner caspase, caspase-3. This effect should strengthen activation of the intrinsic apoptotic pathway. Additionally, RRD2 and RRD4 reduce expression of the IAPs, livin and survivin, respectively. IAP inhibition may also contribute to the efficacies of RRD2 and RRD4 in platinum-resistant cancer cells. IAP overexpression contributes to platinum resistance in several cell lines . Overexpression of XIAP, for instance, generates cisplatin resistance in ovarian cancer cells . Cisplatin sensitivity is restored in these cells when XIAP is inhibited via RNA interference . Similarly, overexpression of survivin causes cisplatin resistance that can be reversed by RNA interference . Survivin-dependent resistance also occurs in vivo . Elevated expression of survivin in tumor cells isolated from human patients correlates with resistance to platinum chemotherapy . Future study should examine IAP expression in the A2780/C30 cell line to determine if they contribute to the platinum resistance exhibited by these cells. B. Activation of Extrinsic Apoptotic Pathways In addition to activating the intrinsic apoptotic pathway, RRD2 and RRD4 produce changes in protein expression that should induce extrinsic apoptotic pathways. 75 Both RRD2 and RRD4 enhance expression of the TNF receptors, sTNF-R1 and sTNF- R2. Elevated expression of these receptors should sensitize cells to apoptotic signaling by their ligand, TNF. Similarly, RRD4 causes increased expression of the TRAIL receptors, TRAILR-1 and TRAILR-3. Increased expression of TRAILR-1 should also enhance sensitization to apoptotic signals. This effect is particularly interesting because several TRAILR-1 agonists are currently undergoing clinical trials in the treatment of ovarian cancer . The effect of enhanced TRAILR-3 expression by RRD4, however, is questionable. TRAILR-3 cannot induce an apoptotic signaling cascade . This protein may act as a decoy TRAIL receptor, but its true function is currently unknown . Enhanced expression of the TNF and TRAIL-1 death receptors should allow RRD2 and RRD4 to induce apoptosis by sensitizing cancer cells to extracellular apoptotic signals. Interestingly, RRD2 and RRD4 do not produce changes in the expression of Fas and FasL proteins. This seemingly contradicts immunofluorescence experiments that show a robust increase in Fas expression and coclustering of Fas receptors at the plasma membrane of A2780 cells following one-hour pyrodach-4 treatment . However, activation of the acid sphingomyelinase enzyme suggests that RRD2 and RRD4 could activate Fas-mediated apoptosis without affecting expression levels of the Fas and FasL proteins. C. Involvement of Acid Sphingomyelinase The robust activation of acid sphingomyelinase by RRD2 and RRD4 should result in marked changes in the composition of the plasma membrane. These changes 76 should encourage formation of ceramide-enriched macrodomains, creating an environment that favors coclustering of Fas receptors. This could result in ligand- independent induction of the Fas death receptor pathway without altering expression levels of the Fas receptor. This mechanism would be similar to the ligand-independent activation of Fas signaling that mediates cisplatin-induced apoptosis in some cell lines. Cisplatin stimulates the Fas death receptor pathway via rapid activation of the acid sphingomyelinase enzyme . Acid sphingomyelinase function is necessary for the induction of apoptosis by cisplatin in certain cell lines . Knockout of the gene expressing acid sphingomyelinase protects the gastrointestinal cells of mice from cisplatin-induced apoptosis . FasL expression, however, is not necessary for the activation of the Fas death receptor pathway by cisplatin . The acid sphingomyelinase-dependent, FasL-independent activation of Fas death receptors may contribute to the cytotoxicity of RRD2 and RRD4. Coclustering of Fas receptors in ceramide-enriched macrodomains could enhance Fas signaling without affecting protein expression levels. Immunofluorescence experiments should be conducted to determine if RRD2 and RRD4 induce colocalization of Fas receptors at the plasma membrane. While acid sphingomyelinase activation probably plays an important role in RRD2- and RRD4-induced apoptosis, the results obtained in A2780/C30 cells demonstrate that acid sphingomyelinase activation is not required for the function of RRD2 and RRD4. RRD2 and RRD4 are effective against A2780/C30 cells despite the 77 absence of sustained activation of the acid sphingomyelinase enzyme in these cells. Therefore, treatment with RRD2 and RRD4 must involve molecular mechanisms that are distinct from acid sphingomyelinase pathways. RRD2 and RRD4 must cause apoptosis through the activation of other pathways, possibly including the TNF, TRAIL, and intrinsic apoptotic pathways. Alternatively, RRD2 and RRD4 may continue to activate Fas signaling in the A2780/C30 cell line in a FasL-dependent manner. The requirement for FasL in RRD2- and RRD4-induced apoptosis should be investigated. Gene expression studies show that 24-hour RRD4 treatment causes increased expression of the FasL transcript . This suggests that activation of Fas signaling by acid sphingomyelinase may augment the conventional activation of Fas receptors by their ligands. D. Other Pro-Apoptotic Effects RRD2 and RRD4 cause other pro-apoptotic changes in A2780 cells. In particular, inhibition of the p21 and p27 proteins may enhance RRD2-induced apoptosis. Both p21 and p27 exert anti-apoptotic effects and can be oncogenic if overexpressed . p21 is overexpressed in many human cancers, including breast, cervical, and prostate cancers . The inhibition of both p21 and p27 by RRD2 would alleviate this protection against apoptosis, enhancing the efficacy of RRD2. 78 II. RRD2 AND RRD4 MODIFY GROWTH AND SURVIVAL SIGNALS A. RRD2 and RRD4 Modulate IGF Signaling RRD2 and RRD4 alter IGF signaling by modifying the expression of several proteins. Both RRD2 and RRD4 influence expression of IGF binding proteins, which could alter growth and survival signaling. RRD4 downregulates expression of IGFBP- 1 and -3, a curious effect considering that these IGF binding proteins antagonize IGF signaling. This suggests that RRD4 treatment enhances IGF signaling, which would counter apoptosis. Interestingly, RRD2 alters the expression of five different IGF binding proteins. This could result in contradictory effects, as RRD2 inhibits expression of some IGFBPs that reduce IGF signaling and also inhibits expression of other IGFBPs that enhance IGF signaling. Therefore, it is important to determine the overall effect of RRD2 on IGF signaling. Enhanced IGF signaling is oncogenic, so it is likely that RRD2 causes an overall reduction in IGF signals. The IGF signaling pathway causes oncogenic transformations by encouraging growth and protecting cells from apoptosis . Cancerous cells frequently overexpress the gene encoding IGF-2, leading to increased IGF signaling . Overexpression of IGF binding proteins that enhance IGF signaling is also oncogenic in some cancer cell lines . Additionally, overexpression of IGFR-1 is oncogenic . Thus, inhibition of the IGF signaling pathway by RRD2 could reverse oncogenic transformations. The most important change induced by RRD2 may be inhibition of the IGF receptor, IGFR-1. IGFR-1 plays an important role in oncogenic IGF signaling . The inhibitory effect of RRD2 on IGFR-1 could be relevant clinically. In fact, an IGFR-1 79 antibody that antagonizes receptor function is currently undergoing phase II clinical trials in ovarian cancer patients . The efficacy of this antibody as a chemotherapeutic agent demonstrates the significance of IGFR-1 inhibition in cancer therapy. Considering this information, the inhibition of IGFR-1 by RRD2 probably plays an important role in the anticancer effects of this drug. B. RRD2 and RRD4 Alter PI3K Signaling Characterization of protein expression demonstrates that both RRD2 and RRD4 inhibit growth and survival signaling through the PI3K pathway. In the A2780 cell line, both RRD2 and RRD4 cause increased expression of PTEN. This should negatively regulate growth and survival signaling through the PI3K pathway. This result coincides with gene expression data showing that RRD4 causes increased expression of the PTEN gene . Enhanced activation of this tumor suppressor protein is probably important in the induction of cytotoxic effects by RRD2 and RRD4. The importance of amplified PTEN expression on downstream targets is evidenced by altered expression of AKT and phospho-AKT. Overexpression of PTEN inhibits growth and survival signaling, resulting in diminished activation of the AKT protein via phosphorylation . Therefore, an increase in levels of dephosphorylated AKT and a decrease in levels of phospho-AKT are expected. Western blots show that both RRD2 and RRD4 cause increased expression of the dephosphorylated form of AKT, as expected, in the A2780 cell line. RRD2 and RRD4 do not, however, cause a decrease in expression of phospho-AKT in A2780 cells. This result appears contradictory. 80 However, it is possible that the ratio of phosphorylated to dephosphorylated AKT impacts growth and survival signaling through the PI3K pathway. Identical results are not observed in the A2780/C30 cell line. RRD4 does not cause any increase in the expression of PTEN and RRD2 enhances PTEN expression during only one of four treatment times. Nonetheless, both RRD2 and RRD4 produce similar effects on AKT and phospho-AKT expression in the A2780/C30 cell line. RRD2 and RRD4 cause increased expression of the AKT protein but do not alter levels of phospho-AKT. These results conflict with PTEN expression, but may be explained by the activity of PI3K. The effects of RRD2 and RRD4 on PI3K expression and activity have not been examined. It is possible that RRD2 and RRD4 cause an increase in dephosphorylated AKT by inhibiting PI3K expression or activity. Therefore, examining the effects of RRD2 and RRD4 on the PI3K enzyme is an important aim for future study. Whatever the mechanism, RRD2 and RRD4 cause increased dephosphorylation of AKT in both the A2780 and A2780/C30 cell lines. This suggests that inactivation of AKT may be an essential mediator of the cytotoxicities of RRD2 and RRD4. Interestingly, inactivation of AKT could account for many of the changes in protein expression observed during RRD2 and RRD4 treatment. AKT inactivation may explain the reduction in p21 and p27 expression that occurs during RRD2 treatment. Phosphorylation of p21 and p27 by AKT results in the cytosolic accumulation of these proteins . A reduction in the activity of AKT could thus account for the reduced accumulation of p21 and p27 observed following RRD2 81 treatment. Inactivation of AKT could also explain diminished expression of the IAPs, livin and survivin. AKT stabilizes IAPs through phosphorylation . Therefore, a reduction in AKT activity could account for the reduced expression of various IAPs. The far-reaching effects of AKT activity suggest a central role for this protein in mediating the effects of RRD2 and RRD4. Interestingly, protein expression in the A2780/C30 cell line demonstrates that RRD2- and RRD4-induced alteration of PI3K signaling is transient. During 12- and 24-hour treatment, RRD2 and RRD4 do not cause any alteration of PTEN or dephosphorylated AKT expression. This result is curious and warrants future study to determine if similar transiency occurs in the A2780 cell line. III. RRD2 OR RRD4? A POSSIBLE PREFERENCE BASED ON MECHANISTIC DIFFERENCES Apparent differences in the mechanisms of RRD2 and RRD4 suggest that RRD2 may be the more rational choice for continued preclinical study. Previous studies suggest that RRD2 is more potent than RRD4, both in vitro and in vivo (S. Moghaddas, pers. comm.). Variations in the cellular effects of RRD2 and RRD4 may account for this difference in potency. RRD2 exhibits several effects on protein expression that should confer an advantage over RRD4. RRD2 causes more pro-apoptotic changes in the expression of intrinsic apoptotic pathway proteins. While both RRD2 and RRD4 induce expression of Bad and inhibit expression of Bcl-2 and an IAP, RRD2 also enhances expression of Bax 82 and may inhibit expression of Bcl-w. This indicates that RRD2 is a more potent activator of the intrinsic apoptotic pathway. Additionally, RRD2 is more likely than RRD4 to exert an inhibitory effect on IGF signaling. Changes in the expression of IGF binding proteins suggest that RRD4 may cause a counteractive enhancement of IGF signaling. RRD2, however, downregulates expression of the IGF receptor, IGFR-1, in addition to altering IGF binding protein expression. This probably results in an overall inhibition of IGF signaling by RRD2. The importance of IGF signaling in oncogenesis suggests that this effect could greatly augment the anticancer activity of RRD2. Finally, RRD2 inhibits expression of p21 and p27 while RRD4 has no effect on the expression of these proteins. This represents another effect that may confer a pro- apoptotic advantage to RRD2. Further study should be conducted before a final decision is made between these two compounds. Nonetheless, current data suggest that RRD2 is a better choice for the treatment of ovarian cancer. IV. CLOSING COMMENTS In conclusion, it is evident that RRD2 and RRD4 effect a variety of changes in protein expression and activity. 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