Human Prostate Cancer Cell Xenografts Research Articles

Docetaxel enhances the therapeutic efficacy of PSMA-specific CAR-T cells against prostate cancer models by suppressing MDSCs.

Prostate cancer can undergo curative effects by radical prostatectomy or radical radiotherapy. However, the best treatment for more aggressive high-risk prostate cancer remains controversial. Insufficient infiltration capacity and dysfunction are commonly occurrences in engineered T lymphocytes expressing chimeric antigen receptor (CAR-T), characterizing cancer immunotherapy failure. We conducted this study to investigate whether the combinative application of docetaxel and PSMA-CAR-T cells could be a more effective treatment to prostate cancer. Expressions of prostate specific membrane antigen (PSMA) on prostate cancer cells were examined by Flow cytometry. The efficaciousness of PSMA-CAR-T was evaluated in vitro using ELISA and RTCA. The effect of intermixed therapy was assessed in vivo utilizing a human prostate cancer liver metastasis mouse model and a human prostate cancer cell xenograft mouse model. The outcome of cytokine discharge and cell killing assays demonstrated that PSMA-CAR-T cells have characteristic effector capacity against PSMA+ prostate cancer cells in vitro. Additionally, collaborative treatment of PSMA-CAR-T cells and docetaxel have cooperative efficacy in a mouse model of human prostate cancer. The merged strategy could be seen as an undeveloped avenue to augmenting adoptive CAR-T cell immunotherapy and mitigating the adverse side effects of chemotherapy. Cooperation of PSMA-specific CAR-T cells and the chemotherapy drug docetaxel can impressively ameliorate antitumor effectiveness against an installed metastatic human prostate cancer model in NPG mice.

Host versus cell-dependent effects of β-arrestin 1 expression in prostate tumorigenesis.

Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is significantly overexpressed (>4-fold) in PCa cells relative to βarr2. In this study, we investigated the effect of βarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in β-arrestin depletion of βarr1 in TRAMP mice (TRAMP/βarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/βarr2-/- animals. Prostate tissues from TRAMP/βarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of βarr1 in TRAMP-C1 (TRAMP-C1-βarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of βarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-βarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-βarr1-GFP and MDA PCa 2b-βarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of βarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.

Research Evidence on High-Fat Diet-Induced Prostate Cancer Development and Progression.

Although recent evidence has suggested that a high-fat diet (HFD) plays an important role in prostate carcinogenesis, the underlying mechanisms have largely remained unknown. This review thus summarizes previous preclinical studies that have used prostate cancer cells and animal models to assess the impact of dietary fat on prostate cancer development and progression. Large variations in the previous studies were found during the selection of preclinical models and types of dietary intervention. Subcutaneous human prostate cancer cell xenografts, such as LNCaP, LAPC-4, and PC-3 and genetic engineered mouse models, such as TRAMP and Pten knockout, were frequently used. The dietary interventions had not been standardized, and distinct variations in the phenotype were observed in different studies using distinct HFD components. The use of different dietary components in the research models is reported to influence the effect of diet-induced metabolic disorders. The proposed underlying mechanisms for HFD-induced prostate cancer were divided into (1) growth factor signaling, (2) lipid metabolism, (3) inflammation, (4) hormonal modulation, and others. A number of preclinical studies proposed that dietary fat and/or obesity enhanced prostate cancer development and progression. However, the relationship still remains controversial, and care should be taken when interpreting the results in a human context. Future studies using more sophisticated preclinical models are imperative in order to explore deeper understanding regarding the impact of dietary fat on the development and progression of prostate cancer.

Metformin combined with quercetin synergistically repressed prostate cancer cells via inhibition of VEGF/PI3K/Akt signaling pathway

The aim of present study was to examine whether metformin in association with quercetin has any synergistically anti-tumor effects on prostate cancer. Our findings showed that metformin in combination with quercetin synergistically inhibited the growth, migration and invasion of both PC-3 and LNCaP cells. Co-treatment of these two agents induced more apoptosis than single agent treatment. The co-treatment-induced apoptosis was caspase-dependent and accompanied by the down-regulation of Bcl-2 family members. Our data also indicated that co-treatment of metformin and quercetin strongly inhibited the VEGF/Akt/PI3K pathway. Moreover, these two agents acted synergistically to repress the growth of human prostate cancer cell xenograft in vivo in nude mice. In conclusion, our findings indicate that the combination therapy of metformin and quercetin exerted synergistic antitumor effects in prostate cancers via inhibition of VEGF/Akt/PI3K pathway. Thus, combination treatment of metformin and quercetin would be a promising therapeutic strategy for prostate cancer patients.

FSH supplementation increases the growth of PC-3 human prostate cancer cell xenograft in gonadotropin-suppressed nude mice

FSH supplementation increases the growth of PC-3 human prostate cancer cell xenograft in gonadotropin-suppressed nude mice

Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells.

Standard treatment for primary prostate cancer includes systemic exposure to chemotherapeutic drugs that target androgen receptor or antihormone therapy (chemical castration); however, drug-resistant cancer cells generally emerge during treatment, limiting the continued use of systemic chemotherapy. Patients are then treated with more toxic standard therapies. Therefore, there is an urgent need for novel and more effective treatments for prostate cancer. The cholesterol biosynthetic pathway is an attractive therapeutic target for treating endocrine-dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as the metabolic precursor of endogenous steroid hormones. In this study, we have examined the effects of RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate; Roche Pharmaceuticals internal reference: RO0488071) (RO), which is an inhibitor of 2, 3-oxidosqualene cyclase (a key enzyme in the cholesterol biosynthetic pathway), on prostate cancer cells. Exposure of both hormone-dependent and castration-resistant human prostate cancer cells to RO reduced prostate cancer cell viability and induced apoptosis in vitro. RO treatment reduced androgen receptor protein expression in hormone-dependent prostate cancer cells and increased estrogen receptor β (ERβ) protein expression in both hormone-dependent and castration-resistant prostate cancer cell lines. Combining RO with an ERβ agonist increased its ability to reduce castration-resistant prostate cancer cell viability. In addition, RO effectively suppressed the growth of aggressive castration-resistant human prostate cancer cell xenografts in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells in vitro. Our study is the first to demonstrate that the cholesterol biosynthesis inhibitor RO effectively suppresses growth of human prostate cancer cells. Our findings suggest that cholesterol biosynthesis inhibitors such as RO, when used in combination with commonly used chemotherapeutic drugs or ERβ specific ligands, could represent a novel therapeutic approach to prevent the growth of prostate cancer tumors.

Effect of FSH supplementation on the GnRH antagonist suppressed growth of PC-3 cell xenografts in nude mice.

e630 Background: One of the fundamental differences between GnRH agonist and antagonist treatment is the permanent suppression of FSH by antagonist, while a rebound in FSH secretion has been documented upon agonist treatments. To assess the potential beneficial effects of permanent FSH suppression in the hormone ablation treatment of prostate cancer we studied the effect of the GnRH antagonist degarelix in the absence and presence of FSH supplementation on the growth of PC-3 human prostate cancer cell xenografts in nude mice. Methods: Intact (n = 20) and gonadectomized (n = 20) nude male mice were inoculated with 2 x 106PC-3 cells. Half of each group received degarelix treatment at 10 mg/kg body weight s.c. In a second experiment (n = 10/group), degarelix treatment was supplemented with recombinant human FSH at10 IU/kg/day using i.p. ALZET osmotic minipumps. Tumor growth in both experiments was monitored for 4 weeks by external inspection and caliper measurement, after which the mice were sacrificed. Results: The first experiment demonstrated that degarelix treatment significantly reduced tumor growth by 33% and 35%, respectively, in intact and gonadectomized mice as compared to non-treated controls (p<0.0001 for each). In the second experiment, FSH reversed the inhibitory effect of degarelix in intact mice. Conclusions: These results demonstrated that the suppression of PC-3 xenograft growth by GnRH antagonist treatment was completely inhibited by concomitant FSH treatment. The findings prove experimentally the principle that combined FSH and LH suppression by GnRH antagonist could offer an advantage over the isolated LH suppression by GnRH agonist upon prostate cancer treatment. [Table: see text]

Quercetin inhibits angiogenesis through thrombospondin-1 upregulation to antagonize human prostate cancer PC-3 cell growth in vitro and in vivo

The rapid growth, morbidity and mortality of prostate cancer, and the lack of effective treatment have attracted great interests of researchers to find novel cancer therapies aiming to inhibit angiogenesis and tumor growth. Quercetin is a flavonoid compound that widely exists in the nature. Our previous study preliminarily demonstrated that quercetin effectively inhibited human prostate cancer cell xenograft tumor growth by inhibiting angiogenesis. Thrombospondin-1 (TSP-1) is the first reported endogenous anti-angiogenic factor that can inhibit angiogenesis and tumorigenesis. However, the relationship between quercetin inhibiting angiogenesis and TSP-1 upregulation in prostate cancer has not been determined. Thus, we explored the important role of TSP-1 upregulation in reducing angiogenesis and anti-prostate cancer effect of quercetin both in vitro and in vivo for the first time. After the selected doses were used for a certain time, quercetin i) significantly inhibited PC-3 and human umbilical vein endothelial cells (HUVECs) proliferation, migration and invasion in a dose-dependent manner; ⅱ) effectively inhibited prostate cancer PC-3 cell xenograft tumor growth by 37.5% with 75 mg/kg as compared to vehicle control group, more effective than 25 (22.85%) and 50 mg/kg (29.6%); ⅲ) was well tolerated by BALB/c mice and no obvious toxic reactions were observed; ⅳ) greatly reduced angiogenesis and led to higher TSP-1 protein and mRNA expression both in vitro and in vivo in a dose-dependent manner. Therefore, quercetin could increase TSP-1 expression to inhibit angiogenesis resulting in antagonizing prostate cancer PC-3 cell and xenograft tumor growth. The present study can lay a good basis for the subsequent concrete mechanism study and raise the possibility of applying quercetin to clinical for human prostate cancer in the near future.

Abstract 777: Effects of Src inhibitors and soy isoflavones on human prostate cancer cells

Abstract Background: Src family kinases are often over-expressed and highly active in solid tumors, including prostate cancer. This phenotype is associated with a poor prognosis partly because Src is a key factor in important signaling pathways involved in cell proliferation, angiogenesis, and initiation of metastasis. As a result, several promising small molecule targeting agents have been developed to prevent phosphorylation of key tyrosine residues that produce Src activation. However, these agents are not without side effects. The purpose of this study was to determine if adding a cytostatic soy isoflavone extract (ISF) to the treatment would produce optimal results at lower doses of the Src inhibitor. The ISF used in these studies has been shown to inhibit tumor growth in mice carrying human prostate cancer cell xenografts without causing toxicity to the host. Methods: Aggressive PC-3ML cells (a gift from A. Fatatis, Drexel University), and weakly tumorigenic LNCaP cells were treated for 24 hr with various concentrations of Src inhibitors (dasatinib, saracatinib), ISF (200 ug/ml, NovaSoy), or a combination. To evaluate effects on the metastatic potential of the cells, functional assays of cell growth and motility were performed, including those that assess clonogenic cell survival, cell cycle progression, and transwell migration and invasion activity. Results: Src inhibitors and ISF alone produced very little reduction in cell viability, but significant cytostatic effects, as determined by a reduction in clonogenicity, the number of cells able to form 50-cell colonies. This was likely due, in part, to changes in cell cycle progression. Both dasatinib and saracatinib caused an accumulation of cells in the G1 phase. As expected, ISF treatment resulted in higher numbers of cells in the G2/M phase. When the treatments were combined, cells exposed to dasatinib were observed to accumulate in both phases, with a significant decrease in S-phase cells. The results were not significant for saracatinib-treated cells. Exposure to either a Src inhibitor or ISF significantly reduced the migration of cells in a transwell chamber and their ability to invade through a Matrigel-coated 8 micron-pore membrane towards a chemoattractant (media containing 10% FBS). Dasatinib produced much greater effects, and at lower concentrations, than saracatinib. When combined with ISF, the effects were enhanced, particularly with dasatinib. Conclusions: In vitro studies suggested that combining a Src inhibitor and ISF resulted in greater inhibition of metastatic potential than either alone. This may indicate that including ISF in treatment regimens may allow a lower dose of the targeting agent to be used to achieve optimal response and also decrease toxicity. Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Effects of Src inhibitors and soy isoflavones on human prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2015-777

Increase in apoptotic effect of Panax ginseng by microwave processing in human prostate cancer cells: in vitro and in vivo studies

BackgroundGinseng, which is widely used in functional foods and as an herbal medicine, has been reported to reduce the proliferation of prostate cancer cells by mechanisms that are not yet fully understood. MethodsThis study was designed to investigate the changes in ginsenoside content in ginseng after treatment with a microwave-irradiation thermal process and to verify the anticancer effects of the extracts. To confirm the anticancer effect of microwave-irradiated processed ginseng (MG), it was tested in three human prostate cancer cell lines (DU145, LNCaP, and PC-3 cells). Involvements of apoptosis and autophagy were assessed using Western blotting. ResultsAfter microwave treatment, the content of ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd in the extracts decreased, whereas the content of ginsenosides 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5 increased. Antiproliferation results for the human cancer cell lines treated with ginseng extracts indicate that PC-3 cells treated with MG showed the highest activity with an half maximal inhibitory concentration of 48 μg/mL. We also showed that MG suppresses the growth of human prostate cancer cell xenografts in athymic nude mice as an in vivo model. This growth suppression by MG is associated with the inductions of cell death and autophagy. ConclusionTherefore, heat processing by microwave irradiation is a useful method to enhance the anticancer effect of ginseng by increasing the content of ginsenosides Rg3, Rg5, and Rk1.

Abstract 5378: Actions of nanoparticulate tetraiodothyroacetic acid (Nanotetrac) on human prostate carcinoma xenograft growth, vascularity and integrin response to radiation

Abstract Tetraiodothyroacetic acid (tetrac) covalently bound to a nanoparticle (poly[lactic-co-glycolic acid]) (= Nanotetrac) has anti-cancer and anti-angiogenic properties that are initiated exclusively at a cell surface receptor on integrin αvβ3 of cancer cells and dividing blood vessel cells. The anti-cancer activities are pro-apoptotic by multiple mechanisms, disorder gene expression in multiple cancer cell survival pathways and are radiosensitizing. The anti-angiogenic effects involve disruption of actions of VEGF, bFGF, PDGF and EGF. In the present studies, susceptibility of human prostate cancer cell xenografts to activity of Nanotetrac was determined in grafts in the nude mouse or to the chick chorioallentoic membrane (CAM) model and radiosensitivity was estimated in the CAM system. Androgen-sensitive LNCaP cell xenografts in the CAM exposed to Nanotetrac (1 μg/CAM) showed 40% decreases in tumor weight and vascular branch points and 50% reduction in hemoglobin content at 7 d (P < 0.01). LNCaP xenograft volumes in the male mouse were reduced by 50% at 5 days (P < 0.01), approximating the volume of the tumor cell implant. In vivo imaging system (IVIS) scans of orthotopic androgen-independent PC3 cell xenografts in the nude mouse showed substantial reduction in tumor size vs. control (PBS) at 7 d (50% reduction) and at 17 d (75% reduction) post-implantation. Radiation studies in PC3 cell tumors established in the CAM system demonstrated that within 1 h of exposure to 1-2 Gy radiation, there was apparently defensive activation (assumption of ‘open conformation’) of αvβ3. Receptor activation response to radiation was blocked by Nanotetrac. We have shown in other cancer cell lines that Nanotetrac inhibits repair of radiation-induced double-stranded DNA breaks and propose that, based on the current observations, this DNA repair effect is due to inhibition of radiation-caused activation of αvβ3. In summary, examined in two models and acting at the cell surface, Nanotetrac substantially reduces volume/weight and vascularity of human prostate cancer xenografts. The agent also inhibited activation of integrin αvβ3 that is induced by radiation exposure and is seen to be a component of the radioresistance response. Citation Format: Shaker A. Mousa, Sudha Thangirala, Murat Yalcin, John T. Leith, Aleck Hercbergs, Hung-Yun Lin, Heng-Yuan Tang, Mary K. Luidens, Paul J. Davis. Actions of nanoparticulate tetraiodothyroacetic acid (Nanotetrac) on human prostate carcinoma xenograft growth, vascularity and integrin response to radiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5378. doi:10.1158/1538-7445.AM2014-5378

Abstract B67: Inhibition of NFκB and proapoptotic Effect of Green Tea in Prostate Tumor Tissue: A Phase II clinical Trial in Men with Prostate Cancer

Abstract Background: Green tea polyphenols (GTPs) have been shown to play a role in the regulation of cell proliferation, apoptosis and inflammation in prostate cancer cells in vitro and in animal models using human prostate cancer cell xenografts. While there is extensive epidemiological and basic scientific evidence for these effects of GTPs, data from human intervention studies is much more limited. Objective: We conducted a phase II intervention study to evaluate the anticarcinogenic effects of green tea in prostate cancer by administering green tea in men prior to prostatectomy. Design: 79 men, diagnosed with prostate cancer and scheduled for prostatectomy, were randomized to either 6 cups of brewed green tea or water daily for 3-8 weeks prior to surgery. 67 men (GT group, N=34; control group, N=33) completed the intervention. Blood and urine samples were collected before and after the intervention and sections of prostate tissue were frozen as OCT blocks following the pathology evaluation. Serum prostate specific antigen (PSA) concentration was determined by ELISA assay and prostate tissue PSA protein concentration was measured by Western blot. The prostate concentration of GTPs and their metabolites was measured using high-performance liquid chromatography (HPLC) with CoulArray electrochemical detection. The protein expression of Ki67 (proliferation), Bcl2, Bax (apoptosis) and nuclear factor kappa B (NFκB) (inflammation) were determined by immunostaining in prostate tissue of confirmed tumor areas. Results: Statistical analysis within each treatment arm showed that the serum PSA concentration was significantly decreased in the GT group (final vs. baseline blood) (P<0.01), while no change was observed in the control group. Prostate tissue PSA protein expression was lower in men consuming GT compared to water control (p=0.06). Bioactive GTPs including epigallocatechin gallate (EGCG) and its methyl metabolite 4″-O-methyl EGCG and epicatechin gallate (ECG) were detected in prostate tissue from 31 of 34 men consuming GT but were absent in prostates in the control group. Intranuclear NFκB staining was decreased significantly while there was no change in cytoplasmic NFκB staining in prostate tumors from men drinking GT by comparison to tumors from the control group. The ratio of cytoplasmic staining of pro-apoptotic Bax to Bcl-2 was increased significantly while cytoplasmic Bcl2 and Bax staining were not changed. Nuclear Ki67 staining was not changed with GT consumption. Conclusion: Green tea consumption may contribute to the inhibition of prostate tumor growth through reducing inflammation and stimulating apoptosis. Analyses of additional markers are needed to confirm these findings. Citation Format: Susanne M. Henning, Piwen Wang, William J. Aronson, Catherine L. Carpenter, David Heber. Inhibition of NFκB and proapoptotic effect of green tea in prostate tumor tissue: A phase II clinical trial in men with prostate cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B67.

Abstract B68: Antioxidant activity of green tea: A phase II clinical trial in men with prostate cancer

Abstract Background: Green tea (GT) contains green tea polyphenols (GTPs) which have been shown to play a role in the regulation of cell proliferation and apoptosis in prostate cancer cells in vitro and in animal models using human prostate cancer cell xenografts. GTPs are also potent scavengers of reactive oxygen species (ROS) which play a role in tumorigenesis. While there is extensive epidemiological and basic science evidence for these effects of GTPs, data from human intervention studies is much more limited. Objective: We conducted a phase II intervention study to evaluate the anticarcinogenic and antioxidant effects of green tea in prostate cancer by administering green tea in men prior to prostatectomy. Design: 79 men, diagnosed with prostate cancer and scheduled for prostatectomy, were randomized to either 6 cups of brewed green tea (GT group, N=34) or water (control group, N=33) daily for 3-8 weeks prior to surgery. 67 men completed the intervention. Blood and urine samples were collected before and after the intervention, and a section of prostate tissue was obtained following the pathology evaluation. Serum prostate specific antigen (PSA) concentration was determined by ELISA assay and prostate tissue PSA protein concentration was measured by Western blot. The prostate concentration of GTPs and their metabolites was measured using high-performance liquid chromatography (HPLC) with CoulArray electrochemical detection. The concentration of 8-hydroxydeoxyguanosine (8-OHdG), a biomarker for oxidative DNA damage, was measured in urine by HPLC. In addition, gene expression of the antioxidant enzymes superoxide dismutase (SOD) and catalase were determined in normal and tumor prostate tissue using real-time quantitative PCR. Results: Statistical analysis within each treatment arm showed that the serum PSA concentration significantly decreased in the GT group (final vs. baseline blood) (P<0.01), while no change was observed in the control group. The comparison of percent change in serum PSA between GT and control group showed a decreasing trend (p=0.085). Prostate tissue PSA protein expression was lower in men consuming GT compared to water control (p=0.06). The major bioactive component of GTPs, epigallocatechin gallate (EGCG) and its methyl metabolite 4″-O-methyl EGCG were found in prostate tissue in similar amounts, along with a small amount of epicatechin gallate. As compared to the pre-intervention level there was a significant decrease in urinary 8-OHdG concentration after GT intervention (83 vs. 50 nmol/g creatinine), while there was no change in the control group (54 vs. 55 nmol/g creatinine). Gene expression of SOD (p=0.087) and catalase (p=0.15) in the prostate tumor tissue normalized to normal prostate tissue in each individual showed a decreasing trend in men consuming GT as compared to the water control. Conclusion: The consumption of 6 cups of GT provided antioxidant protection, which may contribute to an inhibitory effect on prostate tumor growth in humans. Large-scale human intervention studies are needed to confirm these findings. Citation Format: Piwen Wang, William Aronson, Narine Abgaryan, Catherine L. Carpenter, Jaydutt V. Vadgama, David Heber, Susanne M. Henning. Antioxidant activity of green tea: A phase II clinical trial in men with prostate cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B68.

Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation

In prostate cancer, the signals that drive cell proliferation change as tumors progress from castration-sensitive (androgen-dominant) to castration-resistant states. While the mechanisms underlying this change remain uncertain, characterization of common signaling components that regulate both stages of prostate cancer proliferation is important for developing effective treatment strategies. Here, we demonstrate that paxillin, a known cytoplasmic adaptor protein, regulates both androgen- and EGF-induced nuclear signaling. We show that androgen and EGF promoted MAPK-dependent phosphorylation of paxillin, resulting in nuclear translocation of paxillin. We found nuclear paxillin could then associate with androgen-stimulated androgen receptor (AR). This complex bound AR-sensitive promoters, retaining AR within the nucleus and regulating AR-mediated transcription. Nuclear paxillin also complexed with ERK and ELK1, mediating c-FOS and cyclin D1 expression; this was followed by proliferation. Thus, paxillin is a liaison between extranuclear MAPK signaling and nuclear transcription in response to androgens and growth factors, making it a potential regulator of both castration-sensitive and castration-resistant prostate cancer. Accordingly, paxillin was required for normal growth of human prostate cancer cell xenografts, and its expression was elevated in human prostate cancer tissue microarrays. Paxillin is therefore a potential biomarker for prostate cancer proliferation and a possible therapeutic target for prostate cancer treatment.

Design, synthesis, and evaluation of 5-methyl-4-phenoxy-5 H-pyrrolo[3,2- d]pyrimidine derivatives: Novel VEGFR2 kinase inhibitors binding to inactive kinase conformation

We synthesized a series of pyrrolo[3,2- d]pyrimidine derivatives and evaluated their application as type-II inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase. Incorporation of a diphenylurea moiety at the C4-position of the pyrrolo[3,2- d]pyrimidine core via an oxygen linker resulted in compounds that were potent inhibitors of VEGFR2 kinase. Of these derivatives, compound 20d showed the strongest inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC). The co-crystal structure of 20d and VEGFR2 revealed that 20d binds to the inactive form of VEGFR2. Further studies indicated that 20d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhibited PDGFR and Tie-2 kinases. Oral administration of the hydrochloride salt of 20d at 3 mg/kg/day showed potent inhibition of tumor growth in a DU145 human prostate cancer cell xenograft nude mouse model.

Abstract B203: Mitochondria-targeted superoxide spin-traps for treatment of cancers with high oxidative stress

Abstract Many human malignancies such as prostate cancer (CaP) and primary brain tumor (GBM) exhibit high oxidative stress. Developing a successful therapeutic drug to treat advanced metastatic CaP and GBM is a major unmet medical need. It has been demonstrated that in certain chemo- and/or radiation resistant cancer cells with high oxidative stress, cells depend on reactive oxygen species (ROS) for survival and proliferation. Therefore, reducing ROS can be a successful strategy for developing chemotherapeutic drugs for use against chemo- and/or radiation-resistant tumors. The mitochondria are a major source of superoxide and other ROS. We focused on targeting spin-trapping nitroxide analogs at the mitochondrial interstitial space with appropriate linker length in order to remove the superoxide in order to reduce the cellular ROS levels without interfering with the mitochondrial electron transport chain. Our lead compound in this new class of drugs is Mito-tempol-C10. The drug successfully blocked ROS production in human CaP and GBM cell lines as determined by DCF dye oxidation assay. It exhibited marked growth inhibitory effect against androgen-dependent as well as androgen-independent cultured human CaP cells (IC50 < 1 µM) and similar growth inhibitory activity against several human GBM cells lines as well as cultured primary brain tumor stem cells as determined by DNA fluorescence measurement assay. Preliminary pharmacokinetic (PK) study show that the drug has serum half-life of approximately 45 minutes, but is retained by the tumor and other animal tissue for more than 24 hours. The drug is tolerated by ICR white mice, nude mice and NOD-SCID mice at a dose of 15 mg/kg i.p. given once every week for four weeks without any overt systemic toxicity. Preliminary efficacy study at this treatment condition showed marked effect on the growth of intracranially implanted, radiation-resistant U-87 MG human GBM cells in NOD-SCID mice and a small, but significant effect on the growth of androgen-independent PC-3 human prostate cancer cell xenografts growing on the flanks of nude mice. These results confirm that mitochondria-targeted spin-traps that reduce cellular ROS can be therapeutically effective in treatment of advanced, hard-to-treat human tumors with high oxidative stress. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B203.

Complex formation between human prostate‐specific antigen and protease inhibitors in mouse plasma

When secreted from the prostate, most of prostate-specific antigen (PSA) is free and enzymatically active. Upon reaching circulation, active PSA is inactivated by complex formation with protease inhibitors. To justify the use of mouse models for evaluation of the function of PSA and for studies on therapeutic modalities based on modulation of PSA activity, it is important to know whether PSA complexation is similar in mouse and man. To characterize the circulating forms of PSA in mouse, we used subcutaneous LNCaP and 22RV1 human prostate cancer cell xenograft tumor models. We also added PSA directly to mouse serum. Free and total PSA were measured by immunoassay, and PSA complexes were extracted by immunopurification followed by SDS-PAGE, in-gel trypsin digestion and identification of signature peptides by mass spectrometry. In mice bearing xenograft tumors, 68% of the immunoreactive PSA occurred in complex, and when added to mouse serum, over 70% of PSA forms complexes that comprises alpha(2)-macroglobulin and members of the alpha(1)-antitrypsin (AAT) family. In mouse plasma, PSA forms complexes similar to those in man, but the major immunoreactive complex contains AAT rather than alpha(1)-antichymotrypsin, which is the main complex forming serpin in man. The complex formation of PSA produced by xenograft tumor models in mice is similar to that of human prostate tumors with respect to the complexation of PSA.

Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

Pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells.

We completed a multicenter study of the effects of pomegranate cold-pressed (Oil) or supercritical CO(2)-extracted (S) seed oil, fermented juice polyphenols (W), and pericarp polyphenols (P) on human prostate cancer cell xenograft growth in vivo, and/or proliferation, cell cycle distribution, apoptosis, gene expression, and invasion across Matrigel, in vitro. Oil, W, and P each acutely inhibited in vitro proliferation of LNCaP, PC-3, and DU 145 human cancer cell lines. The dose of P required to inhibit cell proliferation of the prostate cancer cell line LNCaP by 50% (ED(50)) was 70 microg/mL, whereas normal prostate epithelial cells (hPrEC) were significantly less affected (ED(50) = 250 g/mL). These effects were mediated by changes in both cell cycle distribution and induction of apoptosis. For example, the androgen-independent cell line DU 145 showed a significant increase from 11% to 22% in G(2)/M cells (P <.05) by treatment with Oil (35 microg/mL) with a modest induction of apoptosis. In other cell lines/treatments, the apoptotic response predominated, for example, in PC-3 cells treated with P, at least partially through a caspase 3-mediated pathway. These cellular effects coincided with rapid changes in mRNA levels of gene targets. Thus, 4-hour treatment of DU 145 cells with Oil (35 microg/mL) resulted in significant 2.3 +/- 0.001-fold (mean +/- SEM) up-regulation of the cyclin-dependent kinase inhibitor p21((waf1/cip1)) (P <.01) and 0.6 +/- 0.14-fold down-regulation of c-myc (P <.05). In parallel, all agents potently suppressed PC-3 invasion through Matrigel, and furthermore P and S demonstrated potent inhibition of PC-3 xenograft growth in athymic mice. Overall, this study demonstrates significant antitumor activity of pomegranate-derived materials against human prostate cancer.

Progression to androgen-independent LNCaP human prostate tumors: cellular and molecular alterations.

Lethal phenotypes of human prostate cancer are characterized by progression to androgen-independence and metastasis. For want of a clinically relevant animal model, mechanisms behind this progression remain unclear. Our study used an in vivo model of androgen-sensitive LNCaP human prostate cancer cell xenografts in male SCID mice to study the cellular and molecular biology of tumor progression. Primary tumors were established orthotopically, and the mice were then surgically castrated to withdraw androgens. Five generations of androgen-independent tumors were developed using castrated host mice. Tumor samples were used to determine expressions of cellular and molecular markers. Androgen-independent tumors had increased proliferation and decreased apoptosis compared to androgen-sensitive tumors, outcomes associated with elevated expression of p53, p21/waf1, bcl-2, bax and the bcl-2/bax ratio. Blood vessel growth in androgen-independent tumor was associated with increased expression of vascular endothelial growth factor. Overexpression of androgen receptor mRNA and reduced expression of androgen receptor protein in androgen-independent tumors suggest that the androgen receptor signaling pathway may play an important role in the progression of human prostate cancer to androgen-independence. The in vivo orthotopic LNCaP tumor model described in our study mimics the clinical course of human prostate cancer progression. As such, it can be used as a model for defining the molecular mechanisms of prostate cancer progression to androgen-independence and for evaluating the effect of preventive or therapeutic regimens for androgen-independent human prostate cancer.