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Characterization of novel nitroplatinum(iv) complexes for the treatment of cancer
h [electronic resource] /
by Jeannette Lo.
[Tampa, Fla.] :
b University of South Florida,
Thesis (MSPH)--University of South Florida, 2004.
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
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ABSTRACT: Many types of chemotherapeutic agents have been developed to target specific mechanisms within the body that control the progression of cancer, though few have been able to circumvent the existing problems associated with the treatments. The current remedies entail grueling drug regimens and toxic side effects that may undermine the effectiveness of the drugs. Cisplatin, a common nitroplatinum(II) drug widely used to treat a variety of cancers, is administered intravenously and circulates systemically, affecting healthy regions of the body as well. Resistance to cisplatin is increasing and the need for new, less toxic medication must be met for future success in cancer therapy. Our lab has synthesized novel nitroplatinum(IV) cisplatin complexes (PH1-14) that may evade these problems.We examined the effects of these compounds on cell viability, as well as effects on cancer-specific mechanisms such as nitric oxide (NO) production, angiogenesis, and the STAT signaling pathways. In vitro studies demonstrated that PH1-11 and PH14 demonstrated greater efficacy at inhibiting cell proliferation with lower IC50 values that ranged from 41-58 uM (as compared with cisplatin IC50 = 66 uM). Data from NO assays were inconclusive, though there was elevated expression of inducible nitric oxide synthase in cells treated with PH3 and PH11. We also found that PH9 was able to inhibit STAT dimerization at concentrations as low as 0.3 uM. PH9 also decreased VEGF and HIF-1α expression, thereby inhibiting angiogenesis. The activity of the PH complexes was also studied in C57BL/6 mice inoculated with murine bladder MB49 tumors. The experimental group showed significantly slower tumorigenesis and smaller tumors as compared with the control group.
Adviser: Kay, Heidi.
x Public Health
t USF Electronic Theses and Dissertations.
Characterization of Novel Nitroplatinum(IV) Complexes for the Treatment of Cancer by Jeannette Lo A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health Department of Global Health College of Public Health University of South Florida Major Professor: Heidi Kay, Ph.D. Boo Kwa, Ph.D. Ann DeBaldo, Ph.D. Date of Approval: July 15, 2004 Keywords: Cisplatin, resistance, STAT, nitric oxide, angiogenesis Copyright 2004, Jeannette Lo
DEDICATION I dedicate this to Dr. Neil Rowland, the professor that gave me a chance and inspired my initial interest in research; to my parents, whom have instilled the values of education and diligence into me; and to Dan Dauer, who gives me purpose for everything I do.
ACKNOWLEDGEMENTS The author would like to thank her major professor Dr. Heidi Kay for her guidance during the research and writing process, and the members of the Kay lab for their contribution in the data collection. The author would also like to recognize the Jove and Yu labs for their assistance with the EMSA assay and animal toxicity studies, Ed Haller for his efforts on x-ray analysis, Laura Pendleton for her work in the Western Blot, the Moffitt clinical labs for running toxicity profiles, and Dr. George Blanck for providing cells and advice. The author also wishes to express gratitude to Dr. Boo Kwa and Dr. Ann DeBaldo for their advice as comm ittee members. Finally, the author would like to express her gratitude to Justin Kerr, her friend and fellow collaborator on this project, who kept her both entertained and grounded, and for his hard work during the most difficult periods of the experimentation.
i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT v LIST OF ABBREVIATIONS vi CHAPTER 1: BACKGROUND AND SIGNIFICANCE 1 1.1 Chemotherapy 1 1.2 Platinum Complexes 2 1.3 Targets and Mechanisms of Cancer Management 5 1.3.1 Angiogenesis 5 1.3.2 Si gnal Transducers and Activators of Transcription (STAT) 6 1.3.3 Biological Effects of Nitric Oxide 7 1.3.4 Novel Nitroplatinum(IV) Complexes 9 CHAPTER 2: EXPERIMENTAL DESIGN 10 2.1 In vitro studies 10 2.1.1 Drug Synthesis 10 2.1.2 Cell Culture 10 2.1.3 Drug Treatment 10 2.1.4 XTT Cell ViabilityAssay 11 2.1.5 MTT Cell Viability Assay 13 2.1.6 Nitric Oxide Production 13 2.1.7 Western Blotting 15 2.1.8 Energy-dispersive X-Ray Analys is of Platinum-Treated Cells 16 2.1.9 Electrophoretic Mobility Shift Assay (EMSA) 18 2.2 In vivo studies 18 2.2.1 Animal Housing and Treatment 18 2.2.2 Toxicology 20 2.2.3 Angiogenesis 21 CHAPTER 3: RESULTS 23 3.1 Cell Viability 23 3.2 Nitric Oxide Production 30 3.3 Nitric Oxide Synthase Expression 34 3.4 Toxicology 35 3.5 X-Ray Diffraction 41 3.6 Inhibition of STAT Dimerization 42
ii 3.7 Angiogenesis 44 3.8 Tumor Growth Inhibition 45 CHAPTER 4: CONCLUSION 47 REFERENCES 50
iii LIST OF TABLES Table 2.1 Schema of drug treatment at various dilutions. 11 Table 3.1 Relative survival for A549 cells treated with 50 uM PH compounds as a function of XTT incubation time. 24 Table 3.2 Calculated IC50 values for PH1-4 and cisplatin from XTT Assay. 25 Table 3.3 Calculated IC50 values for PH1-11 and cisplatin from XTT Assay. 27 Table 3.4 Calculated IC50 values for PH3,4,9-14 and cisplatin from XTT Assay. 28 Table 3.5 Calculated IC50 values for PH1,3,4,7-11 and cisplatin from MTT Assay. 29 Table 3.6 Summary of calculated IC50 values from XTT and MTT Assays. 29 Table 3.7 Biochemical blood serum profiles for the assessment of toxicity. 36 Table 3.8 Creatinine and BUN values in m ouse serum cisplatin or PH9 treatment. 41 Table 3.9 Calculated IC50 values of PH compounds for STAT dimerization. 42 Table 4.1 Comparison of the results of various assays on PH compounds. 48
iv LIST OF FIGURES Figure 1.1 Example structures of platinum(II) and platinum(IV) complexes. 3 Figure 1.2 Platinum complexes designed for the treatment of cancer. 4 Figure 1.3 Schematic of STAT signaling. 7 Figure 2.1 Mechanism of DAF-FM assay. 14 Figure 3.1 Relative survival for cells treated with PH1-4 and cisplatin. 24 Figure 3.2 Relative survival for cells treated with PH4-8 and cisplatin. 26 Figure 3.3 Relative survival for cells treated with PH-3,9-11 and cisplatin. 26 Figure 3.4 Relative survival for cells treated with PH3,4,9-14 and cisplatin. 28 Figure 3.5A Trial 1 results of NO production. 31 Figure 3.5B Trial 2 results of NO production. 33 Figure 3.5C Trial 3 results of NO production. 33 Figure 3.5D Trial 4 results of NO production. 34 Figure 3.6 iNOS expression of PH treated cells. 35 Figure 3.7 Biochemical profiles of mice one day after 5 mg/kg drug injection. 38 Figure 3.8 Biochemical profiles of mice tw o days after 5 mg/kg drug injection. 39 Figure 3.9 Biochemical profiles of mice thr ee days after 5 mg/kg drug injection. 40 Figure 3.10 Inhibition of STAT dimerization by PH compounds. 43 Figure 3.11 Determination of HIF-1 VEGF, and STAT3 ex pression by Western blot. 44 Figure 3.12 Matrigel plugs of MCF-7 tumors. 45 Figure 3.13 Tumor size (mm3) of control and PH treated mice. 46
v LIST OF ABBREVIATIONS A adenine ALT alanine aminotransferase AST aspartate aminotransferase BUN blood urea nitrogen C Celsius CA California cm centimeter CO2 carbon dioxide (gas) cps counts per second DAF-FM 4-amino-5-methylamino2',7'difluorofluorescein dept. department DMEM DulbeccoÂ’s Modified Eagle Medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid EDAX energy dispersive x-ray analysis EDS energy disper sive spectroscopy EMSA electrophoretic mobility shift assay etOH ethanol G guanine h hour HCl hydrochloric acid HIF-1 hypoxic inducible factor-1 hSIE high affinity sis-inducible element IACUC Institutional Animal Care and Use Committee IC50 50% inhibitory concentration iNOS inducuble nitr ic oxide synthesis kg kilogram kV kilovolts M molar mg milligram mL milliliter mM millimolar MMR mismatch repair MTT 3-(4,5-dimethylthaizol-2-yl)2,5-diphenyltetrazolium bromide NCI National Cancer Institute nm nanometer NO nitric oxide (gas) NOS nitric oxide synthesis O2 oxygen (gas)
vi OR Oregon P phosphate PBS phosphate buffered saline PMS N-methyl dibenzopyrazine methyl sulfate Pt platinum RNA ribonucleic acid S phase synthesis phase SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM scanning electron microscope siRNA small interfering RNA STAT signal transducers and activators of signaling TEM transmission electron microscope U unit ug microgram uL microliter uM micromolar USF University of South Florida UV ultraviolet VEGF vascular endothelial growth factor XTT 2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5carboxanilide, disodium salt
vii Characterization of Novel Nitroplatinum (IV ) Complexes for the Treatment of Cancer Jeannette Lo ABSTRACT Many types of chemotherapeutic agents ha ve been developed to target specific mechanisms within the body that control the progression of ca ncer, though few have been able to circumvent the existing problems asso ciated with the treatments. The current remedies entail grueling drug regimens and t oxic side effects that may undermine the effectiveness of the drugs. Cisplatin, a common nitroplatinum(II) drug widely used to treat a variety of cancers, is administered intravenously and circulates systemically, affecting healthy regions of th e body as well. Resistance to ci splatin is increasing and the need for new, less toxic medication must be met for future success in cancer therapy. Our lab has synthesized novel nitroplatinum(IV ) cisplatin complexes (PH1-14) that may evade these problems. We examined the e ffects of these compounds on cell viability, as well as effects on cancer-specific mechanisms such as nitric oxide (NO) production, angiogenesis, and the STAT signaling pathways. In vitro studies demonstrated that PH111 and PH14 demonstrated greater efficacy at inhibiting cell prolif eration with lower IC50 values that ranged from 41-58 uM (as compared with cisplatin IC50 = 66 uM). Data from NO assays were inconclusive, though th ere was elevated expr ession of inducible nitric oxide synthase in cel ls treated with PH3 and PH11. We also found that PH9 was able to inhibit STAT dimeri zation at concentrations as low as 0.3 uM. PH9 also decreased VEGF and HIF-1 expression, thereby inhibiting a ngiogenesis. The activity of
viii the PH complexes was also studied in C57B L/6 mice inoculated with murine bladder MB49 tumors. The experimental group showed significantly slower tumorigenesis and smaller tumors as compared with the contro l group. Toxicological analyses of the blood via metabolic assays showed that no nephrotox icity was observed in dosages of less than 7 mg drug/kg. We conclude from these re sults the potential for the use of novel mechanisms in the treatment of cancers. This work will guide future investigations of these drugs in further preclinical trials and also introduce an alternative to the traditional chemotherapeutic agents.
1 CHAPTER 1: BACKGRO UND AND SIGNIFICANCE 1.1 Chemotherapy Chemotherapy is the use of chemothera peutic agents for the cure, control, and palliation of cancer. Its us e has been documented since the 16th century, when heavy metals were used systemically to treat cance rs, often causing severe toxicity with limited clinical improvement. Today, chemothera py is one of the most commonly used treatments for cancer. A wide range of ch emotherapeutic drugs have been discovered and are available as treatment, including plan t alkaloids, alkylating agents, hormones, and antibiotics. . Continuous research has allowed new drugs to emerge frequently, undergoing clinical trials before being accessible to the public. Many types of chemotherapeutic agen ts have been developed to target specific mechanisms within the body that cont rol the progression of cancer. Cell-cycle specific agents, such as Paclitaxel, can arrest metaphase by interferi ng with the formation of the mitotic spindle. Antimetabolites, such as Floxuidane, are another group of cellcycle specific agents that act by replaci ng components essential to the metabolic synthesis of DNA during the S phase. Alkylati ng agents, such as ci splatin, interrupt replication of genetic material by cross-li nking and strand-breaki ng DNA, leading to cell lysis. Antitumor antibiotics, such as Doxi rubicin, prevent cell division by damaging the cell and interfering with DNA and RNA synthese s. Hormones or hormone-like agents,
2 such as estradiol, inhibit tumor growth by antagonizing the otherwis e naturally occurring ligands from their receptors and in itiating tumor proliferation . 1.2 Platinum Complexes Cisplatin, a widely used chemothe rapeutic agent for the treatment of testicular, ovarian, head and neck, stomac h, and bladder carcinomas, is a platinum(II) complex that was discovered in 1972 . Ci splatin can bind to RNA, proteins, and other sulfur-containing biomolecules, t hough its main biological target is DNA [3-5]. It forms 1,2-intrastrand guanine-guanine, and guani ne-adenine crosslinks, accounting for approximately 90% of the re sulting adduct. The remain ing interactions involve 1,3intrastrand and interstrand adducts. Its primar y crosslink structures serve as a recognition motif for an array of biological molecules, including DNA repair elements, histones, and serum proteins [4, 6]. The formation of th ese adducts result in blocked transcription, replication inhibition, and apoptosis . Howe ver, there are major lim itations to cisplatin in anticancer therapy . As a platinum(II) complex, cisplatin circulates systemically in a chemically active state, causing significant side effects including nausea and vomiting, renal toxicity, and bone marrow damage [2, 4, 79]. Another limitation to cisplatin is the emergence of resistance to cisplatin during tr eatment. Studies ha ve reported several modes of resistance such as reduced drug upt ake, increased drug inactivation [10, 11], altered drug targets, altered gene expre ssion [12-14], and loss of the DNA mismatch repair (MMR) mechanism . The MMR system plays a cr itical role in ensuring genomic stability by correcting damaged DNA. MMR is mediated by directing a nick
3 Figure 1.1 Example structures of pl atinum(II) and platinum(IV) complexes. located 5Â’ or 3Â’ to the mismatch. Exonucleat ed degradation is initiated from the nick to the mismatched base. The DNA that cont ains the mismatch is then removed, resynthesized, and sealed. In normal cells MMR recognizes DNA damage and initiates DNA repair or apoptosis. In resistance cells, the MMR system is lost. This decrease in signaling for repair or apoptosis results in increased survival for drug-treated cells . The loss of DNA MMR resulting in cisplatin -resistant cell lines has been documented [16-18]. Improved, platinum-based treatments have emerged. Carboplatin, another platinum(II) drug, was developed to decrease systemic toxicity and resistance observed with cisplatin. However, as compared to cisplatin, carboplatin has inferior efficacy in treating head and neck, bladde r, and oesophageal cancers [ 19]. An alternative to the existing toxic platinum(II) complexes was needed, leading to the development of platinum(IV) complexes. The octahedral pl atinum(IV) complex are more inert to serum blood proteins as they travels through the body. Then, upon reaching the tumor site,
4 reduction to a square-planar platinum(II) complex allows the complex to bind to DNA . Ideally, a drug maintaining these propert ies can be a potent anti-tumor agent with reduced toxic side effects. Additionally, some studies have shown that platinum(IV) complexes can bind to DNA wit hout prior reduction [20, 21]. JM216 (Satraplatin), a platinum(IV) co mplex, entered clinical trials in 1992 on the basis of possessing several promising preclinical features. It demonstrated potent in vitro and in vivo growth inhibitory properties against se veral tumor varieties, and has relatively mild toxicity profile w ith myelosuppression being dose-limiting . Satraplatin is now undergoing investigation in Phas e III clinical trails. Zak et al  tested another platinum(IV) drug, LA-12, against cisp latin resistant tumor lines and found that LA-12 demonstrated lower IC50Â’s as compared with cisplatin, but had not yet established Figure 1.2 Platinum complexes designed fo r the treatment of cancer [2, 7, 9]. studies for toxicity yet. Tetraplatin, a nother platinum(IV) analogue, entered Phase I clinical trials to assess toxicities and to determine a maximum tolerated dose. Nausea, vomiting, and myelosuppression were moderate, but neurotoxicity was symptomatic inall patients and caused significant functional impairment some pa tients, thus ending further
5 clinical trials . Inves tigations of additional platinum(IV) complexes are currently in progress. 1.3 Targets and Mechanisms of Cancer Management 1.3.1 Angiogenesis A cell must have fundamental met hods of obtaining nourishment from the body to maintain its health and vitality. Bl ood vessels facilitate this by carrying oxygen and nutrients to a cell, while removing waste pr oducts. Angiogenesis, or the formation of new blood vessels, is usually unique to new or developing tissues. In a healthy adult, emergence of vessels may occur for repair or reproduction but has a limited role. However, in cancer, angiogenesis is integral to its growth. A tumor acquires its own blood supply to sustain its development. Vascular endothelial grow th factor (VEGF) is a pr o-angiogenic factor that has been implicated in tumor angiogenesis . Hypoxic inducible factor-1 (HIF-1) is also known to mediate transcrip tion of the gene for VEGF  HIF-1 also plays a role in tumor angiogenesis by mediating P 13K/AKT-induced VEGF expression [26-28], pathways that are often found to aberrant in cancers. HIF1 is a heterodimer comprised of the oxygen-regulated i nducible subunit HIF-1 and a constitutively active HIF-1 . Overexpression of HIF-1 is associated with tumor angiogenesis and tumor cell proliferation and invasion. Drugs that promot e inhibition of VEGF have been studied as potential anti-cancer agents [29-31]. Bevacizumab, a recombinant humanized monoclonal antibody directed to VEGF, has been shown in clinical studies to inhibit tumor neovascularization, and thus tumor grow th . This suggests that development
6 of further chemotherapeutic agents targeting VEGF and its related regulators may be useful in cancer therapy. 1.3.2 Signal Transducers and Activator s of Transcription (STAT) Growth factors are secreted extrace llularly and bind to receptors on cell membranes to initiate messages to grow or di fferentiate. Once the growth factor binds, it sets off a cascade of intracellular signali ng that may dictate th e production of specific protein or gene products, eventually leading to transcription. In cancer, a disruption of this communication system can lead to e rrors in the under/over production of protein products, causing aberrant signaling. Key si gnaling pathways have been implicated in many cancers. Signal transducers and activators of transcription (STAT) proteins are a family of cytoplasmic transcription factors that function as downstream effectors of cytokine and growth factor receptor signaling. These protei ns transmit signals to the nucleus where STATs bind to DNA and induce gene expression [32-36]. Seven STAT family members have been identifi ed: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 . Nume rous studies have shown that constitutively activated STAT1, STAT3, and STAT5 are present in many human cancers . Futhermore, aberrant STAT3 signaling contributes to tumor development through mechanisms of increased cell proliferation. STAT activation occurs when tyrosine kinase activities phosphorylate receptor-bound STATs. The phosphorylated STAT proteins undergo dimerization and translocate to the nucleus, where they bind to specific DNA promoter sequences and induce gene expression . Constitutively active STAT proteins
7 continue this cascade, resu lting in uncontrolled cell pro liferation. The use of a chemotherapeutic agent to inhibit STAT dimerization can potentially be a useful technique to treat cancer. Figure 1.3 Schematic of STAT signaling . 1.3.3 Biological Effects of Nitric Oxide Nitric oxide (NO) plays a biological role in many pathologies, including diabetes, hypertension, and male impotence. Increasing evidence suggests that NO may also have an effect on cancer biology . Activation of ni tric oxide synthase (NOS) and its subsequent release of NO can cause cytostas is [41-43]. It has also been reported that NOS represents a significant macrophage ant itumor mechanism . Peroxynitrite, a reaction product of NO with O2 can damage cells  and induce apoptosis in a
8 concentration-dependent manner [46, 47]. Xie and Fidler  found that tumor cells that were capable of producing very high levels of NO died in vivo while those cells that produced or were subjected to low levels of NO liv ed on to undergo clonal selection. There are several mechanisms for th e action of NO, some of which are mediated through the inhibition of DNA synthe sis and mitochondrial respiration . This is achieved via interaction with intr acellular iron-sulfur pr osthetic groups of Complex I and II of the mitochondrial electron tr ansport system together with the citric acid enzyme aconitase, and non-heme iron of ribonucleotide reductase [50-52]. Conflicting data indicate that macropha ge infiltration into malignant tissue has been correlated with both a decrease in me tastasis and an increase in cell survival [43, 53, 54] However, recent studies suggest th at NO may also assist macrophages in their battle against cancer. Several groups f ound that NO production was a major cytotoxic effector mechanism of macropha ges [55-57]. It allowed macrophages to acquire potent antitumor activities after exposure to cytokines. Additionally, NO can augment the effect s of chemotherapy and radiation. Wink et al  found that NO can enhance the effect of cisplatin in Chinese hamster V79 cells by inhibiting the DNA repair mechanism. This data supports results from a study by Azizzadeh , who also found improved cytotoxicity in Chinese hamster lung fibroblasts using long-acting NO donors. Effect s of NO with other drugs have also been reported. Cook et al  found that the an ticancer action of melphalan improved with NO, while Adams et al  discovered that cytotoxic effects of the antimetabolite fludarabine was more potent in human chroni c lymphocytic leukemia cells when used
9 with a NO-donating drug. Radia tion therapy has also been found to be enhanced when used in conjunction with NO, due to improved sensitivity to radiation from the cells . 1.3.4 Novel Nitroplatinum(IV) Complexes Our aim in the development of novel nitroplatinum(IV) complexes was to decrease systemic toxicity and resistance, and increase anti-tumor activities through the addition of two ligands to the current squa re-planar cisplatin structure. Oxidation produces a platinum(IV) complex that has b een reported to be able to evade the mechanisms of multi-drug resistance [63-66] and reduce clinical toxicity [2, 3, 65, 67, 68]. This new group of platinum(IV) com pounds can circumvent the drug-resistance commonly observed with traditional anti-cance r agents such as cisplatin. They may prevent the direct extrusion of cytotoxic drugs from the cell, or may act by inhibiting the sequestering of the drugs into intracellular compartments, thereby reducing effective intracellular drug concentrations . The prevention of these self-preserving actions can lead to successful drug administration.
10 CHAPTER 2: EXPERIMENTAL DESIGN 2.1 In vitro studies 2.1.1 Drug Synthesis In compliance with the regulations of patents and licensing, the details of synthesis of these platinum(IV) comple xes under patents #03 B100 and #03B005 cannot be disclosed at this time. 2.1.2 Cell Culture The human lung carcinoma cell line A549 was generously provided by Dr. George Blanck (Dept. of Biochemistry, Colle ge of Medicine, USF). Frozen cells were thawed at 23 C and grown in 45% DulbeccoÂ’s Modi fied Eagle Medium:45% HamÂ’s F12 Medium:10% Newborn Calf Serum (Fisher Scientific). L-glutamine, penicillinstreptomycin, and sodium pyruvate (Fisher Sc ientific) were added to media for final concentrations at 3 mM, 100 U/mL, and 1 mM, respectively. Media wa s filter-sterilized using vacuum filtration through a polyethersu lfone membrane of 0.2 micron pore size. Cells were grown in canted-neck vented-cap tissue culture flasks (BD Falcon) in a 37 C incubator (NuAire) with 7.5% CO2 and seeded at confluence. 2.1.3 Drug treatment Cells in log phase were seeded at 90-100% confluence. Cells were washed twice with 1x phosphate buffered salin e (PBS), then lysed from culture flasks
11 with trypsin-versene (Cambrex Bio Science) Cells were seeded into 96-well tissue culture plates (growth area per well = 0.32 cm2) at a density of 2.5 x 105 cells per well. Drug and media were added to the wells in varying concentrations for a final total volume of 200 uL per well. Culture plat es were then incubated for 48 hours at 37 C with 7.5% CO2. Each treatment condition was performe d in triplicate. Controls contained only cells and media. Table 2.1. Schema of drug trea tment at various dilutions. 2.1.4 XTT Cell Viability Assay Cell viability was determined usi ng the tetrazolium salt XTT [2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium -5carboxanilide, disodium salt] Cell Proliferation Kit (MD Biosciences, Switzer land). The XTT assay is a biochemical procedure that allows for th e assessment of viable cells Mitochondrial dehydrogenases in metabolically active cells cleave the tetrazolium ring, pr oducing a water-soluble Treatment Condition Cell volume (uL) Media (uL) Drug volume (uL) Control 100 100 0 10 uM drug 100 90 10 20 uM drug 100 80 20 25 uM drug 100 75 25 30 uM drug 100 70 30 40 uM drug 100 60 40 50 uM drug 100 50 50 60 uM drug 100 40 60 70 uM drug 100 30 70 75 uM drug 100 25 75
12 orange formazan dye. The amount of dye produ ced is proportional to the number of live cells. This dye can then be measured using a spectrophotometer . After drug treatment as described in section 2.1.3, the XTT reagent was prepared by adding 0.2 ml of the PMS (N-m ethyl dibenzopyrazine methyl sulfate) activation reagent to 10 ml of the XTT reagent. This produced enough reaction mixture for the analysis of one 96-well plate. One-hundred microliters of the XTT reaction solution was added to each well (final XTT concentration = 0.3 mg/ml). Plates were incubated for an additional 2-5 hours at 37 C with 7.5% CO2. The plates were then agitated on a plate shaker for approximately 5 minutes and immediately analyzed for absorbance at 475 nm using a UV-Visible sp ectrophotometer fitted with a fiber optic probe (Varian, CA). Control wells were run with each tria l and contained cells, media, and XTT reagent. The spectrophotometer generates absorb ance values in arbitrary units. Our % survival (relative to controls) values were determined by calculating the average absorbance values of triplicate wells, then dividing that average by the average control absorbance value for that trial. The 50% inhibitory concentration (IC50) values were determined using a linear trendline genera ted by the Excel program (Microsoft). A scatter plot of the cell viabil ity data was created as a func tion of drug concentration, then a linear trendline equation for each drug was determined by the software such that the xaxis depicted drug concentration values, and th e y-axis depicted % survival (relative to controls). The value of Â“50Â” was substituted for the Â“yÂ” variable in the equation to obtain the IC50 value.
13 2.1.5 MTT Cell Viability Assay Cell viability was also determined using the tetrazolium salt MTT Cell Proliferation Assay Kit (Molecular Probes). The MTT assay operates under the same principles as the XTT. The assay involves the conversion of the water soluble MTT (3(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazo lium bromide) into an insoluble purple formazan dye. The amount of dye produced is proportional to the number of live cells. The aqueous contents are then removed and the dye is then dissolved in DMSO and quantified using a spectrophotometer. After drug treatment as described in section 2.1.3, 20 uL of the MTT reagent (5 mg/ml) was added to each well. Plates were incubated for an additional 3 hours at 37 C with 7.5% CO2. The plates were then agitated on a plate shaker for approximately 5 minutes. The liquid conten t of the wells were aspirated out and discarded. To dissolve the dye product, 200 uL of DMSO was added to each well. The plates were agitated again until the dye crysta ls were completely dissolved. Samples were immediately analyzed for absorbance at 570 nm using a UV-visible spectrophotometer (Varian, CA) with fiber optic probe. Control wells were run with each trial containing cells, media, and MTT reagent. Relative survival and IC50 values were calculated as described in section 2.1.4. 2.1.6 Nitric Oxide Production The quantity of nitric oxide produ ced was determined using the DAF-FM (4-amino-5-methylamino-2',7'difluorofluor escein) Diacetate Reagent Kit (Molecular Probes, OR). The DAF-FM diacetate assay is based on the reactivity of aromatic vicinal
14 diamines with nitric oxide (NO) in the presence of oxygen . The non-fluorescent DAF-FM diacetate passively diffuses through th e cell membrane and is de-acetylated by intracellular esterases to beco me DAF-FM. The reaction of the weakly fluorescent DAFFM with NO transforms DAF-FM to an inte nsely fluorescent benzot riaole derivative, which can be measured for fluorescence using a fluorimeter [70, 71]. Figure 2.1 Mechanism of DAF-FM assay. Schema adapted from Molecular Probes . Drug treatment was performed as de scribed in section 2.1.3 using phenolred free DMEM media (Biowhittaker). The DAF-FM diacetate reagent was prepared by dissolving 50 ug of the reagent powder with 20 uL of molecular biology grade DMSO [dimethylsulfoxide] (Fisher Scientific). Th e solution was then diluted with media to produce a 105 uM working solution. Ten microliters of the working solution was added to each well. The plates were incubate d with the working solution (final DAF-FM diacetate concentration = 5 uM) for 30-120 minutes at 37 C with 7.5% CO2. The plates were then agitated on a plate shaker and im mediately analyzed for fluorescence via a fluorescence spectrophotometer (Varian, CA). Fluorescence spectrophotometer settings were as follows: excitation wavelength = 495 nm, emission wavelength = 515 nm,
15 excitation slit = 5 nm, and emission slit = 5 nm Each treatment condition was performed in triplicate. Control wells containing cel ls, media, and DAF-FM acetate were run with each trial. 2.1.7 Western Blotting To determine the expression level of inducible nitric oxide synthase (iNOS) in cells, a Western Blot was performed. Cells were grown and treated at 40 uM as described in sections 2.1.2 and 2.1.3. Cells were harvested by tryps inization and lysed with a hypotonic buffer. Equivalent quantit ies of protein from each sample were separated by SDS-PAGE on 4-15% Tris-H Cl Ready Gels (Bio-Rad). Following electrophoresis, proteins were transferred to Immobilon-P me mbranes (Millipore) using a wet transfer method (Bio-Rad). Membranes were bloc ked for 1 h in 5% nonfat dry milk in Tris-buffered saline-Tween-20 and subse quently washed. Membranes were incubated with polyclonal iNOS antibody (Santa Cruz, SC-651) in 5% nonfat dry milk for 1 h. Following washing, membranes were incubated with a secondary antibody in 5% nonfat dry milk for 1 h. The signal was visualized by chemiluminescence using ECL reagent (Amersham Biosciences) and then exposed to film. Band intensities were quantitated using ImageQuant software (Molecular Dynamics ) . This blot was graciously run by Laura Pendleton (Dept. of Biochemistry, USF). To determine the levels of HIF-1 and VEGF expression, DU145 prostate cancer cells were serum-starved for 20 h in serum-free media and treated with PH9 for 6 hours. Fifty g of nuclear or whole-cell extracts was used. HIF-1 rabbit polyclonal antibody (H-206) (1:500 dilu tion) and anti-VEGF monocl onal antibody (1:1,000 dilution)
16 were used for the Western blot. Horserad ish peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit or anti-goat secondary antibodies were used at 1:2,000 and 1:5,000 dilutions, respectively. The signal was developed with SuperSignal West Pico Chemiluminescent Substrate (PIERCE). Thes e blots were kindly run by the Yu lab (Dept. of Interdisplinary On cology, Moffitt Cancer Center). 2.1.8 Energy-dispersive X-Ray Anal ysis of Platinum-Treated Cells Localization of the intracellular platinum-based drugs was attempted with electron microscopy. Cells were grown to confluence (~2-6 x 106 cells) as described in section 2.1.2 in a sterile 10 cm2 glass Petri dish. Cells were incubated with 55 uM of drug for 48 hours. The cells were then wash ed twice with 1x PBS and fixed overnight with 2.5% glutaraldehyde in 0.1 M phosphate bu ffer at room temperature. The fixative was aspirated and saved. The cells were rins ed with PBS, then dehydrated in a graded series of ethanol as follows: 35% ethanol, 70% ethanol and 95% ethanol, 5 minutes each at room temperature. Cells were then scra ped from the Petri dish and infiltrated with a 50:50 mix of 95% ethanol:L R White acrylic resi n in a microcentrifuge tube for 1 hour at room temperature. The cells were infiltrat ed in two 1-hour changes of 100% L R White at room temperature, then infiltrated overnight in L R White at 4 C. The following day, cells were incubated twice in fresh changes of L R W Localization of the intracellular platinum-based drugs was attempted with el ectron microscopy. Cells were grown to confluence (~2-6 x 106 cells) as described in se ction 2.1.2 in a sterile 10 cm2 glass Petri dish. Cells were incubated with 55 uM of drug for 48 hours. The cells were then
17 washed twice with 1x PBS and fixed overn ight with 2.5% glutaraldehyde in 0.1M phosphate buffer at room temperature. The fixative was aspirated and saved. The cells were rinsed with PBS, then dehydrated in a graded series of ethanol as follows: 35% ethanol, 70% ethanol and 95% ethanol, 5 minutes each at room temperature. hite at room temperature. Following the last resin change cells were placed in a gelatin capsule, which was then filled with the resi n. The resin was polymerized at 50 C overnight (~15 hours). Following polymerization, the block of cells was sectioned on a Reichert Ultracut ultramicroto me using a diamond knife. Sectio ns at 0.25 microns and 90 nm, were obtained. The sections were picked up on 200 mesh nickel grids and examined without further staining with a Philips CM10 transmission el ectron microscope equipped with an EDAX 9900 energy-dispersive x-ray an alyzer. The microscope was operated at 60 kV. A spot size of 100 nm was used to examine the cells. X-ray spectra from the nuclei, cytoplasm and cell membrane were co llected at a count rate of 250 cps. A platinum aperture for the microscope was inse rted into the beam to serve as a positiveplatinum control. Additional cells were desiccated from either ethanol or distilled water onto carbon sample holders and examined as bulk samples by scanning electron microscope (SEM) with EDAX analysis. Dried cells we re examined with and without a carbon coating at 30 kV in a Philips 515 SEM with the EDAX analyzer. A spot size of 100 nm was also used to collect x-ray data in the SEM. The cell nuclei and cytoplasm were examined in this study. A drop of the fixativ e was also air-dried on a sample holder and
18 examined by SEM-EDS to detect for possible le vels of platinum that may have diffused out of the cells. The EDAX analyzer is able to detect concentrations of 0.5% for heavy elements such as platinum. Under ideal conditions, the analyzer may detect levels as low as 0.25% concentration of an element in a sample. 2.1.9 Electrophoretic Mobility Shift Assay (EMSA) Cells were grown and treated with drugs as described in section 2.1.2 and 2.1.3. Nuclear extracts from the cells were used for electrophoretic mobility gel shift assays (EMSA) as described by Turkson et al . Briefly, nuclear extracts containing STAT3 and STAT1 were prepared from hu man lung non-small cell line A549, and preincubated with various concentrations of PH compounds for 30 minutes. Volumes containing equal amounts of total pr otein were then incubated with 32P-labeled hSIE oligonucleotide probe, which expres ses high affinity sis-inducible elements that selectively bind STAT1 or STAT3. The resulting DNA-protein complexes were analyzed by EMSA for the STAT-related DNA-binding factors. This work was performed by the Jove lab (Dept. of Interdisciplinary Oncology, Moffitt Cancer Center). 2.2 In vivo studies 2.2.1 Animal Housing and Treatment Male, 8-week-old athymic mice used in these experiments were obtained from Jackson Laboratories and were cared fo r in accordance with the guidelines of IACUC under protocol #R2426 Animals were caged in groups of four and housed in
19 conventional condition in a temperature-co ntrolled vivarium (23C) on a reverse light/dark cycle. Food a nd water were available ad libitum. The mice were acclimated to the housing environment for 7 days prior to testing. At week 2 of the study, mice received subcutaneous injections of A549 cells in PBS (1 x 107 cells/ flank) into the right flank. Tumors grew to a palpable size by week 3. Mice were randomly distributed into four groups of 12 mice. Three treatment and one control group were defined as: (1) Group 1: drug treatment with 7 mg PH9/kg body weight, (2) Group 2: drug treatm ent with 7 mg PH14/kg body weight, (3) Group 3: drug treatment with 7 mg PH12/ kg body weight, and (4) Group 4: control treatment with saline. The treatment groups received a series of th ree 0.2 mL injections of the drug dissolved in DMSO (20%) with sa line vehicle (80%) into the tail vein. The control group received only saline:DMSO ve hicle. Blood from the saphenous vein was collected from the animals weekly into hepa rin-gel tubes. Urine was collected during routine weighings. All procedures were pe rformed during the dark phase of the lightdark cycle. Animals were euthanized at th e 8th week of the study and the organs were harvested and frozen for future work. One compound, PH9, was selected for further in vivo toxicity testing. Yu et al carried out mouse studies as previously described  Briefly, 7 to 8 week-old C57BL/6 male mice (NCI, Frederick, MD) were maintained under pathogen-free conditions in accordance with established inst itutional guidance and approved protocols. The animals received subcutaneous injections of murine bladder tumor MB49 cells (5 x 105 cells/ flank) into the right flank. The tumors were allowed to grow to 3-5 mm in diameter. Tumor sizes were recorded weekly with Vernier calipers. Over the course of
20 two weeks, four tail vein injec tions of PH9 (5 mg/kg) were administered to the treatment group. The control group received only ve hicle (10% DMSO/PBS). Animals were euthanized within 1-3 days of the last injection using a CO2 chamber. Blood was immediately collected via cardiac puncture using a 1 cc syringe, and transferred in heparin-gel tubes. 2.2.2 Toxicology Analyses of toxicity were determin ed by quantifying levels of enzymes and proteins present in blood and urine. The detection of glucose and glutamic acid concentrations in the urine was mediated by an Amplex Red Glucose Assay Kit and Amplex Red Glutamic Acid Assay Kit, resp ectively (Molecular Probes). All procedures were conducted in accordance with the instru ctions provided by the manufacturer [76, 77]. Briefly, stock and working solutions were prepared by combining 1X reaction buffer, horseradish peroxidase, and assay r eagent. A standard curve was created by diluting glucose in 1X reaction buffer. Each urine sample was diluted in 1X reaction buffer. Fifty microliters of the worki ng solution was added to microplate wells containing 50 uL of sample or control specim en. The plates were incubated at either room T in ambient air or at 37 C with 7.5% CO2 depending on kit instructions. Plates were agitated briefly on a plate shaker a nd then immediately meas ured for fluorescence using a fluorescence spectrophotometer. Fluo rescence spectrophotometer settings were as follows: excitation wavelength = 530 nm emission wavelength = 590 nm, excitation slit = 5 nm, and emission slit = 5 nm. B ackground fluorescence was accounted for by
21 subtracting control abso rbance values from sample values. Glucose/glutamic acid values were determined from the absorbance readi ngs by comparison to the standard curve. The detection of albumin, alanine am inotransferase (ALT), and blood urea nitrogen (BUN) concentrations from seru m samples was mediated by Amplex Albumin Reagent, ALT Reagent, and BUN Reagent as say kits, respectively (Amresco). All procedures were conducted in accordance with the instructions provided by the manufacturer[78-80]. Brie fly, blood samples were coll ected in heparin-gel blood collection microtubes. The tubes were cen trifuged at 2,000 xg for 10 minutes. Serum was separated from the clot imme diately and kept on ice or at 4 C until analysis. Assays were performed within 24 hours of blood coll ection. Working solutions were prepared by reconstituting stock reagent powder with 50 uL deionized water. Working solution was incubated for the designated time in a 37 C water bath. Sample s and controls were added to the solution and incubated again. The absorbencies were measured by a UVvisible spectrophotometer with fiber optic probe at wavelengths unique to each assay kit. Toxicological analysis of the 17 blood samples collected from C57BL/6 mice was performed using the Vitros 950 auto mated chemical analyzer (Ortho Clinical Diagnostics). 2.2.3 Angiogenesis To assess for the effect of PH9 on angiogenesis, athymic mice (NCI) were maintained by Yu et al as described in sect ion 2.2.5. Matrigel assays were performed as described previously . Briefly, 2 x 106 MCF-7 tumor cells stably transfected with either an empty control vector or Stat3siR NA expression vector were suspended in 100 l
22 PBS and mixed with 0.5 ml of Matrigel (Collaborative Biochemical Products) on ice, followed by injection subcutaneously into the abdominal midline of nude nice. Matrigel plugs were harvested for photography and assaying hemoglobin contents. Hemoglobin quantification was carried out by the Drabkin method.
23 CHAPTER 3: RESULTS AND DISCUSSION 3.1 Cell Viability Cell viability is a critical factor to consider when determining the effect of a chemotherapeutic agent on cells. The use of XTT in the enumeration of viable cells after drug treatment can indicate if the agent is effective in killing cells, but can also indicate cytotoxicity as we ll [67, 82]. To identify the optimal experimental conditions, preliminary data was generated from a range of treatment parameters at the beginning of the study. An analysis of 14 novel nitroplati num (IV) compounds and cisplatin at various treatment concentrations revealed that cel l viability was not markedly affected at concentrations below 30 uM for all incubati on times measured (data not shown). Cell viability data for these compounds was also co llected for a range of incubation times with the XTT reagent. Table 3.1 summarizes the aver age relative survival values and standard deviations for the drugs at in cubations times of 2-5 hours at drug treatment concentration of 50 uM. Absorbance data of treated cells is presented as % survival (treated absorbance / control absorbance). It was determined that incubation of the cells with the XTT reagent between 3-5 hours yielded reliable data with the low standard deviations between triplicate wells of the same treatment. This data lie wi thin the range of the suggested incubation times as recommended by the reagent manufacturer. These drug concentrations and reagent inc ubation times were used in subsequent experimentations.
24 Table 3.1 Relative survival for A549 cells treated with 50 uM PH compounds as a function of XTT incubation time. Figure 3.1 Relative surv ival for cells were treate d with PH1-4 and cisplatin. XTT Cell Viability0 20 40 60 80 100 120 5075 Drug Concentration (uM)% survival (relative to control) cisPt PH1 PH2 PH3 PH4 Drug 2h 3h 4h 5h Cisplatin 62.0 +/4.1 63.4 +/1.6 62.2 +/0.4 72.1 +/0.7 PH1 57.5 +/1.3 57.6 +/1.0 56.7 +/1.0 61.6 +/1.4 PH2 77.7 +/4.1 73.6 +/2.5 73.4 +/1.1 80.0 +/1.4 PH3 53.6 +/2.3 53.4 +/0.6 52.0 +/0.7 56.2+/1.0 PH4 57.3 +/0.7 57.3 +/0.5 55.6 +/0.6 60.7 +/0.9 PH5 54.0 +/2.5 54.8 +/0.6 53.5 +/0.7 58.4 +/1.1 PH6 55.8 +/0.5 55.6 +/0.3 54.3 +/0.5 59.2 +/0.8 PH7 57.5 +/1.6 57.7 +/0.3 56.8 +/0.7 62.9 +/0.7 PH8 54.7+/1.4 54.7 +/1.1 54.2 +/0.7 58.8 +/1.2 PH9 54.9 +/0.7 55.6 +/0.9 54.2 +/0.9 58.6 +/1.0 PH10 54.0 +/0.5 53.6 +/0.3 52.7 +/0.9 56.5 +/0.8 PH11 56.1 +/0.1 55.6 +/0.0 55.2 +/0.1 59.3 +/0.2
25 Twelve of the fourteen compounds were better cell prolif eration inhibitors than cisplatin when compared at the same c oncentration. This observation is in agreement with other studies of plat inum(IV) compounds [2, 83, 84]. As demonstrated in Figure 3.1, all cells treated with PH 1-PH4 at concentrations of 50 uM and 75 uM had lower survival percentages than ci splatin. Their calculated IC50 were also lower than that of cisplatin. Table 3.2 shows the IC50 values for these drugs. This data suggests that lower dosages of the PH compounds can be used to inhibit tumor cell growth when compared to dosages of cisplatin. It is evident that the calculated IC50 value for PH4 is unlikely to be a valid value. This error may be due to in correct drug dosing or im proper cell distribution during experimentation. Analys es for PH4 were repeated in later experiments. Table 3.2 Calculated IC50 values for PH1-4 and cisplatin from XTT Assay. Subsequent trials run with thes e and other PH compounds resulted in similar inhibition effects as comp ared to cisplatin. Figures 3.2 and 3.3 depict the percent survival percentages for compounds PH4-11. Again, the novel compounds performed significantly better than cisplatin. At concentrations of 60 uM or greater, there is almost no survival of cells. Table 3.3 summarizes the IC50 values for these drugs. Cisplatin has an IC50 of 71 uM, while the PH compounds inhib ited 50% of the cell growth at concentrations near 45 uM. The IC50 values for the PH compounds are consistently lower than that of cisplatin. Drug Calculated IC50 (uM) Cisplatin 80 PH1 31 PH2 41 PH3 -405 PH4 76
26 Figure 3.2 Relative survival for cells we re treated with PH4-8 and cisplatin. XTT Cell Viability0 20 40 60 80 100 120 3040506070 Drug Concentration (uM)% survival (relative to control) cisPt PH4 PH5 PH6 PH7 PH8 Figure 3.3 Relative survival for cells we re treated with PH1-3, 9-11 and cisplatin. XTT Cell Viability0 20 40 60 80 100 120 3040506070 Drug Concentration (uM)% survival (relative to control) cisPt PH1 PH2 PH3 PH9 PH10 PH11
27 Table 3.3 Calculated IC50 values for PH1-11 and cisplatin from XTT Assay. Figure 3.4 depicts the relative surv ival percentages for four of the previously tested compounds and three ot her compounds PH12-14. This experiment gathered conflicting data as compared to the previous studies. Th e activity of cisplatin appeared to be better than that of both the previously characterized novel compounds and the untested compounds. IC50 values for the nitroplatinum (IV) compounds were also significantly higher than values previously shown, though cisplatin actually demonstrated a lower IC50 value. Table 3.4 shows these results. The ligands of compounds PH12 and PH14 are structures which may potentially have a proliferative affect on cells, in agreement with the results. However, in this set of data, better pro liferation is observed for the ot her PH compounds that had previously been found to be e fficient inhibitors of tumor ce ll growth. This observation, suggests that the discordant data from this tr ial may is questionable. Possible sources of error include equipment malfunction, technical mistakes, and unfavorable environmental conditions for study. It can al so be considered that the data from Figure 3.1 Â– 3.3 may Drug Calculated IC50 (uM) Cisplatin 71 PH1 44 PH2 48 PH3 43 PH4 47 PH5 43 PH6 46 PH7 48 PH8 47 PH9 43 PH10 46 PH11 47
28 Figure 3.4 Relative su rvival for cells were treated with PH3,4,9-14 and cisplatin. XTT Cell Viability0 20 40 60 80 100 120 304050 Drug Concentration (uM)% survival (relative to control) cisPt PH3 PH4 PH9 PH10 PH11 PH12 PH13 PH14 Table 3.4 Calculated IC50 values for PH3,4,9-14 and cisplatin from XTT Assay. not be reliable, and further studies need to be conducted to produce repeatable, reliable results for all compounds. Assays of cell viability performed wi th MTT showed similar results to the earlier XTT assays. Table 3.5 summarizes representative data from the MTT assays. The calculated IC50 values for most compounds closel y resemble those from the XTT Drug Calculated IC50 (uM) Cisplatin 47 PH3 90 PH4 68 PH9 545 PH10 57 PH11 56 PH12 191 PH13 107 PH14 58
29 Table 3.5 Calculated IC50 values for PH1,3,4,7-11 and cisplatin using the MTT assay. assay, suggesting that there is agreement between the two assay types and thus that the data is reliable. This agreement can implicat e key drugs that should be further explored. A summary of the IC50 values for the XTT and MTT assays are shown in Table 3.6. Despite the conflicti ng results from XTT3 (data from third XTT trial), the overall data suggests that some of these PH compounds may ha ve the potential to serve Table 3.6 Summar y of calculated IC50 values from XTT and MTT assays. XTT1 XTT2 XTT3 MTT Avg +/SD Cisplatin 80 71 47 63 65.3 +/12.1 PH1 31 44 50 41.7 +/7.9 PH2 41 48 44.5 +/3.5 PH3 43 90 41 58.0 +/22.6 PH4 76 47 68 56 61.8 +/11.1 PH5 43 43 PH6 46 46 PH7 48 61 54.5 +/6.5 PH8 47 506 276.5 +/229.5 PH9 43 545 45 211 +/236.2 PH10 46 57 54 52.3 +/4.6 PH11 47 56 53 52.0 +/3.7 PH12 191 191 PH13 107 107 PH14 58 58 Drug Calculated IC50 (uM) Cisplatin 63 PH1 50 PH3 41 PH4 56 PH7 61 PH8 506 PH9 45 PH10 54 PH11 53
30 as potent anti-tumor agents at low doses. Th e enhanced activity of the PH compounds as compared to cisplatin may be due to the action of the additional ligands on the novel compounds which are not present in cispla tin. The additional activities of these compounds are investigated in subsequent sections. 3.2 Nitric Oxide Production As the platinum(IV) compounds reduce to a platinum(II) state in the cell, the release of its axial nitro-ligands is expected . These ligands eventually form nitric oxide, which has been shown to have potent an ti-tumor activities  An assessment for the presence of NO was mediated by the DAF-FM diacetate assay. Initial trials of this assay were performed with media containi ng phenol-red. It was la ter discovered that phenol-red media may interfere with fluorescen ce readings. These data sets were discarded. Unexpected problems also arose w ith the alignment of the plate reader of the fluorimeter during this study. Efforts were ma de to rectify the fl uorescence interference by replacing the standard media with phenol -red free media during the drug treatment phase of the experiment. The plate reader was re-aligned and tested for operation using fluorescein (excitation wavelength = 492 nm, emission wavelength = 519 nm, excitation slit = 5 nm, and emission slit = 5 nm). Results from the DAF-FM diacetate assays are inconclusive. Figure 3.5A shows the results of a DAF-FM diacetate assay after incubati on with the reagent for 120 minutes. The level of NO production does not appear to have a dose-response relationship until the drug concentration reac hes 40 uM. After this point, the amount of NO production correlates with drug dose. This phenomenon may be related to the data
31 from proliferation assays showing that at drug concentrations of 30 uM and below, cell survival is relatively unaffected. This s uggests that the PH compounds had little effect on NO production at concentrations below 40 uM Thus, if these compounds are causing NO release, the % NO production should be co mparable to the control NO production, or near 100%. However, most levels fall belo w 100%. At higher drug concentrations, the Figure 3.5A Trial 1 results of NO production. NO Production Trial 10 50 100 150 200 1020304050 Drug Concentration (uM)% NO production (relative to control) cisPt PH3 PH9 PH10 PH11 PH12 PH13 PH14 PH compounds will begin to play a role in the production of NO. As drug concentration increases, cell survival drops. Since the da ta of NO production repr esents each well and not the rate per cell, it is expected that the relative NO production percentages underestimate true values because there are fe wer cells per treated well as compared to control wells. Therefore, th e dose-response relationship is ex pected to be even stronger than shown in Figure 3.5A. Relative to ci splatin, only cells treat ed with PH3, PH11, and PH13 seem to exhibit greater NO release. It can not be determined at this time if these
32 compounds are effective NO producers since their values as compared to controls have not been correlated with cell number. It is evident that the standard deviat ions of the data points as depicted by the error bars are large. This variation is be lieved to be due to the extreme sensitivity of the assay reagent, and the possi bility of small variations in the number of cells between wells. This standard deviation was observed in the controls as well. It is important to note that fluorescein, which was included in th e fluorimeter readings to control for read error, showed very little de viation between triplicate wells (data not shown). This observation demonstrates that inaccuracy of fl uorimeter readings can be eliminated as a source of error. Figures 3.5B-D show the results of additional DAF-FM assays. The data here does not provide insight in to the abilities of these comp ounds to induce NO release. Figures 3.5B and 3.5C indicate that cells tr eated with all compounds including cisplatin had higher NO release than untreated control cel ls. However, results depicted in Figure 3.5D concurs with the data from Figure 3.5A in that the PH com pounds were ineffective in eliciting NO release. It is apparent that there is li ttle agreement for % NO production values between the various trials, but there is evidence that cisplatin treated cells generate higher NO values than the PH compounds. This outcome was not expected since some of the PH compounds were designed for this purpose. However, these results do not conclusively indicate that th e PH compounds are ineffective ge nerators of NO, but rather that further studies need to be conducted to accurately measure NO production.
33 Figure 3.5B Trial 2 results of NO production. NO Production Trial 20 50 100 150 200 250 300 350 1020304050 Drug Concentration (uM)% NO production (relative to control) cisPt PH3 PH9 PH10 PH11 PH12 PH13 PH14 Figure 3.5C Trial 3 results of NO production. NO Production Trial 30 100 200 300 400 500 600 700 1020304050 Drug Concentration (uM)% NO production (relative to control) cisPt PH3 PH9 PH10 PH11 PH12 PH13 PH14
34 Figure 3.5D Trial 4 results of NO production from cells. NO production Trial 40 50 100 150 200 250 1020304050 Drug Concentration (uM)% NO production (relative to control) cisPt PH3 PH9 PH10 PH11 PH12 PH13 PH14 3.3 Nitric Oxide Synthase Expression A Western blot analysis was perf ormed to detect the expression of inducible nitric oxide synthase (iNOS) in treated and untreated cells. Figure 3.6 summarizes the relative iNOS expression (normalized to Bactin) after 48 hour treatment with PH compounds and cisplatin. All cells treated with PH compounds and cisplatin demonstrated higher iNOS values than cont rol cells. iNOS is known to produce a high concentration of NO in tissues. Tumor cel ls that produce high levels of NO die in vivo . Furthermore, studies have reported that an inverse relationshi p exists between the expression level of iNOS and the metastatic potential of murine tu mor cells [48, 85-87].
35 These reports, in conjunction with our results, suggest that high levels of cellular NO production resulting from PH treatment ma y contribute to an titumor activity and decreased metastatic potential. Figure 3.6 iNOS expressi on of PH treated cells. iNOS Expression0.0 0.5 1.0 1.5 2.0 2.5Control P H 3 PH11 P H 1 4 PH10 P H 1 2 PH9 P H 1 3 PH4 c is pl a t iniNOS protein level (relative to B-actin) 3.4 Toxicology Due to mechanical errors, sample contamination, inconsistent collection practices, and reagent sensitivity, the blood serum data collected from the nude mice were deemed unreliable and thus excluded fr om analysis for this study. This decision was based on the generation of many negativ e values when assessing for blood enzymes, which is clearly erroneous. Toxicological pr ofiles were thus developed from the animal groups from Yu et al. Serum obtained at euthanasia was analyzed by the Vitr os 950 automated chemical analyzer (Ortho Clinical Diagnosti cs). The biochemical profile of Mouse PH9-
36 1.1 from Day 1 was excluded from analysis due to a hemolyzed blood sample. C57BL6 mice from Day 1 showed the most abnormal blood chemistry values (Table 3.7). This was expected as these animals have the highest dose of drug in their bodies. As the drug is eliminated from their systems, the bioche mical profiles of mice fr om Days 2-3 return Table 3.7 Biochemical blood serum profiles for the a ssessment of toxicity. BUN albumin Creatinine AST ALT Normal 14-252.6-3.30.5-0.966-170 24-140 Day 1 Control 1.1 18.0 1.8 0.496.0 78.0 Control 1.2 18.7 1.7 0.4 325.3 98.7 Control 1.3 16.0 1.60.5 192.0 101.3 PH9-1.1 60.8 1.3 1.0 339.2 224.0 PH9-1.2 16.0 1.8 0.4 252.0 272.0 Day 2 Control 2.1 21.02.6 0.381.0 46.0 Control 2.2 18.0 1.80.6122.0 66.0 Control 2.3 15.0 2.3 0.2119.0 88.0 PH9-2.1 20.0 1.6 0.478.0 76.0 PH9-2.2 15.4 2.1 0.3 190.3 68.6 Day 3 Control 3.1 20.0 2.3 0.2 54.0 35.0 Control 3.2 19.0 2.5 0.287.0 61.0 PH9-3.1 18.0 2.3 0.2161.0 70.0 PH9-3.2 22.0 2.4 0.2 273.0 259.0 PH9-3.3 20.0 1.9 0.284.0 44.0 PH9-3.4 21.0 2.4 0.2139.0 53.0 PH9-3.5 14.0 2.3 0.2 347.0 101.0 closer to normal values. Normal ranges we re determined from a variety of literature sources, so it should be noted that values will vary according to breed, age, sex, sampling technique, and quality of blood sample . Therefore, inte rpretations are not definitive. Blood urea nitrogen (BUN) and albumi n levels are used to assess kidney function. Elevated BUN levels may indicate kidney failure, disease, and dehydration. Elevated albumin levels may indicate dehydration. Thus, if high albumin levels are noted in conjunction with other elevat ed values, it should be considered that the out-of-range
37 values are due to dehydration and not necessarily organ damage. Creatinine is the end product of phosphocreatine metabolism, an enzyme associated with muscle contractions. If elevated creatinine levels are observed with elevated BUN, kidney disease is often implicated. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels are used to assess liver damage. AST is an indicator of the breakdown and elimination of nitrogen. Low AST levels ma y indicate starvation or malnutrition, while high levels may suggest liver damage, muscle damage, and inflammation. ALT is also involved with nitrogen metabolism and is most often associated with liver function. Low levels suggest starvation or malnutrition, while high levels indicate liver damage, toxin ingestion, and various metabolic disorders. Lo w creatinine levels indi cate liver disease or starvation, while high levels may suggest dehy dration, or kidney failu re or disease. Figure 3.6 graphically displays the biochemistry profiles of the blood collected from mice one day after the treatment injecti on. Although the control mice demonstrated individual blood chemistry valu es that may indicate toxicity, there was insufficient data across the various blood panels that could collectivel y suggest that there was any organ disease or damage. It is also evident that low albumin levels are observed for nearly all control and treated mice. One of albuminÂ’s functions is to transport drugs within the blood stream [89, 90]. It has been shown that albumin is reactive to platinum(II) complexes due to its thiol group . Thus, low albumin levels may be anticipated with platinum tr eatment. Mouse PH9-1.2 had high levels of AST and ALT, and low albumin and creatinine. This findi ng may suggest that th e administration of 5 mg/kg of PH9 to the animals may cause undesira ble toxic side effects, and that a lower
38 Figure 3.7 Biochemical blood profiles of mi ce one day after 5 mg/kg drug injection. dose may need to be considered to preven t organ damage while sustaining the drugÂ’s anti-tumor action. Figure 3.8 displays the bi ochemistry profiles of the blood collected from mice two days after the la st injection. Creatinine and al bumin levels were again low for both treated and control mice, but other data did not support toxicity concerns. It was concluded that low creatinine le vels were not suggestive of li ver disease. However, the blood profile of PH9-2.2 of Day 2 demons trates low creatinine and albumin in conjunction with high AST (but normal ALT), possibly indica tive of liver damage.
39 Figure 3.8 Biochemical blood profiles of mi ce two days after 5 mg/kg drug injection. Figure 3.9 displays the biochemistry profiles of the blood collected from mice three days after the treatment injecti on. Again, only the data from the treated animals indicate possible liver toxicity. PH9-3.2 and PH9-3.5 had low values of creatinine and albumin, and high values of liv er enzymes that suggested liver damage. The control animals did not exhi bit overall sign s of toxicity.
40 Figure 3.8 Biochemical blood profiles of mice three days after 5 mg/kg drug injection. Cisplatin has been known for its indu ction of renal toxicity. Preclinical studies of cisplatin performed on mice re ported that significan t kidney damage was associated with the treatment [91, 92]. Leleiveld et al found levels of BUN and creatinine in cisplatin -treated C57BL mice (6.7-10.0 mg/kg) to be nearly five times the levels found in untreated animals on the four th day after treatment . These PH9 treated animals demonstrated no signs of nephrotoxicity. Table 3.8 compares our BUN
41 Table 3.8 Creatinine and BUN values of mice after cisplati n or PH9 treatment. BUN Creatinine Cisplatin nmol/L Nmol/L Control 8.6 +/0.1 58.5 +/0.6 6.7 mg/kg cisplatin 55.0 +/15.0 281.0 +/88.0 10.0 mg/kg cisplatin 72.0 +/12.0 282.0 +/55.0 PH9 mg/Dl mg/dL Control 18.2 +/2.0 0.4 +/0.2 5 mg/kg PH9 18.3 +/2.9 0.3 +/0.1 and creatinine values with thos e values reported from cisplati n. It is evident that PH9 treated mice did not exhibit any measurable signs of kidney disease. Values of the treated animals are similar to values of untreated animals. This evidence suggests that PH9 may reduce toxicity relative to cisplatin. 3.5 X-Ray Diffraction X-ray analysis of the platinum-t reated cell block from the TEM was unable detect a platinum signal anywhere in the cells. A spectra generated from the positive-platinum control confirmed that the x-ray detector was functional. Energy emission signals from the analysis of trea ted cells showed the presence of sulfur, phosphorus and chlorine cells. The emission spect ra also indicated the presence of silica in the resin. X-ray analysis of the platinum-treated bulk sample from the SEM was unable to detect a platinum signa l anywhere in the cells. Analysis of the fixative and the drug also suggested that there wa s no platinum in the sample. This data indicates that the level of platinum in the cells and culture medium after treatment and fixation was below the minimum level of detectability for the
42 x-ray analyzer. Another mode of analysis with lower limits of detection that may be more sensitive may be necessary to detect a nd quantify the amount of platinum present in the cells. Alternatively, platinum localiza tion may require the analysis of cellular organelles though the lysis of large numbers of treated cells This may be mediated by atomic absorption or inductively-coupled plasma. 3.6 Inhibition of STAT Dimerization The PH compounds were able to in hibit acellular dimerization of STAT proteins at concentrations as low as 0.3 uM. At concentrations of 30 uM, complete inhibition of STAT dimerization was observed for all PH compounds. The IC50 for STAT dimers by these compounds are summarized in Table 3.9. Table 3.9 Calculated IC50 values of PH compounds for STAT dimerization. Previous studies with other inhibito rs of STAT report that much higher concentrations are necessary to achieve the sa me effect. Blaskovich et al found that JSI124 (Ccurbitacin I) had an IC50 value of 500 nm in the hum an lung carcinoma A549 cell line . Grandis et al used antisense oligonu cleotides to inhibit STAT at 12.5 uM. It is evident that the IC50 values of the PH compounds are e ffective at lower concentrations, suggesting that these PH compounds may be s uperior STAT inhibitors. However, since Compound STAT3:STAT3 (uM) STAT1:STAT3 (uM) STAT1:STAT1(uM) PH4 0.5 1.0 1.0 PH1 0.5 1.0 1.0 PH10 2.0 2.2 0.5 PH7 0.4 0.4 0.4 PH11 0.3 0.4 0.4
43 this assay was conducted with nuclear extracts and not whole cells, cellular transport and other mechanisms and other mechanisms will activities in vivo. Figure 3.10 Inhibition of STAT dimerization by PH compounds. Assessment of the effects of the PH compounds on other STAT dimers is critical in understanding th e compoundsÂ’ effects on other si gnaling cascades. Though the PH compounds were effective at pr eventing STAT3:STAT3 binding, the STAT1 homodimer is less affected at concentrations below 10 uM (Figure 3.10). It has been shown that STAT1 activation is elevated in a few cancers, but its function has mostly been observed as growth suppression rather than malignant transformation  Thus, the preservation of the STAT1 dimerization function is desirable in maintaining its actions as a potential tumor suppressor. The STAT1:STAT3 heterodimer has been weakly linked to similar effects of constitutively active STAT3, so its inhibition continues to be a desired effect.
44 3.7 Angiogenesis As shown in Figure 3.11, YuÂ’s group at Moffitt revealed that Western Blot analysis of cells treated with PH9 resulted in decreased HIF-1 and VEGF expression. Lanes 1-4 denote the concentra tion of PH9 administered. Bactin is a housekeeping gene Figure 3.11 Determination of HIF-1 VEGF, and STAT3 expressi on by Western blot. PH9 affects HIF-1 and VEGF expression in a dose-de pendent manner (top gel). This reduction of expression can be corr elated with STAT3 e xpression (bottom gel). that normalizes the amount of protein in each well to maintain consistency for analysis between lanes. The intensity of HIF-1 and VEGF bands fades across the lanes, indicating that PH9 affects the expression of these proteins in a dose-dependent manner. This reduction in expression of HIF-1 and VEGF correlates with the expression of STAT3, suggesting a relationship. As STAT3 expression decreases, HIF-1 and VEGF expression decreases as well. This data is in agreement with the data found in section
45 3.7, where PH9 was found to reduce STAT3 activity. Since the over-expression of VEGF and HIF-1 is associated with tumor angioge nesis, tumor cell proliferation and invasion, PH9 may be an inhibitor of critical angiogenic proteins, possibly related to STAT3 inhibition. Further in vivo studies verified that treat ment the blocking with PH9 resulted in reduced microvessel density. Matrigel-implanted hu man breast carcinoma MCF-7 tumors from PH treated mice were excised and analyzed for angiogenesis. Figure 3.12 Matrigel plugs of MCF-7 tumors. Figure 3.12 shows control and PH9 treated Matr igel plugs of MCF-7 tumors that were harvested 5 days after implant ation. Mice treated with th e DMSO vehicle demonstrate increased neovasculature as compared to mice treated with PH9. Further studies identifying mechanistic pathways are in pr ogress in collaborations with the Yu lab. 3.8 Tumor Growth Inhibition In vitro studies conducted by YuÂ’s gr oup indicated that MB 49 cells did not respond to PH9 treatment . However, mi ce treated with PH9 demonstrated inhibited
46 MB49-tumor growth. Within 5 days of tu mor induction, a palpable mass was detected for control and experimental mice. Tumo rs continued to grow throughout the drug Figure 3.13 Tumor size (mm3) of control and PH treated mice. regimen for both groups. Animals treated wi th PH9 exhibited significantly slower growth and smaller tumors as compared with control animals (Figure 3.13). Because the same cell line used in vitro did not respond to PH9 treatment, it is believed that the tumor inhibition was due to an immunological or sy stemic response to the drug. These facts suggest that PH9 may be used as an effec tive chemotherapeutic ag ent to inhibit tumor growth.
47 CHAPTER 4: CONCLUSION In an effort to overcome common pr oblems associated with the traditional methods of cisplatin cancer treatment, we have synthesized novel chemotherapeutic agents designed to circumvent drug-resistan ce and toxic side effects, while achieving improved activity by targeting unique path ways unique to cancer. We focused on obtaining preliminary data to characterize th ese compounds in order to elucidate potential compounds for further study. In vitro and in vivo studies were performed to describe their activities. Table 4.1 summarizes the results of key assays performed on the PH compounds. Data not shown in this table include toxicity and angiogenesis findings, since only PH9 was examined in those assays Cell proliferation assays performed from XTT and MTT tetrazolium salt reagents s howed that all PH compounds, with the exception of PH12 and PH13, exhibited lower IC50 values than that of cisplatin. Western blot analyses of iNOS expression de monstrated that PH3 and PH11 treated cells had greater iNOS expression than cisplati n. All compounds that were tested for inhibition of STAT3 dimerization had IC50 values of 2 uM or lower. Contrary to the nephrotoxicity commonly observed in cisplatin-tr eated mice, our toxicological analysis of PH9 treated mice indicated that no kidney damage was evident. However, elevated livr enzymes were measured, warranting further st udies. Additionally, PH9 exhibited anti
48 Table 4.1 Comparison of the results of various assays on PH compounds. Compound Avg IC50 (uM) Relative iNOS expression STAT3 IC50 (uM) Cisplatin 65.3 +/12.1 1.64 ---PH1 41.7 +/7.9 ---0.5 PH2 44.5 +/3.5 ------PH3 58.0 +/22.6 2.19 ---PH4 61.8 +/11.1 1.56 0.5 PH5 43 ------PH6 46 ------PH7 54.5 +/6.5 ---0.4 PH8 276.5 +/229.5 ------PH9 211 +/236.2 1.23 ---PH10 52.3 +/4.6 1.55 2.0 PH11 52.0 +/3.7 1.99 0.3 PH12 191 ------PH13 107 1.37 ---PH14 58 1.10 ---angiogenesis properties, as demons trated by the inhibition of HIF-1 and VEGF expression. These assays have identified PH3, PH9, and PH11 as the most promising candidates for further studies. These nitroplatinum(IV) compounds are al so expected to circumvent the resistance seen with cisplatin since their novel mechanisms may not involve DNA binding and therefore evades the DNA mismatch repair mechanism. These data suggest that these novel nitroplatinum compounds may serve as effectiv e alternatives to cisplatin. Further studies will refine the data established in this study. Future work will include the determination of the spectrum of activity of the PH compounds in various cell lines. We also hope to identify the optimal dose by con ducting further cell viability assays and toxicology studies. Additionally, the structures of the platinum complexes must be verified using x-ray crystallography. Nitric oxide production may be assessed with further Western blotting for iNOS. La stly, we wish to explore STAT3 inhibition
49 and anti-angiogenesis mechanisms for PH3 and PH11. This additional work may reveal future directions into clinical trials.
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