TRAMP-C2 Cells Research Articles

Abstract 1998: Inhibition of hedgehog signaling in prostate cancer by extracts of Sutherlandia

Abstract Prostate cancer possesses long latency periods and is responsive to dietary mediators, making it a target for phytochemoprevention. Many botanical compounds that have been proposed to prevent cancer may potentially work via inhibition of the hedgehog signaling pathway. Here we investigated the potential of Sutherlandia frutescens (also called Lessertia and “cancer bush” in South Africa) to inhibit hedgehog signaling. We hypothesize that the anti-cancer effects of Sutherlandia are due to its inhibition of hedgehog signaling pathway activity. Methods: We evaluated Sutherlandia's effects of growth inhibition in vitro both in the human prostate cancer cell line PC3 and mouse prostate cancer cell line TRAMP-C2 by exposing the cells for 72hrs to plant extracts before protein concentrations were measured to evaluate inhibition of growth. To determine hedgehog pathway inhibitory activity, we treated two different cell lines (an NIH3T3 cell line and a TRAMPC2 cell line) stably transfected with a firefly luciferase reporter having Gli response elements in the firefly luciferase promoter. After 48hr exposure to treatment with multiple doses of Sutherlandia, a firefly substrate was added to the cell lysate and the luciferase signal was measured using a Biotek Synergy plate reader. Finally, Sutherlandia was incorporated into a diet and fed to TRAMP (Transgenic Adenocarcinoma Mouse Prostate) mice to examine whether the plant can delay or even inhibit prostate cancer incidence in the TRAMPmouse model. Results: Sutherlandia extracts inhibited growth of both PC3 and TRAMPC2 cells. In addition, hedgehog signaling was inhibited by Sutherlandia in both NIH3T3 and TRAMPC2 reporter cells. Interestingly, in vivo the lowest concentration of Sutherlandia diet had the greatest effect on inhibition of poorly differentiated adenocarcinoma in the TRAMP mice. Conclusions: Sutherlandia's inhibition of prostate cancer growth may be via inhibition of hedgehog signaling. Future work to identify in Sutherlandia the active compound having this effect, and the ideal concentration for use in vivo may lead to a means whereby dietary intervention for prostate cancer prevention and/or treatment is possible. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1998. doi:1538-7445.AM2012-1998

Abstract 609: Benzyl isothiocyanate inhibits prostate cancer development in the transgenic adenocarcinoma mouse prostate model

Abstract Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has also been shown to have anti-tumor properties. In the present study, we attempted to determine whether BITC inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. Male TRAMP mice and their nontransgenic (normal) littermates at 5 wk of age were randomly divided into control and BITC-treatment groups and gavage-fed with 0 (vehicle), 5, or 10 mg/kg of BITC every other day. At the time of sacrifice (24 wk of age), BITC did not affect body weight of normal or transgenic animals. The genitourinary tract weight of TRAMP mice was increased markedly as compared to normal mice, and this increase was suppressed significantly via the oral administration of 5 or 10 mg/kg of BITC. H&E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the 24 week old vehicle-fed TRAMP mice, whereas number of lobes with WDC was reduced and the number of lobes with prostatic intraepithelial neoplasia was increased by feeding with 5 or 10 mg/kg of BITC. BITC feeding reduced the number of cells expressing the Ki67 (a proliferation marker) in the prostatic tissue. Additionally, BITC feeding reduced the expression of cyclin E, cyclin D1, cyclin A, and cyclin-dependent kinase (CDK)2. Our in vitro cell culture results revealed that BITC decreased DNA synthesis and CDK2 activity in TRAMP-C2 mouse prostate cancer cells. However, BITC did not affect apoptosis either in transgenic mice in vivo or in TRAMP-C2 cells in vitro. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in animals treated with BITC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 609. doi:1538-7445.AM2012-609

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Oral delivery of particulate prostate cancer vaccine: In vitro and in vivo evaluation

Background: Various approaches have been evaluated for generation of efficient immune response against tumor antigens. Our approach exploits usage of particulate delivery to generate immune response against prostate cancer antigens.Purpose: The aim of this study was to evaluate the efficacy of prostate cancer vaccine derived from a murine prostate cancer cell line, TRAMP C2 in murine model via oral route using aleuria aurantia lectin as a targeting ligand for M-cells in the intestinal Peyer’s patches.Methods: The whole cell lysate (WCL) was obtained from TRAMP C2 murine prostate cancer cell line and was formulated into particles using one step spray drying process. For in vivo studies, 4–6 week old C57BL/6 male mice were vaccinated orally biweekly for 10 weeks. Serum samples were analyzed at regular intervals to determine serum IgG levels. The mice were then challenged with live TRAMP C2 cells to determine efficacy of the vaccine.Results: The serum IgG levels of vaccinated animals were higher compared to that of the controls. Moreover, the tumor growth was retarded significantly in the vaccinated mice compared to that of controls (p < 0.001).Conclusions: The above findings suggest that oral particulate WCL vaccine can trigger an immune response against prostate cancer antigens.

Activation of Nod1 and Nod2 induces innate immune responses of prostate epithelial cells

Nod1 and Nod2 are cytosolic receptors which are responsible for sensing bacterial peptidoglycan derivatives. In this study, we determined whether Nod1 and Nod2 are involved in the innate immune responses of prostate epithelial cells. The expression of Nod1 and Nod2 was examined by RT-PCR and immunohistochemistry. ELISA was performed to determine the production of cytokines/chemokines. Activation of NF-κB and MAPK was examined using western blot analysis. The Nod1 gene was distinctly expressed in all tested cells including DU145, PC3, and TRAMP-C2 cells, whereas Nod2 expression was weak. Both Nod1 and Nod2 proteins were expressed in normal mouse prostate epithelia with difference of expression levels. Tri-DAP (Nod1 agonist), but not MDP (Nod2), increased the production of IL-8 (or KC) and IL-6 in prostate epithelial cells. Tri-DAP and MDP could upregulate the gene expression of COX-2 and activate NF-κB and MAPK. In addition, Tri-DAP and MDP synergized with TLR agonists to induce the production of IL-8/KC or IL-6 in PC3 and TRAMP-C2 cells. We finally showed that Nod1 and Nod2 were also expressed in a wide range of prostate lesions including prostate intraepithelial neoplasm (PIN), phyllodes-like tumor, and adenocarcinoma in TRAMP (transgenic adenocarcinoma of the mouse prostate) mice, even though the expression level of Nod1 and Nod2 was different. These results indicate that Nod1 and Nod2 may play important roles in the innate immune response of prostate epithelial cells and the development and progression of prostate cancer.

Growth and Progression of TRAMP Prostate Tumors in Relationship to Diet and Obesity

To clarify effects of diet and body weight on prostate cancer development, three studies were undertaken using the TRAMP mouse model of this disease. In the first experiment, obesity was induced by injection of gold thioglucose (GTG). Age of prostate tumor detection (~33 wk) and death (~43 wk) was not significantly different among the groups. In the second study, TRAMP-C2 cells were injected into syngeneic C57BL6 mice and tumor progression was evaluated in mice fed either high-fat or low-fat diets. The high fat fed mice had larger tumors than did the low-fat fed mice. In the third study, tumor development was followed in TRAMP mice fed a high fat diet from 6 weeks of age. There were no significant effects of body weight status or diet on tumor development among the groups. When the tumors were examined for the neuroendocrine marker synaptophysin, there was no correlation with either body weight or diet. However, there was a significant correlation of the expression of synaptophysin with earlier age to tumor detection and death. In summary, TRAMP-C2 cells grew faster when the mice were fed a high-fat diet. Further synaptophysin may be a marker of poor prognosis independent of weight and diet.

Human prostate cancer ZIP1/zinc/citrate genetic/metabolic relationship in the TRAMP prostate cancer animal model

Prostate cancer is the second leading cause of cancer deaths among men. The availability of animal models that represent the events and factors that exist in the natural history and biology of human prostate cancer is essential in dealing with prostate cancer. In recent decades and presently, emphasis has been directed at the development and employment of prostate cancer induced in transgenic mice. However, the important consistent hallmark characteristic and event of decrease in zinc and citrate and downregulation of ZIP1 zinc transporter in prostate malignancy has not been studied or identified in any animal model. We investigated the status of these parameters in TRAMP tumors as compared with human prostate cancer. The results show that citrate levels are markedly decreased in the developing and advancing stages of malignancy in TRAMP. Zinc levels are also decreased and ZIP1 transporter is lost in TRAMP tumors. In vitro studies show that zinc treatment of TRAMP C2 cells exhibits cytotoxic effects. Collectively, these results mimic the ZIP1, zinc, and citrate status and relationship that exist in human prostate cancer. This is the first report that establishes the existence of the human prostate zinc/citrate hallmark characteristic and relationship in an animal model. It now appears that the TRAMP model will be suitable for studies relating to the implications and role of zinc- and citrate-related metabolism in the development and progression of human prostate cancer.

The Hexane Extract of Saussurea lappa and Its Active Principle, Dehydrocostus Lactone, Inhibit Prostate Cancer Cell Migration

Saussurea lappa has been used in Chinese traditional medicine for the treatment of abdominal pain, tenesmus, nausea, and cancer; previous studies have shown that S. lappa also induces G(2) growth arrest and apoptosis in gastric cancer cells. In this study, we investigated the effects of hexane extracts of S. lappa (HESLs) on the migration of DU145 and TRAMP-C2 prostate cancer cells. DU145 and TRAMP-C2 cells were cultured in the presence of 0-4 μg/mL HESL with or without 10 ng/mL epidermal growth factor (EGF). HESL inhibited the basal and EGF-induced migration of prostate cancer cells in a dose-dependent manner, whereas HESL did not influence the viability of these cancer cells under the conditions used in this study. Active fractions of HESL were separated via column chromatography, and the structure of the active principle was determined using (1)H and (13)C nuclear magnetic resonance spectroscopy. The active compound, dehydrocostus lactone (DHCL), in fraction 7 dose-dependently inhibited the basal and EGF-induced migration of prostate cancer cells. HESL and DHCL reduced matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 secretion but increased TIMP-2 levels in both the absence and presence of EGF. Our results demonstrate that the inhibition of MMP-9 secretion and the stimulation of TIMP-2 secretion contribute to reduced migration of DU145 cells treated with HESL and DHCL. These results indicate that HESL containing its active principle, DHCL, has potential as an antimetastatic agent for the treatment of prostate cancer.

Regulation of neuroendocrine differentiation by AKT/hnRNPK/AR/β‐catenin signaling in prostate cancer cells

Current diagnostic tools cannot predict clinical failure and androgen-independent disease progression for patients with prostate cancer (PC). The survival signaling pathways of prostate cells play a central role in the progression of tumors to a neuroendocrine (NE) phenotype. NE cells demonstrate attributes that suggest that they are an integral part of the signaling cascade leading to castration-resistant PC. In this study, making use of in vitro neuroendocrine differentiation (NED) of human LNCaP and mouse TRAMP-C2 cells after androgen withdrawal, and of the transgenic adenocarcinoma of mouse prostate (TRAMP) model, we characterized a sequence of molecular events leading to NED and identified a number of markers that could be detectable by routine analyses not only in castration resistant PC but also in hormone naïve PC at the time of initial diagnosis. We found that NED associates with AKT activation that in turn regulates heterogeneous nuclear ribonucleoprotein K (hnRNP K), androgen receptor (AR) and β-catenin levels. Addition of molecules targeting membrane-bound receptors and protein kinases blocks NE differentiation in LNCaP and TRAMP-C2 cells. The extent of AKT phosphorylation and hnRNP K, AR and β-catenin levels may have a potential value as prognostic indicators discriminating between androgen-responsive and unresponsive cells and could be used as molecular targets to monitor the anti-tumor action of new therapeutic protocols based on antireceptor agents and/or neuroendocrine hormone antagonists.

Inhibition of tumor cell migration and metastasis by the proton-sensing GPR4 receptor

GPR4 is a member of the proton-sensing G protein-coupled receptor family. Within tumor microenvironments, the interstitial acidic pH may activate GPR4 to regulate the behavior of tumor cells. Mouse B16F10 melanoma cells and TRAMP-C1 prostate cancer cells, genetically engineered to overexpress GPR4 or the control vector, were subject to a series of cell migration, invasion and metastasis assays. Upon GPR4 overexpression and activation in an acidic pH, the migration of B16F10 and TRAMP-C1 cells was substantially inhibited in comparison to the vector control. Similar results were observed in the Matrigel invasion and transendothelial invasion assays. At the molecular level, stimulation of GPR4 by acidosis induced the activation of RhoA and the formation of actin stress fibers. In addition, treating B16F10 cells with the known Rho activator CN01 (calpeptin) strongly inhibited cell migration, recapitulating the acidosis/GPR4-induced motility inhibition phenotype. To examine the biological effects in vivo, B16F10 melanoma cells were intravenously injected into syngeneic C57BL/6 mice and pulmonary metastasis was inhibited by approximately 80% in GPR4-overexpressing B16F10 cells in comparison to the vector control. Upon treatment with the Rho activator CN01, the phenotype of the B16F10 vector cells paralleled that of the GPR4-overexpressing cells in cell migration and metastasis assays. These findings suggest that GPR4 activation by an acidic pH inhibits tumor cell migration and invasion, and the Rho GTPase is at least partly responsible for this phenotype.

Prevention of Prostate Cancer with Oleanane Synthetic Triterpenoid CDDO-Me in the TRAMP Mouse Model of Prostate Cancer

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), a synthetic analog of oleanolic acid, and its C28 methyl ester derivative (CDDO-Me), have shown potent antitumorigenic activity against a wide range of cancer cell lines, including prostate cancer cells in vitro, and inhibited the development of liver and lung cancer in vivo. In the present study, we examined the efficacy of CDDO-Me in preventing the development and progression of prostate cancer in the transgenic adenocarinoma of the mouse prostate (TRAMP) model. CDDO-Me inhibited the growth of murine TRAMPC-1 prostate cancer cells by inducing apoptosis through the inhibition of antiapoptotic p-Akt, p-mTOR and NF-κB. Early intervention with CDDO-Me (7.5 mg/kg) initiated at five weeks of age for 20 wk inhibited the progression of the preneoplastic lesions (low-grade PIN and high-grade-PIN) to adenocarcinoma in the dorsolateral prostate (DLP) and ventral prostate (VP) lobes of TRAMP mice. Even delayed administration of CDDO-Me started at 12 wk of age for 12 wk inhibited the development of adenocarcimona of the prostate. Both early and late treatment with CDDO-Me inhibited the metastasis of tumor to the distant organs. Treatment with CDDO-Me inhibited the expression of prosurvival p-Akt and NF-κB in the prostate and knocking-down Akt in TRAMPC-1 tumor cells sensitized them to CDDO-Me. These findings indicated that Akt is a target for apoptoxicity in TRAMPC-1 cells in vitro and potentially a target of CDDO-Me for inhibition of prostate cancer in vivo.

Chronic administration of green tea extract to TRAMP mice induces the collapse of Golgi apparatus in prostate secretory cells and results in alterations of protein post-translational processing

Considering its long latency, prostate cancer (PCa) represents an ideal target for chemoprevention strategies. Green tea extract (GTE) has been proved to be one of the most promising natural substances capable of inhibiting PCa progression in animal models (transgenic adenocarcinoma of mouse prostate), as well as in humans. However, the cellular targets of the GTE action are mostly unknown. The main objective of this work was to investigate whether the endoplasmic reticulum (ER) and the Golgi apparatus (GA), known to be actively involved in sensing stress stimuli and initiating and propagating cell death signalling, may represent the subcellular targets of GTE action. To this end, 42 TRAMP mice were divided into four experimental groups: groups II and IV, received GTE in tap water (0.3 g/100 ml solution) starting at 8 weeks of age and up to the time of sacrifice. Groups I and III were respective age-matched water-fed controls. The animals were sacrificed after 4 weeks (groups I and II) or 40 weeks of treatment (groups II and IV). We also treated TRAMP-C2 cells with GTE (20 µg/ml for 7 days) to check the expression profile of clusterin (CLU), a protein involved in prostate tumourigenesis, extensively processed through ER-GA before being secreted through the plasma membrane. In vivo we found that chronic administration of GTE in TRAMP mice results in collapse of ER and GA in prostate epithelial cells. Consistently, in vitro we found that the mature, fully processed form of CLU, sCLU, is strongly reduced by GTE treatment in TRAMP-C2 cells. Taking into account the sCLU biogenesis dependence on the ER-GA integrity and the proposed anti-apoptotic role of sCLU, the possibility for GTE to counteract PCa progression by interfering with sCLU biogenesis is suggested.

Pharmacodynamics of curcumin as DNA hypomethylation agent in restoring the expression of Nrf2 via promoter CpGs demethylation

Prostate cancer (PCa) is one of the most deadly malignancies among men in the United States. Although localized prostate cancer can be effectively treated via surgery or radiation, metastatic disease is usually lethal. Recent evidence suggests that the development and progression of human prostate cancer involves complex interplay between epigenetic alterations and genetic defects. We have recently demonstrated that Nrf2, a master regulator of cellular antioxidant defense systems, was epigenetically silenced during the progression of prostate tumorigenesis in TRAMP mice. The aim of this study is to investigate the potential of curcumin (CUR), a dietary compound that we have reported to be able to prevent the development of prostate cancer in TRAMP mice, as a DNA hypomethylation agent. Using bisulfite genomic sequencing (BGS), treatment of TRAMP C1 cells we showed that CUR reversed the methylation status of the first 5 CpGs in the promoter region of the Nrf2 gene. Methylation DNA immunoprecipitation (MeDIP) analysis revealed that CUR significantly reduced the anti-mecyt antibody binding to the first 5 CpGs of the Nrf2 promoter, corroborated the BGS results. Demethylation of Nrf2 was found to be associated with the re-expression of Nrf2 and one of its downstream target gene, NQO-1, one of the major anti-oxidative stress enzymes, both at the mRNA and protein levels. Taken together, our current study suggests that CUR can elicit its prostate cancer chemopreventive effect, potentially at least in part, through epigenetic modification of the Nrf2 gene with its subsequent induction of the Nrf2-mediated anti-oxidative stress cellular defense pathway.

Synergistic potentiation of antitumor immunity with light-activated drug therapy and anti-CTLA4 antibody treatment.

e13034 Background: Talaporfin sodium (TS) is a light-activated, locally cytoreductive drug in Phase 3 clinical trials for several different cancers. It is activated intratumorally with a thin, flexible LED array. TS treatment has been shown in preclinical studies to activate CD8+-dependent anti-tumor immunity in a model of metastatic breast cancer. Anti-CTLA4 antibody is an immunotherapy that acts by suppressing negative regulation of immune effectors. It has recently completed a successful Phase 3 clinical trial in melanoma patients in which it was shown to prolong survival.TS and anti-CTLA4 antibody have great potential to synergistically reduce primary tumor burden and generate systemic anti-tumor immunity. Methods: This study examined combination treatment with these two therapies in a murine prostate cancer tumor model (C57BL/6 + TRAMP C2). A large, established tumor on the left side of each mouse was treated with light-activated TS (or light-only control). One day after treatment, mice were injected intradermally with 0.5 x 106 TRAMP C2 cells on the opposite flank. Anti-CTLA4 (or vehicle control) was given on days 1, 4, 7 and 10. Progression of the untreated tumor was measured. Results: Growth of untreated tumors was prevented or significantly delayed in 55% and 33% of combination-treated animals, respectively (n = 9). In addition, contralateral tumor growth was prevented in 1 of 9 animals given talaporfin sodium alone while another animal’s tumor showed growth delay. None of the untreated controls or anti-CTLA4 only animals showed a similar response. Individual combination-treated mice with delayed tumor growth often showed an atypical tumor growth curve in which progression either tapered off or began to reverse between 30 and 40 days post treatment. Conclusions: Reduction of tumor using local treatment with a light-activated drug could potentiate the anti-tumor immune response resulting from anti-CTLA4 treatment and prevent growth of secondary tumors in a preclinical prostate cancer model.

Macrophages induce neuroendocrine differentiation of prostate cancer cells via BMP6‐IL6 Loop

Frequently associated with hormone refractory prostate cancer are neuroendocrine cells. Because these cells do not express androgen receptors and are castration-resistant, further understanding the mechanism of neuroendocrine differentiation (NED) of prostate cancer cells may yield novel intervention methods in hormone refractory prostate cancer. In this regard, the present study investigated the effect of macrophages on prostate cancer NED. THP-1 and LNCaP or RAW264.7 and TRAMP-C2 cell line co-cultures were used to investigate NED-macrophage interactions. Also interleukin-6 (IL-6) knockout mice and macrophage-depleted mice were used to test NED in vivo. We found that co-culturing with THP-1 human monocytic cell line and RAW 264.7 murine macrophage cell line led to the NED of LNCaP and TRAMP-C2 prostate cancer cells, respectively. Specifically, the conditioned media of activated macrophages stimulated the expression of parathyroid hormone-related peptide (PTHrP), a marker of NED, in both LNCaP and TRAMP-C2 cells. Mechanistically, bone morphogenetic protein-6 (BMP-6) derived from prostate cancer cells increased the expression of IL-6 in macrophages. Subsequently, IL-6 induced the NED of prostate cancer cells. When this feedback loop was disrupted with neutralizing antibodies to either BMP-6 or IL-6, NED was no longer observed. In human prostate cancer tissues, neuroendocrine cells frequently co-localized with macrophages and BMP-6. In mice, the removal of IL-6 or macrophages blocked the BMP-6-induced NED of prostate cancer cells. Therefore, we propose that BMP-6 secreted by prostate cancer cells induces IL-6 expression in macrophages; IL-6, in turn, stimulates the NED of prostate cancer cells.

Synergistic potentiation of anti-tumor immunity with light-activated drug therapy and anti-CTLA4 antibody treatment.

27 Background: Talaporfin sodium (TS) is a light-activated, locally cytoreductive drug in phase III clinical trials for several different cancers. It is activated intratumorally with a thin, flexible LED array. TS treatment has been shown in preclinical studies to activate CD8+-dependent anti-tumor immunity in a model of metastatic breast cancer. Anti-CTLA4 antibody is an immunotherapy that acts by suppressing negative regulation of immune effectors. It has recently completed a successful phase III clinical trial in melanoma patients in which it was shown to prolong survival. TS and anti-CTLA4 antibody have great potential to synergistically reduce primary tumor burden and generate systemic anti-tumor immunity. Methods: This study examined combination treatment with these two therapies in a murine prostate cancer tumor model (C57BL/6 + TRAMP C2). A large, established tumor on the left side of each mouse was treated with light-activated TS (or light- only control). One day after treatment, mice were injected intradermally with 0.5 × 106 TRAMP C2 cells on the opposite flank. Anti-CTLA4 (or vehicle control) was given on days 1, 4, 7 and 10. Progression of the untreated tumor was measured. Results: Growth of untreated tumors was prevented or significantly delayed in 55% and 33% of combination-treated animals, respectively (n = 9). In addition, contralateral tumor growth was prevented in 1 of 9 animals given talaporfin sodium alone while another animal's tumor showed growth delay. None of the untreated controls or anti-CTLA4 only animals showed a similar response. Individual combination-treated mice with delayed tumor growth often showed an atypical tumor growth curve in which progression either tapered off or began to reverse between 30 and 40 days post treatment. Conclusions: Reduction of tumor using local treatment with a light-activated drug could potentiate the anti-tumor immune response resulting from anti-CTLA4 treatment and prevent growth of secondary tumors in a preclinical prostate cancer model. [Table: see text]

Abstract A104: 3’, 4’, 5’-Trimethoxyflavonol (TMFol), a novel putative prostate cancer chemopreventive agent: In vitro and in vivo preclinical activity

Abstract Flavonoids occurring in many edible fruits and vegetables, typified by fisetin and quercetin, have been suggested to prevent malignancies, including those of the prostate. Only a handful of flavonoids have thus far been subjected to exhaustive pharmacological investigation, and it is unknown whether analogues exist with pharmacological properties superior to those of flavonoids studied to date. We identified TMFol as a potent inhibitor of growth of TRAMP C2, LNCaP, and PC-3 prostate cancer cells, with IC50 values of 1.3-5µM. For comparison, the respective IC50 values for fisetin and quercetin are 8-10 fold higher. Studies in mice hint at the possibility that TMFol may be useful as prostate cancer chemopreventive agent, because TMFol levels in prostate tissue after single oral administration of 240 mg/kg reached 18 nmol/g, and steady state levels after dietary intake (0.2%) were 5 nmol/g These concentrations are consistent with those eliciting growth inhibition in prostate cells in vitro. Mechanistic studies have shown TMFol to arrest cells in the G2M phase of the cell cycle and induce apoptosis. In addition, TMFol inhibited androgen receptor signalling. When nude mice bearing the TRAMP C2 cell xenograft were exposed to TMFol (0.2% in the diet) tumor volume at termination of the experiment was significantly reduced by 41% in comparison to control (p&amp;lt;0.001). Quercetin and fisetin had no effect, at equimolar doses, in the same mouse model. Compared to other flavonoids thus far tested as putative prostate cancer chemopreventive agents, TMFol seems superior in terms of growth-inhibitory potency, and in vivo efficacy, probably related to its metabolic stability. It therefore warrants further preclinical and clinical investigation. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A104.

Regression of prostate tumors upon combination of hormone ablation therapy and celecoxib in vivo

Hormonal ablation is the standard of treatment for advanced androgen-dependent prostate cancer. Although tumor regression is usually achieved at first, the cancer inevitably evolves toward androgen-independence, in part because of the development of mechanisms of resistance and in part because at the tissue level androgen withdrawal is not fully attained. Current research efforts are focused on new therapeutic strategies that will increase the effectiveness of androgen withdrawal and delay recurrence. We used a syngeneic pseudo-orthotropic mouse model of prostate cancer to test the efficacy of combining androgen withdrawal with FDA-approved COX-2 inhibitor celecoxib. GFP-tagged TRAMP-C2 cells were co-implanted with prostate tissue in the dorsal chamber model and tumors were allowed to establish and vascularize. Tumor growth and angiogenesis were monitored in real-time using fluorescent intravital microscopy (IVM). Androgen withdrawal in mice was achieved using surgical castration or chemical hormonal ablation, alone or in combination with celecoxib (15 mg/kg, twice daily). Celecoxib alone decreased the growth of prostate tumors mostly by inducing mitotic failure, which resulted in increased apoptosis. Surprisingly, celecoxib did not possess significant angiostatic activity. Surgical or chemical castration prevented the growth of prostate tumors and this, on the other hand, was associated with disruption of the tumor vasculature. Finally, androgen withdrawal combined with celecoxib caused tumor regression through decreased angiogenesis and increased mitosis arrest and apoptosis. Celecoxib, a relatively safe COX-2-selective anti-inflammatory drug, significantly increases the efficacy of androgen withdrawal in vivo and warrants further investigation as a complement therapy for advanced prostate cancer.

LL‐37 as a therapeutic target for late stage prostate cancer

The antimicrobial peptide, leucine-leucine-37 (LL-37), stimulates proliferation, angiogenesis, and cellular migration, inhibits apoptosis and is associated with inflammation. Since these functional processes are often exaggerated in cancer, the aim of the present study was to investigate the expression and role of LL-37 in prostate cancer (PCa) and establish its value as a therapeutic target. We evaluated the expression of LL-37 and the murine orthologue, cathelicidin-related antimicrobial peptide (CRAMP) in human and murine prostate tumors, respectively. Compared to normal/benign prostate tissue, both LL-37 and CRAMP were increasingly over-expressed with advancing grades of primary PCa and its metastasis in human tissues and in the transgenic adenocarcinoma mouse prostate (TRAMP) model, correspondingly. We subsequently knocked-down CRAMP in the highly tumorigenic TRAMP-C1 cell line via a RNA interference strategy to examine the importance of CRAMP on cellular proliferation, angiogenesis, invasion, apoptosis, activation of signaling pathways and tumor kinetics. Abrogation of CRAMP expression led to decreased proliferation, invasion, type IV collagenase, and the amount of phosphorylated Erk1/2 and Akt signaling in vitro. These results were paralleled in vivo. Syngenic implantation of TRAMP-C1 cells subjected to CRAMP knock-down resulted in a decreased tumor incidence and size, and the down-regulation of pro-tumorigenic mechanisms. CRAMP knock-down in a murine PCa model analogously demonstrated the tumorigenic contributions of LL-37 in PCa and its potential as a novel therapeutic target for the treatment of PCa and potentially, other cancers over-expressing the peptide.

Abstract 1436: Interleukin-17A stimulates expression of chemokines and cytokines in prostatic epithelial cells via nuclear factor-kappaB and extracellular signal-regulated kinase pathways

Abstract T helper 17 (Th17) cells secret interleukin-17A (IL-17A) and interleukin-17F. Th17 cells and IL-17A are increased in prostate tumors. The role of IL-17A in prostate cancer is not clear. The objective of this in-vitro study was to investigate the effects of IL-17A on prostatic epithelial cells. Cell growth assay, Western blot, and real-time quantitative reverse transcription- polymerase chain reaction (qRT-PCR) analysis were used to measure the effects of IL-17A on the immortalized normal human prostatic epithelial cell lines (RWPE-1 and pRNS-1-1), human high-grade prostatic intraepithelial neoplasia (PIN) cell line, human prostate cancer LNCaP cell line, and mouse prostate cancer TRAMP-C1 cell line. We found that recombinant human IL-17A (20 ng/ml) did not affect cell growth rate in any of the prostatic cell lines studied. IL-17A did not significantly activate Nuclear Factor-kappaB (NF-kappaB) or Extracellular signal-Regulated Kinase (ERK) signaling pathway in RWPE-1, pRNS-1-1, or PIN cells. IL-17A activated NF-kappaB and/or ERK signaling pathways in LNCaP and TRAMP-C1 cells. When IL-17RC (receptor of IL-17A) was overexpressed in PIN and LNCaP cells, activation of NF-kappaB or ERK signaling pathway by IL-17A was significantly enhanced. IL-17A modestly induced mRNA expression of chemokines (CXCL1 and CXCL2) in RWPE-1, pRNS-1-1, and PIN cells. IL-17A induced mRNA expression of chemokines (CXCL1, CXCL2, CCL2, CCL5, CCL7, and CCL20) and cytokine IL-6 in LNCaP cells. In mouse TRAMP-C1 cells, IL-17A induced mRNA expression of chemokines (CXCL1, CXCL2, CXCL5, CCL2, and CCL5) and cytokine IL-6. When IL-17RC was overexpressed in PIN and LNCaP cells, chemokine expression was significantly enhanced. Our findings suggest that although IL-17A has no effects on cell growth in-vitro, it may modulate the stroma-epithelial interaction in tumor microenvironment in-vivo via stimulation of expression of chemokines and cytokines. Acknowledgement: DoD W81XWH-05-1-0567, NIH/NCRR 2P20 RR020152-06, and LCRC Fund. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1436.