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ln compliance with the Canadian Privacy Legislation sorne supporting forms may have been removed from this dissertation. While these forms may be included . in the document page count, their removal does not represent any loss of content from the dissertation. • " j eutic agent for Evaluation of prostate secretory protein (PSP-94) as a novel therap nancy blocking prostate cancer progression and hypercalcemia of malig Nicholas Shukeir BSc Department of Physiology McGill University Montreal, Quebec November, 2002 in partial fulfillment A thesis submitted to the Faculty of Graduate Studies and Research of the requirement of the degree of Master in Sciences. © Nicholas Shukeir, 2002 1+1 National Library of Canada Bibliothèque nationale du Canada Acquisitions and Bibliographie Services Acquisisitons et services bibliographiques 395 Wellington Street Ottawa ON K1A ON4 Canada 395, rue Wellington Ottawa ON K1A ON4 Canada Your file Votre référence ISBN: 0-612-88295-0 Our file Notre référence ISBN: 0-612-88295-0 The author has granted a nonexclusive licence allowing the National Library of Canada to reproduce, loan, distribute or sell copies of this thesis in microform, paper or electronic formats. L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou aturement reproduits sans son autorisation. Canada • Abstract Human prostate cancer is one of the most common malignancies affecting men. It is associated with a high degree of mortality and morbidity due to the development of non-skeletal and skeletal metastases. A common complication in patients suffering from prostate cancer is the development of hypercalcemia of malignancy. While detennination of PSA and PSP-94 levels can serve as diagnostic/prognostic markers, PSP-94 can also serve as a therapeutic agent. The efficacy of PSP-94 to block tumor progression and hypercalcemia of malignancy was testcd in our syngcneic il! vivo rat modcl of prostate cancer. Rat prostate cancer Mat Ly Lu cells were transfected with full length cDNA encoding PTHrP [Mat Ly LuPTHrP] which is known to be the main factor responsible for hypercalcemia of malignancy. Mat Ly Lu-PTHrP cells werc inoculated subcutaneously into the right flank or via intracardiac injection into the left ventricle of male Copenhagen rats. AnimaIs were treatcd with differcnt doses of PSP-94. Tumor volume, time of hindlimb paralysis, plasma calcium and plasma and tumoral PTHrP levels were determined. PSP-94 caused a significant delay in the development of hind-limb paralysis, reduction in tumor volume, plasma calcium levels, plasma and tumoral PTHrP levels as compared to control animaIs receiving vehicle alone. These effects were due to the induction of tumor cell apoptosis. In conclusion, our studies illustrate the ability ofPSP-94 to block prostate cancer growth and skeletal metastases . • • Résumé Le cancer de la prostate est un des cancers masculins le plus fréquemment diagnostiqué et présente une incidence élévée de mortalité et de morbidité à cause du développement de métastases à distance osseuses et non-osseuses. Les patients atteints du cancer de la prostate développent souvent l'hypercalcémie maligne. Bien que les dosages sériques des marqueurs tumoraux PSA et PSP-94 soient de bons outils diagnostiques et pronostiques, PSA-94 s'avère aussi un agent thérapeutique. L'efficacité du PSP-94 à bloquer la progression tumorale et 1'hypercalcémie maligne a été testée en utilisant notre modèle syngénique in vivo du cancer de la prostate chez le rat. Les cellules cancéreuses de protaste de rat Mat Ly Lu ont été transfectées avec l'ADNe pleine longueur de la PTlIrP [Mat Ly Lu-PTHrP], principal agent causal de l'hypercalcémie maligne. Les cellules Mat Ly Lu-PTI-IrP ont été inoculées à des rats mâles Copenhague par voix sous-cutanée dans le flanc droit ou par injcction intracardiaque dans le ventricule gauche. Les animaux ont ensuite été traités avec différentes doses de PSP-94. Le volume tumoral, le temps d'apparition de la paralysie des membres postérieurs, le taux de calcium sérique ainsi que la quantité sérique et tumorale de la PTHrP ont été mesurés. Comparativement aux animaux contrôles ayant reçu la substance véhicule seulement, l'inoculation de PSP-94 a retardé de façon significative le développement de la paralysie des mémbres postérieurs, réduit le volume tumoral ainsi que le taux de calcium sérique et les niveaux de PTHrP sériques et tumoraux. Ces effets sont attribuables à l'induction de • l'apoptose chez les cellules tumorales. En conclusion, notre étude démontre que PSP- 11 • 94 peut bloquer la progression de la croissance tumorale du cancer de la prostate ainsi que ses métastases osseuses . • 111 .' Acknowledgments 1 would like to thank the people who with their guidance and support made this work possible. 1 would like to extend my gratitude to Dr. Shafaat A. Rabbani for giving me the opportunity to work in his laboratory allowing me to pursue my graduate studies and for providing me with helpful scientific guidance. 1 am also thankful for the wonderful people whom 1 met and had the privilege of working alongside. Notables include Julienne Gladu, Pouya Pakneshan, Parissa Khalili-Bourjumani, Jing Guo, Mike Macoritto and Lucie Canaff. 1 am extremcly grateful to Ani Arakelian for teaching me the essentials of working in an acade111ic laboratory and for providing me with guidance and insightful hclp throughout the course ofmy graduate studies. Finally, 1 would like to thank my parents and brother for believing in me and • encouraging me with decisions that 1 have made . • IV Abbreviations • ~-MSP beta-microseminoprotein BMPs Bone morphogenetic proteins Ca+2 Calcium CPA Cyproterone CRE Cyclic AMP response clement DNA Deoxyribonucleic acid DRE Digital Rectal Examination ECM Extracellular Matrix EGF Epidermal Growth Factor FBS Fetal Bovine Serum FGF Fibroblast Growth Factor FSH Follicle Stimulating Hormone HCG Human Chorionic Gonadotropin I.C. Intracardiae I.P. Intraperitoneal LH Luteinizing Hormone LHRH Luteinizing Hormone Releasing Hormone MGA Megestrol Acetate MMPs Matrix Metalloproteinases mRNA Messenger Ribonucleic Acid PAP Prostatic Acid Phosphatase v l' PDGF Platelet Derived Growth Factor PRLR Prolactin Receptor PSP-57 Prostate Secretory Prote in of 57 amine acids PSP-94 Prostate Secretory Protein of 94 amino acids PTHrP Parathyroid Honnone Related Peptide s.c. Subcutaneous TGF-a Transfom1ing Growth Factor Alpha TGF-p Transfonning Growth Factor Beta TRUS Transrcctal Ultrasound uPA Urokinasc VEGF Vascular Endothelial Growth Factor VI • Table of Contents Abstract. ............................................................................................ i Résun1é ............................................................................................. .ii Acknow ledgell1ents ............................................................................. .iv Abbreviations ...................................................................................... v Table of Contents ................................................................................ vii List of Figures ............... '" ., ..................... , ......................................... .ix List of Tables ..... '" ...................... '" ....................... " .......................... .ix Charter 1: Introduction Prostate Cancer .......................................................................... 1 Risk Factors for Prostate Cancer ...................................................... 2 , Screening and Grading of Prostate Cancer .......................................... 4 Biological Progression of Prostate Cancer. ......................................... 7 Prostate Cancer and Metastases ...................................................... 10 Prostate Cancer and PTHrP ......................................................... .. 13 Treatment of Prostate Cancer ......................................................... 14 Discovery of Prostate Secretory Protein of 94 amino acids ...................... 17 . Gene Organization of Prostate Secretory Protein ................................. 18 Regulation of PSP-94 .................................................................. 19 DifferentiaI Expression of PSP-94 ................................................... 21 • Actions of PSP-94 ...................................................................... 22 vii • Receptor for PSP-94 ................................................................... 25 Objectives of Thesis ................................................. ~ ................. 27 Chapter 2: Prostate Secretory Prote in (PSP-94) decreases tumor growth, metastases and hypercalcemia of malignancy in a syngeneic in vivo model of prostate cancer .................................................................................... 28 Introduction ............................................................................. 29 Materials and Methods ................................................................ 32 Materials ........................................................................ 32 Cells and Cell Culture ........................................................ 32 Animal Protocols .............................................................. 33 Histological Analysis ......................................................... 34 Othcr Analytical Mcthods ................................................... 35 Statistical Analysis ............................................................ 35 Results ............................................ '" .................................... 36 Discussion .............................................................................. 57 Chapter 3: General Discussion ................................................................ 61 . Chapter 4: References ....................................................................................... 70 Appendix ...................................................................................................................... 91 • VIII • List of Tables 1. Clinieai Staging of Primary Tumor (T) ...................................................................... 6 2. Staging by Regional Lymph Node Involvement (N) ................................................. 6 3. Staging by Presence of Distant Metastases (M) ......................................................... 6 List of Figures Chapter 1. 1. Biological Progression of Prostate Cancer ..................................................... 9 2. Proeess of Metastases ................................................................................... 12 3. Modei of Osteoblastie Bone Metastases eaused by Prostate Caneer........... l3 Chapter 2. 1. Effeet ofPSP-94 on Mat Ly Lu-PTHrP eeU growth ............................ 45 2. Effeet ofPSP-94 on Mat Ly Lu-PTHrP tumor volume ....................... .47 3. Effeet of PSP-94 on animal weight.. ............................................ .49 4. Effeet ofPSP-94 on Mat Ly Lu-PTHrP tumor weight.. ...................... 51 5. Effeet ofPSP-94 on spinal metastases ........................................... 53 6. Effeet of PSP-94 on plasma PTHrP and Calcium in tumor bearing animaIs .................................................................................. 55 7. Effeet ofPSP-94 on PTHrP production by Mat Ly Lu-PTHrP tumors ...... 57 • ix 1; 8. Effect ofPSP-94 on DNA fragmentation of Mat Ly Lu-PTHrP cells in vitro and in vivo ........................................................................................................ 59 • x • Chapter 1 Introduction Prostate Cancer Carcinoma of the prostate has developed into a major and escalating international health problem. In many developed countries, prostate cancer is one of the most commonly diagnosed cancers in men and is the second leading cause of cancer mortality following lung cancer (l ,2,4). It is estimated that 18,200 Canadians will be diagnosed with prostate cancer in 2002 with 4,300 people dying from the disease (3). Insuflicient data from many developing countries has made it difficult to accuratcly estimate the worldwide prevalence of prostate cancer. However, the general incidence has been, on the rise over the last few years with an increasing number of men bcing diagnosed with prostate cancer. This has been attributed to an increase in the awarencss of the disease as weil as to bettcr screening programs in placc. Although there are many risk factors for developing prostate cancer, age is the primary factor. Autopsies carried out on prostate cancer patients have shown that prostate cancer is present in 50% of men in their 50s with the number jumping to 70% of men who are over 80 years of age (2). Enhanced screening programs and improved treatment modalities for the disease have maintained the mortality rate from prostate cancer at a steady rate despite the higher incidence in the number of people being • diagnosed with this common cancer. • Risk Factors for Prostate Cancer There are many risk factors associated with the development of prostate cancer. The strongest predisposing factor is age. Men under the age of 50 rarely exhibit signs of the disease with prostate cancer being quite common in older men with symptoms ranging from urinary obstruction to bone pain. Other predisposing factors inc1ude hormones, growth factors, diet, vitamins, dietary supplements, environ mental factors and viruses (2, 4-19). A balanced production and regulation of growth factors can therefore control cell proliferation and cell death in the nom1al prostate (4). Any deviation from this well controlled balance results in either involution of the prostate or the dcvc10pment of prostate cancer. Some growth factors that have been shown to be involved include the insulin-like growth factor family (IGF) (7,9), epidermal growth factor (EGF), the fibroblast growth factor family (FGF) (16,17), the endothelial growth factors, plateletderived growth factor (PDGF), vascular endothelial growth factor (VEGF) (17) as weIl as transforming growth factors (TGF) a and p (18,19). Most of the growth factors mentioned above exhibit a stimulatory role on the proliferation of the normal prostate. To date, the role of TGF-p remains eontroversial in prostate cancer. Sorne studies have attributed a stimulatory role to TGF -p (19) while others have shown TGF -p to exhibit an inhibitory role (18). In addition, androgens also play a major role in the carcinogenesis of the prostate. Several studies have shown higher levels of circulating testosterone in patients suffering from prostate cancer. Whereas the • precise role of androgens in the induction and initiation of prostate cancer is not yet 2 fully understood, once prostate cancer has been established androgens play a role in advancing the cancer to its highly invasive phenotype. The consumption of certain vitamins and dietary supplements appear to offer sorne degree of protection against prostate cancer. Recent studies have shown that higher levels of vitamin E and selenium is associated with lower risks of advanced prostate cancer (10,13,14). Furthermore, vitamin D has becn shown to have a differentiative and an anti-proliferative effect on human prostate cancer cells thus promoting its role as a chemotherapeutic agent for malignancy (8, Il,12,15). People who consume a vegetarian, low fat diet are at lower risk for prostate cancer. Higher fat intake is not only associated with higher risk of cardiac problems and strokes but is also associated with increased incidence of developing prostate cancer (5). One possible mechanism by which high fat levels increase the risk of prostate cancer is by rcducing the absorption of vitamin A. Higher levcls of beta-carotene, directly rclated ; to the amount of vitamin A absorption, appear to be protective against the development of some cancers including prostate cancer (2). Furthermore, phytoestrogens, such as genistein and diadzein found in vegetables, may counteract the effects of androgens on the prostate. Another possible mechanisms by which these substances exhibit their anti-carcinogenic effects is by inhibition of angiogenesis as weil as the inhibition of the tyrosine-specific protein kinase associated with the respective growth factor receptors (2) . • 3 Screening and Grading of Prostate Cancer Early detection of this common disease leads to appropriate intervention and hence a better prognosis. It is highly recommended, by man y cancer societies aIl over the world, that aH men over the age of 50 undergo annual screening tests in order to facilitate early detection. There are a number of screening tests available for prostate cancer. Historically, a digital rectal examination (DREs) was the most commonly uscd test for detection (2,20,21). However, the utilization of prostate speci fic antigen (PSA) lcvcls for the early detection of prostate cancer has provided a less invasive, highly practical altemative screening option. Despite the fact the PSA levels offer the best performance characteristics by providing a significantly higher predictive value, these two screening tests should be carried out simultaneously to complement each othcr since some individuals exhibiting characteristics of prostate cancer, as , diagnosed by DRE, have low levels of PSA and viee versa (2). A more reliable method to screen for prostate cancer is by performing a transrectal ultrasound (TRUS) (2,22), which has the added advantage ofbeing able to identify suspieious lesions that are non-palpable by a DRE. However, many factors inc1uding high costs render TRUS an unsuitable screening modality for the early detection of prostate cancer. Whereas screening for prostate cancer aIlows for the early detection of the disease, grading of prostate cancer can serve as an important prognostic marker and a valuable tool to design therapeutic strategies (23). Over the years, a number of grading systems have been used but the most commonly applied system is the Gleason grading system (2,23). It has been shown that not only does the Gleason 4 l, grading system provide signifieant prognostic information but it has also exhibited highly reproducible results (2). The Gleason grading system is based on the gIanduIar pattern of the tumor as identified by microscopic examination of prostatic tissue un der low magnification (2,23). A 5-step grading system is used with the two grades being added together to yield the so-called "Gleason grade" which ranges from 2-10. The higher the Gleason grade the worse the prognosis and vice versa. Since the treatment options for prostate cancer can vary, diagnosis as well as staging of prostate cancer is essential for the selection of the most appropriate therapy. The CUITent tumor staging system used is based on the Tumor (T), Node (N), Metastasis (M) staging system, also known as the TNM staging (2,24). An example of the TMN staging system is shown in tables 1-3 (2). As mentioned earlier, ORE and PSA levels arc very useful tool for sereening for the disease but they arc also hclpful in determining the stage of the cancer. Combination of PSA levels, clinical stage (TNM) and Gleason , score greatly enhances the prediction of the stage at which the cancer is . • 5 Table 1 Clinical staging of primary tumor (T) . TX TO Primary tumor cannot be assessed No evidence of primary tumor T1 Clinically inapparent tumor not palpable or visible by imaging T1 a Tumor incidental histological finding in 5% or less of tissue resected Tumor incidental histological finding in more th an 5% of tissue resected Tumor identified by needle biopsy (e.g. because of elevated PSA) T1 b T1c Table 2 Staglng by regional Iymph node involvement (N) NX Regional Iymph nodes cannot be assessed NO No regional Iymph node metastasis N1 Metastasis in regional node or nodes T2 T2a T2b Tumor confined wlthln the prostate Tumor involves one lobe Tumor involves both lobes T3 Tumor extends through the prostate capsule • MX Presence of regional metastasis cannot be assessed T3a T3b T3c Unilateral extrapsular extension Bilateral extrapsular extension Tumor invades the seminal vesicle(s) MO No distant metastasis T4 Tumor invades any of bladder neck, external sphincter, or rectum M1 M1 a M1 b M1c Distant Metastasis Non-regionallymph node(s) metastasis Bone(s) metastasis Other sites metastasis T4a Tumor invades any of bladder neck, external spincter or rectum T4b Tumor invades levator muscles and/or the pelvic wall ,Table 3 Staglng by presence.ofdlstant metastasls (M)';' " , lB) Roger Kirby et al. Prostate Cancer, Second Edition, 2001 • • Biological Progression of Prostate Cancer ssion of As with other homlOne dependent malignancies, the biological progre development of prostate cancer is the result of many events that ultimately lead to the of the prostate highly advanced, metastatic prostate cancer (figure 1). Carcin oma ps into a develo ps when the norma l epithe lium of the prosta te gland develo c mutations histological cancer as a result of many factors. These include geneti There are no causing the inactivation or silencing of tumor suppressive genes (2,29). diagnosed by symptoms experienced by the patient at this stage and the cancer is only enic effects histological examination of the prostatic tissue. Persistence of these mutag addition to the leads to additional tlll110r suppressive genes being silenc ed in enes that have activation of tllmor promoting genes, the proto-oncogencs. Some oncog Her-2 /neu and been shown to be upregu lated in prosta te cance r includ e the lasia or a PTPC AAX2 oncogenes. (30,31 ). As a result benign prosta tic hyperp enced by men, localized primary tumor develops. The most common symptom experi w obstruction, when the cancer is at this stage, is associated with bladde r outflo plete emptying namely reduced urinary outflow and frequency accompanied by incom (2). Up until this of the bladder as a result ofpros tatic tissue obstruction of the urethra dent state. stage of prostate cancer progression, the cancer is in a hormo ne depen development of However, if the cancer is left undiagnosed and untreated then the Sorne of the androg en-ind epend ent and metastatic prostate cance r is inevitable. • gens) (1,25) factors which facili tate this progression include sex steroids (andro nes (28). A very growth factors (4), angiogenic factors (27), proteases (26) and cytoki 7 common feature in prostate cancer is the development of skeletal and non-skeletal metastases (32,33). As a result of systemic metastases, the patient will complain about signs oflethargy, weight loss and cachexia and hemorrhage (2). If the cancer reaches the bone, th en pain is the primary complaint of patients with ultimate development of paraplegia as a result of spinal cord compression (34). 8 • • • Prostate Cancer and Metastases. Primary prostate tumors which are localized within the capsule are rarely associated with mortality. Rather it is the development of tumor metastases at distant sites of the body that is the deadly factor. Metastasis to the bone is a frequently observed complication in patients who suffer from advanced prostate cancer (32,33). While skeletal metastases can be of the osteolytic or ostcoblastic variety those observed in patients with prostate cancer are usually of the osteoblastic phenotype (35,36,37). It is believed that these osteoblastic metastases are the result of cancer cells producing factors that stimulate osteoblast proliferation, diffcrentiation and cvcntually bone formation (36). As shown in figure 2, the process of bone metastascs is very similar to that of metastases to any other site in the body (36). Once the primary tumor rcaches a certain size, a phenomenon known as the "angiogcnic switch" occurs which is éharacterized by the recruitmcnt of new blood vessels. Primary tumor ceUs growing in the site of origin invade into their surrounding tissue by producing proteolytic enzymes which help breakdown the extracellular matrix (ECM). Such proteolytic enzymes inc1ude urokinase (uPA) and matrix metaUoproteinases (MMPs) (26,36). Breakdown of the ECM aUows tumor ceUs to traverse the waUs of smaU blood vessels present in the primary organs or the newly , recruited ones and enter the circulation allowing the tumor cells to reach distant sites. This process is quite inefficient and many circulating tumor ceUs do not survive the normal protective mechanisms. The few ceUs that manage to survive and reach their secondary organ, bone or other tissues, extravasate from the blood vessels into the 10 •• stroma of the organ. There they begin groW1l1g aga1l1 with the whole process beginning reinitiated resulting in the development of tumor metastases at multiple sites. There is increasing evidence to indicate that certain growth factors are involved in the stimulation of bone formation which is associated with metastatic prostate tumors. Patients with prostate cancer have increased levels of endothelin-l which is known to stimulate bone formation and osteoblast proliferation (38,39). The TGF-p superfamily as weIl as the bone morphogenetic proteins (BMPs), namely BMP-2, BMP-3, BMP-4 and BMP-6 have been shown to be stimulators of bone formation (36). Othcr growth factors that have been shown to be increased in patients with prostate cancer include the FGFs and PDGFs (4). Furthcrmore, while an amino terminal fragment of uPA has becn shown to cxhibit mitogenie activity for osteoblasts (40), the carboxy terminal protcolytic domain mediates tumor invasiveness and growth factor activation (36). In addition, studies have shown that expression of uPA by prostate cancer ecUs leads to an increase in the metastatic ability of the cells leading to an increased number of osteoblastic bone leasions (41). A model for osteoblastic bone metastases caused by prostate cancer is summarized in figure 3 . • Il - ,le The Process of Metastasis Prirnary malignant neoplasrn Neovascularization Invasion ~ " • ~. ~ ""'" ""'" Adherance Arrest in distant organ capillary bed t ft ~ Response to rnicroenvironrnent .. Turnor cell proliferation Ernbolisrn Multicell aggregates ~ ,- Extravasation Metastasis ® Gregory Mundy, Nature Reviews, 2, 2002 '. ~ BMPs latentTGF-p uPA ~~ active TGF-p • ~ PTHrP ~ Proteases J --- ~ Inactive PTHrP fragments + Bone Formation ® Gregory Mundy, Nature Reviews, 2, 2002 FGFs • Prostate cancer and PTHrP ion of A common observation in prostate cancer patients is the over-product that PTHrP is PTHrP in cases of advanced prostate cancer (42,43). It is now believed seen in many the major factor respon sible for hyper calcem ia of malig nancy and prosta te carcin omas inc1uding breast carcin omas, renal cell carcin omas with maximal carcinomas (44, 45). The expression ofPTH rP increases progressively expression in highly malignant prostate cancer cells (46). te cancer The growth promoting effects of PTHrP were studied in many prosta the rat prostate cclI lines including the hum an prostate cancer PC-3 cell line as weB as by exogenous cancer Mat Ly Lu cell line. Increased expression of PTHrP either resulted in addition of PTHrP peptides or stable transfection of PTHrP cDNA growth (47,48). increased tumor cclI growth as weB as accelerated primary tumor tory role of Examination of the mechanism of action of PTHrP revealed an inhibi apoptotic agents PTHrP on apoptosis causing increased ceU survival after exposure to (47,48 ). ration, ln addition to PTHrP s role in osteoblast differentiation and prolife ial (36,49). PTHrP has also been associ ated with increa sed metas tatic potent tases from Immunohistochemical and in situ hybridization analysis of bone metas cant number of prostate cancer patients revealed the expression of PTHrP in a signifi expressed not samples (46). Furthermore, the PTHrP receptor was also shown to be from prostate only in the primary prostate tumor but also in the bone metastases • cancer patients (46). 14 • Treatment of Prostate Cancer in The stage at which prostate cancer is at can serve as the primary factor patients with choosing an effective treatment modality (50). Treatment options for on therapy and localized prostate cancer inc1ude watchful waiting, surgery, radiati the prostatic brachytherapy which involves the insertion of radioactive pellets within gical prostate tumor to deliver a localized form of radiotherapy. Patients with histolo are carefully cancer and suffering from no symptoms usually receive no treatment and regularly by monitored for any symptoms that might develop. Patients are monitored rate of disease DREs and PSA levels to determine the patien t's condit ion and progression, and furthcr decisions are made accordingly. Since these kinds of age of 70, the prostate cancer are diagnosed accidentally with patients being over the ly die with rationalc for using such an approach is that the patients will most probab prostate cancer ratbcr tban afthe disease. ete Radical prostatectomy is a surgical procedure which involves the compl excision of the prostat~ gland, the seminal vesic1es and adjacent tissues (2,50). to afford a Currently, radical prostatectomy is used in patients in whom it is likely ate any chance cure, in effect, only in those where the surgery will completely elimin of the surgery, of metastatic disease developing. In order to improve the outcome androgen patient with larger tumor volumes undergo a regim e of preoperative . y inc1uding depriv ation (50,52). Many disadv antage s of radica l prosta tectom the use of impotence, incontinence, rectal injury and potential mortality have limited this radical procedure . • 15 • An alternative treatment approach for localized prostate cancer that has proven efficacy is radiation therapy. The objective of radiation therapy is to achieve the highest possible tumor dose with minimal radiation injury to the surrounding nom1al tissue. Generally, the criteria for patients considered for radiation therapy are similar to those being considered for radical prostatectomy. Brachytherapy is a procedure that involves the insertion of radioactive pellets into the prostatic tumor to localize the effects (50,51). The advantages of brachytherapy over radiation therapy include the usage of low dose radiation as weIl as conformai seed plantation which can be carried out in one setting as opposed to multiple fractions required for external bcam radiation. The absence of surgery is a major bene fit for patients sclecting radiation therapy which aIlows this form of therapy to be applied to paticnts with less favorable, general medical condition. Duc to the established role of androgens on the initiation and progression of prostate cancer, hormonal' therapy is the trcatmcnt option for locally advanccd prostate cancer. The basis of hormonal therapy is the dependence of the prostate gland as weIl as prostate cancer celIs on androgens (2,50,52). Several methods of achieving hormonal therapy are available. Bilateral orchiectomy leads to symptom relief in a large percentage of symptomatic patients. Bilateral orchiectomy leads to the removal of most of the circulating testosterone at once. However these modalities are associated with side effects such as loss of libido and psychological effects (2,50). Other less invasive methods of hormonal therapy include the administration of , leutinizing hormone releasing hormone (LHRH) agonists. Although low doses of LHRH agonists mimic the actions of LHRH, superphysiological doses were found to 16 • downregulate the LHRH receptor with subsequent decrease in LH and circulating testosterone levels (50). Anti-androgen agents are another form of hormonal therapy. These agents compete for androgen receptors minimizing the effects of androgens. Two types of anti-androgen agents exist: steroidal which have significant progestational activity and nonsteroidal (50). Examples of steroidal anti-androgen agents include cyproterone (CP A) and megestrol acetate (MGA), whereas flutamide and nilutamide make up non-steroidal agents. Prostatic tu mors are composed of a heterogeneous subpopulation of cells with some being hormone-dependent while others are hormone-independent. Hormone deprivation abolishes the hormone-dependent subpopulation but lacks the similar desirable effect on the hormone-indepcndent subpopulation. As a result the hormoneindependent cells survive, begin to proliferate and repopulate the prostatic tumor. Consequcntly hormonal therapy is almost always followed by a recurrence of the ( disease (2). Despite advanees in diagnosis and treatmcnt modalities, prostate cancer continues to pose challenging therapeutic problems. This has led to the evaluation of additional novel therapeutic agents to control disease progression. Among these approaches gene therapy (53), protease and growth factor inhibitors and chemical agents which can block various intracellular signaling pathways have shown promising effects and can be developed for patients with advanced hormone refractory form ofprostate cancer (2) . • 17 • Discovery of Prostate Secretory Protein of 94 amino acids The notion that a nonsteroidal, gonadally derived inhibitory substance, an "inhibin" that is capable of inhibiting pituitary FSH levels was first put forward by McCullagh in 1932 (54). This notion came about wh en castration of male rats resulted in prostatic hypertrophy which could be reversed by the introduction ofwater soluble testicular extracts. This observation as well as reduction of FSH levels in men following castration or irradiation of the testis led to the original thought that the testes were the primary site of production of "inhibin". However it became clear that the production of "inhibin" extended beyond the testes when serum FSH levels were suppressed following administration of ovarian follicular fluid into proestrous rats (55). Dcspite mounting evidencc of the existence of such an inhibitory hormone it was not until llluch later, with the development of sensitive and specifie radioimmunoassays for inhibin (56), that the isolation and purification of inhibin took place. At that time, inQibin was purified from bovine and porcine follicular fluid; however isolation of inhibin from the human followed shortly thereafter (57). It was shown that inhibin was formed through a dimeric assembly of an a subunit and one oftwo c10sely related ~ subunits (~A or ~B) (58). Dimerization of the a subunit with either the ~A or ~B yielded inhibin A and inhibin B respectively (58). When the inhibin genes were isolated and c10ned it was realized that inhibin purified from human seminal fluid was not related to the dimeric inhibin. The • distinction stemmed from prostatic inhibin being a single polypeptide chain of 94 amino acids instead of being a dimer of a and ~ subunits (59,60). Despite this 18 difference between the two inhibins, they both share the same mode of action of inhibiting FSH levels without altering the levels of LH (62). Alternative names used to describe prostatic inhibin include prostate secretory protein of 94 ami no acids (PSP-94), ~-microseminoprotein W-MSP) and sperm-coating antigen (103). Since its discovery it has been shown that PSP-94 is one of the tluee predominant proteins secreted by the prostate gland along with PSA and P AP (61). The gene eneoding PSP94 as well as the complete amino acid sequence of PSP-94 have since been determined (63,64,65,66,67). Furthermore, sinee its first isolation from the prostate gland it has been demonstrated that PSP-94 expression is not restrieted to the prostate, as the protcin is present in other tissues of the human body (68). Partieular tissues that have becn shown to express PSP-94 include mueosal structures stlch as the tracheobronchial tract and the stomaeh (66,68). Morcover, female reproductive tissues have also been del1lonstrated to express PSP-94 particularly breast, ovaries . and endomctrial tissues (69). Dcspite the vast array of tissues that express PSP-94 the prostate glad expresses ~he majority of PSP-94 as eompared to other tissues (69). Gene Organization of Prostate Secretory Protein Human PSP is eneoded by a single gene that resides on chromosome 10 (q 11.2) (70,71). The human PSP gene spans approximately l4kb long and consists of four exons (J, II, III and IV); alternative splieing of these exons yields either PSP94 or PSP-57 (72). Three out of the four exons are present in both transcripts: exon l, exon II and exon IV (72). Complete dcletion of exon III yields PSP-57 (72). The • upstream region of the human PSP gene spans about 2.8kb, however the majority ofit 19 li is not necessary for basal promoter activity as determined by luciferase activity (73,74). The promoter reglOn contains many putative transcription regulatory elements for ubiquitous transcription factors (73,74). Sorne of these regulatory elements include a TATA box, a cAMP response element (CRE) and three glucocorticoid response elements (73,74). The structural organization of the gene with its promoter and multiple transcription regulatory elements along with the potential for alternative splicing suggests tissue specific expression of the different isofonns of PSP. Of these two isoforms, the 94 amino acid isofonn is the common isoform in humans as determined by mRNA lcvcls (72). Furthcrmore, it appcars that PSP-57 expression is more tissue specific than that of PSP-94. Other th an the prostate gland whcre both isoforms arc cxpressed, it appcars that the expression of PSP-57 and that of PSP-94 follow an inverse relationship whereby the majority of PSP-57 is expressed in tissues that do not express PSP-94 and vice versa. Such examples include PSP-57 expression in kidney and bladder tissues where no PSP-94 expression ean be detected and PSP-94 expression in lung and breast where PSP-57 is not expressed (72). PSP94 and PSP-57 share the same 5' untranslated region as weIl as an amine acid sequence corresponding to a signal secretion peptide which is cleaved before secretion of the proteins (72). Regulation of PSP-94 With the cloning of the PSP gene, the regulatory mechanism(s) of human PSP-94 production are being elucidated. The regulation of PSP-94 has been mostly 20 • are androgen studied in benign hyperplastic prostatic tissues and LnCAP cells which are found to responsive, non-invasive prostate cancer celllin es (74,75,76). Honno nes with G-protein be the potent regulators of PSP-94 expression through interaction an increase in coupled receptors leading to activation of adenylyl cyclase resulting in levels lead cAMP (74). Most honnones that lead to an increase in intracellular cAMP n levels in to a transient increase in the levels of both PSP-94 mRNA and protei were shown to human prostate ceUs. Specifical1y, the levels of PSP-9 4 protei n tic tissues were increase in a dose-dependent manner when benign hyperplastic prosta ted prostatic incubated in vitro in the presence of FSH or LH as compared to untrea l hormones tissues (75,76). Furthe rmore treatment of LnCAP cells with severa l has been inc1uding epincphrine, vasoactive intestinal peptide (VIP) and isoprotereno r through the shown to stimulatc the expression of PSP-94 in a dose-depcndent manne of cAMP was increased produ ction of PSP-9 4 mRNA (74). The precis e role ction were elucidated wh en the same effect of increases in PSP-94 mRNA produ forksolin(74). reproduced upon incuba tion of LnCAP cells in the presen ce of of PSP-94, the Whereas the majority of hormones lead to an increase in the levels manner when levels of PSP-94 protein were shown to be reduced in a dose-dependent presen ce of benign hyperplastic protatic tissues were incubated in vitro in the (75,76) honnones such as prolactin and human chorionic gonadotropin (HCG) ned for Sex steroids such as testosterone and estrogen have also been exami lastic prostatic any possible effect on PSP-94 gene expression in both benign hyperp • of these sex tissues and LnCAP cells. However, it has been demonstrated that none en-independent steroids has an effect on PSP-94 expression leading to the androg 21 • nature of PSP-94 expression. (75,76). Furthermore immunohistological examination ofPSP-94 levels in benign prostatic tissues coming from patients that have undergone androgen deprivation therapy and those who have not been exposed to the therapy revealed that the production of PSP-94 was not under the influence of androgens (77). Differentiai Expression of PSP-94. PSA and PAP levcls were the standard prostate cancer markers due to their differential levels of expression as the prostate cancer progresses (78). Utilizing a number of different techniques including in situ hybridization, immunohistochemistry and detcrmination of PSP-94 mRNA levels, it has been demonstrated that the expression of PSP-94 is diffcrcntial depending on the stage of prostate cancer (77,79,80,81). The levels of PSP-94 arc quite high is normal prostatic tissue and , exhibit a progressive dccline as the prostate cancer advances from a low invasive, highly differentiated, aI~drogen-dependent state to a poorly differentiated, androgen- independent state with complete lack of PSP-94 expression in highly advanced prostate cancer. This di fferenti al expression has allowed PSP-94 to have a diagnostic/prognostic value comparable to that of PSA and P AP (82,83). While PSA and P AP levels are androgen dependent, PSP-94 levels are not dependent on the . levels of circulating androgen (77,79). This provides PSP-94 with an added advantage as a diagnostic/prognostic marker in tumors that have been previously exposed to • androgen ablating agents . 22 • The expression ofPSP-94 in other tissues of the human body allows for its use as a biochemical diagnostic/prognostic marker in cases other than prostate cancer. Although less common th an prostate cancer, gastric carcinoid tumors affect as much as 10% of the population and are quite often associated with poor prognosis as a result of difficulty in diagnosing the disease (84). The expression of PSP-94 in gastric carcinoid tu mors follows an opposite course to its expression in prostate cancer. In a study carried out by Femlund et al it was shown that the expression of PSP-94 in tumor tissues collected from patients with gastric tumors correlated with tumor diameter and tissue invasion depth and subsequently with tumor progression where highly invasive, metastatic tumors stained positive for PSP-94 whilc less invasive tumors of smaller diametcr stained negative for PSP-94 (84). With PSP-94 levels bcing easily measured by the use of radioimmunoassays, PSP-94 can serve as a useful marker in gastric carcinoid tUll1ors. Furthermore PSP-94 is expressed in man y female tissues including ovarian, éndometrial and brcast tissues (69). Just like in prostate cancer, PSP-94 levels decrease progressively as breast cancer progresses with lack of PSP-94 in late stage breast cancer. This differential expression of PSP-94 allows it to be a diagnostic/prognostic marker in breast cancer. Actions of PSP-94 . The main biological function of PSP-94 is the suppression of FSH levels (62). Although the pituitary gland is the main biological site for production of FSH, it has been demonstrated by Garde et al that the prostate gland is an extrapituitary source of • FSH (85). Furthermore, increased levels of FSH levels in cases of benign prostatic 23 • prostate gland hyperplasia cornbined with the discovery of FSH recept ors on the prostate gland suggest an autocr ine/pa racrin e mode of action of FSH on the and through (86,87,88). In the normal prostate, PSP-94 production is quite significant gh the precise a negative feedback loop FSH levels are kept at constant levels. Althou r, a study mechanisrn by which PSP-94 acts to reduce FSH levels remains unclea sis of the 3-D carried out by Jyothi et al shed sorne insight into this matter (89). Analy en regions in structure of PSP-94 revealed structural and functional similarity betwe ility that the PSP-94 and FSH (89). A hypothesized rnechanism favors the possib for the FSH function-mimicking domain of PSP-94 might trigger a false recognition ce of PSPreleasing system and thus lead to a suppression of FSH levels in the presen 94. ds. Traditionally, women were the major targets for most contraceptive metho d at men with Condoms and vasectomies are the main contraceptive methods directe ch to male each of them having their own limitations. A hormonal, reversible approa ception. The contraception would all<;>w for a safe and non-invasive method of contra on the optimal basis of hormonal contraception is that spennatogenesis is dependent n of PSP-94 concentration of gonadotrophin secretion, FSH (90,91,92). Administratio spermatogenic results in suppression in the levels of circulating FSH causing loss of (91,92). On the activity in the testis resulting in a condition known as azoospermia levels of FSH other hand, severa! studies carried out have also shown that increased d testicular lead to impairment of spermatogenesis, agglutination of sperm and reduce • resulting in a weight as a result of a decrease in serniniferous tubule diame ter of FSH were drarnatic decrease in fertility (93,94,95,96). Increases in the levels 24 brought about by immunization against PSP-94 with antibodies as well as introduction of synthetic peptides corresponding to regions of PSP-94. lt is speculated that the mechanism by which increased levels of FSH lead to the above-mentioned effects is by down regulation of FSH receptors on the testis following persistent elevation of serum FSH levels (93,94). In addition the antibodies raised against PSP94 were found to affect sperm function by damaging the membrane integrity of the spermatozoa (95). Although the mam function of PSP-94 is the inhibition of FSH, PSP-94 possesses other functions as weil. As mentioned previously, although the majority of PSP-94 is synthesized by the prostate gland in the male, PSP-94 is found in brcast, endometrial and ovarian tissues in the female (69). PSP-94 has been found to suppress levcls of circulating prolactin in the female (97). Since prolactin is known to be the hormone responsible for lactation, PSP-94 leads to suppression in lactation by . acting on the hormone prolactin. Prolactin is also iI1volved in the proper maturation and functioning of the corpus lutcum (98,99). Excess PSP-94 leads to improper preparation of the uterus for embryo implantation which is dependent on the continuous secretion of estrogen and progesterone by the corpus luteum (98,99). Interestingly, prolactin has been shown to be involved in proper ossification of bone. Histomorphometric analysis of bone in prolactin receptor (PRLR) knockout animais revealed lower bone formation rate and reduced bone density (99). Thus PSP94 should exhibit an indirect effect on bone morphology through inhibition of prolactin resulting in decreased osteoblastic activity, although the precise role of PSP- • 94 on bone turnover has not yet been elucidated. 25 • The mode of action of the prolactin suppression effect of PSP-94 rernained unclear until the recent study carried out by Jyothi et al which provided sorne insight explaining how PSP-94 carries outs it effect (89). By carrying out docking experiments, it was discovered that more than 70% of the residues between the PSP94/prolactin and prolactin/prolactin receptor complexes were similar. Moreover, the remaining 30% of the residues on prolactin have low affinity to the prolactin receptor. Thus it appears that PSP-94 suppress lactation by directly acting on the crucial binding sites of prolactin and preventing its binding to the prolactin receptor (89). Briefly, other physiological actions that have been associated with PSP-94 inc1ude the inhibition of phytohacmagglutinin (PHA) stimulatcd proliferation of pcriphcral blood mononuc1ear cells (100), suppression of DNA synthesis in the prostate gland (101) and anti-prolifcrativc cffects on fibroblastic cclllincs spccifically NRK-49F and Balb/c 3T3 cells (102). Receptor for PSP-94 Although a great deal is known about the regulation of PSP-94 production and its function in the reproductive as well as other systems, very little is known about how PSP-94 exerts its effects on the cellular and molecular lcvels. The failure of identification and characterization of a putative PSP-94 receptor(s) is rcsponsible for this lack of knowledge. With advances in molecular biology techniques as well as cloning procedures, failure to identify and clone a PSP-94 receptor would lead one to speculate that none exist. As a result, PSP-94 actions may be via perturbation of the • signaling cascade of other messengcrs as opposed to PSP-94 possessing a separate 26 • signaling mechanism. The recent study carried out by Jyothi et al whereby PSP-94 domains were shown to cxhibit structural and functional homology to that of FSH leads to the speculation that reduction of circulating FSH levels would be due to alterations in the homeostasis of FSH rather than PSP-94 exhibiting a direct effect on the levels of FSH (89). Furthermore, it was also shown that PSP-94 reduces circulating prolactin levels by directly interacting with crucial binding sites of prolactin preventing its binding to its receptor. So could it be that PSP-94 exerts its effects solely by interrupting the signaling cascade of other messengers as opposed to initiating its own separatc signaling cascade? Several studics carricd out sinee the discovery of PSP-94 indicate otherwise. Utilizing competitive inhibition binding assays, it was shown th nt addition of unlabelled PSP-94 inhibited specifie binding of labelcd PSP-94 to LNCaP cclls (103). Furthermorc the same binding inhibition pattern was also observed in PC-3 cells . (104). This indicated the presence of binding proteins specific for PSP-94. The presence of specific "binding proteins" for PSP-94 was also shown on the prostate gland (105). However, does the presence of these 'binding proteins" on the surface of the cells imply that they are the receptors for PSP-94? A number of criteria have to be met before one can indeed confirm the presence of such binding proteins (106). First, it needs to be established that these binding proteins are expressed in known PSP-94 target tissues. In particular, they must be expressed in the prostate gland since it is the gland that expresses the highest amount of PSP-94 compared to the other tissues that express PSP-94. Second, the binding proteins must bind PSP-94 with high affinity. • Third, PSP-94 must exhibit high specificity for the binding protein, however, other 27 .\ ligands can have low-affinity association with the binding prote in. Although results from several studies have indicated that these three criteria have been satisfied, the final criteria that has to be met before these "binding proteins" can be identified as ptoteins involved in PSP-94 mediated effects is their ability to initiate a PSP-94 signaling pathway to direct it's biological effect in target tissues. Objective of Thesis Developmcnt of novel thcrapeutic modalities for prostate cancer is important in being able to combat this common disease. The objectives of the thesis are to evaluate the efficacy of prostate secretory protein of 94 amino acids (PSP-94) in blocking prostate cancer progression and its associated hypercalcemia of malignancy. In addition, a mcchanistie basis for the action(s) of PSP-94 was cxplorcd . • 28 ·) Chapter 2 Prostate Secretory Protcin (PSP-94) decreases tumor growth, metastases and hypercalcemia of malignancy in a syngeneic in vivo model of prostate cancer. Nicholas Shukeir,l Ani Arakelian,l Salam Kadhim 2, Seema Garde2 and Shafaat A. Rabbani 1 IOepartment of Medicine, Physiology and Oncology, McGill University Health Centre, Montreal, Quebcc, Canada and 2Procyon BioPharma Inc, Montreal, Quebec, Canada The study in this chapter is aimed at evaluating the ability of PSP-94 to block prostate cancer progression and its associated hypercalemia of malignancy in a syngeneic in vivo model of prostate cancer. The study is presented in the form of a paper to be submitted for publication. 1 was responsible for a11 the experimental work present in this chapter. 1 appreciate the advice of Dr. Rabbbani and Ani Arakelian during the study. In addition, many thanks and appreciation goes out to Sa11am Kadhim and Seema Garde for providing PSP-94 as we11 as their insightful advice . • , 29 • Abstract Prostate cancer is a common malignancy affecting men, which is often associated with skeletal metastases to cause high incidence of morbidity and mortality. In this hormone-dependent cancer, prostate specific antigen (PSA) and prostate secretory protein of 94 amino acids (PSP-94) are known to serve as prognostic markers for disease progression. In the CUITent study we have examined the effect of PSP-94 on prostate cancer growth and metastases to the skeleton. For these studies, Mat Ly Lu rat prostate cancer cells were transfected with full length cDNA encoding parathyroid hormone rc1ated protein (PTHrP) [Mat Ly Lu-PTHrP], which is known to be the major pathogenetic factor for malignancy associated hypercalcemia. Mat Ly LuPTl-IrP cells were inoculatcd subcutaneously (S.c.) into the right flank or via intracardiac route (I.c.) into the left ventricle of syngcneic male Copenhagen rats. I.C. ~ inoculation of Mat Ly Lu cells routinely results in tumor metastases ta the lumbar vertebrae rcsulting in hind-limb paralysis. AnimaIs were infused with different doses of PSP-94 (0.1, 1.0 & 10.0 )..tg/kg/day) starting on the day of tumor cell inoculation. Time of hind-limb paralysis and tumor volume were determined and comparison was made between PSP-94 treated animaIs and control animaIs receiving vehicle alone. At the end of the study animaIs were sacrificed and plasma calcium, plasma PTHrP and tumoral PTHrP levels were determined. While the highest dose of PSP-94 caused a modest but statistically significant delay in the development of hind-limb paralysis a marked dose-dependent decrease in primary tumor volume was seen in experimental • animaIs receiving PSP-94 due to its ability to promote tumor celI apoptosis. 30 1\ Furthermore, while control animaIs routinely developed hypercalcemia due to PTHrP production, treatment with PSP-94 led to a near normalization of plasma calcium and a marked reduction in PTHrP production as determined by radioimmunoassay and immunohistochemistry. Collectively, these results demonstrate the ability of PSP-94 to be an effective treatment modality for prostate cancer where decrease in plasma PTHrP and calcium levels can serve as useful biochemical markers for monitoring the efficacy of this novel anti-tumor agent. • 31 Introduction Prostate cancer is one of the most commonly diagnosed cancers in men and is the second leading cause of cancer mortality following lung cancer (107). A distinct feature of prostate cancer is its ability to cause osteob1astic skeletal metastases which contributes to the high rate of morbidity and mortality associated with this hormone dependent malignancy (32). Additionally, a significant number of patients with prostate cancer exhibit an increase in their plasma calcium levels due to the production of PTHrP by tumor cells (42,43). Several studies have provided convincing evidence that indeed PTHrP is the major pathogenetic factor responsible for hypercalcemia of malignancy whieh is observed in 15-20% of ail cancer patients (44,108). The progression of clinical prostate cancer can be blockcd at its early stage whcn the cancer is weil confined within the prostate gland (109). However, increased production of many factors including growth factors, sex steroids, angiogcnic factors, , and proteases such as urokinasc (uPA) and matrix metalloproteinases (MMPs) by tumor ceUs and their surrounding stroma results in progression towards a highly invasive hormone-independent state which ultimately leads to metastatic prostate cancer associated with high mortality (1,25,41,49). Despite recent advances in the therapeutic modalities for prostate cancer including surgery and radiotherapy, limited suc cess has been obtained in treating hormone-independent prostate cancer (110). PSP-94, a 94 amino acid, cysteine rich non-glycosylated prote in, is one of the three predominant proteins secreted by the prostate gland and found in human seminal fluid along with prostate specifie antigen (PSA) and prostatic acid • phosphatase (P AP) (61,111). Alternative names used to describe PSP-94 inc1ude 32 .\ prostatic inhibin (~-inhibin) and ~-microseminoprotein (l03). One of the mam 1 biological functions of PSP-94 is the inhibition of follic1e stimulating hormone (FSH) (112). While the majority of FSH is produced and secreted by the pituitary gland it has been demonstrated that the prostate gland is an extrapituitary source of FSH (85). Elevated levels of FSH, in cases of benign prostatic hyperplasia, along with the presence of FSH receptors on the prostate gland suggest an autocrine/paracrine regulation of this hormone on prostate proliferation (86,87). Decreased levels of PSP94 in patients suffering from prostate cancer as weIl as the development of multiple gonadal tumors have implicated a tumor suppressive role for PSP-94 (82). Several studies have demonstrated a progressive decrcase in PSP-94 expression as prostate cancer progresses from a hormone-depcndent to a hormonc-independent state with complete lack of PSP-94 production in highly advanced metastatic prostate cancer (80). This differential expression has allowed PSP-94 to have a prognostic value for prostate cancer (79,83). One added advantage of PSP-94 as a prognostic marker is our ability to, determine its level of production in the hormone-independent stage of the disease that allows for higher degree of sensitivity in tumors that have been previously been exposed to androgen ablating agents (77). In the present study, we have evaluated the effect of PSP-94 to decrease prostate cancer tumor growth and metastases. For these studies we have used our weil characterized syngeneie in vivo model of rat prostate cancer using the rat prostate cancer ceU line Dunning R3227 Mat Ly Lu transfected with the full length cDNA encoding rat PTHrP (49). In this model, S.c. inoculation of tumor cells into the right • flank of male Copenhagen rats routinely results in the development of primary tumors 33 • whereas I.e. inoculation of tumor ceUs leads to skeletal metastases at lumbar vertebra causing hind limb paralysis (41,49,113). Following S.e. and I.e. inoculation of Mat Ly Lu-PTHrP cells, the ability of different doses of PSP-94 to reduce tumor growth, metastases, tumoral PTHrP production, plasma calcium and PTHrP was evaluated . • 34 Materials and Methods Materials. PSP-94 was a gift from Procyon BioPharma Inc. (Montreal, Quebec, Canada) (57). CeUs and cell culture. The Dunning R3327 Mat Ly Lu ceU hne was obtained from Dr. J. T. Isaacs (John Hopkins School of Medicine, Baltimore, MD) and transfected with full length cDNA encoding rat PTHrP as previously described (49). One of the three weIl characterised monoclonal cell hnes Mat Ly Lu-PTHrP-8 was used throughout the course of these studies. Cells were maintained in vitro in RPMI 1640 supplcmented with 2 mM L-glutamine (Life Technologies, Ine. Grand Island, N.Y.), lO cYo fetal bovine serum (FBS), 100 units/ml penicillin-strcptomycin sulphatc (Life Technologies, Inc.), and 250nM dexamcthasonc and G418 (600mg/ml) according to previously established mcthods of culture of these experimental ecUs (Il ). Morphological analysis of control and experimental Mat Ly Lu-PTHrP cells treated with PSP-94 was carried out by plating 5x 104 ceUs/ weU in 6-well plates (Falcon Plastics, Oxnard, CA) in the presence of 10% FBS. CeUs were examined daily for any change in their morphology and photographed (114). Effect of PSP-94 on Mat Ly Lu-PTHrP tumor ceU invasive capacity was examined by 2-compartment Boyden Chamber (Transwell, Cu star, Cambridge, MA) and basement membrane Matrigel (Becton Dickinson Labware, Bedford MA) as previously described (115) . • 35 For growth curves, Mat Ly Lu-PTHrP cells were plated in 6-well plates (Falcon Plastics, Oxnard, CA) at seeding densities of 5x103 cells/well. For 3 days, cells from triplicate wells were cultured in the presence of different doses of PSP-94 (0.1, 1.0 & 10.0 Ilg/ml), trypsinized, resuspended, and counted in a model Z Coulter counter (Coulter Electronics, Beds, UK). Medium was changed every two days. For DNA fragmentation, Mat Ly Lu-PTHrP ceUs were plated in 6 well plates (Falcon Plastics, Oxnard, CA). CeUs were treated with PSP-94 (10.0 Ilg/ml) for up to 72 hours. DNA from treated cells incubated with PSP-94 and ceUs treated with vehicle alone was extracted using a Phenol:Choloroform:Isoamyl alcohol solution (50:48:2). Equal amounts of DNA were subjected to gel electrophoresis on a 2% agarosc gel. DNA fragmentation was visualised by UV light using a transilluminator. Animal Protocols. lnbrcd male Copenhagcn rats weighing 200 - 250g were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Before inoculation, Mat Ly Lu-PTHrP tumor ceUs growing in serum-containing medium were washcd with Hanks buffer, trypsinized, and collected by centrifugation at 1500 rpm for 5 minutes (41,49,114). Cell pellets (10 x 103 cells) were resuspended in lOOll1 saline and injected using 1ml insu lin syringes into the 1eft ventricle of rats anaesthetised with a ketamine/xylazine cocktail. AnimaIs were divided into control groups which received vehicle al one (PBS) and experimental groups which were infused I.P. with different doses (0.1 - 10.0 Ilg/kglday) of PSP-94 starting at the time of tumor ceU inoculation (day 0) until the day of skeleta1 metastases development. The time after tumor cell • inoculation which was required to develop hind limb para1ysis (an index of spinal 36 cord compreSSlOn due to lumbar vertebrae metastases) was detemlined and percentage of starting number of animaIs developing hind-limb paralysis was plotted. Altematively, cell pellets (1 x 106 cells) were resuspended in 100 III saline and injected using 1ml insulin syringes into the right flank of rats. From the time of tumor cell inoculation, experimental animaIs were treated with different doses (0.1, 1.0 or 10.0 Ilg/kg/day) of PSP-94 via S.C. injections for 15 consecutive days. Control animaIs received PBS alone as vehicle control. AH animaIs were numbered, kept separately and monitored daily for the development of tumors. The tumor mass was measurcd in 2 dimensions by calipers and tumor volume was calculated according to the cquation (l x w 2 )/2 (/=Icngth, w=width) (49,114). Ali control and expcrimental animais werc weighed cvcry alternatc day to dctcrminc any adverse ciTeet of PSP-94. Both control and cxpcrimental animais wcrc sacri ficcd on day 16 post tumor ccli inoculation and their tumors wcre rcmovcd and wcighcd. Additionally, thcsc tumors wcre used for histological analysis as dcscribcd bclow. Blood from ail control and expcrimental animaIs was collccted on day 16 for determination of plasma Ca+2 and PTHrP levels (49). Histologie Analysis. For immunohistological analysis, primary tumor samples were dewaxed by heating at 60°C and rehydrated in a graded alcohol series (100%70%). Anti-rat antibody against PTHrP was used as the primary antibody. Tumor sections were incubated overnight at 4°C followed by further incubation with biotinylated universal antibody (Vector Laboratories, Burlingame, CA) for 45-60 • minutes. Sections were rinsed with TBST followed by incubation with Vectastain ABC-AP Reagent (Vector Laboratories, Burlingame, CA) for 30 minutes. These 37 l,: sections were agam washed with TBST and incubated with a Napthol AS-Mix Phosphate/Fast Red solution (Sigma-Aldriche, OakviUe, ON). The sections were finaUy counterstained with Methyl Green (Vector Laboratories, Burlingame, CA) and mounted. For TUNEL assay, tissue sections were dewaxed by heating at 60°C foUowed by washing in xylene and rehydrated through a graded series of ethanol and water. Tissues were incubated with proteinase K for 30 min at 37°C and fixed, blocked and permeabilized. Apoptotic cells were detected by TUNEL assay (in situ cell death detection kit, Boehringer Mannheim, Indianapolis, IN) according to the manufacturcrs' instructions. Positive TUNEL staining was visualised by fluorescence microscopy (116). In other experiments following TUNEL assay, tissue sections were counterstained with Hocchst 33258 (Sigma-Aldrich, Oakville, Canada). Hoechst staining was addcd to tissues at a final concentration of 24ug/ml in PBS and incubated for 15 minutes at room temperature. Tissue sections were washed and visualized by fluorescence microscopy using a blue screen (116). AU results of immunohistochemistry and TUNEL assay were evaluated and interpreted by two independent examiners. Other Analytical Methods. Plasma calcium levels were determined by atomic absorption spectrophotometry (model 703, Perkin-Elmer, Norwalk, CT). For plasma PTHrP, aU samples were tested in two dilutions in PTHrP R.I.A. kit (Nichols Institute • Diagnostics, San Juan Capïstrano, CA.) according to the manufacturers instructions . 38 Statistical Analysis. Results are expressed as the mean ± SEM of at least triplicate detenninations, and statistical comparisons are based on the Student's t-test or analysis of variance. A probability value of <0.05 was considered to be significant (117) . • 39 .\ Results Effeet of PSP-94 on MatLyLu-PTHrP Cell Growth, Morphology and Invasion. Mat Ly Lu-PTHrP ceUs were grown in the presence of 0.1, 1.0 & 10.0 ).lg/ml of PSP-94 or vehicle alone for up to 3 days and the ability of PSP-94 to alter cell doubling time was evaluated daily. Comparison was also made with doubling time of wild type untransfected Mat Ly Lu cells. Transfection of Mat Ly Lu with PTHrP cDNA resulted in reduced doubling time and increase in tumor cell growth due to the growth promoting effects of PTHrP. A significant decrease in Mat Ly LuPTHrP ccli growth was scen following treatmcnt with 10.0 ~Lg/ml of PSP-94 for 72 hrs (Figure 1A). Lower doses of PSP-94 (0.1 and 1.0 ).lg/ml) failed to exhibit any significant changes on tumor cell growth (data not shown). Treatmcnt of Mat Ly LuPTl-IrP cells with 10.0 ).lg/ml of PSP-94 for 3 days rcsuItcd in a noticcablc change in tumor celI morphology whére tumor cells were found to change their normal spindlclike shape to a more rounded and condensed appearance (data not shown). Using a Boyden Chamber Matrigel invasion assay, aIl doses of PSP-94 failed to alter the invasive capacity of Mat Ly Lu-PTHrP cells.(data not shown). Effect of PSP-94 on Mat Ly Lu-PTHrP tumor growth. Male Copenhagen rats were inoculated with Mat Ly Lu-PTHrP cells via S.c. route of injection into the right flank. Starting from the day of tumor ceU inoculation animaIs were infused S.c., below the tumor ceU inoculation site, with different doses of PSP-94 (0.1-10.0 ).lg/kg/day) for up to 15 days. • \ Effect of PSP-94 on reducing tumor growth was evaluated by daily determination of tumor volume with comparison being made to control tumor-bearing animaIs receiving vehicle alone. Control animaIs showed a 40 • progressive increase in tumor volume throughout the duration of the study. In contrast to this, experimental animais receiving PSP-94 showed a marked dose-dependent reduction in tumor volume throughout the course of this study (Figure 2). Both control and experimental animais were monitored for any noticeable side effects and cachexia resulting in weight loss. AU animais were weighed at timed intervals throughout the duration of the study and did not show any significant changes in the weight of control and experimental groups of animais that can be attributed to any potcntial side effect ofPSP-94 treatment (Figure 3). Effeet of PSP-94 on Mat Ly Lu-PTHrP tumor weight. In order to determine the effect of PSP-94 on tumor weight, animaIs inoculated with Mat Ly Lu-PTHrP via S.c. route of injection wcre sacrificed at the end of the study (day 16) and their tumors excised and weighed. Control animais recéiving vehicle alone exhibited large tumors while treatment with different doses of PSP-94 (0.1-10.0 Ilg/kg/day) resulted in a significant dose-dependent decrease in tumor weight (Figure 4). Effeet of PSP-94 ,on the development of skeletal metastases. Mat Ly Lu- PTHrP cells were inoculated into male Copenhagen rats via I.e. injection into the left ventricle. Starting from the day of tumor ceU inoculation (day 0), animais were administered with different doses ofPSP-94 (0.1-10.0 llg/kg/day) via I.P. route. The effect of PSP-94 on delaying the development of skeletal metastases was evaluated by daily monitoring of the animaIs for the development of hind-limb paralysis. AIl of control animais (100%) inoculated with Mat Ly Lu-PTHrP ceUs and receiving vehicle • alone developed hind-limb paralysis by day 13. While 0.1 and 1.0 Ilg/kg/day of PSP94 had no significant effect on the time of hind limb paralysis (data not shown), 41 treatment with 10.0 j.lg/kg/day of PSP-94 resulted in a statisticaUy significant delay in the number of animaIs developing hind limb paralysis. Percentage of total number of animaIs not developing hind limb paralysis at different days is shown in Figure 5. Effect of PSP-94 on plasma PTHrP and calcium levels and tumoral PTHrP production. In order to detelmine the effect of PSP-94 on plasma PTHrP and calcium levels animais inoculated with Mat Ly Lu-PTHrP ceUs via S.c. route were sacrificed at the end of the study and plasma was coUected and PTHrP levels were analyzed using a radioimmunoassay. Comparison was made between plasma collected from normal, non-tumor bearing animais, control tumor bearing animais receiving vehicle alone and plasma collected from experimental animais recciving different doses of PSP-94 (0.1-10.0 j.lg/kg/day). Normal non-tumor bcaring animaIs showed basal Icvels of plasma PTHrP whereas animais inoculatcd with Mat Ly LuPTI-IrP cells and rccciving vehicle alone showcd marked clcvatcd Icvcls of immunorcactive plasma PTHrP lcvels. Treatmcnt of tumor bearing animaIs with PSP94 resulted in a dose-dependent decrease in plasma PTHrP levels (Figure 6A). Analysis of plasma collected from normal non-tumor bearing animaIs and. tumor bearing animaIs receiving vehicle alone revealed a marked increase in plasma calcium levels of control tumor bearing animaIs at the time of sacrifice on day 16 past tumor ceU inoculation. In contrast, experimental groups of animaIs receiving different doses of PSP-94 exhibited a significant reduction in their plasma calcium levels. The highest dose of PSP-94 (10.0 j.lglkg/day) resulted in near normalization of plasma • calcium ofthese experimental group of animais (Figure 6B) . 42 • Tumors from control group treated with vehicle al one and experimental groups treated with different doses of PSP-94 (0.1- 10.0 ~g/kg/day) were excised and analyzed for tumoral PTHrP production by immunohistochemical reaction specifie for PTHrP (1-34). Intense color staining of tumors from control groups of animaIs receiving vehicle alone was observed. In contrast a dose dependent decrease in PTHrP immuno-staining was observed in experimental tumors from animaIs receiving different doses of PSP-94 (Figure 7). Effect of PSP-94 Mat Ly Lu-PTHrP tumor ccli apoptosis ill vitro and i1l vil'o. In order to investigate the underlying molecular mechanism of action of PSP-94 in rcducing tumor growth Mat Ly Lu-PTl-IrP cells wcrc culturcd in the presence of PSP94 (10.0 pg/ml) or vchic\c alone for diffcrcnt time intcrvuls. Genomic D;--~A \'. ·~s extracted from cells cultured in the presence of vehicle alonc or PSP-94. Equal quanti tics of DNA were subjected to elcctrophoresis on a 2% agarosc gel and assessed for the degree of DNA fragmentation. Control Mat Ly Lu-PTHrP cells cultured with vehic\e alone exhibited no signs of DNA fragmentation. However, experimental Mat Ly Lu-PTHrP eeUs cultured in the presence ofPSP-94 (10.0 ~g/ml) exhibited marked DNA fragmentation after 72 hours of treatment (Figure 8A). The degree of DNA fragmentation was also analyzed in vivo using TUNEL assay whieh can serve as a marker for apoptosis. Tumor sections treated with PSP-94 (10.0 ~g/kg/day) were significantly more TUNEL positive as compared to vehic1e treated control tumors (Figure 8B). Counterstaining with Hoechst reagent revealed the • presence of apoptotic bodies in tissue sections from animaIs treated with PSP-94 . Control, vehicle treated tumors exhibited normal DNA staining patterns (Figure 8B). 43 • These in vitro and in vivo findings demonstrate that indeed reduction in tumor volume following treatment wiî.h PSP-94 is due to its ability to promote tumor cell apoptosis . • 44 • - E Cl ::t o o ,... Q) E .1- { } f o M o N (&o~x) Jaqwnu lIaO • o • Fig. 1. Effect of PSP-94 on Mat Ly Lu-PTHrP cell growth. Mat Ly Lu ceUs transfected with vector alone (CMV) or PTHrP were ~~cded at a density of 5x 103 cens/weIl in 6-well plates. Mat Ly Lu-PTHrP ceUs were treated with PSP-94 and were trypsinized and counted using a coulter counter as described in "Materials and Methods". Change in ceU number foI1owing treatment with 10.0 Ilg/ml of PSP-94 for 72 hrs is shown. Each point represents the mean of 3 different experiments. Significant differences from control cells and PTHrP transfectcd ccll are reprcsented by asterisks (p<0.05) . • 46 - • 25 ;;-- 20 E o -o> CTL ~ PSP-94 0.1 ug/kg/day PSP-94 1.0 ug/kg/day PSP-94 10.0 ug/kg/day CJ • "-" Q) E :::l o * 15 J- O E 10 ~ 5 o 7 9 11 Time (days) 14 16 • Fig. 2. Effeet of PSP-94 on Mat Ly Lu-PTHrP tumor volume. Male Copenhagen rats were injected S.c. into the right flank with lx106 Mat Ly Lu~"!"~~p cells. Starting on the time of tumor ceU inoculation animaIs were infused daily with different doses of PSP-94 for fifteen consecutive days as described in "Materials and Methods". Tumor volume was measured at timed intervals and comparison was made with that of tumor-bearing animaIs receiving vehicle alone as control (CTL). Results represent the mean ± SEM of 5 animaIs in each group in 3 different experiments. Significant differences from control tumor-bearing animaIs receiving vehicle alone are rcpresented by asterisks (p<O.05) . • 48 - re 240 1 - - CTL -6PSP-94 0.1 ug/kg/day - PSP-94 1.0ug/kgday - .. -. PSP-94 10.0ug/kg/day 2301- -a - en E ca 1- C) ---...., 220 ..c: C) - Q) 3: ...., ca 210 a: 200 . ~/ o 1 1 5 7 9 11 Time (Days) 1 1 1 13 15 17 • Fig. 3. Eilect of PSP-94 on animal weight. Mal~ l'openhagen rats werp i!1jr:'cted S.C. into the right flank with lxl0 6 Mat Ly Lu- PTHrP cells. Starting on the time oftumor cell inoculation animaIs were infused with different doses of PSP-94 for fifteen consecutive days as described in "Materials and Methods". AH animaIs were weighed at timed intervais and comparison was made with that oftumor-bearing animaIs receiving vehicle aione as control (CTL). Results represent the mean ± SEM of 5 animaIs in each group in 3 different experiments . • 50 • l~::s tn o o . or- Cl ~ ....... Cl ::s . ==============~O ~----------------------------~~ ~ o ____________________________ C\I • Ln ,.. o or- Ln 1- ~o o ~ Cl 1 a. Cl) a. • Fig. 4. Effect of PSP-94 on Mat Ly Lu-PTHrP tumor we!ght. Male Copenhagen rats were inoculated with lxl0 6 Mat Ly Lu-PTHrP cells via subcutaneous injection into the right flank. Starting from the day of tumor cell inoculation animaIs were administered with different doses of PSP-94 for fifteen consecutive days as described in "Materials and Methods". At the end of the study tumors from control (CTL), vehicle treated animaIs and PSP-94 treated animaIs were excised and weighed. Results represent the mean ± SEM of 5 animaIs in each group in 3 different experiments. Significant differences from control tumor-bearing animaIs receiving vchicle al one are represcnted by asterisks (p<O.05) . • 52 • -te r-- ---_ Lt') ~ o::t Cl 1 -la.. I-cn Ua.. -te r __________ :!.\ 1 : (1) E .t- \ 1 0 0 1 J 1 1 0 CO 0 0 0 N (0 ~ ~ • ". . SIBW !UB paZÂ IBJB d-UO U JO 0/0 ~ ~ ): 0 • Fig 5. Effeet of PSP-94 on spinal metastases. Male Copenhagen rats were inoculated via I.e. route into the left ventricle with IOxJ0 3 M~t L~' !..~-PTHrP ceUs. Starting on the time oftllm,-,r ('cH inoculation (day 0) animaIs were infused with different doses of PSP-94 (0.1 - 10.0 Ilg/kg/day) until the day of development of hind-limb paralysis as described in "Materials and Methods". AnimaIs receiving vehicle alone as control (CTL) or PSP-94 were monitored daily for the development of hind-limb paralysis and % of animaIs not paralyzed at different time points in each group was calculated. Results represent the mcan ± SEM of 5 animaIs in each group in 3 diffcrent cxpcrimcnts. SignifiC'ant diffcrcnccs in O~I of non-pClrn!yzcd :mimals from control tUlllor-bcaring animaIs rccciving vchiclc alone arc rcprcscntcd by astcrisks (p<0.05) . • 54 • A. - 100 ~ ~ M -- 80 ca 60 1 ..- - ..J en s:: Q,) > * :l C'" Q,) 0 40 E a. ........ a. '- J: 20 r- a. .- 0 N CTL 0.1 ug/k g 1.0u g/kg 10.0 ug/k g PSP-94 B. 5.0 -E -E :E 4.5 :l 4.0 ë::.; * ca 0 ct! E en 3.5 ct! a. • 3.0 01 [1 C4 N CTL 0.1 ug/k g 1.0u g/kg 10.0 ug/k g PSP -94 • Fig. 6. Effeet of PSP-94 on plasma PTHrP and Calcium in tumor bearing animaIs. Panel A: Male Copenhagen rats were inoculated S.c. with lxl0 6 Mat Ly Lu-PTHrP cells. Starting on the time of tumor cell inoculation animaIs were administered with different doses of PSP-94 for fifteen consecutive days as described in "Materials and Methods". All animaIs were sacrificed at the end of the study (day 16) and plasma \Vas collected from control (CTL), vehicle treated animaIs and PSP-94 treated animaIs and analysed for immunoreactive plasma PTHrP (iPTHrP) levels using radioimmunoassay as described in "Materials and Methods". Plasma PTHrP levels in normal non-tumor bearing animais is also shown (N). Panel B: Male Copenhagcn 6 rats were inoculated S.c. with 1x 10 Mat Ly Lu-PTl-IrP cells. Starting on the time of tumor cell inoculation animais \Vere infused \Vith different doses of PSP-94 for fifteen consecutive days as described in "Muterials and Methods". All animais were sacrificcd at the end of the study (day 16) and plasma was collected from vehicle treated control (CTL) a;td animaIs and PSP-94 treated animaIs for analysis of plasma calcium levels as described in "Materials and Methods". Plasma calcium from normal, non-tumor bearing animaIs is also shown (N). Results represent the mean ± SEM of 5 animaIs in each group in 3 different experiments. Significant differences from control tumor-bearing animaIs receiving vehicle a10ne are represented by asterisks (p<O.05) . • 56 • • CTL PSP94 (0.1 ug/kg) PSP94 (1.0ugfkg) PSP94 (10.0ug/kg) • Fig. 7. s. Effect of PSP-94 on PTHrP production by Mat Ly Lu-PT HrP tumor 6 Lu-PT HrP ceUs. Male Copenhagen rats were inoculated S.c. with lxl0 Mat Ly ç:t~rtinQ: differe nt on the time of tumor ceU inoculation animaIs were infuse d with "Mate rials and doses of PSP-9 4 for fifteen consec utive days as descri bed in their prima ry Metho ds". AlI animaIs were sacrificed at the end of the study and ns of tumor s tumors removed, paraffin embedded, and sectioned. Histol ogical sectio of PSP-9 4 were from animaIs receiv ing vehicle along (CTL) or differ ent doses "Mate rials and stained with and antibody specifie for PTHrP (1-34) as descri bed in sections were Methods". Three animaIs \Vere present in each group and three tumor three differe nt anaIyzed for each animal. Results repres ent the mean ± SEM of ments is shown. expcriments. A representative photomicrograph of three such experi Magnification 200X . • 58 A. ~ en 1 ..J tU a. CI) a. B. CTL TUNEL Assay Hoechst Staining • PSP-94 • Fig. 8. Effect of PSP-94 on DNA fragmentation of 1\1at Ly Lu-PTHrP cells in vitro and in vivo. Panel A. Mat Ly Lu-PTHrP cells were cultured in the presence of vehicle al one or PSP-94 (1 0.0 ~g/ml) for up to 72 hours. DNA was isolated [rom control, vehicle treated cells and PSP-94 treated cells as described in "Materials and Methods". Isolated DNA was subjected to electrophoresis on a 2% agarose gel and visualized under UV light. A representative photograph of three such experiments is shown. 6 Panel B. Male Copenhagen rats were inoculated with 1x 10 Mat Ly Lu-PTHrP cells. Starting on the time of tumor ceIl inoculation animaIs were infused with different doses of PSP-94 for fifteen consecutive days as describcd in '"Materials and Methods". AIl animais were sacrificed at the end of the study and their primary tU1110rs removed, para ffi ï' cmbeddcd, scctioncd and proccsscd by TUNEL assay as described in "Matcrials and Methods" (upper panel). Following TUNEL, they were . counterstained with Hoechst reagent (lower pancl). A rcprescntative photomicrograph for thrce such expcrimc~ts in each group is shown. Magnification 200X • 60 Discussion ln this study we have used a syngenelc model of rat prostate cancer to demonstrate the ability of PSP-94 to reduce prostate cancer growth and metastases. High degree of amino acid sequence homology between human and rat PSP-9.1 allowed the use of human PSP-94 for these studies (11.s~ ~ of thi&:~1Glo-gous model for prostate cancer allows for full interaction between the host environment and growth factors (EGF, TGF -~) (4) and proteases (uP A, MMPs) (26,116) secreted by tumor cells. In order to evaluate the efficacy of PSP-94, Mat Ly Lu cells transfected with full-Iength cDNA encoding PTHrP (Mat Ly Lu-PTHrP) ('clIs were utilizeu. Thcse prostate cancer cells are hormone-indepcndent allowing for the evaluatioll of the effeet of PSP-94 on late stage prostate cancer. Due to the high levcls of PTl-IrP production these animais routinely develop hypercaJcemia, a common complication in many paticnts suffering from prostate cancer (42,43). Mat Ly Lu.. PTHrP cells had a higher rate of cell proliferation as compared to control Mat Ly Lu cells transfected with v~ctor aJone. Treatment of Mat Ly Lu-PTHrP cells with PSP-94 for 72 hrs resulted in a significant effect on tumor cell proliferation, morphology and DNA fragmentation which is considered to be an apoptotic marker. Inoculation of male Copenhagen rats with Mat Ly Lu-PTHrP cells into the right flank via S.C. injections resulted in the development of primary tumors. Whereas control, vehicJe trcated animaIs developed large primary tumors, treatment with different doses of PSP-94 resulted in a dose-dependent decrease in their tumor mass. These anti-tumor effects were not associated with any noticeable side effects or • weight loss of experimental animaIs. TUNEL analysis carried out on tumoral sections 61 from control and experimental animais revealed that PSP-94 treated tumors are more TUNEL positive as compared to control tumors, indicating a higher degree of apoptosis in PSP-94 treated animais. In addition to this, counterstaining with Hoechst reagent revealed condensed; apoptotic chromatin in PSP-94 treated tumors whereas control tumors exhibited normal DNA staining. Upon sacrifice of animais plasma was collcctcd and analyzed for plasma PTHrP and calcium levcls. Nom1al, non-tumor bearing animaIs have undetectable levels of plasma PTHrP whereas inoculation of animais with Mat Ly Lu-PTHrP cells resulted in marked increase in their plasma PTHrP lcvels. In contrast to this, treatmcnt with the differcnt doses of PSP-94 resulted in a dose-dcpcndent dccrcase in plasma PTllrP levcls. In addition, th(' <;;1111(' dn<;('- dcpc11llcnt dccrcase was obscrvcd in tumoral PTHrP production whcn tumor samplcs [rom control, yehicle trcatcd and PSP-94 trcatcd animaIs were subjected to immunohistochcmical analysis. Bcing the major pathogcnctic factor of hypcrcalccmia of malignancy, plasma calcium Icvels corrclatcd with that of plasma PTHrP lcvcls (42,43,44). Inoculation of Mat Ly Lu-PTHrP cclls into thc animaIs resultcd in a marked increase in their plasma calcium levels as compared to serum from nom1al, non-tumor bearing animaIs. Administration of different doses of PSP-94 resulted in a dose-dependent decrease in plasma calcium levels with the highest dose of PSP-94 leading to a near normalization of plasma calcium levels. Since the major cause of prostate cancer related mortality is the development of metastases, evaluation of the effect of PSP-94 on delaying the development of skeletal metastases was carried out by inoculating male Copenhagen rats with Mat Ly • Lu-PTHrP cells via I.e. route into the left ventric1e. 62 Routine injection of Mat Ly Lu • ceIls into the !eft ventricle results in the deve!opment of skeletal metastases causing compression of the spinal cord lcading to hind-limb paralysis (49). Whereas all control, vehicle treated animais developed hind-limb para1ysis by day 13, administration of the highest dose of PSP-94 starting from the time of tumor cell inoculation resulted in modest delay in skeletal metastases. Such results suggest 10w bioavailability of PSP-94 to the skeleton, a common drawback associated with developing effective therapeutic agents for skeletal metastases (119). Using this mode! we were not only able to demonstrate the anti-tumor effects of PSP-94 by reduction in tumor volume and weight but also biochcmica1 parameters like plasma calciulll and PTI-lrP lcvcls <11so showcd a markcd dccrease following thcrapy. A significant finding in these studics was tl1at while dccrease in tumor volume was dose-dependent, 10.0 Ilg/kg/day of PSP-94 did not show a marked dccrcase in tumor volume as compared to 1.0 ~lg/kg/day PSP-94. In contrast, the ability of PSP-94 to rcduce plasma calcium, plasma PTHrP and tumoral PTHrP continued to show a dose-dcpcndcnt cffcct \Vith 10.0 Ilg/kg/day PSP-94 causing near normalization of plasma calcium and PTHrP leve1s. These findings allow. us to specu1ate that PSP-94 may also have addition al effects including its ability to regulate PTHrP production by tumor cells or alter calcium homeostasis. lndeed PSP-94 has been shown to suppress follicle stimulating hormone (FSH) which is kno,wn to regulate intracellular calcium (120). Suppression of FSH by PSP-94 may serve as an additional mechanism to cause anti-tumor effects due to the growth-promoting effects • of FSH_~n prostate cancer (86). Furthem10re, although cloning and characterization of a putative PSP-94 receptor has not been estab1ished severa1 studies have provi,ded 63 • evidence for the existence of PSP-94 binding proteins on prostate cancer ceUs. Binding of PSP-94 to these proteins might initiate a signaling cascade that results in the anti-tumor effects observed (103,104). Collectively, the results of this study demonstrate PSP-94 to be an effective inhibitor of hormone-independent, late stage prostate cancer growth and its associated hypercalcemia of malignancy without manifesting any noticeable cytotoxic effects. Further studies will define the minimum sequence requirement to obtain maximum level of efficacy. These synthetic peptides, their peptidomimetic analogues al one or in combination with currcntly available chemotherapeutic agents will provide unique opportunitics to block prostate cancer progression with highly effective non-toxic biotherapeutic agents which can be dclivered over a long period of time without any drug associated side effects. Thcse approaches will go a long way in reducing prostate cancer associated morbidity and mortality . • 64 • Chapter 3 General Discussion Despite advances in the staging and therapeutic modalities for prostate cancer it still remains one of the most çommonly diagnosed cancers in men associated with high morbidity and mortality rates (32,107). The depcndence of the prostate gland on androgens leads to the utilization of hom10nal therapy resulting in the ablation of circulating androgens (2,50,52). Consequently, a reduction in prostate tumor volume is achieved; eventually however, ihc rccurrence o~ the disease is imminent as androgen-indepcndent cells proliferate and compose the majority of the tumor. Dcvelopmcnt ofboth skclctal and non-skcletal mctastases is the primary factor for the high mortality rate associated with advanced prostate canccr (32). However there arc many rcasons to be cheerful. Improvemcnts in many therapcutic modalities for prostate cancer including chemoprevcntion, pharmacotherapy, potential for gene therapy and the development oftumor vaccines are grounds for optimism (53). PSP-94, an inhibin, is one of the three predominant proteins that are secreted by the prostate gland and has been shown to be associated with prostatic tumors (61). While the majority of PSP-94 is produced by the prostate gland, other tissues such as breast, ovary, endometrium and trachea have also been shown to express PSP-94 but to a lesser extent (66,68,69). The differential expression ofPSP-94 in prostatic tumors as the disease advances from an early to late stage prostate cancer has allowed for its • use as a prognostic/diagnostic marker (82,83) . 65 • Whereas h0n110neS su ch as FSH, VIP, epinephrine and isoperentol have been shown to upregulate the expression of PSP-94, in benign hyperplastic tissue and androgen-dependent, low invasive LneaP prostate cancer cell lines (74), androgens such as testosterone and estrogen do not alter the expression of PSP-94 (75,76). The androgen-independent nature of PSP-94 has allowed for its superior use as a prognostic/diagnostic marker in tumors that have been exposed to androgen ablating agents. This thesis dealt with evaluating the potential use of PSP-94 as a thcrapeutic modality for reducing prostate cancer growth, metastases and hypcrcalcemia of malignancy. While prostate cancer cells treated with PSP-94 cxhibitcd an incrcase in their doubling time hence a reduction in their growth rate, administration of PSP-94 ill vivo resulted in significantly smallcr tumors with corrcsponding lowcr tumor weights. ln addition, biochcmical markers such as scrum PTllrP and calcium Icvcls wcre also reduccd following trcatmcnt with PSP-94. Furthcn11orc, PSP-94 causcd a rcduction in the lcvels of PTHrP production by tumor cells. No drug-associated side effccts were manifcsted leading to the possible use of PSP-94 on prostate cancer patients over a prolonged period of time to block the progression of prostate cancer. Administration of PSP-94 yielded a mode st but significant delay in the development of skeletal metastases, suggesting low bioavailability to the skeleton. Such results are a common drawback in development of anti-metastatic agents. Although the results obtained throughout the course of this thesis point to a valuable therapeutic option in PSP-94 several future studies should be explored . • Structure functions studies whereby PSP-94 is broken down into many different 66 • peptides should be carried out in order to define the minimum amino acid sequence required to elicit the anti-tumor effects of this protein. Generation of su ch peptides would lead to not only increased efficacy in terms of reducing tumor volume but might also result in increased bioavailability to the skeleton as a result of the smaller peptides being able to readily penetrate into the skeleton. Such an effect would lead to a more effective response in delaying the development of skeletal metastases, as se en through the delaying of development ofhind-limb paralysis. The degree of tumor reduction obtained through administering PSP-94 alone is sufficient to warrant its use as a stand alone drug for combating this disease; however, one has to keep in mind that the Mat Ly Lu-PTl-irP modc1 uti\ized in these studies rcprcsents late stage, highly invasive, androgen-independent prostate cancer. It can be postu\atcd that the cfficacy of PSP-94 to block tumor progression will be significantly highcr in a mode\ which represents early stage. low invasive prostate cancer. One would expect that administration of PSP-94 will elicit a greater response wh en tested in such a system due to the androgen dependent nature of the disease at its early stage. As mentioned previously, one of the main physiological functions of PSP-94 is the inhibition of FSH (62) which has been shown to be a growth factor involved in the progression of prostate cancer (86,87). Administration of PSP-94 in such a model will have a dual effect whereby PSP-94 elicits its own apoptotic response as well as inhibiting crucial growth factors involved in the progression of prostate cancer one of which is FSH. Furthermore, combination studies should be carried out whereby PSP- • 94 is administered in combination with various commonly utilized therapeutic 67 • approaches for prostate cancer which have shown promising results. Of interest is whether the combinatorial approach yields an additive or a synergistic response. As previously mentioned, the reduction in tumor volume levels off after 1.0 Ilg/kg/day while 10.0 Ilg/kg/day proved to be more effective at reducing plasma calcium and PTHrP levels leading to near normalization ofboth biochemicalmarkers. FSH has been shown to regulate intracellular calcium (120) and inhibition of FSH by PSP-94 might provide for a mechanism which explains the more pronounced reduction in both plasma calcium and PTHrP levels as compared to reduction in tumor volume. Therefore studies should be carried out to evaluate the direct ability of PSP-94 to alter intraccllular calcium transport. Sincc PSP-94 is expressed in tissues other th an the prostate including ovarian, cndomctrial and breast tissues (66,68,69), evaluation of the efficacy of PSP-94 in blocking the progression of the respective cancers. The regulation of the PSP-94 gene by agents specifically hormones is weB elucidated. However the precise mechanism of the differential expression of PSP-94 in prostate cancer as the cancer progresses from an carly stage to a late stage with complete lack of PSP-94 production in highly advanced prostate cancer is not yet understood. One possible mechanism behind this differential expression is through epigenetic regulation of gene transcription by DNA methylation. DNA methylation, the addition of methyl groups to a cluster of CpG islands, has been shawn to be involved in the regulation of man y proto-oncogenes as well as tumor suppressor • genes. Addition of the methyl groups to CpG islands interfcres with the interaction between the promoter binding protein and the respective promoter region. 68 • suppreSSIOn of Conse quentl y, hyper -meth ylatio n of the promo ter result s in protei n leve1s. transcription of the DNA leading to lower levels of mRNA and hence ined, howev er, The methy lation status of the PSP-9 4 gene is yet to be determ Risbridger et al have demonstrated the hypen nethyl ation of the inhibin Cl subunit in tion of other prosta te carcin oma (121). Such observ ations as weIl as to the regula lation might genes through DNA methylation leads to the speculation that DNA methy theref ore highly be one factor involv ed in the regulation of the PSP-9 4 gene. It is status of the desirable to carry out in depth analysis to determine the methy lation ities for this PSP-9 4 gene as this can lead to the development of therap eutic modal disease. one possible The work presen ted in this thesis demonstrated that apopto sis is tumor volume. mechanism by which admin istratio n of PSP-94 results in reduci ng precis e molec ular Additional detailed studies need to be carried out to identif y the evalua ting other basis by which PSP-9 4 induce s apoptosis. Furthe rmore , studie s volum e, such as mecha nisms by which PSP-9 4 leads to a reduct ion in tumor inhibition of angiogenesis, should be carried out n to this Although a great deal about the regulation of PSP-9 4 is known in additio ity for prostate thesis providing evidence for the use of PSP-94 as a therapeutic modal hypercalcemia cancer and its associated complications namely skeletal metast ases and ined. This is of malignancy, the precise mode of action of PSP-94 is yet to be detenn been c10ned and particularly important since the putative PSP-94 receptor has not yet • provid e further characterized. Comp lete charac terizat ion of a PSP-9 4 recept or will for the anti-tumor potential insight as to the potential signaling pathways respon sible 69 1 effects of PSP-94. Modulation of these signaling cascades in order to provide an increased effect should prove useful in combating this common disease. In conclusion, PSP-94 has been shown to be an effective treatment modality for prostate cancer and its associated complications. While administering PSP-94 alone is a first major step in providing a novel biotherapeutic modality without any noticeable side effects, combination studies with other therapeutic agents that have been shown promising results as well as development of smaller synthetic peptides or peptidomimitics agents will result in enhancement of its efficacy and add to our existing arsenal of therapeutics against prostate cancer. • 70 Chapter 4 References 1. Feldman, BJ. and Feldman, D. The development of androgen-independent prostate cancer. Nature Reviews Cancer. 1: 34-45, 2001 2. Kirby, RS., Christmas, T J., Brawer, MK. Prostate cancer, second edition, 2001 3. Health Canada. Canadian Cancer Statistics, 2002 4. lIellawell, GO. And Brewster, SF. Growth factors and thcir rcccptors in prostatc cancer. BJU International, 89: 230-240, 2002 5. Moyad, MA. 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Research Personnel and QU:llifications: List the names of ail individuals who will be in contact with animais in this study (including the Principal lnvestigator) and their employment classification (investigator, technician, research assistant, undergraduate/graduate student, fellow). Indicate any training received (e.g workshops, lectures, etc.). The PI certifies that an personnellisted here have suitable training and/or experience, or will be provided with the specifie training which qualifie:i'them to perform the procedures described in the protocol. Each person listed in this section must sign to indicate that slhe has read this protocol. (Space will expand as needed.) Name Signnture Classilication Training Information 1 A\' S.A. Rabbani Investigator M.D., University Animal Course Julie Gladu Technician University Animal Course Ani Arakelian Technician University Animal Course Nicholas Shukeir Graduate studt!nt University Animal Course Parissa Khalili Graduate student University Animal Course • Enter the tirst name, press 'enter', then the 2" Dame ... complete the tirst calumn, then the 2"', th en the 3rd •• If 3n undergradu3te studenl is involycd . the rok of the student and the suoervision recei\'d must be describcd. 5. 1 Summary (In language that will be understood by members of the general public) a) RATIONALE: Dcscribc, in 3 short paragraph, the o\'eral1 3im of the study and its potential benefitto human/animal hcalth or to the advancement of scientilic knowled!!e. Development of novel therapeutic strategies to control hypercalcemia of malignancy. b) SPECIFIC OBJECTIVES OF THE STUDY: Summarize in point form the primary objectives of this study. To examine the chemical and biologieal charactcristics ofparathyroid hormonc-like factors rcleased by tumers associated with the hypcrcalcemia of malignancy syndrome. c) l'ROGRESS REPORT: If thb b a rcnewal of an ongoing project, BRlEFLY summarizc wh:lt was accomplishcd during the prior approval period and inùicate if anù holV the currcntl?,o:ds diffcr from those in the original application. Invcstigation of thc role of PTHRP in tumor biology and to examine various strategies to control tumor progression. \ d) SUl\l:\lARY OF l'ROCEDL'RES FOR A:"iI:\L\L USE REPORT TO THE COC: Using KEY \YORDS O;--;L Y, list the procedures used (c.g. anacsthesia, brecding culony, injection Il', g3va~e, drug administration, major su rvival su rger;', eu tlwnasia b~' e'l:5a n~uination. behavioural studies). Rcfcr to Appendix lof the Guidelines for a more complete list or su!!!!ested kev words. - Polyclonal antibody production, c:.lthar.asia by exsanguinotion - Subeutaneous injection of tumor ce Ils/caudal artery collection ofblood'bleeding from eor.·passive immunization - Anaesthesia, intracardiac injection of tumor cells, drug ac:ministration intrape:itoneally.'succ:.ltaneously, saphenous vcin blood collection. mini-pumo implantation. 6_ AnimaIs To Be l"sed e) \. Purpose of ...... :l:;;;al L'se (Chee;; Ùn.,): Studies of a fundamentai nature:'basic research Iiances for human/veterinarv medicine Will the project involYe breeding animaIs? NO X YES Will the projec! involve the generation of genetically altered animaIs? Will field studies be conducted? NO X 'l'ES 1\0 X YES c) Description of AnimaIs Splstrain 1 \ Rats 1 • Species & strain Supplier/Source Strain Sp/strain 2 Rats 1 \ Copenhagen \ Sp/strain 3 \ Rats Splstrain \ Rabbits ~ Splstrain 5 \ Nude mice 1 1 Sp/strain 6 \ Nude mice 1 Fischer \ SpragueDawley 91 NZ\V Balb C Balb C