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Toxicological and chemotherapeutic effects of oral cesium chloride in PC-3 and LNCaP prostate Cancer… Low, Jonathan Cedric 2007

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T O X I C O L O G I C A L A N D C H E M O T H E R A P E U T I C EFFECTS OF O R A L C E S I U M C H L O R I D E I N PC-3 A N D L N C A P P R O S T A T E C A N C E R H U M A N X E N O G R A F T S  by JONATHAN CEDRIC L O W B S c , The University o f British Columbia, 2004  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FORTHE DEGREE OF M A S T E R OF SCIENCE  in T H E F A C U L T Y OF G R A D U A T E STUDIES  (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH C O L U M B I A A p r i l 2007 © Jonathan Cedric Low, 2007  ABSTRACT Purpose: H i g h p H Therapy is an alternative cancer therapy involving the daily ingestion of concentrated dosages o f cesium chloride (CsCl). Although the U . S . Food and Drug Administration has not approved the use o f C s C l as a cancer treatment, individuals seeking this therapy need only search the internet to purchase C s C l solutions or tablets. Companies market these C s C l treatments by presenting the most recent C s C l cancer research, the majority o f which is outdated and incomplete. The purpose o f this study was to assess the therapeutic and toxicological effects o f C s C l administration in mice bearing human prostate cancer tumors. M e t h o d s : Three C s C l dose titration studies were completed in tumor bearing and nontumor bearing athymic nude mice. A l l mice were administered either vehicle (controls), 150mg/kg, 300 mg/kg, 600 mg/kg, 800 mg/kg, 1000 mg/kg or 1200mg/kg o f C s C l once daily by oral gavage for 30 consecutive days. Body mass was measured daily, food and water consumption were measured every two days, and tumor volume was measured twice weekly. Histopathological analysis was conducted on tissues collected from each o f the studies. Serum A S T , A L T and creatinine were also measured. Results: Chronic administration o f 800mg/kg to 1200mg/kg C s C l significantly reduced PC-3 tumor growth but had no effect on L N C a P tumor growth. In addition, tumor bearing and non-tumor bearing animals receiving these concentrated C s C l dosages (800mg/kg-1200mg/kg) developed bladder crystals and showed an increase in water consumption. A n observed loss in body mass and an increased prevalence o f postmortem cardiac fibrin clots appeared to be dependent on the xenograft type and  concentration o f C s C l administered. C s C l did not affect serum A S T , A L T and creatinine levels i n any o f the treatment groups. Conclusions: Although C s C l appears to have some therapeutic benefit in treating the PC-3 prostate cancer model, a number o f toxicological effects were also observed linked to its administration, including loss of body mass, increase o f water consumption, fibrin clot formation and bladder crystal formation.  iii  TABLE OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST O F T A B L E S  vii  LIST O F F I G U R E S  viii  LIST O F ABBREVIATIONS  xi  ACKNOWLEDGEMENTS  xii  DEDICATION  xiii  S E C T I O N 1 Introduction  1  1.1. Project Overview  2  1.2. Background 1.2.1. Cancer . 1.2.2. Prostate Cancer '.. 1.2.2.1. Epidemiology 1.2.2.2. Oncogenes and Tumor Suppressor Genes 1.2.2.3. Androgens and The Androgen Receptor 1.2.2.4. Diagnosis and Prostate Cancer Stages 1.2.2.5. Conventional Treatment 1.2.3. Alternative Cancer Treatments 1.2.4. The Cesium Chloride Alternative Cancer Treatment 1.2.4.1. Historical use o f Cesium Chloride: Brewer's " H i g h p H Therapy" 1.2.4.2. Reported Therapeutic Effects o f Cesium Chloride 1.2.4.3. Reported Toxicological Effects o f Cesium Chloride 1.2.4.4. Reported Biodistribution o f Cesium Chloride ;  4 4 4 4 .....6 7 9 12 13 14 14 15 17 19  1.3. Rationale  20  1.4. Hypothesis  21  1.5. Specific A i m s  22  S E C T I O N 2 Materials and Methods....  23  2.1. Materials and Reagents 2.1.1. Instrumentation 2.1.2. Animals 2.1.3. Chemicals and Reagents  24 24 24 25  2.2. Tumor growth and toxicity in the PC-3 and L N C a P human prostate tumor xenograft models, after administration o f escalating doses o f C s C l 2.2.1. A n i m a l Husbandry  25 26  iv  2.2.2. 2.2.3. 2.2.4. 2.2.5. 2.2.6. 2.2.7.  C e l l Lines and Tumor C e l l Inoculation Integration o f Animals into Each Study Dose Selection and Drug Administration Measuring the Therapeutic Response o f C s C l Monitoring Acute Toxicity from C s C l A n i m a l Euthanization and Tissue Harvesting  28 30 34 .35 36 37  2.3 Investigation o f organ and tissue specific toxicity from C s C l (histopathology and serum marker tests) in human prostate tumor xenograft bearing nude mice 2.3.1. Histopathology 2.3.2. Serum A S T and A L T Analysis 2.3.3. Serum Creatinine Analysis  39 39 40 41  2.4 Comparisons between Control Animals  44  2.5 Statistical Analysis and Removal o f Outliers  45  S E C T I O N 3 Results  46  3.1. Tumor growth and toxicity in the P C - 3 and L N C a P human prostate tumor xenograft models, after administration o f escalating doses o f C s C l 3.1.1. P C - 3 Solid Tumor Xenograft Study 3.1.2. L N C a P Solid Tumor Xenograft Study 3.1.3. Non-tumor Bearing Nude Mouse Study 3.2. Investigation o f organ and tissue specific toxicity from C s C l (histopathology and serum marker tests) in human prostate tumor xenograft bearing nude mice 3.2.1. Histopathology 3.2.2. Serum A S T / A L T 3.2.3. Serum Creatinine 3.2.4. Bladder Crystals and Viscous Mucosal Fluid 3.3. Comparison o f Control Animals  47 47 52 57  61 61 68 68 ....75 77  S E C T I O N 4 Discussion  88  4.1. Tumor growth and toxicity in the P C - 3 and L N C a P human prostate tumor xenograft models, after administration o f escalating doses o f C s C l ".  89  4.2. Investigation o f organ and tissue specific toxicity from C s C l (histopathology and serum marker tests) in human prostate tumor xenograft bearing nude mice  91  4.3. Comparisons between Control Animals  93  4.4. Conclusion  95  4.5. Significance and Limitations o f the Results 4.6. Future Studies  ,  v  ....95 98  4.7. Synopsis  REFERENCES  LIST OF TABLES Table 1  Summary o f the animal grouping within the PC-3 study  31  Table 2  Summary o f the animal grouping within the L N C a P study  32  Table 3  Summary o f the animal grouping within the non-tumor study  33  Table 4  Dosage correction o f vehicle and C s C l solutions to compensate for differences i n animal body mass  35  Parameters o f the high performance liquid chromatography protocol developed to assess creatinine concentrations in murine serum  43  Table 5  vii  LIST OF FIGURES Figure 1  Actions o f the A R once bound by D H T  Figure 2  Progression o f prostate cancer as described by the T N M staging system  10  Timeline illustrating some o f the specific procedures undertaken within the three animal studies  27  Analysis o f tissues and serum obtained from the animals studies o f Aim 1 ,  38  Figure 3  Figure 4  Figure 5  8  A S T and A L T catalyzed chemical reactions that take place once serum is added to the A S T and A L T kinetic assay kits  42  Figure 6  Pathways involved in the production and excretion o f creatinine  44  Figure 7  Percent average initial body mass over time o f nude mice bearing a PC-3 tumor xenograft for control and treatment groups  48  Average food consumption over time o f nude mice bearing a P C - 3 tumor xenograft for control and treatment groups  49  Average water consumption over time o f nude mice bearing a P C - 3 tumor xenograft for control and treatment groups  50  Figure 8  Figure 9  Figure 10 Percent average initial tumor volume over time o f nude mice bearing a PC-3 tumor xenograft for control and treatment groups  51  Figure 11 Percent average initial body mass over time o f nude mice bearing a L N C a P tumor xenograft for control and treatment groups  53  Figure 12 Average food consumption over time o f nude mice bearing a L N C a P tumor xenograft for control and treatment groups  54  Figure 13 Average water consumption over time o f nude mice bearing a L N C a P tumor xenograft for control and treatment groups  55  Figure 14 Percent average initial tumor volume over time o f nude mice bearing a L N C a P tumor xenograft for control and treatment groups Figure 15 Percent average initial body mass over time o f tumor free nude mice for control and treatment groups  vin  ...56  58  Figure 16 Average food consumption over time o f tumor free nude mice for control and treatment groups  59  Figure 17 Average water consumption over time o f tumor free nude mice for control (vehicle) and treatment groups  :60  Figure 18 Cardiac fibrin clots discovered post-mortem in a nude mouse o f the PC-3 study  62  Figure 19 Percentage o f nude mice from the three studies with the presence o f post-mortem fibrin clots for control and treatment groups  63  Figure 20 Abnormalities o f the small intestine discovered post-mortem in a nude mouse o f the PC-3 study  64  Figure 21 Percentage o f nude mice from the three with the presence o f atrophy o f mucosal epithelium o f the small intestine for control and treatment groups  65  Figure 22 Percentage o f nude mice from the three studies with the presence o f atrophy o f villi o f the small intestine for control and treatment groups  .....66  Figure 23 Percentage o f nude mice from the three studies with the presence o f inflammatory infiltrate o f the small intestine for control and treatment groups  67  Figure 24 Serum A S T and A L T levels o f nude mice bearing a PC-3 xenograft for control and treatment groups '.  69  Figure 25 Serum A S T and A L T levels o f nude mice bearing a L N C a P xenograft for control (vehicle) and treatment groups Figure 26 Serum A S T and A L T levels o f tumor free nude mice for control and treatment groups  ....70  71  Figure 27 Chromatograms o f a creatinine spiked standard and a serum sample using Hydrophilic Interaction Chromatography  72  Figure 28 Standard curve for the serum creatinine H P L C assay  73  Figure 29 Serum creatinine levels o f nude mice from the three studies for control and treatment groups  74  Figure 30 Crystal structures removed from the bladders o f nude mice treated with 600mg/kg-1200mg/kg C s C l daily  75  ix  Figure 31 Percentage o f nude mice with the presence o f bladder crystals or viscous mucosal fluid in the P C - 3 , L N C a P and non-tumor studies  76  Figure 32 Average tumor volume over time o f nude mice representing a PC-3 or L N C a P xenograft  78  Figure 33 Percent average initial tumor volume over time o f nude mice representing a PC-3 or L N C a P xenograft  79  Figure 34 Average food consumption over time o f nude mice from the control groups o f the three C s C l dose titration studies  81  Figure 35 Average water consumption over time o f nude mice from the control groups o f the three C s C l dose titration studies  ...82  Figure 36 Percent average initial body mass over time o f nude mice from the control groups o f the three C s C l dose titration studies  83  Figure 37 Serum A S T and A L T levels o f nude mice from the control groups o f the three C s C l dose titration studies :  84  Figure 38 Serum creatinine levels o f nude mice from the control groups o f the three C s C l dose titration studies .„  85  Figure 39 Presence o f atrophy o f the intestinal mucosal epithelium in nude mice from the control groups o f the three C s C l dose titration studies  85  Figure 40 Presence o f atrophy o f the intestinal villi in nude mice from the control groups o f the three C s C l dose titration studies  86  Figure 41 Presence o f intestinal submucosal infiltrate in nude mice from the control groups o f the three C s C l dose titration studies  86  Figure 42 Presence o f cardiac fibrin clots in nude mice from the control groups of the three C s C l dose titration studies  87  x  LIST OF ABBREVIATIONS ANOVA  analysis o f variance  AR  androgen receptor  AST  aspartate aminotransferase  ALT  l-alanine:2-oxoglutarate aminotransferase  A A —.  atomic absorption  Cs  cesium  CsCl  cesium chloride  DHT  dihydrotestosterone  DMEM  dulbecco's modified eagle medium  DRE  digital rectal examination  FBS  fetal bovine serum  HPLC  high performance liquid chromatography  IL-6  interleukin-6  IL-lp  interleukin-lp  LOD  limit o f detection  LOQ  limit o f quantification  NAD  nicotinamide adenine dinucleotide  NADH  reduced nicotinamide adenine dinucleotide  %CV—  percent coefficientof variance  PSA  prostate specific antigen  RPMI  roswell park memorial institute  STD  standard deviation  TNF-a  tumor necrosis factor a  xi  ACKNOWLEDGEMENTS I would like to thank my supervisors, Dr. E m m a Guns and Dr. Kishor Wasan. D r . Guns provided a great environment to conduct my experiments and to learn new techniques. In addition, her enthusiasm, open mindedness and overall support have been crucial throughout m y research. Dr. Wasan's vast experience and knowledge in pharmaceutical sciences have been pivotal to the success and rigor o f m y project. Thank you both for your contributions. I would like to thank m y committee members, Dr. Brian Rodrigues, Dr. Helen Burt, Dr. Marcel Bally, Dr. Stelvio Bandiera and Dr. U r s Hafeli for their suggestions and contributions to m y project. Lastly, I would like to thank my coworkers, past and present, at the Prostate Centre at V G H for their guidance and friendship: Dr. Simon Cowell, A n d y Eberding, Y a n g Zhou, Catherine Wood, A l a i n Musende, Hans Adomat, M a r y Bowen, Jennifer Locke, D r . Ladan Fazli and Michael Blicker.  xn  DEDICATION This work is dedicated to... M y parents, Cedric and Pamela L o w , my siblings, Christopher and Rachel L o w , and to my girlfriend, Sarb Tatla. Their unwavering support has been crucial throughout m y studies.  xm  Section 1 Introduction  i  1.1 Project Overview Prostate cancer is the most prevalent form o f cancer in North American men. L i k e most cancers, the mechanism by which individuals develop this disease is poorly understood; likely there is some interplay between genetics and environment. The search for alternate treatments for advanced prostate cancer remains elusive. Although techniques exist to cure early stage, localized (stage T1-T3), hormone dependent prostate cancer, current treatments for advanced prostate cancer (stage T 4 - M ) typically only increase life expectancy by months to one or two years. A number o f alternative cancer treatments exist, whose proponents often claim curative effects. However, most o f these treatments have not been rigorously researched to support these claims. Regardless, a large proportion o f cancer patients often purchase these alterative therapies once diagnosed with their disease. These alternative treatments are often self administered by the patient either while receiving their conventional therapy or alone as a single agent. Co-administration o f alternative treatments with conventional cancer therapies may be dangerous, as possible interactions between the two therapies may lead to higher levels o f toxicity than administration o f either agent alone. Cesium chloride (CsCl) is the main component o f an alternative cancer treatment known as " H i g h p H Therapy". According to Dr. Keith Brewer, the creator o f this therapy, administration o f C s C l can reduce tumor volume by increasing intracellular tumor p H in both man and mice (1). However, to date there is no published work by Dr. Brewer illustrating C s C l ' s ability to increase intracellular p H . Although the U S . Food and Drug Administration has not approved the use o f C s C l as a cancer treatment, individuals seeking this therapy need only search the internet to  2  purchase C s C l solutions or capsules. Most o f the companies that advertise on the internet, promote their products by citing the most current C s C l cancer research, the majority o f which is outdated or incomplete (2-4). There have been a limited number o f preclinical and clinical studies conducted evaluating the therapeutic effects o f C s C l . For the most part, the results from these studies provide support that C s C l may be able to treat different forms o f cancer (5-10). However, the archaic cancer models and suboptimal experimental designs used in these studies reduce their credibility. Studies investigating the toxicity o f C s C l have revealed some inherent side effects, but have not produced a rigorous toxicological profile. In addition, the severity o f these reported side effects appear to vary considerably from study to study; cardiac abnormalities in man and canines are the most severe effects attributed to C s C l administration (11-23). It should be noted that only a few case studies have reported patients with these cardiac abnormalities (15-23). The lack o f C s C l research is detrimental to the safety o f consumers taking C s C l products, because the efficacy and toxicity o f C s C l has not been fully delineated. The purpose o f this research project was to assess the therapeutic effect o f chronic daily administration o f C s C l in mice bearing human prostate tumor xenografts. In addition, the toxicological profile o f C s C l was also determined using tumor bearing and non-tumor bearing nude mice through histopathology and serum based toxicity assays.  3  1.2 Background  1.2.1. C a n c e r Cancer is a disease which is typically characterized by unregulated cellular growth and proliferation that disrupts the function o f normal bodily systems, potentially leading to the death o f the host. The transition o f a normal cell to that o f a population o f cancerous cells requires numerous rounds o f mutation and proliferation i n the midst o f evading cellular regulatory circuitry. Hanahan and Weinburg described six physiological alterations that normal cells must undertake to become cancerous: growth signal selfsufficiency, antigrowth signal insensitivity, avoidance o f apoptosis, infinite replicative capability, angiogenesis and the ability to metastasize (24). To date more than 100 different types o f cancer have been identified (24). In 2002, it was predicted worldwide that 10.9 million people were diagnosed with cancer and 6.7 million people died o f the disease (25).  1.2.2. Prostate C a n c e r  1.2.2.1 E p i d e m i o l o g y Prostate cancer is the second most prevalent form o f cancer i n men and ultimately the fifth most prevalent form o f cancer worldwide (25). In most developed countries, the incidence o f prostate cancer has increased over time, while the mortality rate from this disease has decreased (26). These same trends are not observed in developing countries as a result o f under funded health care systems and limited access to these health care  4  systems (26). Interestingly, the incidence o f prostate cancer is quite low in most Asian countries including Japan (25). However, an increase in incidence is observed in individuals who emigrate from a low-risk country like Japan to a high-risk country like the United States (25). These results indicate the potential role o f environmental factors influencing this disease (25). In North America, prostate cancer is the most prevalent form o f cancer in men as it contributes to 33% o f all new cancer cases (26-29). It is now predicted that one in every six American men w i l l develop prostate cancer sometime during their lifetime (30). Individuals with this disease have approximately a 9% mortality rate (29). The main risk factors that have been associated with prostate cancer are genetics, race, age and diet (26, 27). The genetic predisposition o f prostate cancer is not well understood. Although certain genes have been implicated as being important predisposing factors for prostate cancer, large inter-individual differences in the frequency o f these genes provide evidence that prostate cancer is genetically a heterogeneous disease (26). Studies have suggested that genetic predisposition contributes to a large proportion (just under 50%) o f prostate cancer cases (31, 32). In the United States, African American men have consistently displayed the highest incidence o f prostate cancer compared to men o f other ethnicities (26, 27). This greater incidence observed in one race cannot be attributed to any one factor but rather has both genetic and environmental influences. Prostate cancer has typically been described as a disease that affects elderly individuals; individuals less than 50 years old rarely display this disease (26). It has been  5  hypothesized that aging provides the time required for sufficient maladaptive cellular events to occur which contribute to the formation o f cancer (33). Diet has been linked with the incidence o f prostate cancer. Specifically, it has been argued that the high fat North American diet increases the risk o f prostate cancer. However, this idea has yet to be definitively proven (27).  1.2.2.2 Oncogenes and Tumor Suppressor Genes The impact o f specific oncogenes and tumor suppressor genes on the propagation o f prostate cancer still remains poorly defined due to the genetic complexity o f the disease. Oncogenes that have been linked to the development o f prostate cancer include c-myc, E R B B 2 and bcl-2. C-myc is transcription factor involved i n the regulation o f apoptosis, cellular proliferation and differentiation (34, 35). C-myc over-expression by gene amplification has been observed i n some prostate cancer tumors (36). E R B B 2 is a transmembrane phosphoprotein which is also involved in the regulation o f apoptosis, cellular proliferation and differentiation (37, 38). E R B B 2 over-expression has been observed in some prostate tumors, but it is likely not the product o f gene amplification (36, 39). bcl-2, an antiapoptotic gene, was reported to be over-expressed in 33-50% o f prostate tumors; much more frequently than c-myc and E R B B 2 (40, 41). Tumor suppressor genes that have been linked to the progression o f prostate cancer are P T E N and P53. One should recall that both alleles must be silenced for complete inactivation o f a tumor suppressor gene. Chromosome loss and point mutations have been observed in tumors for both P T E N and P53 (42-44). In the case o f P T E N , i f one allele is  6  inactivated by a point mutation, the inactivation o f the second allele occurs by an unknown mechanism (45).  1.2.2.3 Androgens and the Androgen Receptor Androgens are responsible for the growth, development and functioning o f the prostate (46-48). Testosterone, the most prevalent androgen in males, is converted by 5areductase to 5a-dihydrotestosterone ( D H T ) , which is the main ligand for the androgen receptor (46). Once bound to the androgen receptor in the prostate, D H T promotes cell proliferation and differentiation, while thwarting cell death (39). Most androgens are produced by the Leydig cells o f the testes. The adrenal glands also produce a small amount o f androgen. The androgen receptor ( A R ) is expressed mainly by the luminal epithelial cells o f the prostate (46). When no ligand is present, the A R remains in the cytoplasm (Figure 1). However, once the A R is bound to D H T , it relocates to the nucleus and is a transcription factor for androgen regulated genes (Figure 1) (49). Specifically, the receptor-ligand complex binds to androgen response elements, which are located upstream o f the target genes' coding sequences. The activation o f these target genes are not only regulated by the receptor-ligand complex but by other coactivators and repressors (50). Due to the dependence o f prostate cancer cells on androgens for growth, a treatment strategy known as androgen ablation therapy is administered to individuals with advanced prostate cancer. This therapy is not a cure as it ultimately selects for prostate cancer cells that can survive under low androgen levels. There are three postulated mechanisms described leading to the survival o f these androgen independent cells. The  7  first mechanism describes that the cancer cells might not be completely androgen independent at all, but rather able to produce more androgen receptors v i a amplification o f the androgen receptor gene (51). Greater numbers o f A R would allow these cells to survive under low androgen levels. The second mechanism describes cancer cells with mutated androgen receptors that are able bind non-androgen molecules present in circulation (52-54). Once bound to the A R , these hon-androgen molecules would initiate the same proliferate effects as when androgen binds the A R . The final mechanism proposes that non-cancer cells, like neuroendocrine cells, secrete neuropeptides that support the growth o f the cancer cells (51).  Growth  Survival  Differentiation  Figure 1. Image o f the actions o f the A R once bound by D H T . Once bound to D H T , A R dissociates from heat shock proteins, becomes phosphorylated and dimerizes (50). The D H T - A R complex binds to A R E with cofactors resulting in the transcription o f specific genes (50). (Figure modified from Javidan et al., 2005 (50))  8  1.2.2A Diagnosis and Prostate Cancer Stages The prostate is a walnut-shaped gland located under the bladder that envelops the urethra. The prostate is divided into five different lobes: anterior, median, posterior, and two lateral lobes. The primary function o f the prostate is to produce fluid that is added the semen upon ejaculation (55). The prostate is surrounded by four distinct tissue areas: connective and fatty tissue (located on the rectal, lateral and anterior sides o f prostate), seminal vesicles (located at the base o f the prostate), bladder neck (located at the base o f the prostate), and pelvis musculature (located on the apical side o f prostate) (56). There are two main tests used by physicians to diagnose prostate cancer; prostate specific antigen ( P S A ) tests and digital rectal exams ( D R E ) (57). The P S A test involves measuring P S A concentration in the blood. P S A , which is secreted by prostatic epithelial cells, tends to increase in individuals with prostate cancer due to the enlargement o f the prostate (58). D R E is a more invasive test compared to the P S A test. This test requires access to the prostate by way o f the rectum, in order to determine any structural irregularities indicative o f prostate cancer. Only the two lateral and posterior lobes can be palpated using a D R E (57). If a positive result is obtained from either o f these tests, a biopsy is normally performed to determine the presence and the extent o f the cancer (57). The Gleason grading system is used by pathologists to determine the extent o f cellular differentiation. According to this system, well differentiated cells are given a score o f one, while poorly differentiated cells are given a score o f five. The T N M staging system was developed for physicians, to assess prostate cancer progression. The T stage refers to the form o f cancer present within the prostate (Figure 2). The N stage refers to the progression o f the cancer into the pelvic lymph nodes.  9  T 1 Stage  Figure 2. Image representing the progression o f prostate cancer as described by the T N M staging system. (Figure modified from US. National Cancer Institute P D Q database website (59))  Finally, the M stage refers to the level o f metastases that has occurred. Each o f the stages described are further subdivided into sub-stages. The T stage is further divided into the T l , T 2 , T3 and T 4 sub-stages, each o f which has their own sub-stages. The T l a and T i b stages refer to prostate cancer that is discovered by accident during a transurethral resection o f the prostate in individuals who have benign prostate hyperplasia (56, 60). U s i n g histopathological analysis, tumors which occupy <5% o f the prostate are classified as T l a and tumors which occupy >5% o f the prostate a classified as T i b (60). The T i c classification refers to elevated serum P S A levels (positive P S A test) (56, 60). Digital rectal exams are unable to detect tumors that are in the T l stage (56). The T2 sub-stages T2a, T2b and T2c refer to tumors that are detectable by the digital rectal exam (56, 60). T2a refers to a tumor that is constrained within a single lobe o f the prostate and occupies less than 50% o f the lobe (56). T2b refers to a tumor that is constrained within a single lobe o f the prostate and occupies more than 50% o f the lobe (56). T 2 C refers to the presence o f cancer in both lobes o f the prostate (56). The T3 and T4 sub-stages refer to the invasion o f the surrounding tissue o f the prostate by the tumor. T3a and T3b sub-stages refer to a tumor that has spread to the connective and fatty tissue on one side or both sides o f the gland respectively (60). The T3c substage indicates that the tumor has invaded the seminal vesicles (60). The T 4 sub-stage indicates that the tumor has progressed and invaded the bladder neck, rectum and/or the pelvis musculature (56, 60). The N and M stages and sub-stages refer to various levels o f invasion o f the prostate cancer in pelvic lymph nodes and other sites outside the prostate. The N sub-stages refer  11  to gradual increases i n the number o f lymph lobes affected but also the size o f tumors present within the lymph lobes. The M sub-stages refer to the presence o f the cancer in bone, a main site for prostate cancer metastases, but also the presence o f cancer in other distant sites.  1.2.2.5 Conventional Treatment The treatment regimen administered to an individual with prostate cancer depends on a number o f different factors including the progression o f the cancer, health and age o f patient, and degree o f risk associated with each procedure. Early stage, localized (T1-T3), hormone dependent prostate cancer is normally treated with either external radiation therapy or a radical prostatectomy (55). Both o f these procedures can essentially cure a patient i f performed correctly, however, each runs the risk for possible mortality and morbidity (55, 61). T w o additional procedures which are starting to be used more frequently for treating early stage prostate cancer are brachytherapy and cryotherapy. Brachytherapy involves the insertion o f radioactive seeds (iodine-125 or iridium-192) into the prostate and other affected tissues rendering the death o f the cancer cells over time (62). Cryotherapy involves the insertion o f 17-gauge needles directly into the affected areas. These needles undergo several freeze-thaw cycles, resulting in the death o f the cancer cells and surrounding tissue (63). A final option for patients with early stage prostate cancer is known as "watchful waiting", in which no treatment is administered and the cancer is monitored with subsequent P S A tests and D R E s . Hormone therapy, specifically androgen ablation therapy, is typically administered to treat advanced prostate cancer that has metastasized. Prostate cells are dependent on  12  androgens for proliferation, protein synthesis initiation and cell death inhibition (57). Thus, the removal o f androgens through bilateral orchiectomy or administration o f luteinizing hormone-releasing hormone agonists results in cell death (57). It should be noted that although androgen ablation therapy reduces androgen levels significantly, it does not remove androgens completely. The removal o f androgens induces a high initial mortality rate i n the cancer cell population, but ultimately selects for androgen independent (hormone refractory) cancer cell growth (64). Once the prostate cancer has become hormone refractory, chemotherapy is often administered as a last resort, often with limited therapeutic effect. Docetaxel in combination with prednisone is currently the gold standard chemotherapeutic cocktail for advanced prostate cancer (51, 65, 66). In phase III clinical trials, patients administered the docetaxel based therapy showed greater survival compared to patients treated with a mitoxantrone based therapy (51, 65). It is believed that docetaxel binds directly to (3tubulin subunits promoting polymerization, which ultimately results in microtubule stabilization (51). A s the cellular cytoskeleton no longer responds normally, the cell undergoes apopto si s (51).  1.2.3 Alternative Cancer Treatments Advanced prostate cancer often does not respond well to chemotherapy. F o r this reason, along with the risks associated with other standard treatments, a number o f prostate cancer patients turn to complementary or alternative self-treatments. Sometimes patients w i l l even self medicate with these alternative therapies while receiving conventional therapy. A recent survey in British Columbia o f 1108 men with prostate  13  cancer indicated that 39% o f these individuals had used a complementary or alternative therapy (67). Unfortunately, many alternative products have not been investigated rigorously, and it is relatively unknown whether they provide any benefit to the individual who is selfmedicating. Fueled by the increased use o f alternative therapies by cancer patients, a number o f research facilities have begun to investigate these products. The purpose o f these studies is to elucidate the therapeutic and toxicological effects o f these agents, as well as determine their possible use in combination therapies with conventional treatments for cancer. " H i g h p H Therapy" is an alternative cancer therapy that is built around the compound C s C l . Currently, the effects o f C s C l alone and in combination with chemotherapeutic drugs are relatively unknown.  1.2.4 The Cesium Chloride Alternative Cancer Treatment  1.2.4.1 Historical use of Cesium Chloride: Brewer's "High p H Therapy" In the early 1980's an alternative cancer therapy involving the use o f C s C l was developed by the late Dr. Keith Brewer. The theoretical premise o f this therapy was that C s C l could combat cancers through elevating intracellular tumor p H (1). Due to technological constraints, this C s C l induced change in tumor p H was never scientifically measured. However, firm on his theory, Brewer marketed this C s C l therapy under the name " H i g h p H Therapy" (1). Dr. Brewer began investigating C s C l after realizing its prevalence in several geographical areas with low cancer incidence (1). The groups o f people living in these  14  areas had traditionally eaten food grown i n the volcanic soil that was rich in cesium (Cs), and had inadvertently included Cs in their diets for generations (1). The proposed mechanism o f C s C l starts with the uptake o f C s by the cancer cells. Once inside the cells, Brewer believed that the Cs was able to facilitate an increase in the intracellular p H o f the cells; that is, change the neutral to slight alkaline p H o f normal cancer cells, to a basic p H o f 7.5-9.0 (1). Brewer described that this proposed change in intracellular p H was not well tolerated by the cancer cells, resulting in many cells undergoing cell death (1). To date, there have been no epidemiological studies conducted to determine the number of people who have used or are currently using C s C l . However, judging from the ample availability o f C s C l products offered on the internet through multiple companies around the world, one could assume that a large number o f people have tried to self-treat using this therapy (2-4). Dr. Abdul-Haqq Sartori, one o f the main supporters o f C s C l , has claimed to have treated more than 750 patients with his Enhanced C s C l therapy (68).  1.2.4.2 Reported Therapeutic Effects of Cesium Chloride There have been only a few published studies evaluating the therapeutic effects o f C s C l against cancer. The methodologies o f most o f these studies were not rigorously conducted or were hindered due to technology barriers. In the one published clinical study on C s C l treatment, 50 terminal patients with generalized metastatic disease were given 6-9g o f C s C l each day for three years (5). The results showed that 50% o f these individuals were still alive at the end o f the treatment (5). Pain also diminished in all patients within the first three days o f the study (5).  15  Furthermore, autopsies o f a few patients that had died during the treatment indicated very low levels o f cancer tissue present (5). However, there was no control group in this study; every patient was treated with C s C l . In vivo C s C l studies reported to date have used mice with surgically implanted murine sarcoma I, colon-38, or M T 2 9 6 mammary rumors, or BZP-induced skin carcinoma. In these studies, a reduction in tumor volume was observed in animals that were administered daily intraperitoneal injections o f 505.5mg/kg C s C l ( l . l m g C s C l in combination with zinc and vitamin A for the animals with colon tumors) (6-10). One issue that arises when implanting or inducing tumors in animals is that some tumors fail to propagate due to factors independent o f the drug; tumors fail to propagate in animals of treatment and control groups. W i t h experimental designs that involve pretreatment o f the animals with drug prior to tumor implantation or induction (as in the studies listed above), the researcher would not know whether the reduction i n tumor volume observed in the treatment groups was completely related to the drug. Furthermore, there is no way of normalizing treatment groups in terms o f tumor growth characteristics at the beginning of each o f these studies; one would have to assume that all the tumors implanted or induced were the same size and had the same growth potential. A few in vivo studies have also examined the impact o f C s C l on mortality in tumor bearing animals. A study involving mice with SR-implanted tumors that received ' pretreatment with C s C l (505.5mg/kg) for 7 days prior to tumor inoculation, observed a reduction in tumor induced mortality rate compared to control animals (69). However, similar experiments conducted in rats bearing N o v i k o f f Hepatoma showed opposite  16  results; there was less mortality i n control animals compared to C s C l treated animals (70, 71). Recently, our laboratory conducted the first C s C l study in a human derived cancer xenograft mouse model. M i c e bearing a L N C a P prostate cancer xenograft treated with 150mg/kg C s C l daily via oral gavage had no significant reduction in tumor volume compared to control mice (72). N o other dosages o f C s C l were examined i n this study. A reduction in tumor volume was only observed when 150mg/kg C s C l was administered in combination with vitamin D (72).  1.2.4.3 Reported Toxicological Effects of Cesium Chloride In terms o f toxicity, there appear to be some inherent side affects attributed to C s C l . treatment, although the severity o f these effects has not been resolved. Historically, the only adverse effects reported by patients were nausea, diarrhea, paresthesia (tingling o f the skin), and hypokalemia (potassium deficiency) (5, 73). The consumption o f high potassium foods was shown to reduce the onset o f nausea and diarrhea (73). Studies in canines have shown a link between C s C l administration and impaired cardiac function. It has been reported that canines administered concentrated dosages o f C s C l experience long Q T syndrome (abnormally long delay between ventricle depolarization and ventricle repolarization), torsades de pointes (ventricular tachycardia with irregular ventricle depolarization and ventricle repolarization) and various other cardiac arrhythmias (11-14). Interestingly, a few recent case studies have reported similar cardiac problems in patients self-administering C s C l (15-23). In one case study, a 43 year old woman who had been recently diagnosed with a brain tumor was admitted to hospital  17  after having two seizures (16). O n arrival to the hospital, she had a third seizure and then suddenly collapsed due to ventricular tachycardia, for which defibrillation was administered (16). During her stay at the hospital she displayed ventricular tachycardia again on two separate occasions (16). U p o n examination, the physicians found no evidence o f heart disease (16). It was discovered that one day prior to her admittance to the hospital she had just finished a 9g/day 10 day regimen o f C s C l treatment (16). In mice, the acute L D  5 0  dose o f intraperitoneal administered C s C l was determined to be  ~1.7g/kg (10, 74, 75). The acute  LD o dose 5  of orally administered C s C l was determined  to be 1.9-2.3g/kg (76, 77). Animals that died from the concentrated intraperitoneal injection o f C s C l reportedly experienced respiratory failure followed by convulsions (10, 74). Animals that did not perish experienced copious salivation, increased urination, piloerection, central nervous system depression, and shallow, irregular breathing (10, 74). Additional studies in mice investigated the impact o f C s C l on development by replacing mothers' drinking water with 1 M C s C l solution after giving birth. M a l e pups whose mothers consumed C s C l , had a decrease in brain mass and had lower body mass after breast-feeding was discontinued (weaning period), as compared to control pups (7880). Similarly, male pups whose mothers consumed C s C l during pregnancy had a reduction in brain mass, but had increased spleen and testis mass (81, 82). Studies investigating the effects o f C s C l on murine bone marrow metaphase cells in vivo revealed that C s C l administration elicits clastogenic effects (chromosomal breakage) (83, 84). 125-500mg/kg C s C l administered orally resulted in chromosomal breaks and dicentrics in a dose dependent fashion within 6, 12, 18 or 24h (83, 84). A dose o f 500mg/kg C s C l also reportedly reduced the mitogenic index o f these bone marrow cells  18  24h after administration (83, 84). Several studies have demonstrated that the clastogenic effects o f C s C l could be reduced, i f animals were pretreated with vitamin C or Phyllanthus emblica fruit extract, or i f C s C l was co-administered with chlorophyllin or calcium chloride (85-87). Two additional studies analyzed the effects o f C s C l on bone marrow erythrocytes. A dose o f lOOOmg/kg orally administered C s C l increased the prevalence o f micronuclei in these cells 24 hours after administration (88). This same effect was not observed in animals administered 500mg/kg C s C l (88, 89).  1.2.4.4 Reported Biodistribution of Cesium Chloride There have been a number o f C s C l distribution studies completed in both man and animals. The average half life o f C s C l was reported as 83-96 days in adult males and 65.5-77.2 days in adult females (90-93). One should note that half life appears to increase with body mass, indicating a possible rationale for the differences observed between males and females (91, 94). Studies in humans reported that approximately 78% o f ingested C s C l is absorbed into the blood (95). It appears that C s C l pharmacokinetics can be described by a twocompartment model; 6% is rapidly excreted with a half-life o f 0.3 days, while the remaining 94% is slowly excreted over time (93, 95). According to a published pharmacokinetic model developed for C s C l distribution, C s C l is most concentrated in the heart, kidneys and liver after ingestion or intravenous injection (90). C s C l levels quickly drop in these organs within the first 24h and most o f the C s C l is sequestered in the skeletal muscle for long term storage (19, 90).  19  The average half-life o f C s C l in mice was determined to be 6.1 days (96). A subsequent experiment demonstrated that the pharmacokinetics o f C s C l i n mice could be described by a two-compartment model where 90% o f C s C l is rapidly excreted in mice with a halflife o f 2.83 days, while the remaining 10% is excreted more slowly with a half-life o f 16.3 days (97). This is considerably different than the pharmacokinetic profile observed in humans. C s C l retention studies in animals have shown varied results as different tissues have been examined within different studies. C s C l was reported to sequester mostly i n the kidneys, small intestine and tumors, 14 m i n after intraperitoneal administration in mice bearing M T 2 9 6 mammary tumors (98). However, when C s C l was administered orally to athymic nude mice bearing L N C a P tumors, it was concentrated mostly in the heart, liver, and kidneys within the first hour (72). Similar results have also been observed in studies involving rats (99, 100). C s C l concentrations in nude mice increased in the brain, spleen, prostate and tumors, and decreased in the heart, liver and kidneys over a 24h period (72). In non-tumor bearing mice chronically administered C s C l for 442 days, it was determined that C s C l concentrated in the muscle, heart and kidneys (101).  1.3 Rationale A large number o f patients have tried an alternative therapy to treat their cancer. The C s C l alternative cancer therapy requires patients to self-treat using high dosages (3-9g) o f C s C l each day for extended periods o f time (weeks to months). However, there have been no studies examining the therapeutic and toxicological effects o f high (greater than 150mg/kg), repeated doses o f orally administered C s C l in humans or human xenograft  20  models. Previous experiments involving tumor bearing mice, demonstrated that C s C l may have some therapeutic effect over a specific dose range. However, the design o f these studies has failed to mimic the conditions under,which cancer patients would administer this treatment; for example, patients would likely begin treatment after the diagnosis o f cancer and not before. In addition, previous studies examining the toxicological effects C s C l have been very specific; the overall toxicity profile o f C s C l throughout the body has not been documented, especially for chronic administration o f C s C l over the long term. The lack o f research investigating C s C l as an alternative cancer treatment is deleterious to the safety o f consumers self administering C s C l products. This study w i l l provide the medical community with a comprehensive, rigorous evaluation o f the therapeutic and toxicological effects o f C s C l in human prostate tumor xenograft models. Furthermore, it w i l l provide insight into the effects o f this treatment against androgen dependent and androgen independent cancer.  1.4 Hypothesis D a i l y a d m i n i s t r a t i o n o f o r a l c e s i u m c h l o r i d e w i l l h a v e a t h e r a p e u t i c effect a g a i n s t h u m a n prostate t u m o r xenografts while eliciting some toxicity in the n u d e mouse model  21  1.5 Specific Aims  A i m 1: To evaluate tumor growth and toxicity in the P C - 3 and L N C a P human prostate tumor xenograft models, upon administration o f escalating doses o f C s C l to athymic nude mice.  A i m 2: To analyze tissues obtained from the studies in A i m 1 to further investigate organ and tissue specific toxicity (histopathology and serum marker tests) in human prostate tumor xenograft bearing nude mice.  22  Section 2 Materials and Methods  23  2.1. Materials and Reagents  2.1.1. Instrumentation Tumors volumes were measured using digital calipers. The mass o f food, water and the mice themselves were monitored with an Acculab top loading balance (Bradford, M A , U S A ) . Tissue sections, for histopathological analysis, were examined with a R T colorS P O T high resolution digital camera from Diagnostic Instrument. Inc. (Tampa, F L , U S A ) , mounted on Olympus System light microscope model B X 5 1 (Markham, O N , Canada). Semi-quantitative image analysis was performed using Image-Pro Plus version 4.5.1.22 from Media Cybernetics, (San Diego, C A , U S A ) . A S T and A L T assays were analyzed using a Bio-Tek Instruments Power Wave X with K C 4 Kineticalc Version 2.6 Revision 3 software (Winooski, V T , U S A ) . Serum creatinine was analyzed using a Waters 2695 Separations Module with a 3um bore, 2.1x 50mm Waters Atlantis™ H I L I C Silica column paired with a Waters 996 Photodiode Array Detector (Mississauga, On, Canada). The H P L C and detector were run using Empower Pro version 5.0 2002 software. A l l statistical analysis was conducted using SigmaStat 3.0 software.  2.1.2 Animals A l l animal studies used 6-8 week old male athymic nude mice (HSD:Athymic NudeF o X N l ) with body weights o f 25-30g from Harlan Sprague Dawley, Inc (Indianapolis, N U  IN, U S A ) .  24  2.1.3 Chemicals and Reagents Mouse LabDiet® 5058 was purchased from Jamiesons Pet Food Dist. (Delta, B C Canada). Cesium chloride (minimum 99%) was purchased from Sigma A l d r i c h (Oakville, O N , Canada). Dulbecco's Modified Eagle M e d i u m ( D M E M ) , Roswell Park Memorial Institute ( R P M I ) M e d i u m 1640, Fetal Bovine Serum (FBS), 0.25% Trypsin, 1 m M ethylenediamine tetraacetic acid were purchased from Invitrogen, Inc (Burlington, O N , Canada). Eosin, Hematoxylin, H P L C grade water, H P L C grade acetonitrile, ammonium formate were purchased from Fischer Scientific (Ottawa, O N , Canada). Ethanol was purchased from Commercial Alcohols (Brampton, O N , Canada). Neutral Buffered Formalin 10% was purchased from E M Science (Fort Washington, P A , U S A ) . The creatinine standard was purchased from Cayman Chemical (Ann Arbor, M I , U S A ) . Infinity™ A S T and A L T kits were purchased from Thermo Electron Corporation (Louisville, C O , U S A ) .  2.2 Tumor growth and toxicity in the PC-3 and LNCaP human prostate tumor xenograft models, after administration of escalating doses of CsCl. The effect o f C s C l products on cancer patients is relatively unknown as there have been no rigorous studies conducted examining the therapeutic and toxicological effects o f C s C l in humans or human xenograft models. Typically, for clinical drug investigation, Phase I and Phase II clinical trials are conducted as rigorous drug assessments in humans. Within these studies, the drug is administered at increasing concentrations to a small group o f diseased or healthy patients to determine its therapeutic and toxicological effects. Although, C s C l must first be analyzed in a preclinical setting to delineate its  25  effects, the experimental design o f these preclinical studies can mimic those o f clinical drug trials. Within this aim, three animal studies were conducted to assess the actions o f C s C l in vivo; the first study involved 56 animals bearing established PC-3 tumors, the second study involved 53 animals bearing established L N C a P tumors and the final study involved 41 non-tumor bearing animals. Figure 3 illustrates the timeline under which specific procedures were undertaken in each o f the three animal studies. C s C l ' s therapeutic response was monitored in the two xenograft studies. A s each o f the chosen xenografts represents a different stage in prostate cancer progression, a response observed in either xenograft could provide pre-clinical evidence for the stage o f progression at which C s C l is most efficacious. Acute toxicity was monitored in all three studies; both in tumor bearing and in non-tumor bearing animals. Acute toxicity observed i n the animals of the two xenograft studies could be contributed to the combined effects o f C s C l and each tumor. Alternatively, any acute toxicity observed in the non-tumor bearing animals was expected to be from C s C l alone as there would not be any influence from tumor burden. Intra-study and inter-study comparisons between the three studies provided insight on the extent o f C s C l ' s anticancer properties and acute toxicological effects relative to the concentration o f C s C l administered and tumor model used. In addition, such comparisons allowed for tumor induced acute toxicity to be investigated.  2.2.1 A n i m a l H u s b a n d r y Once the nude mice arrived at The Prostate Center, they were placed in microisolator cages, 3 mice per cage, and were left to acclimatize to their new environment for two  26  Food and Water Measured Tumors and Body Mass Measured Animals Arrive at Prostate Centre  PC-3 Study  — Animals Arrive at Prostate Centre  LNCaP Study •  Inoculation of PC-3 Cells  •  Study Begins  Animals placed in separate cages  Study Begins: first group of animals integrated into ^^tucly^^^  Study Ends  i  Inoculation of L N C a P Cells  m  Animals Arrive at  m Animals placed in separate cages  •  Non-Tumor S T Study 0  Animals placed in separate cages  Last group of animals integrated into study  Study Begins  First group of animals removed from study  Study Ends: Last animals removed from study  Study Ends  1  8  Week Figure 3. A timeline illustrating some o f the specific procedures undertaken within the three animal studies.  10  11  weeks. The room environment in which the animals were housed had a controlled temperature o f 19-25 °C, a 30-70% humidity and a 12-hour light cycle. Following the acclimatization process, mice were transferred into individual cages, and the husbandry duties (e.g. the changing o f the cages, food and water) were passed from the animal care staff on to the researcher. The purpose o f this exercise was to decrease the level o f disturbance to each cage, in order to accurately assess water and food consumption as measures o f acute toxicity for each mouse.  2.2.2 Cell Lines and Tumor Cell Inoculation After the first week o f the acclimatization period, mice o f the PC-3 and L N C a P studies were inoculated subcutaneously with prostate cancer cells, and tumors were allowed to propagate. The P C - 3 and L N C a P cell lines were chosen for these studies as they have been extensively characterized and are accepted models for prostate cancer both in vitro and in vivo. In addition, these two cell lines have molecular characteristics similar to prostate cancer cells observed clinically at different stages o f the disease. Specifically, L N C a P cells have similarities to prostate cancer cells prior to androgen ablation therapy and P C - 3 cells have similarities to prostate cancer cells post androgen ablation therapy. Using cell lines that represent different stages o f prostate cancer progression is beneficial for this study, as it has not been documented at what stage prostate cancer patients are more likely to use C s C l treatments. The L N C a P cell line originated in 1977 from a supraclavicular lymph node metastasis from a 50-year old Caucasian male and displays all the characteristics o f a hormone responsive prostate carcinoma (102). It is fibroblastoid, androgen sensitive as it has a  28  mutated yet functional A R , and expresses P S A (102, 103). Following injection into athymic nude mice, L N C a P cells form tumors with variable growth rates and morphology. In comparison, the PC-3 cell line is representative o f a hormone insensitive prostate cancer and was derived from a metastatic tumor (104). The PC-3 model has also been well characterized: the cells are androgen independent since they express very low levels o f A R or do not express it at all, and they do not produce P S A (103-105). P C - 3 tumors generally have a conserved shape^ a greater growth rate and are typically less vascularized compared to L N C a P tumors. The athymic nude mouse was used as the sole strain o f mouse for the purposes o f this project. This strain was selected due to its compatibility with the PC-3 and L N C a P cell lines in hosting subcutaneous human tumor xenografts. The PC-3 and L N C a P cell lines respond quite differently when inoculated into the nude mouse model. Tumor propagation is never 100% for either cell line, however, inoculated PC-3 cells tend to form tumors more readily than L N C a P cells. For this reason 60 nude mice were inoculated subcutaneously with 2 x 1 0 PC-3 cells in a single dorsal site. O f 6  these, 56 animals had viable tumors on day 1 o f the PC-3 study. Alternatively, for the L N C a P study, 70 nude mice were inoculated subcutaneously with 2 x 1 0 L N C a P cells in 6  two dorsal sites. O f these, 53 animals were incorporated into the L N C a P study. The P C - 3 and L N C a P prostate cancer cells which were injected into the animals were originally cultured in D M E M and R P M I media, respectively, in the presence o f 5% F B S . One senior animal technician was responsible for inoculating all o f the animals to maintain the consistency o f the method.  29  Once tumors became visible, their volumes were measured rigorously using calipers. This information, in addition to body mass values was crucial for allocating mice into treatment groups and ultimately determining the start dates o f the PC-3 and L N C a P studies.  2.2.3 Integration of Animals into Each Study In order to make comparisons between treatment groups within each study and between each o f the three studies, animals were arranged into treatment groups so that day 1 values for tumor volume and body weight would not be statistically different when treatments groups were compared intra-study and inter-study. In other words, each treatment group had approximately the same tumor volume and body mass on day 1 as all the other treatment groups within the same study and between the three studies. In order to accomplish this standardization, animals o f the three studies were not necessarily integrated into their respective studies on the same day. Animals o f the PC-3 study were integrated into the study 4 weeks after their arrival at The Prostate Centre, since the tumors o f each animal exceeded a predetermined volume (50-70mm ). Alternatively, 3  animals o f the L N C a P study were integrated into the study in a staggered fashion because o f the inter-variability o f the L N C a P tumor's growth kinetics. A s tumors reached a predetermined size (70mm ), the respective animals were integrated into the study: the 3  first animals were integrated into the study 4 weeks after their arrival at T h e Prostate Centre and the last animals integrated 7 weeks after their arrival at The Prostate Centre. Animals o f the non-tumor bearing study were integrated into the study 4 weeks after their  30  arrival at The Prostate Centre, in order to maintain a consistent starting age with animals o f the P C - 3 and L N C a P studies. In the P C - 3 study, the homogeneous growth o f the P C - 3 tumors allowed for all o f the animals to be integrated into the experiment on the same day. M i c e were randomly distributed into the seven treatment groups according to their tumor volume and body mass (n=9 for control group, n=8 for the 150mg/kg, 300mg/kg, 600mg/kg, 800mg/kg, lOOOmg/kg groups and n=7 for the 1200mg/kg group). Each treatment group had an average tumor volume o f 82-107 m m and an average body mass o f 28.4-31.4g. Table 1 3  illustrates the average tumor volumes and body mass o f animals within each o f the seven treatment groups. Statistical analysis b y one way analysis o f variance ( A N O V A ) showed no statistical difference between any o f the treatment groups in regards to average tumor volume or average body mass on day 1 o f the study (P>0.05).  Table 1. Summary o f the animal grouping within the P C - 3 study. Vverage Tumor ' Volume ± S T D (mm ) / Body Mass ± S T D (g), on Day 1  Group  Tumor  CsCl Dose (mg/kg body wt)  Number of Mice/Group  1  PC-3  Vehicle (water)  9  101±57/30.1±2.4  2  PC-3  150.  8  102±68/31.4±2.3  3  PC-3  300  8  107±71 / 2 8 . 4 ± 2 . 1  4  PC-3  600  8  95±61 / 2 8 . 9 ± 1 . 7  5  PC-3  800  8  100±62/29.7±2.4  6  PC-3  1000  8  82±28 / 29.5±2.5  7  PC-3  1200  7  99±44/29.5±1.5  31  3  ''^i  It was generally observed that L N C a P rumors have a highly variable growth rate, therefore mice were integrated into this experiment i n a staggered fashion. Tumor volumes were monitored rigorously, until volumes greater than 7 0 m m were observed, at 3  which time the animals were allocated into one o f the seven treatment groups (n= 8 for the control, 150mg/kg, 800mg/kg, lOOOmg/kg groups, and n=7 for the 300mg/kg, 600mg/kg, 1200mg/kg groups). B o d y mass was also taken into consideration in . determining the distribution o f the animals into the treatment groups. Each treatment group had an average tumor volume o f 96-115mm and an average body mass o f 25.83  28.5g on day 1 o f the experiment. Table 2 illustrates the average tumor volumes and body mass o f animals within each o f the seven treatment groups. Statistical analysis by one way A N O V A showed no statistical difference between any o f the treatment groups in regards to average tumor volume or average body mass on day 1 (P>0.05).  Table 2. Summary o f the animal grouping within the L N C a P study. Average Tumor CsCl DostNumber of Volume ± S T D (mm ) Croup Tumor (nig/k« body wt) / Body Mass Mice/Group ± S T D (g), on Day 1 3  1  LNCaP  Vehicle (water)  8  114±35/26.9±2.3  2  LNCaP  150  8  110±36/26.4±3.1  3  LNCaP  300  • 7  113±50/26±3.5  4  LNCaP  600  7  96±23/25.8±3.2  5  LNCaP  800  8  115±61 / 2 6 . 6 ± 2 . 4  6  LNCaP  1000  8  108±32/28.5±2.1  7  LNCaP  1200  7  98±26/26.3±1.8  32  The 41 mice in the non-tumor bearing study were randomly distributed into the seven treatment groups according to body mass (n=6 for the control, 150mg/kg, 300mg/kg, 600mg/kg, 800mg/kg, lOOOmg/kg groups, and n=5 for the 120 mg/kg group). The average body mass in each treatment group was 26.3-27.6g. Table 3 illustrates the average body mass o f animals within each o f the seven treatment groups. Statistical analysis by one way A N O V A showed no statistical difference between any o f the treatment groups in regards to average body mass on day 1 o f the study (P>0.05).  Table 3. Summary o f the animal grouping within the non-tumor study. (•roup  1 tinior  ( s( 1 Dose (mg/kg body wt)  Number of Mice/Group  A\eraj»e Bodv Mass t STD (g), on Da\ 1  1  None  Vehicle (water)  6  27.6±4.0  2  None  150  6  27.5±2.7  None  300  6  27.1±2.8  4  None  600  6  27.4±2.8  5  None  800  6  26.2±2.3  6  None  1000  6  26.6±2.1  7  None  1200  5  26.6±1.8  • 3  '  Comparison by one way A N O V A showed no statistical difference i n average tumor volume in any o f the treatment groups o f the PC-3 and L N C a P studies on day 1 o f each study (P>0.05). In terms o f average body mass, only the 150mg/kg group o f the P C - 3 study was statistically different from other treatment groups o f the L N C a P (150mg/kg,  33  300mg/kg , 600mg/kg , 800mg/kg and 1200mg/kg) and non-rumor (800mg/kg) bearing studies (P>0.05); all other treatment groups showed no statistical difference from other treatment groups between the three studies.  2.2.4 Dose Selection and Drug Administration Most C s C l product instructions recommend that consumers use the product once or twice daily for months at a time via oral administration. In order to stay consistent with this drug regimen, animals were administered C s C l once daily via oral gavage. However, these animal experiments were limited to a 30 day duration due to growth o f tumors within the control groups o f the P C - 3 and L N C a P studies. The range o f C s C l dosages was determined based on several factors: 150mg/kg appears to exhibit no toxicity i n the nude mouse model based on previous studies in our laboratory; the oral L D  5 0  o f C s C l in mice is an acute dose o f 2306mg/kg (77); the half-  life o f C s C l i s - 3 - 6 days in mice (96, 97); a C s C l distribution study in mice conducted i n our laboratory found that C s C l is retained in major bodily organs 24h after administration, indicating a potential additive accumulation o f C s C l in these organs upon chronic dosing (72); companies advise patients to take 3-9g o f C s C l per day, which in terms o f body surface area translates to 527mg/kg-l 581 mg/kg i n mice (for a 70kg human). Considering these factors, six dosages o f C s C l were chosen to cover a range o f 0-1200mg/kg C s C l to be dosed daily via oral gavage: vehicle (water), 150mg/kg, 300mg/kg, 600mg/kg,. 800mg/kg, 1 OOOmg/kg or 1200mg/kg. Water was chosen as the vehicle for delivery o f C s C l in this study, as it is often used as the vehicle for commercial C s C l products In addition, C s C l is very soluble i n water  34  allowing for homogenous drug administration. Water is also an inert substance, which w i l l not contribute any therapeutic or toxicological effects; theoretically, the only effects observed in this study should be from C s C l . Dosages administered were corrected on a daily basis for changes in body mass by changing the dose volume administered to the animals. Table 4 illustrates the dosage volumes administered according to the weight o f a mouse.  Table 4. Dosage correction o f vehicle and C s C l solutions to compensate for differences in animal body mass Weight of animal (n) Dosage administered (pi) 24±1.5  80  27±1.5  90  30±1.5  100  33±1.5  110  36±1.5  120  2.2.5 M e a s u r i n g the Therapeutic Response of C s C l The therapeutic effect o f C s C l was monitored in the PC-3 and L N C a P studies by measuring tumor volumes. Calipers were used to determine the measurable linear axes and applying the formula 7i/6 x L x W x H (L:major axis, W:width, H:height). Tumor volume was measured twice weekly using calipers; once at the beginning o f the week and once at the end o f the week. In the L N C a P study, total tumor volume was not tracked on animals that had more than one tumor propagate. Only the tumor that was large enough  35  on day 1 o f the study (greater than 70mm ) was tracked, as secondary tumors usually 3  only became measurable as the study progressed. In addition, serum samples were removed from the L N C a P tumor bearing animals weekly via tail vein bleeds. The serum was intended to measure P S A , the levels o f which are normally correlated to changes in tumor volume in the L N C a P xenograft model. However, these tests were not completed due to restrictions in study length resulting from toxicity in the L N C a P bearing animals, and the lack o f an observed therapeutic effect from C s C l administration in this model.  2.2.6 M o n i t o r i n g Acute T o x i c i t y from C s C l Three endpoints representing acute toxicity were measured for within each study: body mass was measured daily prior to dosing and food and water consumption were measured every two days. A l l three o f these measurements were carried out using a top-loading scale. For body mass measurements, animals were placed directly on the scale. Food consumption was monitored by weighing the mass o f all uneaten food pellets. Water consumption was monitored by weighing the mass o f each water bottle; a decrease o f l g water would signify 1 m l o f water consumed assuming that water has a density o f ~ l g / m l . A n y animals displaying substantial decreases in body mass (>20% initial mass), shakiness, lethargy, and a decrease in food and water consumption were euthanized to minimize suffering, and their associated treatment group was no longer tracked throughout the study. Within these treatment groups that were no longer being tracked, animals that had not shown acute toxicity were treated until signs o f toxicity were observed. Thus, not all animals within these groups were euthanized on the same day, but were instead euthanized when a specific threshold o f toxicity was observed.  36  A l l tumor volume, body mass, food and water consumption measurements were conducted solely by the researcher. B y allocating these measurements to a single individual, the data collected from these studies are more concise than i f multiple researchers were making the measurements, as each would have their own measuring techniques.  2.2.7 Animal Euthanization and Tissue Harvesting O n the last day o f each study, mice were euthanized approximately 24 hours after the last administration o f vehicle or C s C l by carbon dioxide asphyxiation. Tumors, brains, hearts, lungs, livers, spleens, kidneys, small intestines, femurs, quadricep muscles and . prostates were removed from each animal. O n removal, each tissue was cut in half. One half was placed in formalin for histopathology and future tissue micro-array studies, and the other half was snap frozen for future C s tissue distribution studies using atomic absorption ( A A ) . Serum was collected from each animal via cardiac puncture for creatinine, A S T (aspartate aminotransferase), A L T (L-alanine:2-oxoglutarate aminotransferase), and A A analysis. Figure 4 illustrates the tissues and organs removed from each o f the animals i n A i m 1, and shows the subsequent experiments both completed and postulated (grey boxes) involving these tissues and organs.  37  Each Animal in Aim 1  Tumor, brain, heart, lung, liver, spleen, kidney, small intestine, femur, quadricep muscle, prostate  Fixed in formalin +. paraffin embedded  Serum  Snap frozen  AA  Histopathology  AST/Creatinine  Immunohistochemistry  Figure 4. Analysis o f tissues and serum obtained from the animals studies o f A i m 1. Experiments designated in the grey boxes are part o f future studies, the data o f which w i l l not be presented within this thesis.  38  2.3 Investigation of organ and tissue specific toxicity from CsCl (histopathology and serum marker tests) in human prostate tumor xenograft bearing nude mice After completing a preclinical study, it is important to further investigate the toxicity o f the compound administered; specifically, on different organs and tissues. The organ specific toxicity profile o f a chemotherapeutic compound is crucial for determining cross reactivity with other agents, for designing potential detoxification measures, and for developing targeted drug carrier molecules (assuming the compound o f interest has a therapeutic effect). A i m 2 was designed to delineate the cause o f potential acute toxicities and to develop an organ toxicity profile for C s C l through histopathological analysis and serum marker tests. A s the use o f the nude mouse model was consistent and conserved between the three studies, comparison could be made between treatment groups within the three studies and also between the three studies. B y making such intra-study and inter-study comparisons, the extent o f toxicity from each tumor alone, from C s C l alone, and from C s C l in combination with each o f the tumor types was investigated.  2.3.1 Histopathology Histopathology is regularly used to determine the toxicological profile o f pharmacological agents and diseases. For histopathological analysis, brains, hearts, lungs, livers, spleens, kidneys, small intestines and quadriceps were collected from each mouse and transferred into 10% formalin. The formalin was removed after 48 hours o f fixation and tissues were dehydrated in ethanol. For paraffin embedding, each tissue sample was  v enclosed in a plastic cassette, and placed i n an ethanol bath. The cassettes were  39  subsequently taken to V G H Pathology Laboratory (Vancouver, B C , Canada) for the embedding process. Once received from the pathology laboratory, the tissue containing paraffin blocks were placed on a cold plate for least one hour, and cut into 5 micron sections using a microtome. Each section was then placed on the surface o f 45°C water bath for 45 m i n before mounting onto slides. Slides were incubated at 37°C over night. The slides were subsequently stained with hematoxylin and eosin. Each slide was analyzed for organ specific changes by a pathologist who was blinded to the treatment groups. Analysis o f all o f the tissues collected would not be feasible in terms o f cost and time constraints; thus, only the tissues o f three animals from the control, 150mg/kg, 800mg/kg and 1200mg/kg groups from each o f the three studies were analyzed initially. If the pathologist recognized any abnormalities in a specific organ or tissue o f a specific animal, that organ or tissue o f interest was analyzed in all animals o f the control, 150mg/kg, 800mg/kg and 1200mg/kg o f all three studies to increase the n-values and determine i f the toxicological phenomenon was real.  2.3.2 S e r u m A S T a n d A L T Analysis Serum Aspartate Aminotransferase ( A S T ) and L-Alanine:2-Oxoglutarate Aminotransferase ( A L T ) levels were measured as secondary measures o f organ and tissue toxicity. A S T is present in the liver, heart, kidney, skeletal muscle and erythrocytes (106). Damage to any o f these organs and tissues results in the release o f A S T into the blood, and thus, increases blood A S T concentrations (106). A L T is concentrated in the  40  liver (107). Damage to the liver results in release o f A L T into circulation, and thus, increases blood A L T concentration. Standard A S T and A L T kinetic assay kits (Infinity™ A S T Liquid Stable Reagent and Infinity™ A L T Liquid Stable Reagent, respectively) were implemented in 96-well plates to quantify A S T and A L T serum levels. Plates were scanned using a Power Wave X microplate reader linked with K C 4 Kineticalc. Both o f these assays measure their respective markers indirectly by monitoring absorbance at 340nm which represents reduced nicotinamide adenine dinucleotide ( N A D H ) , and decreases i n signal intensity as N A D H is oxidized to nicotinamide adenine dinucleotide ( N A D ) over time (Figure 5). In these assays, the rate o f oxidization o f N A D H is dependent on the concentration o f A S T or A L T , which catalyze primary reactions producing Bronstead-Lowry bases (Oxaloacetate in the A S T catalyzed reaction and Pyruvate in the A L T catalyzed reaction), which accept protons from N A D H (Figure 5). Before any o f the serum samples were used, these assays were validated using mouse serum obtained from other experiments. The main variables that were considered were the volumes o f serum and test reagents used and the incubation times. In addition, intraday and interday variability o f the assays were determined.  2.3.3 Serum Creatinine Analysis Serum creatinine was measured as a secondary measure of kidney toxicity. Creatine produced by the liver, kidneys and pancreas is converted to creatinine in muscle; the amount o f creatinine produced depends on the amount o f muscle tissue (Figure 6) (108). In the absence o f disease, kidneys secrete creatinine at a constant rate. However, during  41  end stage renal failure, the glomerular filtration rate w i l l decline leading to a decrease i n creatinine secretion (108).  A S T Assay ^ - A s p a r t a t e •*• 2 - O x o g l u t a r a t e -—->* O x a l o a c e t a t e •#- LHGIutamate O x a l o a c e t a t e -4- N A D H ••  W£>H  >  Mafate * N A D  ALT Assay ALT' -•>L»A l a n i n e + 2 - O x o p l t J t a r a t o Pyrawate* N A D H  -—•  Pyruvato + —».  L-Qlutamato  L-Lactate + N A D  "igure 5. A S T (above) and A L T (below) catalyzed chemical reactions that take place once serum is added to the A S T and A L T kinetic assay kits. A S T and A L T are measured indirectly by monitoring absorbance at 340nm which decreases as N A D H is oxidized to N A D over time. (Figure modified from the Thermo Electron Infinity™ A S T ( G O T ) Liquid Stable Reagent product insert (106) and the Thermo Electron Infinity™ A L T (GPT) Liquid Stable Reagent product insert (107)) Picric acid based creatinine assays are typically used to assess serum creatinine concentrations in a number o f species. However, mouse serum contains chromagens which interfere in these picric acid based creatinine assays, resulting in overestimations o f serum creatinine levels (109, 110). T o assess serum creatinine in the nude mice, a high performance liquid chromatography ( H P L C ) method was developed. Creatinine levels in serum were quantified using an H P L C method developed on a Waters 2695 Separations Module paired with a Waters 996 Photodiode Array Detector. The column chosen for this assay was a 3pm bore diameter 2.1x 50mm Waters Atlantis™ H I L I C Silica column. The mobile phase used was a 97.5% acetonitrile solution  42  containing 0.025% ammonium formate. Creatinine standards (Oug/ml, 5{j,g/ml, 1 Oug/ml, 20ug/ml, 40ug/ml and 80ug/ml) were created in 94.5% acetonitrile from a creatinine 2mg/ml stock solution. For sample preparation, lOul o f serum was added to 190ul o f 99.5% acetonitrile, vortexed, and centrifuged at 15000g at 4 ° C for 5min. The supernatant (-94.5% acetonitrile) from each sample was collected and placed in a H P L C autosample vial for injection. Duplicate injections o f 80ul were performed for each standard and sample. The flow rate was maintained at l m l / m i n resulting in the creatinine peak to elute at ~2min, which was monitored with an ultraviolet absorbance o f 238nm. After every ten injections a gradient elution was run for lOmin to clean the column and a quality control sample was injected to monitor the consistency o f the method. Table 5 is a summary o f the parameters used in this protocol.  Table 5. Parameters o f the high performance liquid chromatography protocol developed to assess creatinine concentrations in murine serum. Column Type 3um 2.1x 50mm Waters Atlantis™ H I L I C Silica column 97.5% acetonitrile solution Mobile Phase containing 0.025% ammonium formate lml/min Flow Rate Sample Volume  80ul  Wavelength Analyzed  238nm  The H P L C method was validated using excess mouse serum collected from other experiments ongoing in the lab. This test serum was used to determine the optimum column type, mobile phase, sample preparation method, concentrations o f creatinine  43  standards, flow rate, ultraviolet absorbance frequency, and column cleaning procedure. In addition, intraday and interday variability o f the assay was determined. Serum from control, 150mg/kg, 800mg/kg and 1200mg/kg animals were analyzed to maintain consistency with the histopathology analysis.  Kidney, liver and pancreas  Brain and muscles  Creatine  Phosphocreatine  Muscles Creatinine  Kidneys  Excreted Figure 6. A summary o f the pathways involved i n the production and excretion o f creatinine.  2.4 Comparisons between Control Animals Once the three animal studies were completed, it was evident that the two different tumor types were contributing different levels o f toxicity in the nude mouse model. This phenomenon was explored by comparing the data from the acute toxicity measurements (body mass, food consumption, water consumption and tumor volume), histopathological analysis and serum marker tests ( A S T , A L T and creatinine) between the three control groups from the three animal studies.  44  These experiments were warranted as all animals within the three animal studies experienced the same living, handling and dosing conditions. In addition, all animals analyzed were terminated on day 30 o f their respective studies at which time tissues and serum were harvested.  2.5 Statistical Analysis and Removal of Outliers A l l o f the data collected from each experiment was analyzed using the SigmaStat 3.0 software. Groups were first compared by one-way A N O V A . If statistical significance was observed (P<0.05), a subsequent Tukey Test was implicated to determine which groups were significantly different from each other. For each o f the parameters measured, treatment groups would sometimes have a single animal, which would not follow the same trends as the rest o f the group. In this study, these animals were considered outliers i f the majority o f results collected from the animal (results from multiple days) did not fall in the designated range: (Group Average + 2 Standard Deviations (STD)) - (Group Average - 2STD). The results collected for identified outlier animals were not included in group averages and calculations o f variance.  45  Section 3 Results  46  3.1 Tumor growth and toxicity in the PC-3 and LNCaP human prostate tumor xenograft models, after administration of escalating doses of CsCl.  3.1.1 P C - 3 Solid T u m o r Xenograft Study The study o f C s C l in PC-3 bearing mice produced some very interesting results at the highest doses tested. M i c e administered lOOOmg/kg and 1200mg/kg C s C l displayed a significant reduction in % average initial body mass compared to the control, from day 5 and day 8 onwards, respectively (Figure 7). However, the magnitude o f changes in body mass observed upon C s C l administration did not extend beyond the designated cutoff considered to be toxic (% average initial body mass was not reduced below 80% for any of the animals); thus, euthanization was not performed. In terms o f food consumption, there was no difference between any o f the treatment groups and the control (Figure 8). Alternatively, water consumption increased significantly in the 1200mg/kg, lOOOmg/kg and 800mg/kg groups compared to the control, from day 3 onwards for the 1200mg/kg and 1000 mg/kg groups and from day 17 onwards for the 800mg/kg group (Figure 9). The 300 mg/kg group also displayed a significant elevation in water consumption compared to the control, from day 3 to day 10 (Figure 9). A significant reduction in % average initial tumor volume was observed in the three highest dosed groups when compared to the control. The 1200mg/kg group displayed a significant reduction in tumor growth compared to the control from day 5 onwards (Figure 10).  47  10% C o n t r o l $  105%  1 5 0 m g / k g  (n=8)  3 0 0 m g / k g  (n=8)  -*- 6 0 0 m g / k g  (n=7)  8 0 0 m g / k g  (n=8)  CO  O 100% .Q  "(5  (n=9)  95% Ui (0  L_ >  0)  90%  1 0 0 0 r r g / k g  (n=8)  1 2 0 0 r r g / k g  (n=7)  CO  -+85%  -|-  1  i — T — i  r — i  1  1  r  13  ~i  1  I  17  Time (days)  1  1  1  21  1  r  25  29  Figure 7. Percent average initial body mass over time o f nude mice bearing a P C - 3 tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg C s C l ) . A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates a significant difference between the control and treatment groups beginning on day 5 and extending to the end o f the study (P<0.05). Pair-wise multiple comparison v i a Tukey test indicates a difference between the 1200mg/kg group and the control from day 5 until the end o f the study (P<0.05), and a difference between the lOOOmg/kg group and the control from day 8 until the end o f the study (P<0.05).  85 . -•- C o n t r o l  (n=9)  80 . 150  m g / k g  (n=8)  300  m g / k g  (n=8)  600  m g / k g  (n=8)  - * - 800  m g / k g  (n=8)  c  .2  75 .  a  E 3  70 .  c o o o  65 .  ° Q) O)  60 .  ©  55 .  >  <  -+-  1000  m g / k g  (n=8)  1200  m g / k g  (n=7)  50 . 1+2  5+6  9^10  13+14  Time (days)  17+18  2 1 + 2 2  2 5 + 2 6  Figure 8. Average food consumption over time o f nude mice bearing a P C - 3 tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg C s C l ) . A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. There was no statistical difference between any o f the groups (P>0.05).  15.0 -•- C o n t r o l  c o  13.0  |  (n=9)  HB- 1 5 0 m g / k g  (n=8)  11.0  3 0 0 r r g / k g  (n=8)  9.0  6 0 0 m g / k g  (n=8)  8 C X D m g / k g  (n=8)  3 CO  c o o  1 o  o O)  2  a)  > <  7.0 5.0 3.0  3+4  7+8  11+12  15+16  19+20  1 0 0 0 r r g / k g  (n=8)  -H- 1 2 0 0 m g / k g  (n=7)  23+24  T i m e (days) Figure 9. Average water consumption over time o f nude mice bearing a PC-3 tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg CsCl). A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates a significant difference between the control and treatment groups beginning on day 3 and extending to the end o f the study (P<0.05). Pair-wise multiple comparison via Tukey test indicates a difference between the 1200mg/kg group and the control from day 3 until the end o f the study (P<0.05), a difference between the lOOOmg/kg group and the control from day 3 until the end o f the study (P<0.05), a difference between the 800mg/kg group and the control from day 17 until the end o f the study (P<0.05), and a difference between the 300mg/kg group and the control from day 3 to day 10 (P<0.05).  1200% C o n t r o l  (n=9)  | 1000% 1 5 0 m g / k g ( n = 8 )  o > o E  800%  3 0 0 m g / k g  (n=8)  3  600%  6 0 0 m g / k g  (n=7)  •-  400%  8 0 0 r r g / k g  (n=8)  0)  O)  (0 o > ro  200%  -•- 1 0 0 0 m g / k g  (n=7)  -+-  (n=7)  1 2 0 0 m g / k g  0% 1  8  12 15 19 Time (days)  22  26  29  Figure 10. Percent average initial tumor volume over time o f nude mice bearing a PC-3 tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg CsCl). A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates a significant difference between the control and treatment groups beginning on day 5 and extending to the end o f the study (P<0.05). Pair-wise multiple comparison via Tukey test indicates a difference between the 1200mg/kg group and the control from day 5 until the end o f the study (P<0.05), a difference between the lOOOmg/kg group and the control from day 26 until the end o f the study (P<0.05), and a difference between the 800mg/kg group and the control from day 12 to the end o f the study (P<0.05).  Likewise the lOOOmg/kg and 800mg/kg groups showed significant reductions in tumor growth compared to the control from day 26 and day 12 onwards, respectively (Figure 10).  3.1.2 L N C a P Solid T u m o r Xenograft Study Administration o f C s C l in combination with the effects o f L N C a P tumor burden was quite toxic in the nude mouse model. The 1200mg/kg, lOOOmg/kg and 800mg/kg treatment groups were removed from the study, when one or more animals in each group saw a reduction in body mass below 80% o f their initial body mass and were subsequently euthanized (Figure 11). The 1200mg/kg and lOOOmg/kg groups were both discontinued on day 15 and the 800mg/kg group was removed on day 19 (Figure 11). The animals in these groups that did not show this significant reduction in body mass on the described days remained in the study until this toxicity point was reached. O n average, animals from the 1200mg/kg and lOOOmg/kg groups remained i n the study for 21 days and animals o f the 800 mg/kg group remained in the study for 27 days. Although the 1200mg/kg, lOOOmg/kg and 800mg/kg groups were removed prior to the completion o f the study, each o f these groups showed a significant reduction in % average initial body mass compared to the control. This reduction in body mass was first observed on day 6 for the 1200mg/kg group, day 7 for the 1000mg/kg group, and day 16 for the 800mg/kg group, and remained significantly different for each group.until their removal from the study (Figure 11). There was no significant difference in food consumption or water consumption between any o f the treatment groups and the control (except on day 3+4 where the 600mg/kg group had a significant increase in food consumption) (Figures 12, 13).  52  110% C o n t r o l  w 105% w to  (n=8)  •*- 1 5 0 r r g / k g  (n=8)  -±-  3 0 0 r r g / k g  (n=7)  6 0 0 m g / k g  (n=7)  -*- 8 0 0 m g / k g  (n=8)  ^ 100% O  £  (0  95% 90%  a) (0  > (0  85% -•- 1 C ) 0 0 r n g / k g  (n=8)  —t— 1 2 C ) 0 r r g / k g  (n=6)  80% 75% Time (days)  Figure 11. Percent average initial body mass over time o f nude mice bearing a L N C a P tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg, lOOOmg/kg and 1200mg/kg treatment groups were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates a significant difference between the control and treatment groups beginning on day 6 and extending to day 19 (P<0.05). Pair-wise multiple comparison via Tukey test indicates a difference between the 1200mg/kg group and the control from day 6 to day 15 (P<0.05), a difference between the lOOOmg/kg group and the control from day 7 to day 15(P<0.05), and a difference between the 800mg/kg group and the control from day 16 to day 19 (P<0.05).  120.0 C o n t r o l  o 10.00 a  E  (n=8)  -m- 1 5 0 r r g / k g  (n=8)  3 0 0 r r g / k g  (n=7)  6 0 0 r r g / k g  (n=7)  8 0 0 m g / k g  (n=8)  3  W c  o 8.00 o ~o o o o  6.00  03  o >  <  -•- 1 0 0 0 m g / k g  (n=8)  1 2 0 0 m g / k g  (n=7)  4.00  />  ^ \>  jP  ^ v>  ^  jP v  $> ^  o* (jf  Q  Time (days) Figure 12. Average food consumption over time o f nude mice bearing a L N C a P tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg, lOOOmg/kg and 1200mg/kg treatment groups were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates a significant difference between the control and treatment groups on day 3+4 (P<0.05). Pair-wise multiple comparisons via Tukey test indicates a difference between the 600mg/kg group and the control (P=0.008).  13 C o n t r o l C  O  11  1 5 0 m g / k g  (n=8)  3 0 0 r r g / k g  (n=7)  6 0 0 r r g / k g  (n=7)  -*- 8 0 0 m g / k g  (n=8)  a E 3  W c  o o o  —  +•1  (n=8)  (0  $  a>  o>  -+-  re i_  1 0 0 0 m g / k g  (n=8)  1 2 0 0 m g / k g  (n=7)  a> >  < Time (days) Figure 13. Average water consumption over time o f nude mice bearing a L N C a P tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg, lOOOmg/kg and 1200mg/kg treatment groups were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%. Results are represented as mean value ± standard error o f the mean. There was no statistical difference between any o f the groups (P>0.05).  600% C o n t r o l  £ 500%, O  t 400% o  £  r  3oo%  (n=7)  1 5 0 m g / k g  (n=7)  3 0 0 r r g / k g  (n=6)  6 0 0 r r g / k g  (n=7)  8 0 0 r n g / k g  (n=8)  CO  •- 200% <o § 100%) — — +  (0  1 C O 0 m g / k g  (n=8)  1 2 0 0 r r g / k g  (n=7)  0% 8  12 15 19 Time (days)  22  26  29  Figure 14. Percent average initial tumor volume over time o f nude mice bearing a L N C a P tumor xenograft for control (vehicle) and treatment groups (150mg/kg-1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg, lOOOmg/kg and 1200mg/kg treatment groups were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%. Results are represented as mean value ± standard error o f the mean. There was no statistical difference between any o f the groups (P>0.05  In terms o f water consumption, there appears to be a trend that suggests an increase in water consumption in the two groups that received the highest concentrations o f C s C l , four days prior to their removal from the study (Figure 13) Unlike the P C - 3 xenograft, the L N C a P xenograft was less responsive to the C s C l treatment. Only on day 5 did any o f the treatment groups have a significant reduction in % average initial tumor volume compared to the control group (lOOOmg/kg, 800mg/kg and 600mg/kg groups) (Figure 14).  3.1.3 Non-tumor Bearing Nude Mouse Study The third study involving non-tumor bearing mice provided insight into the toxicity o f C s C l alone without a confounding tumor burden. When C s C l was chronically administered to these animals, it was discovered that none o f the treatment groups displayed any significant difference in % average initial body mass when compared to the control (Figure 15). Even animals receiving the highest concentrations o f C s C l , showed a similar increase in body mass compared to the control mice, which was not observed in either o f the previous two studies. There was no difference in food consumption between any o f the treatment groups and the control animals (Figure 16). However, water consumption was significantly elevated beginning on day 15 in animals receiving 1200mg/kg and lOOOmg/kg C s C l (Figure 17). Water consumption was also elevated in animals receiving 800mg/kg C s C l from day 21 to 27 (Figure 17).  57  125% C o n t r o l  g 120% E  •§ 115% JQ  1  110%  o (0  a5 105% > CO  (n=6)  1 5 0 m g / k g  (n=6)  3 0 0 m g / k g  (n=6)  6 0 0 m g / k g  (n=6)  8 0 0 m g / k g  (n=6)  -•- 1 0 0 0 r r g / k g  (n=6)  1 2 0 0 m g / k g  (n=5)  100% Time (days) Figure 15. Percent average initial body mass over time o f tumor free nude mice for control (vehicle) and treatment groups (150mg/kg1200mg/kg C s C l ) . A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates no difference between the control and treatment groups for the duration o f the study (P>0.05).  12.00  3  11.00  C o n t r o l  •**  10.00  1 5 0 m g / k g ( n = 6 )  a E  9.00  3 0 0 m g / k g  (n=6)  (0  8.00  6 0 0 m g / k g  (n=6)  o  7.00  o o  8 0 0 m g / k g  (n=6)  6.00  c O  3  c o  •a  a> O)  «j k.  o  >  <  (n=6)  -•- 1 0 0 0 r r g / k g  (n=6)  — 1 2 0 0 m g / k g  (n=5)  5.00 4.00 SI  Jp \  Time (days)  Figure 16. Average food consumption over time o f tumor free nude mice for control (vehicle) and treatment groups (150mg/kg1200mg/kg C s C l ) . A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. There was no statistical difference between any o f the groups ( P > 0 . 0 5 ) .  15.00 -•- C o n t r o l  14.00  c 13.00  O a E  l_  o o O) (0  o >  <  -*• 1 5 0 m g / k g  (n=6)  3 0 0 m g / k g  (n=6)  6 0 0 m g / k g  (n=6)  8 0 0 m g / k g  (n=6)  12.00  w c 11-00  O o  (n=6)  10.00 9.00 8.00 7.00  1 0 0 0 r r g / k g  (n=6)  6.00  1 2 0 0 m g / k g  (n=5)  5.00  Time (days) Figure 17. Average water consumption over time o f tumor free nude mice for control (vehicle) and treatment groups (150mg/kg1200mg/kg C s C l ) . A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. Statistical analysis by way o f one way A N O V A indicates a significant difference between the control and treatment groups beginning on day 15 and extending to the end o f the study (P<0.05). Pair-wise multiple comparison via T u k c y test indicates a difference between the 1200mg/kg group and the control from day 15 until the end o f the study (P<0.05), a difference between the 1 OOOmg/kg group and the control from day 15 until the end o f the study (P<0.05), and a difference between the 800mg/kg group and the control from day 21 to day 27 (P<0.05).  3.2 Investigation of organ and tissue specific toxicity from CsCl (histopathology and serum marker tests) in human prostate tumor xenograft bearing nude mice  3.2.1 Histopathology Tissues were collected from each o f the animals in the three C s C l dose titration studies for histopathological analysis. O f the eight tissues examined, abnormalities were only observed in the hearts and small intestines. Fibrin clots present in the aorta, atrium or ventricles were found in animals from all three studies (Figure 18). Although fibrin clots can form post-mortem, there appears to be a higher incidence o f fibrin clots in animals receiving 800-1200mg/kg C s C l compared to control animals in both the P C - 3 and L N C a P studies (Figure 19). This same trend was not observed in mice without tumors; fibrin clots were present i n 33% o f the animals within in each treatment group (Figure 19). Atrophy o f the mucosal epithelium and v i l l i , as well as the presence o f inflammatory infiltrate o f the submucosa o f the small intestine were observed in both control and C s C l treated mice o f the three studies (Figure 20). There appeared to be no evident trend in the incidence o f these abnormalities across the different treatment groups and the control group in each o f the three studies (Figures 21, 22, 23).  61  F  Figure 18. Images o f cardiac fibrin clots discovered post-mortem in a nude mouse o f the PC-3 study.  = r  ilc»r in ,  J  C l o t  PC-3 bearing  mice  1  I  150mg/kg  800mg/kg  1200mg/kg  T r e a t m e nt  LNCaP bearing mice  V  150mg/kg  800mg/kg  1200mg/kg  Treatm ent  Mice without  V  tumors  150mg/kg  800mg/kg  1 200mg/kg  Treatment  Figure 19. Percentage o f nude mice from the three studies (PC-3, L N C a P , non-tumor bearing) with the presence o f post-mortem fibrin clots for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, 1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg and 1200mg/kg treatment groups o f the L N C a P study were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%  63  S m a l l Intestine  I n f l a m m a t o r y Infiltrate 2  Atrophic Figure 20. Image o f abnormalities o f the small intestine discovered post-mortem i n a nude mouse o f the P C - 3 study  PC-3 bearing  mice  100% 90% 80% 70% 60% 50% 40% 30% 20°/o 10% 0% V  150mg/kg  800mg/kg  1200mg/kg  Treatment  LNCaP bearing mice 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%  lllllllliliP^  l^plBJi BiiiSiP^  (jjljlB  800mg/kg  1200mg/kg  BilBiBillil V  150mg/kg  T r e a t m e nt  Mice without  tumors  100% 80% 60% 40% 20% 0%  JL  v  150mg/kg  800mg/kg  1200mg/kg  Treatment  Figure 21. Percentage o f nude mice from the three studies (PC-3, L N C a P , non-tumor bearing) with the presence o f atrophy o f mucosal epithelium o f the small intestine for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, 1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg and 1200mg/kg treatment groups o f the L N C a P study were removed halfway through the study because t h e % average initial body mass o f the mice dropped below 80%. '  65  PC-3 bearing mice 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% V  1 50mg/kg  800mg/kg  1200mg/kg  Treatment  LNCaP bearing mice 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% V  150mg/kg  800mg/kg  1200mg/kg  Treatm ent  Mice without  tumors  100% 80% 60% 40% 20% 0% V  150mg/kg  800mg/kg  1 200mg/kg  Treatm ent  Figure 22. Percentage o f nude mice from the three studies (PC-3, L N C a P , non-tumor bearing) with the presence o f atrophy o f villi o f the small intestine for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, 1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg and 1200mg/kg treatment groups o f the L N C a P study were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%.  66  PC-3 bearing  mice  100% 80% 60% 40% 20%  0% V  150mg/kg  800mg/kg  1200mg/kg  Treatment  LNCaP bearing mice 100% 90%  5  80% 70% 60% 50% 40% 30% 20% 10% 0% V  150mg/kg  800mg/kg  1200mg/kg  Treatment  Mice without  V  tumors  150mg/kg  800mg/kg  1200mg/kg  Treatment  Figure 23. Percentage o f nude mice from the three studies (PC-3, L N C a P , non-tumor bearing) with the presence o f inflammatory infiltrate o f the small intestine for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, I200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg and I200mg/kg treatment groups o f the L N C a P study were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%.  67  3.2.2 Serum A S T / A L T Serum collected from animals in the three C s C l dose titration studies was analyzed for A S T and A L T levels as secondary measures o f cellular and tissue damage. Specifically, these two markers were used to assess damage to the heart, liver, skeletal muscle, kidney and erythrocytes. Serum from the C s C l treated P C - 3 , L N C a P and non-tumor bearing mice showed no difference i n the levels o f these markers as compared to the control animals within each study (Figures 24, 25, 26).  3.2.3 Serum Creatinine Serum collected from the animals was also analyzed for creatinine levels as a measure o f kidney toxicity. Figure 27 illustrates the chromatograms o f a creatinine spiked standard and o f a serum sample. In each chromatogram, the peak eluting just after 2 minutes is creatinine (Figure 27). The peak that elutes before creatinine is cytosine (~1.69 min), which was intended to be used as an internal standard until problems arose with variable basal levels o f cytosine present i n the serum samples (Figure 27). The two peaks that elute after creatinine in the serum sample are unknown artifacts (Figure 27). Figure 28 represents the standard curve developed for the serum creatinine assay. One can observe the highly linear relationship between peak area and creatinine concentration ( R = 0.9997) (Figure 28). The Limit o f Detection ( L O D ) and Limit o f Quantification 2  ( L O Q ) o f this assay were 1.27ug/ml and 4.23ug/ml, respectively. The intraday and interday Percent Coefficient o f Variance ( % C V ) were 3.16% and 0.93%, respectively.  68  Serum AST 50 45 40 35  3  30  mi Klill  iBlillli  20 15  Sll  ^lillil  25 -  ilffpl 1111  -  JjjjP i l l jlilll llJii iwlit  10 5  IBIlIIiii  0 Control  Billilltill  <HI1H  150mg/kg  800mg/kg  1200mg/kg  800mg/kg  1200mg/kg  Treatment  Serum ALT 35 30 25  3.  20  10  i  5 0 Control  150mg/kg Treatment  Figure 24. Serum A S T and A L T levels o f nude mice bearing a P C - 3 xenograft for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, 1200mg/kg C s C l ) . A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates no difference in A S T or A L T levels between the control and treatment groups (P=0.726, P=0.067, respectively).  69  S e r u m AST 160 140 120  f  100  lllillllllii  80 60 40 20 0  Control  150mg/kg  800mg/kg  1200mg/kg  Treatment  Serum ALT 60 50  £• 3 0 S  5 <  2 0 10 0  Control  150mg/kg  800mg/kg  1200mg/kg  Treatment  Figure 25. Serum A S T and A L T levels o f nude mice bearing a L N C a P xenograft for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, 1200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg and 1200mg/kg treatment groups were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates no difference i n A S T or A L T levels between the control and treatment groups (P=0.736, P=0.569, respectively).  70  Serum AST 80 70 60 ^  50  & 40 > |  30 20 10 0  Control  150mg/kg  800mg/kg  1200mg/kg  800mg/kg  1200mg/kg  Treatment  Serum ALT 40 35 30 3" 25 20 |  1510 5 0 Control  150mg/kg Treatment  Figure 26. Serum A S T and A L T levels o f tumor free nude mice for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, 1200mg/kg C s C l ) . A l l mice were administered vehicle or C s C l daily for 30 days. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates no difference in A S T levels between the control and treatment groups (P=0.085). However, there was a statistical difference in A L T levels between the 150mg/kg and 800mg/kg group (P 0.029).  71  Figure 27. Chromatograms o f a creatinine spiked standard (above) and a serum sample (below) using Hydrophilic Interaction Chromatography. In the two chromatograms, cytosine elutes at ~1.69 m i n and creatinine elutes at -2.06 min. In the chromatogram o f the serum sample, the two substances that elute at -2.39 and - 2 . 8 0 m i n are unknown artifacts.  72  0  10  20  30  40  50  60  70 80  Creatinine (ug/ml) Figure 28. Standard curve for the serum creatinine H P L C assay. Standards were created in 94.5% acetonitrile. The standard curve has R = 0.9997, a L O D = 1.27ug/ml and L O Q = 4.23ug/ml. The intraday and interday % C V for this assay were 3.16% and 0.93%, respectively. 2  73  PC-3 bearing m i c e  i  r8  18 16 14 12 10 8 6 4 2 0 Control  150mg/kg  800mg/kg  1200mg/kg  Treatm ent  L N C a P bearing mice 16 14 12 10 8 6 4 2 0 150mg/kg  800mg/kg  1200mg/kg  Treatment  M i c e without t u m o r s 14 -,  C  150mg/kg  800mg/kg  1200mg/kg  Treatment  Figure 29. Serum creatinine levels o f nude mice from the three studies (PC-3, L N C a P , non-tumor bearing) for control (vehicle) and treatment groups (150mg/kg, 800mg/kg, I200mg/kg C s C l ) . Most mice were administered vehicle or C s C l daily for 30 days; the 800mg/kg and 1200mg/kg treatment groups o f the L N C a P study were removed halfway through the study because the % average initial body mass o f the mice dropped below 80%. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates no difference between the control and treatment groups for any o f the studies (P>0.05).  74  There was no difference in serum creatinine levels between the treatment groups and the controls within each o f the three studies (Figure 29).  3.2.4 B l a d d e r C r y s t a l s a n d V i s c o u s M u c o s a l F l u i d  During the excision o f prostates from the PC-3 animals, it became evident that high levels o f inflammation were present in the bladders o f animals that had received the higher doses o f C s C l . Further investigation revealed the presence o f crystals or viscous mucosal fluid in the bladders o f these animals (Figure 30). On completion o f study two and three, it was realized that the presence o f these crystals or viscous mucosal fluid were not restricted solely to the PC-3 study, but were present in animals from all three studies. These bladder abnormalities only existed in animals that had been administered C s C l at concentrations o f 600mg/kg and higher (Figure 31).  Figure 30. Image o f crystal structures removed from the bladders o f nude mice treated with 600mg/kg-1200mg/kg C s C l daily. These crystals were discovered in animals from the P C - 3 , L N C a P and non-tumor studies. In the PC-3 and non-tumor studies, these crystals were removed from the animals on day 30 during tissue collection. However, in the L N C a P study these crystals were observed as early as day 15 in animals that were euthanized (lOOOmg/kg and 1200mg/kg treatment groups).  75  100%  J I 80% H 0 90%  JS  70%  u  60%  I•o m  3  3  B M i c e without tumour  50%  M i c e with P C - 3 tumour  40% 30% 20%  •  M i c e with L N C a P tumour  10% 0 % -. 300mg/kg  600mg/kg  800mg/kg  1000mg/kg  1200mg/kg  Treatment Figure 31. Percentage o f nude mice with the presence o f bladder crystals or viscous mucosal fluid in the P C - 3 , L N C a P and non-tumor studies. In the PC-3 and non-tumor studies, these crystals were removed from the animals on day 30 during tissue collection. However, in the L N C a P study these crystals were observed as early as day 15 in animals that were euthanized (lOOOmg/kg and 1200mg/kg treatment groups). Results are represented as percentage o f mice per treatment group with the presence o f bladder crystals or pus. Bladder crystals or pus may have been present in the 600mg/kg group from the PC-3 study, but unfortunately were overlooked in these animals.  3.3 Comparison of Control Animals U p o n completion o f the three studies, it was apparent that the PC-3 and L N C a P xenograft models used had different tumor growth characteristics and were contributing to different levels o f toxicity i n the nude mice. The PC-3 xenograft model used was contrived from a single subcutaneous inoculation o f PC-3 cells while the L N C a P xenograft model used was contrived from inoculation in two subcutaneous sites with L N C a P cells. B y the end o f the study, each L N C a P bearing mouse either represented a single tumor, or a large tumor and significantly smaller tumor. The large tumors, which were measurable on day 1 o f the L N C a P study, were the only tumors tracked on animals that had two tumors propagate; the smaller tumors normally only became measurable by the end o f the study, and thus were not measured throughout the study. Through comparison o f tumor results from the control group o f the PC-3 study with the control group o f the L N C a P study, one can observe the different growth characteristics o f each tumor type relative to the other. Figure 32 illustrates the difference in average tumor volume between the PC-3 and L N C a P control groups. Although, there is no significant difference in average tumor volume between the PC-3 and L N C a P tumors when compared over a 29 day period, statistically the average tumor volume o f the PC-3 group begins to depart from that o f the L N C a P group from day 19 as indicated by a decreasing p-value (Figure 32). Figure 33 illustrates the difference in % average initial tumor volume between the PC-3 and L N C a P control groups. The L N C a P group shows a significant reduction in % average initial tumor volume when compared to the PC-3 group by day 29 (P=0.011) (Figure 33)..  77  Figure 32. Average tumor volume over time o f nude mice representing a PC-3 or L N C a P xenograft. Results are represented as mean value ± standard error o f the mean. There was no statistical difference between the two groups (P>0.05).  1200% d)  Time (days) Figure 3 3 . Percent average initial tumor volume over time o f nude mice representing a P C - 3 or L N C a P xenograft. Results are represented as mean value ± standard error o f the mean. Statistical analysis using a Students T-Test indicates a significant difference between the two groups by the end o f the study ( P = 0 . 0 1 1 ) .  Comparison o f the control groups from all three studies provided insight into the systemic burden elicited by each o f the specific tumor models relative to the non-tumor bearing mice. Although there was no difference observed i n food and water consumption between the three control groups (Figures 34, 35), there was a significant difference in % average initial body mass between the L N C a P animals and the animals from the other two studies (Figure 36). A dramatic loss o f body mass was observed i n the L N C a P animals beginning on day 3, which is not observed in the other two control groups (P<0.05). This decrease in body mass appears to be inversely proportional to the increase of the L N C a P tumors in these animals (Figures 32, 36). This same trend was not observed in the P C - 3 bearing animals (Figures 32, 36). Comparison o f the serum from the control animals from the three studies provided some interesting insights on cellular and tissue damage possibly linked to the different tumor models. Serum from the L N C a P animals had significantly elevated A S T levels (P<0.001), but showed no differences in A L T and creatinine levels when compared the serum from the other two control groups (Figure 37, 38). The histopathological results from the control animals o f three studies provide a trend in terms o f intestinal abnormalities. The P C - 3 animals have a consistently higher prevalence o f atrophy to their intestinal mucosal epithelium and v i l l i , as well as a higher prevalence o f intestinal submucosal infiltrate when compared to the other two control groups (Figures 39, 40, 41). In terms o f cardiac abnormalities, the P C - 3 bearing animals had slightly lower prevalence o f the fibrin clots compared to the other two control groups (Figure 42).  80  10  Mice without tumour (n=6)  c o a E  8  3 (A C  Mice with P C - 3 tumour (n=9)  J  o o o o  1/  a>  r  1  Mice with L N C a P tumour (n=8)  O)  >  <  * >°  <y  N  *f  4  X  *  <9  Time (days) Figure 34. Average food consumption over time o f nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as mean value ± standard error o f the mean. There was no statistical difference between any o f the groups (P>0.05).  Time (days) Figure 35. Average water consumption over time o f nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as mean value ± standard error of the mean. There was no statistical difference between any o f the groups (P>0.05).  120%  Time (days) Figure 36. Percent average initial body mass over time o f nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates a significant difference between the treatment groups beginning on day 3 and extending to the end o f the study (P<0.05). Pair-wise multiple comparison via Tukey test indicates a difference between the L N C a P control group and the other two control groups from day 3 until the end o f the study ( P O . 0 5 ) .  Serum A S T  H Mice w ithout Tumors  Mice w ith P C - 3 Tumors  • Mice w ith L N C a P Tumors  Treatment  Serum A L T 40 -, H Mice w ithout Tumors  35 30 zr  3 >»  Act  \>  25 -  Mice w ith P C - 3 Tumors  20 15 • Mice w ith L N C a P Tumors  10 5  n Treatment  Figure 37. Serum A S T and A L T levels o f nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates a significant difference i n A S T between the treatment groups (P<0.05). Pair-wise multiple comparisons v i a Tukey test indicates a difference in A S T levels between the L N C a P group and the other two groups (P<0.001). There is no statistical difference in A L T levels between any o f the treatment groups (P = 0.068).  84  16 14 12 10  8 6  -I  4 2 0  Non-Tumor  PC-3  LNCaP  Treatment  Figure 38. Serum creatinine levels o f nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as mean value ± standard error o f the mean. Statistical analysis using one way A N O V A indicates no statistical difference between any of the treatment groups (P=0.107).  Non-tumor  PC-3  LNCaP  Treatment  Figure 39. Presence o f atrophy o f the intestinal mucosal epithelium in nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as percentage o f mice per treatment group with the presence o f atrophy o f the intestinal mucosal epithelium  85  100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%  Non-tumor  PC-3  LNCaP  Treatment  Figure 40. Presence o f atrophy o f the intestinal villi in nude mice from the control groups of the three C s C l dose titration studies. Results are represented as percentage o f mice per treatment group with the presence o f atrophy o f the intestinal v i l l i  WmmBBmKm  90% 80% 70% 60% 50% 40% 30% 20% 10% 0%  IMIHiilil  Non-tumor  PC-3  LNCaP  Treatment  Figure 41. Presence o f intestinal submucosal infiltrate in nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as percentage o f mice per treatment group with the presence o f intestinal submucosal infiltrate  86  c  100%  'C  90%  n il n  80% 70% 60% 50% 40%  "5  30% 20% 10% 0% Non-tumor  PC-3  LNCaP  Treatment  Figure 42. Presence o f cardiac fibrin clots in nude mice from the control groups o f the three C s C l dose titration studies. Results are represented as percentage o f mice per treatment group with the presence o f fibrin clots.  87  Section 4 Discussion  88  4.1 Tumor growth and toxicity in the PC-3 and LNCaP human prostate tumor xenograft models, after administration of escalating doses of CsCl. In the present study, C s C l was able to significantly slow the growth o f the P C - 3 tumor model when administered at 800mg/kg to 1200mg/kg. This same therapeutic effect was not observed in the L N C a P model. However, it should be noted that the animals in the L N C a P study, which received the highest dosages (800mg/kg-1200mg/kg) o f C s C l were euthanized halfway through the study, and thus, did not receive the same number o f C s C l treatments as the PC-3 bearing animals. A speculative mechanism for C s C T s therapeutic effect may be similar to its mechanism for disrupting cardiac function. In the heart, C s C l has been shown to block a number o f potassium channels including K i r 2 . 1 , Kir3.1/Kir3.4 and channels involved in transient outward potassium current (111-117). C s C l has also been shown to slow the rate o f inactivation o f the H E R G potassium channel, which is involved in the onset o f long Q T syndrome (111, 118). Each o f the described channels play a role in repolarizing cardiac cells back to their resting potential during an action potential (116, 118, 119). Thus, the effects o f C s C l on these channels are likely linked to the propagation o f cardiac arrhythmias which have been observed in animal models and in some humans receiving the C s C l treatment. Like cardiac cells, some o f these inward rectifying potassium channels have been described in cancer cells, where they maintain the resting potential o f these cells at depolarized levels to ensure unlimited tumor growth (120, 121). Although, these channels have not yet been described in prostate cancer cells, their existence could provide a mechanism for the observed effects o f C s C l . If PC-3 cells express higher levels o f these channels than L N C a P cells, one could hypothesize their presence as a possible  89  rationale for C s C l ' s therapeutic effects in reducing the P C - 3 tumor growth while not hindering the growth o f the L N C a P tumor. There appeared to be a number o f acute toxicological effects o f C s C l in the athymic nude mouse model, specifically, increased water consumption, the presence o f bladder crystals and viscous mucosal fluid, and a decrease in body mass. Animals receiving the three highest dosages o f C s C l in both PC-3 and non-tumor study demonstrated a significant increase in water consumption. These results were not observed in the L N C a P study, perhaps due to the early removal o f the animals o f interest. It appears that C s C l could be dehydrating the animals, possibly through dieresis. However, this claim cannot be confirmed as urination was not measured. The presence o f bladder crystals or viscous mucosal fluid in animals from all three studies was an unexpected finding. The formation o f these crystals appears to be directly dependent on the concentration o f C s C l administered; crystals were only observed in animals that were administered 600mg/kg to 1200mg/kg C s C l . However, the number o f animals representing these crystals did not increase with administration o f increased C s C l concentration. Although it is not known when these crystals first began to form in the mice, their presence was observed as early as day 15 in the euthanized animals o f the L N C a P study. Some o f these crystals were occupying up to 60-80% o f the volume within the bladders o f these animals. Unfortunately, these bladder crystals were overlooked in the 600mg/kg dosed animals o f the PC-3 study. A s the P C - 3 study was the first study completed out o f the three animal studies and the 600mg/kg group was the first group euthanized within this study,  90  we did not recognize the presence o f bladder inflammation during the organ harvesting o f these animals. It w i l l be o f interest to determine the composition o f these crystals to help rationalize the role o f C s C l in their formation. Infrared spectroscopy has been performed on the bladder crystals, however, the obtained spectrums are difficult to interpret as they do not match up with known bladder/kidney crystal spectrums. Some o f the crystals have been sent to a collaborating researcher i n the United States for further analysis in regards to their composition. In hindsight, it may have been beneficial to harvest bladder tissue from the affected animals for additional histopathology analysis. A significant decrease in body mass was observed in animals receiving the higher concentrations o f C s C l in both the P C - 3 and L N C a P studies, (Figures 7, 11). However, non-tumor bearing mice appeared to tolerate these concentrated doses. Although there was a dose dependent trend i n loss o f body mass o f these animals, no groups were statistically different from the control by the end o f the study (Figure 15). This result indicates that the significant decreases i n mass o f the nude mouse model, as observed i n the P C - 3 and L N C a P studies, is likely to be due to the additive systemic burden and/or toxicity o f the combination o f both tumor and C s C l (Figures 7, 11).  4.2 Investigation of organ and tissue specific toxicity from CsCl (histopathology and serum marker tests) in human prostate tumor xenograft bearing nude mice Histopathological analysis identified abnormalities in the hearts and small intestines o f animals in the three studies. The abnormalities observed in the hearts manifested as fibrin clots. The prevalence o f animals with fibrin clots increased i n a dose dependent fashion  91  in both the P C - 3 and L N C a P studies. However, in the non-tumor bearing study, there was no difference in the prevalence o f animals with fibrin clots between the controls and the treated animals; 33% o f the animals in each group displayed these clots. Based on this observation, it is difficult to determine i f these fibrin clots were caused by C s C l or formed post-mortem from cardiac punctures, since they were so prevalent in the control group o f the non-tumor bearing study. However, it is difficult to ignore that the P C - 3 and L N C a P bearing animals treated with 800 and 1200mg/kg C s C l , had a consistently higher prevalence o f clots when compared to their control groups, respectively, and with the control o f the non-tumor bearing study. From this observation, it appears that fibrin clot formation shows some dependence on both the presence o f a tumor and the dose o f C s C l administered. Interestingly, the cardiac abnormalities in the human clinical case studies, were caused by C s C l doses that were similar to the 800 and 1200mg/kg doses ( i f human to mouse body surface area dose conversion is implemented), which resulted in the highest prevalence o f animals with fibrin clots in the P C - 3 and L N C a P studies (15-18, 20, 22,23). Abnormalities observed i n the small intestines o f animals from the three studies manifested as atrophy o f the mucosal epithelium and v i l l i as well as inflammatory infiltrate o f the submucosa. There were no consistent trends i n the incidence o f intestinal abnormalities within the three animal studies. Only in the L N C a P study did the incidence o f animals with these abnormalities increase in a dose response fashion with increased C s C l administration. A large number o f animals in all three studies had inflammatory infiltrate o f the intestinal submucosa, which was not conserved to any one treatment group within the studies.  92  There were no differences observed i n serum A S T and A L T levels between any o f the treatment groups as compared with the control group i n the three studies. These results indicate that C s C l treatment at the concentrations administered, did not cause considerable structural damage to the heart, liver, skeletal muscle, kidney and erythrocytes. The H P L C assay developed to assess serum creatinine levels was sufficiently optimized before running the samples, based on the peak separation, the standard curve, L O D , L O Q and % C V achieved. The addition o f an internal standard within this assay may have increased its accuracy; however, we experienced difficulty in identifying a suitable internal standard. The results show that serum creatinine levels were not significantly different between the treatment groups and the control within each o f the three studies. Such results indicate that the C s C l treatment at the concentrations administered did not result in a reduction in glomeruli filtration in the animals.  4.3 Comparison of Control Animals There is a distinctive differential impact o f the two tumor types on body mass. The results suggest that there are clear differences in the system burden manifested i n nude mice bearing P C - 3 and L N C a P prostate cancer tumors. Although tumor growth was less rapid in L N C a P bearing animals as compared to animals bearing P C - 3 tumors, L N C a P bearing animals were visibly thinner and weaker. In addition, a distinct decrease in body mass over time in L N C a P bearing animals was observed, which appears to be inversely related to tumor growth. This phenomenon was not observed in P C - 3 bearing animals as their body mass increased over time similar to control animals regardless o f increase in  93  tumor volume and greater levels o f intestinal abnormalities. Interestingly, there was no difference observed in food consumption in L N C a P bearing mice compared to control animals, despite their loss o f body mass, however, it is possible that the methods used in this study to measure this parameter were not sensitive enough to detect these changes. L N C a P bearing mice had significantly increased serum A S T levels but showed not differences in serum A L T and creatinine levels when compared to P C - 3 and control mice. These results indicate potential cardiac, skeletal muscle or erythrocyte damage in animals which could potentially be linked to the observed toxicity within these animals. The loss o f body mass attributed to the L N C a P tumor has been described previously in other tumor bearing animals with cancer linked cachexia. Cachexia affects more than half o f all cancer patients and is the cause o f 20% o f cancer-related deaths (122). It is characterized as complex metabolic syndrome which incurs anorexic effects followed by a progressive depletion o f adipose and muscle mass which ultimately increases morbidity and mortality amongst cancer patients (122-124). Weight loss is the typical marker for cachexia as it is easy to measure and clinically relevant in terms o f toxicity. To fully characterize the L N C a P xenograft model as a model for cachexia, a more comprehensive in vivo study must be completed, involving the measurement o f adipose and muscle mass post-mortem, immunohistochemistry analysis on adipose and muscle tissue for markers o f cachexia, and serum analysis for markers o f cachexia. Investigation o f the cytokines, interleukin-6 (IL-6), interleukin-lp (IL-1B) and tumor necrosis factor a (TNF-a), would be o f particular interest in the L N C a P bearing mice. These specific cytokines have been associated with cachexia in animal models (125, 126) and have also been shown to regulate growth and are expressed by prostate cancer cells. IL-6 has also  94  been shown to be positively correlated with neuroendocrine differentiation and androgen independent prostate cancer progression (127). Alternatively, IL-1 (3 and T N F - a have been shown to reduce proliferation o f the L N C a P cells in vitro (128, 129).  4.4 Conclusions This study is the first to provide a detailed investigation into the therapeutic and acute toxicological effects o f C s C l in human prostate tumor xenograft models. Although C s C l appears to have some therapeutic benefit in treating prostate cancer, a number o f toxicological effects were also observed in conjunction with its administration, including loss o f body mass, increase o f water consumption, fibrin clot formation and bladder crystal formation. Interestingly, one o f most life threatening o f these toxicological effects from C s C l , specifically, a loss in body mass, was not observed in disease free animals receiving the C s C l treatment. This observation indicates some interplay between C s C l administration and the presence o f a tumor compounding to culminate this effect. Comparison o f control animals from the three studies provided some provoking findings in terms o f tumor specific toxicity. The results provoke further study o f the L N C a P tumor model to rationalize its possible role in the induction o f cachexia-like symptoms and serve as a potential in vivo model for this significant and life threatening cancer-related health problem.  4.5 Significance and Limitations of the Results A s there have not been any epidemiological studies completed in regards to the use o f C s C l by cancer patients, like many other alternative cancer treatments, it is difficult to  95  provide an estimate on the number o f users o f this therapy. However, we conducted this research as warranted by the unregulated availability o f these products through the internet and the potential harm they may be causing due to their potential toxicity. In addition, it is important to assess the potential therapeutic effects o f C s C l and weigh the risk to benefit ratio for its use as an anti-cancer product in the hope o f increasing the limited arsenal o f chemotherapeutic compounds against this devastating disease. The results from this preclinical study provide some insight on the therapeutic and toxicological effects o f concentrated, repeated doses o f orally administered C s C l i n human prostate xenograft models. C s C l was shown to reduce the growth rate o f the P C - 3 human prostate cancer tumor, but was also shown to elicit certain levels o f toxicity in the nude mouse model at the same dosages. This information is important for individuals considering the use o f this alternative therapy. A n additional, serendipitous, finding o f this study was the differences in tumor induced toxicity by the P C - 3 and L N C a P prostate cancer xenografts. These effects have not been described previously in the scientific literature and should be further studied. The limitations o f results o f this study are a product o f the models used and the experimental design. The P C - 3 and L N C a P cell lines used in these experiments were both isolated from metastasized sites and not from primary tumors. In other words, both o f these cell lines represent rapidly dividing, late stage prostate cancer, and thus, do not provide information on the therapeutic effects o f chemotherapeutic agents on early stage prostate cancer. Another limitation o f using these cell lines, and o f cell lines in general, is the formation o f subpopulations o f cells through selection processes as a consequence o f cell culturing techniques. Experimentation, in vitro and in vivo, on different  96  subpopulations o f the same cell line using the same protocol can render different results A s both the PC-3 and L N C a P cells used in these experiments had been passaged at least 25 times prior to inoculation, it is possible that the cells injected into the athymic nude mice in this study were subpopulations o f each respective cell line. The athymic nude mouse prostate cancer xenograft model is routinely used to analyze the therapeutic and toxicological effects o f anticancer agents. However, as this an in vivo model for prostate cancer, the drug effects observed do not always match the effects o f the same drugs in human clinical trials. In some instances, drugs that have an optimal therapeutic effect in the athymic nude mouse prostate cancer xenograft model, show only minimal effects in humans. A n additional limitation o f this in vivo model is the use o f the immunocompromised animals. The immunocompromised status o f these animals allows them to inoculated with human prostate cancer cells without rejection, but prevents the researcher from analyzing the effects o f their therapy on the immune system. Once prostate tumors propagate within these animals, the studies are usually limited to one month due to the aggressive growth rate o f most prostate cancer cell lines. This is a further limitation o f this model, as it prevents long term studies. There were steps in the experimental design that in hindsight, may limit the results o f this study. Firstly, the procedure to measure food and water consumption (weighing the food pellets and water bottles on a top loading scale) may have overestimated these two parameters and may have lacked the sensitivity to detect small differences between groups. Partly uneaten food pellets may have been missed and not weighed, and water may have been spilled either by the animals or the experimenter. Ultimately, the use o f  97  metabolic cages would have increased the accuracy o f the measurements and allowed the experimenter to also collect urine and feces for further toxicological analysis. However, the use o f metabolic cages would have been considerably more expensive and unpractical considering the number o f animals surveyed. Secondly, in each o f the animal studies, bladder tissue should have been removed from controls and animals that displayed bladder crystals or viscous mucosal fluid. W i t h the bladder tissue, histopathological analysis could help further delineate the cause o f these bladder crystals and viscous mucosal fluid. Finally, the dosages o f C s C l that were chosen to administer to the animals may have not covered the range o f human dosages i f a proper conversion is undertaken. Although the doses o f C s C l used in the human clinical case studies are similar to some o f the doses used in our study ( i f human to mouse body surface area dose conversion is implemented), other factors must be considered, such as half-life which is quite different between humans (-65.5-96 days) and mice (-3-6 days) (90-93, 96, 97). For this reason, one has to refrain from making direct inferences from the results in terms o f the dosages administered, to the potential physiological complications in humans at similar dosages.  4.6 Future Studies Although oral administration o f C s C l in solution appeared to elicit some toxicity, it did elicit some therapeutic effect in the PC-3 model. Future studies could investigate the reduction o f C s C l ' s toxicity while maintaining or enhancing its therapeutic effect. The first series o f experiments would need to investigate C s C l ' s distribution within the animal; what tissues is it accumulating in and how does this information relate to the observed therapeutic and toxicological effects observed in this study and in other  98  scientific literature. If C s C l is accumulating in the tumors, experiments could investigate the presence o f potassium channels that regulate cellular resting potential (Kir2.1, Kir3.1/3.4, H E R G ) as a possible mechanism for C s C l ' s therapeutic effect, in the P C - 3 and L N C a P prostate cancer cell lines. In addition, tumor tissue microarrays could be constructed and subsequently probed for specific growth and apoptotic regulating proteins to determine i f C s C l is affecting specific biochemical pathways. Other tissues, in which C s C l is determined to accumulate i n , could also be used in microarray experiments to determine C s C l ' s toxicological effect in these tissues. A further toxicological study would need to investigate the dose effects o f C s C l on the murine cardiac system. It would be important to determine whether the dosages o f C s C l that reduce tumor volume, also affect cardiac output as observed in canine experiments. In addition, this study could further analyze the effects o f C s C l on the renal system to fully understand its role in the production o f the observed bladder crystals. Ultimately, i f C s C l is shown to directly reduce tumor volume, while eliciting toxicity i n specific organs, studies could investigate the integration o f C s C l into drug carrier molecules to reduce its toxicity. I f C s C l could be loaded inside a drug carrier molecule with targeted specificity, it may be possible to implement the direct release o f C s C l at the site o f the tumor. In this mode it is possible that C s C l could be used as a viable chemotherapeutic agent. Finally, a comprehensive investigation into the toxicological effects o f the L N C a P tumor on the nude mouse model could be conducted. Results from the current study provide some evidence that the L N C a P model may elicit cachexic-like symptoms in nude  99  mice. To date, none o f the established human prostate cancer models have been identified and/or reportedly characterized with an associated cachexia.  4.7 Synopsis C s C l has been used by some cancer patients as an alternative treatment to their conventional therapy, however, it has not been previously determined as to whether C s C l consumption is beneficial or deleterious. The purpose o f the research conducted as part o f this thesis was to assess the therapeutic and toxicological effects o f oral C s C l administration in mice bearing prostate cancer tumors. Although there were some confounding factors within this study that may have compromised the accuracy o f the results, the overall design o f this study was very thorough and provides a detailed analysis o f the effects o f C s C l therapy in vivo. The results o f this study have shown that oral C s C l may have some therapeutic effect against advanced prostate cancer, however, its administration also elicits specific toxicities, including loss o f body mass, increase o f water consumption, fibrin clot formation and bladder crystal formation. One should be cautious when translating the therapeutic and toxicological effects observed in the treated animals to humans as establishing C s C l dose conversion from one species to another is difficult. W e have shown that C s C l therapy may be associated with some therapeutic effect while also eliciting significant signs o f toxicity. 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