Refractory Prostate Cancer Cells Research Articles

Claudin-3 Loss of Expression Is a Prognostic Marker in Castration-Resistant Prostate Cancer

Castration-resistant prostate cancer (CRPC) development is the foremost concern after treatment of patients with high risk with locally advanced or metastatic prostate cancer. Androgen receptor (AR) is the main driver of CRPC development, through its interaction with epigenetic modifier genes, placing epigenetics modifications in the forefront of CRPC development. Comparing the DNA methylation and expression profile of androgen-sensitive and -refractory prostate cancer cells, we describe the epigenetic silencing of claudin-3 (CLDN3) in AR positive cells resistant to androgen deprivation (LNCaP-abl). CLDN3 silencing was associated with DNA methylation, loss of histone acetylation and H3K27 methylation, and was re-expressed by the combined treatment with the epigenetic modulators Aza and SAHA. From a functional point of view, CLDN3 loss was associated with increased cellular invasion. Immunohistochemical analysis showed decreased CLDN3 expression in samples from CRPC patients. Interestingly, CLDN3 expression was significantly decreased in samples from patients with high total Gleason score (≥8) and locally advanced tumors. Finally, CLDN3 loss of expression was associated with worse disease-free survival and time to clinical progression. In conclusion, our findings strongly indicate that epigenetic silencing of CLDN3 is a common event in CRPC that could be useful as a molecular marker for the prognosis of prostate cancer patients and to discriminate aggressive from indolent prostate tumors.

Transcriptional suppression of androgen receptor by 18β-glycyrrhetinic acid in LNCaP human prostate cancer cells.

Androgen receptor (AR) plays a pivotal role as a target for amplification/mutation in pathogenesis and tumor progression in prostate, and thus, controlling AR activity or expression might be a feasible therapeutic approach for the treatment of prostate cancer. Here, we report the novel mechanisms by which 18β-glycyrrhetinic acid (GA) targets AR to stimulate cell death in both hormone-responsive and -refractory prostate cancer cells. We found that miR-488, a tumor suppressive microRNA, was markedly induced by GA treatment, resulting in the down-regulation of AR expression and inhibition of cellular responses mediated by androgens. Moreover, GA not only suppressed the expression of androgen target genes (TMPRSS2, PSA, and NKX3.1), but also enhanced the suppressive effect of anti-androgens (bicalutamide and flutamide) on LNCaP cell growth. Our data further provides evidence that down-regulation of AR expression by GA may occur through transcriptional suppression at AR promoter region between - 1014 and - 829. Ectopic expression of SFR and E2F3α reversed the inhibitory effect of GA on AR promoter activity as well as protein expression, suggesting that GA may target transcription factors SRF and E2F3α to regulate AR expression. Taken together, our study provides new insights on AR regulation and GA as a potential therapeutic candidate for human prostate cancer.

Abstract B016: CDK7 inhibition suppresses AR addicted Castration-Resistant Prostate Cancer through MED1 inactivation

Abstract Metastatic castration-resistant prostate cancer (CRPC) is an aggressive disease with high mortality rate, primarily resulting from the transcriptional addiction driven by Androgen Receptor (AR). First-line CRPC treatments typically target AR-signaling, but are rapidly bypassed, resulting in only a modest survival benefit with the FDA approved anti-androgen therapies such abiraterone or enzalutamide. Therefore, molecular approaches that more effectively block the AR-transcriptional program are urgently needed. Here, we present evidence demonstrating that AR transcriptional signaling is activated through a “phosphoswitch” catalyzed by cyclin-dependent-kinase 7 (CDK7). Specifically, CDK7 phosphorylates (in vitro and in vivo) the transcriptional co-activator MED1, a key subunit of Mediator complex, at Threonine 1457 to promote the formation of the AR-transcriptional complex at enhancers and super-enhancer (SE) sites. Furthermore, knockdown or inhibition of CDK7 with the recently developed covalent inhibitor THZ1 abolished T1457 phosphorylation that led to MED1 decruitment from the chromatin, attenuated AR-signaling and eliminated AR-addicted naïve or enzalutamide refractory prostate cancer cells. Interestingly, the reduced AR transcriptional output and cellular phenotypes associated with CDK7 knockdown or inhibition was reversed by T1457D phosphomimic suggesting MED1 as a major effector substrate of CDK7 transcription kinase. Finally, THZ1 demonstrated tumor regression in CRPC xenograft models in vivo. In summary, our identification of MED1 T1457 as a novel CDK7 substrate, which is essential for driving AR-mediated transcription, makes CDK7 a potential “non-oncogene dependency” in AR addicted advanced prostate cancer. Taken together, we will present data that strongly support the clinical evaluation of CDK7 specific inhibitors as a monotherapy or in combination with second generation anti-androgens in refractory castration-resistant prostate cancer. Conflict of Interest: None (P.S. This work is undergoing final revision for publication in Cancer Discovery) Citation Format: Reyaz Rasool, Ramakrishnan Natesan, qu deng, Shweta Aras, Priti Lal, Jessica Posimo, Nallasivam Palanisamy, Goutham Narla, Robert Den, Matthew Freedman, donita brady, irfan a asangani. CDK7 inhibition suppresses AR addicted Castration-Resistant Prostate Cancer through MED1 inactivation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B016. doi:10.1158/1535-7163.TARG-19-B016

Polyprenylated polycyclic acylphloroglucinol: Angiogenesis inhibitor from Garcinia multiflora

A new polyprenylated polycyclic acylphloroglucinol, garcimultiflorone K (1), has been isolated from the stems of Garcinia multiflora, together with two known compounds, garcimultiflorone A (2) and garcimultiflorone B (3). The structure of new compound 1 was determined through spectroscopic methods including 1D and 2D NMR and MS analyses. The anti-angiogenic and anti-cancer effects of compounds 1–3 were evaluated in human endothelial progenitor cells (EPCs) and cancer cells. Of these, garcimultiflorone K (1) displayed the most potent anti-angiogenic property by suppressing cell growth and tube formation of EPCs. Compound 1 also exhibited growth-inhibitory activities against human hepatocellular carcinoma cell line SK-Hep-1 and hormone refractory prostate cancer cell line PC-3 with GI50 values of 4.3 ± 1.6 and 6.6 ± 0.4 μM, respectively.

Highly Selective Photodynamic Therapy with a Short Drug-Light Interval Using a Cytotoxic Photosensitizer Porphyrus Envelope for Drug-Resistant Prostate Cancer Cells

Background: Photodynamic therapy (PDT) is a less invasive cancer treatment using photochemical reactions induced by light irradiation to a photosensitizer (PS). Highly selective PDT with fast accumulation of the PS in target site might be a promising treatment option for drug-resistant prostate cancer facing high incidence rate of elderly men who have no effective treatment options and require a minimally invasive treatment. Hemagglutinating virus of Japan envelope (HVJ-E) allows selective and fast drug delivery to the drug-resistant prostate cancer cells via rapid cell membrane fusion. PS named porphyrus envelope (PE) has been developed by insertion of lipidated protoporphyrin IX (PpIX lipid) into HVJ-E. In this study, we investigated the optimal conditions for PE preparation and laser irradiation for highly selective PDT using PE with a short drug-light interval. Materials and Methods: Human hormon refractory prostate cancer cell line PC-3 and human normal prostate epithelial cell line PNT2 were cultured. PpIX lipid uptake and cytotoxicity of PDT in the cells incubated with PE for 10 min were evaluated by measuring fluorescence intensity and by using a cell counting reagent 24 h after PDT, respectively. Results: PpIX lipid uptake and cytotoxicity of PDT were increased with PpIX lipid concentration. Cytotoxicity of PDT using PE was more than 9 times as strong as that with PpIX lipid and PpIX induced by 5-aminolevulinic acid. Much stronger cytotoxicity was induced in PC-3 cells than PNT2 cells with the ratio of cell death rate for cancer to normal cells up to 4.64 ± 0.09. Conclusions: Fast PS delivery with HVJ-E allows highly selective PDT with a short drug-light interval. Therefore, PDT using PE has a potential to shorten treatment period and reduce side effects of PDT.

MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells

Autophagy and apoptosis are two well-controlled mechanisms regulating cell fate. An understanding of decision-making between these two pathways is in its infancy. Monoamine oxidase A (MAOA) is a mitochondrial enzyme that is well-known in psychiatric research. Emerging reports showed that overexpression MAOA is associated with prostate cancer (PCa). Here, we show that MAOA is involved in mediating neuroendocrine differentiation of PCa cells, a feature associated with hormone-refractory PCa (HRPC), a lethal type of disease. Following recent reports showing that NED of PCa requires down-regulation of repressor element-1 silencing transcription factor (REST) and activation of autophagy; we observe that MAOA is a novel direct target gene of REST. Reactive oxygen species (ROS) produced by overexpressed MAOA plays an essential role in inhibiting apoptosis and activating autophagy in NED PCa cells. MAOA inhibitors significantly reduced NED and autophagy activation of PCa cells. Our results here show MAOA as a new decision-maker for activating autophagy and MAOA inhibitors may be useful as a potential therapy for neuroendocrine tumors.

Heteronemin Is a Novel c-Met/STAT3 Inhibitor Against Advanced Prostate Cancer Cells.

Prostate cancer is one of the most prevalent cancers in men worldwide. Aberrant activation of c-Met/signal transducer and activator of transcription-3 (STAT3) signaling is involved in prostate carcinogenesis, underscoring the demand for developing c-Met/STAT3-targeting drugs. Thus, we first utilized virtual screening strategy to identify STAT3-inhibiting marine compound, heteronemin, and then validated the STAT3-inhibiting function of heteronemin in prostate cancer cells. Human prostate cancer LNCaP, DU145, and PC-3 cell lines were treated with heteronemin for 24 hr, then the cell viability was evaluated by MTT assay. Flow cytometry was performed to analyze the apoptosis in heteronemin-treated cells. Western blot and quantitative real-time PCR were executed to further confirm the c-Met/STAT3 signaling inhibition by heteronemin in DU145 and PC-3 cells. In this study, we employed the virtual screening strategy to identify heteronemin, a spongean sesterterpene, as a potential STAT3 inhibitor from Taiwan marine drugs library. Application of heteronemin potently suppressed the viability and anchorage-independent growth of human prostate cancer cells. Besides, heteronemin induced apoptosis in prostate cancer cells by activation of both intrinsic (caspase-9) and extrinsic (caspase-8) apoptotic pathways. By luciferase assay and expression analysis, it was confirmed that heteronemin inhibited the phosphorylation of c-Met/src/STAT3 signaling axis, STAT3-driven luciferase activities and expression of STAT3-regulated genes including Bcl-xL, Bcl-2, and Cyclin D1. Finally, heteronemin effectively antagonized the hepatocyte growth factor (HGF)-stimulated c-Met/STAT3 activation as well as the proliferation and colonies formation in refractory prostate cancer cells. These findings suggest that heteronemin may constitute a novel c-Met/STAT3-targeting agent for prostate cancer. Prostate 76:1469-1483, 2016. © 2016 Wiley Periodicals, Inc.

Sensitization of androgen refractory prostate cancer cells to anti-androgens through re-expression of epigenetically repressed androgen receptor – Synergistic action of quercetin and curcumin

Epigenetic repression of Androgen Receptor (AR) gene by hypermethylation of its promoter causes resistance in prostate cancer (CaP) to androgen deprivation therapy with anti-androgens. Some dietary phytocompounds like quercetin (Q) and curcumin (C) with reported DNMT-inhibitory activity were tested for their ability to re-express the AR in AR-negative CaP cell lines PC3 and DU145. Combined treatment with Q+C was much more effective than either Q or C in inhibiting DNMT, causing global hypomethylation, restoring AR mRNA and protein levels and causing apoptosis via mitochondrial depolarization of PC3 and DU145. The functional AR protein expressed in Q+C treated cells sensitized them to dihydrotestosterone (DHT)-induced proliferation, bicalutamide-induced apoptosis, bound to androgen response element to increase luciferase activity in gene reporter assay and was susceptible to downregulation by AR siRNA. Bisulfite sequencing revealed high methylation of AR promoter CpG sites in AR-negative DU145 and PC3 cell lines that was significantly demethylated by Q+C treatment, which restored AR expression. Notable synergistic effects of Q+C combination in re-sensitizing androgen refractory CaP cells to AR-mediated apoptosis, their known safety in clinical use, and epidemiological evidences relating their dietary consumption with lower cancer incidences indicate their potential for use in chemoprevention of androgen resistance in prostate cancer.

Abstract C91: Reversible lysine specific demethylase-1 inhibition is synergistic with docetaxel in castrate refractory prostate cancer

Abstract Lysine specific demethylase-1 (LSD-1) expression correlates with poor survival in prostate cancer. LSD-1 is also key in androgen receptor signaling in androgen sensitive and castration refractory prostate cancer. LSD-1 is a chromatin modifying enzyme that in conjunction with its binding partners functions as an ‘erasor’ in the Epigenetic code process and demethylates lysine 4 and 9 residues on H3 protein resulting in gene repression or activation. Specifically in prostate cancer it demethylates H3k9me2 playing an important role in androgen receptor signaling promoting myc expression. Docetaxel chemotherapy is effective in prostate cancer but limited by toxicity and development of resistance. In this study we demonstrate synergy between LSD-1 inhibition and docetaxel in castrate refractory cell lines. We further demonstrate that LSD-1 inhibition is effective in docetaxel resistant cell lines. Cell survival assays were performed with HCI2509 on castrate refractory and docetaxel resistant prostate cancer cell lines. Histone methylation and myc expression were assessed. Colony formation assays, cell cycle analysis, xenograft studies were performed. Synergy studies were performed to assess the combination of 2509 and docetaxel. HCI2509 is cytotoxic to castrate refractory and docetaxel resistant prostate cancer cell lines in low micromolar doses. Treatment with HCI2509 resulted in dose dependent increase in H3K4me2, H3K4me3, H3K9me2 levels and decrease in myc protein. RT-PCR showed a significant decrease in MYC expression upon treatment with HCI2509. Cell cycle progression was arrested at G0/G1 upon treatment with 2509 with inhibition of colony formation at 10 to 100 fold lower concentrations. PC3 xenografts in mice showed significant reduction in tumor burden upon treatment with 2509 with increased H3K9me2 marks and reduced myc protein levels. LSD-1 inhibition with HCI2509 has therapeutic potential in castrate refractory and docetaxel resistant prostate cancer. It inhibits myc expression and sensitizes castrate refractory prostate cancer to docetaxel. Citation Format: Sumati Gupta, Alexis Weston, Jared Bearss, Sunil Sharma. Reversible lysine specific demethylase-1 inhibition is synergistic with docetaxel in castrate refractory prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C91.

Octreotide in combination with AT-101 induces cytotoxicity and apoptosis through up-regulation of somatostatin receptors 2 and 5 in DU-145 prostate cancer cells.

Prostate cancer (PCa) is the most common type of cancer among males. Although survival rate of early-stage PCa is high, treatment options are very limited for recurrent disease. In this study, the possible synergistic cytotoxic and apoptotic effect of octreotide in combination with AT-101 was investigated in DU-145 hormone and drug refractory prostate cancer cell line. To enlighten the action mechanisms of the combination treatment, expression levels of somatostatin receptors 2 and 5 (SSTR2 and SSTR5) were also investigated. Cell viability was measured by XTT assay. Apoptosis was assessed through DNA fragmentation analysis and caspase 3/7 assay. mRNA and protein levels of SSTR2 and SSTR5 were evaluated by qRT-PCR and western blot analysis, respectively. Octreotide in combination with AT-101 inhibited cell viability and induced apoptosis synergistically in DU-145 cells as compared to any agent alone. Combination treatment increased both SSTR2 and SSTR5 mRNA and protein levels in DU-145 cells. The data suggest that this combination therapy may be a good candidate for patients with advanced metastatic PCa do not respond to androgen deprivation.

Abstract 538: Identification and validation of novel prostate cancer biomarkers using the Berg Interrogative Biology™ platform

Abstract Prostate cancer is the most frequent cancer diagnosis among men and the second leading cause of cancer-related death. Despite the widespread use of digital rectal exam (DRE) and blood-based screening of prostate-specific antigen (PSA) for prostate cancer screening, there are significant limitations in their specificity and prognostic value. Biomarkers which distinguish i) PSA-low prostate cancer from benign prostatic hyperplasia (BPH), and ii) indolent versus aggressive disease course represent unmet clinical needs. Experimentally, a panel of prostate cancer cell lines and non-tumorigenic, human primary cells were exposed to in vitro conditions designed to simulate poor oxygenation, low pH, diminished nutrient microenvironments, and metabolic perturbations (24-48 h) followed by iTRAQ proteomic analysis of cell lysates. Using the Berg Interrogative Biology™ platform, proteomic data were then subjected to Bayesian network learning to map molecular interactions, with cytoskeletal and scaffolding proteins Filamin A (FLNA), Filamin B (FLNB), and Keratin 19 (KRT19) identified as candidate prostate cancer biomarkers. To validate biomarker expression, mRNA and protein was quantified in panel of primary human prostate epithelial cells (HPrEC) and androgen-sensitive (LnCAP) or refractory (DU145, PC-3) prostate cancer cells, and each was differentially detected in one or more prostate cancer cell lines compared to HPrEC. Using proteomic analysis, peptides from FLNA, FLNB, and KRT19 were also detected cell culture media conditioned by prostate cancer cells (24 h), indicating they can be secreted. Importantly, unlike PSA expression, global regulation of FLNA, FLNB, and KRT19 expression remained unaltered after treatment with multiple prostate-cancer relevant stimuli (e.g., hypoxia, androgens, and inflammatory stimuli). In vivo validation was next conducted in sera from men (N = 447) with confirmed prostate cancer, benign prostate tumors, or BPH using LDT ELISA assays in a CLIA-certified laboratory. To assess the sensitivity and specificity of FLNA, FLNB, and KRT19 compared to PSA, ROC curve analysis was performed. The individual predictive power of each biomarker alone was comparable to that of PSA. However, the combination of age, levels of FLNA, FLNB, and KRT19, and PSA out-performed PSA alone in identification of patients with prostate cancer stratified compared to benign status, gleason scores and incidence of BPH. Together, these data validate the use of the Berg Interrogative Biology™ platform for biomarker discovery and indicate that FLNA, FLNB, and KRT19 can be used in conjunction with PSA for more sensitive and specific prostate cancer screening, a critical unmet need in the field. Citation Format: Niven R. Narain, Anne Diers, Rakibou Ouro-Djobo, Joyce Chan, Leonardo O. Rodrigues, Vivek K. Vishnudas, Eleftherios P. Diamandis, Viatcheslav R. Akmaev, Rangaprasad Sarangarajan. Identification and validation of novel prostate cancer biomarkers using the Berg Interrogative Biology™ platform. [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 538. doi:10.1158/1538-7445.AM2015-538

A precisely substituted benzopyran targets androgen refractory prostate cancer cells through selective modulation of estrogen receptors

Dietary consumption of phytoestrogens like genistein has been linked with lower incidence of prostate cancer. The estradiol-like benzopyran core of genistein confers estrogen receptor-β (ER-β) selectivity that imparts weak anti-proliferative activity against prostate cancer cells. DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran (BP), a SERM designed with benzopyran core, targeted androgen independent prostate cancer (PC-3) cells 14-times more potently than genistein, ~25% more efficiently than tamoxifen and 6.5-times more actively than ICI-182780, without forfeiting significant specificity in comparison to genistein. BP increased apoptosis (annexin-V and TUNEL labeling), arrested cell cycle, and significantly increased caspase-3 activity along with mRNA expressions of estrogen receptor (ER)-β and FasL (qPCR) in PC-3 cells. In classical ERE-luc reporter assay BP behaved as a potent ER-α antagonist and ER-β agonist. Accordingly, it decreased expression of ER-α target PS2 (P<0.01) and increased expression of ER-β target TNF-α (P<0.05) genes in PC-3. ER-β deficient PC-3 (siRNA-transfected) was resistant to apoptotic and anti-proliferative actions of SERMs, including stimulation of FasL expression by BP. BP significantly inhibited phosphorylation of Akt and ERK-1/2, JNK and p38 in PC-3 (immunoblotting), and thus adopted a multi-pathway mechanism to exert a more potent anti-proliferative activity against prostate cancer cells than natural and synthetic SERMs. Its precise ER-subtype specific activity presents a unique lead structure for further optimization.

Zerumbone inhibits growth of hormone refractory prostate cancer cells by inhibiting JAK2/STAT3 pathway and increases paclitaxel sensitivity.

Zerumbone, a phytochemical isolated from Zingiber zerumbet has been shown previously to exhibit antineoplastic activity. But, the effect of zerumbone in prostate cancer has not been evaluated. Prostate cancer is frequently associated with elevated levels of interleukin-6 (IL-6), which exerts its oncogenic effects through activation of Janus kinase 2 (JAK2) followed by activation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Here, we investigated whether the anticancer effects of zerumbone are mediated through inhibition of the JAK2/STAT3 signaling pathway and whether zerumbone can increase the paclitaxel (PTX) sensitivity of prostate cancer cells. Zerumbone exerted significant cytotoxicity of DU145 versus PC3 prostate cancer cells through cell cycle arrest at G0/G1 phase followed by apoptosis. Zerumbone selectively inhibited JAK2 in both DU145 and PC3 cells. However, the biological axis of IL-6/JAK2/STAT3 was inhibited only in DU145 cells as no STAT3 phosphorylation was detected in PC3 cells even after IL-6 stimulation. Other signaling pathways in DU145 cells remained unaffected. The expression of prostate cancer-associated genes, including cyclin D1, IL-6, COX2, and ETV1, was blocked. Zerumbone also synergistically increased the sensitivity to PTX. Further preclinical study might reveal the potential use of zerumbone as a chemotherapeutic agent for hormone refractory prostate cancer where IL-6/JAK2/STAT3 signaling is aberrantly active and may be combined with PTX.

Anthocyanin induces apoptosis of DU-145 cells in vitro and inhibits xenograft growth of prostate cancer.

PurposeTo investigate the effects of anthocyanins extracted from black soybean, which have antioxidant activity, on apoptosis in vitro (in hormone refractory prostate cancer cells) and on tumor growth in vivo (in athymic nude mouse xenograft model).Materials and MethodsThe growth and viability of DU-145 cells treated with anthocyanins were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was assessed by DNA laddering. Immunoblotting was conducted to evaluate differences in the expressions of p53, Bax, Bcl, androgen receptor (AR), and prostate specific antigen (PSA). To study the inhibitory effects of anthocyanins on tumor growth in vivo, DU-145 tumor xenografts were established in athymic nude mice. The anthocyanin group was treated with daily oral anthocyanin (8 mg/kg) for 14 weeks. After 2 weeks of treatment, DU-145 cells (2×106) were inoculated subcutaneously into the right flank to establish tumor xenografts. Tumor dimensions were measured twice a week using calipers and volumes were calculated.ResultsAnthocyanin treatment of DU-145 cells resulted in 1) significant increase in apoptosis in a dose-dependent manner, 2) significant decrease in p53 and Bcl-2 expressions (with increased Bax expression), and 3) significant decrease in PSA and AR expressions. In the xenograft model, anthocyanin treatment significantly inhibit tumor growth.ConclusionThis study suggests that anthocyanins from black soybean inhibit the progression of prostate cancer in vitro and in a xenograft model.

Pao Pereira Extract Inhibits the Growth of Hormone Refractory Prostate Cancer Cells by Suppressing NFKappaB Signaling

Pao Pereira Extract Inhibits the Growth of Hormone Refractory Prostate Cancer Cells by Suppressing NFKappaB Signaling

The aryl hydrocarbon receptor is constitutively active in advanced prostate cancer cells.

BackgroundDistant prostate cancers are commonly hormone refractory and exhibit increased growth no longer inhibited by androgen deprivation therapy. Understanding all molecular mechanisms contributing to uncontrolled growth is important to obtain effective treatment strategies for hormone refractory prostate cancers (HRPC). The aryl hydrocarbon receptor (AhR) affects a number of biological processes including cell growth and differentiation. Several studies have revealed that exogenous AhR ligands inhibit cellular proliferation but recent evidence suggests AhR may possess intrinsic functions that promote cellular proliferation in the absence of exogenous ligands.Methods/ResultsqRT-PCR and western blot analysis was used to determine AhR mRNA and protein expression in hormone sensitive LNCaP cells as well as hormone refractory DU145, PC3 and PC3M prostate cancer cell lines. LNCaP cells express AhR mRNA and protein at a much lower level than the hormone refractory cell models. Cellular fractionation and immunocytochemistry revealed nuclear localization of AhR in the established hormone refractory cell lines while LNCaP cells are devoid of nuclear AhR protein. qRT-PCR analysis used to assess basal CYP1B1 levels and a xenobiotic responsive element binding assay confirmed ligand independent transcriptional activity of AhR in DU145, PC3 and PC3M cells. Basal CYP1B1 levels were decreased by treatment with specific AhR inhibitor, CH223191. An in vitro growth assay revealed that CH223191 inhibited growth of DU145, PC3 and PC3M cells in an androgen depleted environment. Immunohistochemical staining of prostate cancer tissues revealed increased nuclear localization of AhR in grade 2 and grade 3 cancers compared to the well differentiated grade 1 cancers.ConclusionsTogether, these results show that AhR is constitutively active in advanced prostate cancer cell lines that model hormone refractory prostate cancer. Chemical ablation of AhR signaling can reduce the growth of advanced prostate cancer cells, an effect not achieved with androgen receptor inhibitors or growth in androgen depleted media.

Curcumin Inhibits Prostate Cancer Bone Metastasis by Up-Regulating Bone Morphogenic Protein-7 in Vivo.

A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF-β signaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP- 7). This enhancement of BMP-7 in the context of TGF-βin the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regu- lated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF-βsignaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic setting.

Moniliformediquinone Induces In Vitro and In Vivo Antitumor Activity through Glutathione Involved DNA Damage Response and Mitochondrial Stress in Human Hormone Refractory Prostate Cancer

Hormone refractory metastatic prostate cancer is a major obstacle in clinical treatment. The key focus of this study was the discovery and development of a potential agent for this disease. Several pharmacological and biochemical assays were used to characterize the apoptotic signaling pathways of moniliformediquinone, a natural product, in hormone refractory metastatic prostate cancer. Moniliformediquinone induced cell cycle arrest at the S-phase and subsequent apoptosis in the hormone refractory metastatic prostate cancer cell lines PC-3 and DU-145. Further examination showed that moniliformediquinone induced a DNA damage response associated with Chk1, Chk2, c-Jun and JNK activation. Mitochondrial apoptosis pathways were also activated, including loss of mitochondrial membrane potential, cytochrome c release, and activation of caspase-9 and 3. The antioxidant and glutathione precursor N-acetylcysteine, and the antioxidant Trolox™ completely abolished moniliformediquinone induced generation of reactive oxygen species. However, N-acetylcysteine but not Trolox blocked moniliformediquinone mediated apoptosis and related signaling cascades. Further identification showed that moniliformediquinone alone did not conjugate glutathione but significantly decreased cellular glutathione levels. The in vivo study revealed that moniliformediquinone completely inhibited tumor growth with no weight loss. Our data suggest that moniliformediquinone is a potential anticancer agent for hormone refractory metastatic prostate cancer by decreasing cellular glutathione, leading to a DNA damage response and cell cycle arrest at the S-phase. Mitochondrial stress also occurs due to moniliformediquinone action through loss of mitochondrial membrane potential and cytochrome c release, which in turn induce the activation of caspase cascades and apoptotic cell death.

Peripheral blood microRNAs expression profile: A fingerprint for metastatic prostate cancer.

e22171 Background: We previously have studied differentially microRNA expression levels related to hormone refractory and sensitive prostate cancer cell lines (VCap and LNCap respectively). Now, we investigated circulating miRNAs differentially expressed between metastatic prostate adenocarcinomas (mPA) and healthy controls (HC) that may serve as novel diagnostic and/or prognosis markers. Methods: Using SYBR-green-based custom microRNA RT-qPCR arrays technology (Exiqon), we compared the expression levels of miRNAs in blood samples from 18 HC and 48 mPA. Results: Among a panel of 92 candidates we found 41 underexpressed (UE) and 8 overexpressed (OE) miRNAs. The level of significance was p&lt;0.05. Conclusions: Among the UE miRNAs are miR-203 and 129 which regulate a cohort of metastatic genes. Among the OE is miR-200a, a member of miR-200 family which directly targeted β-catenin mRNA and miR-135b which induce invasion and distant metastasis. The knowledge of miRNAs involved in mPA progression could provide us a profile that could identify patients for adjuvant therapy or even propose new target drugs to avoid disease progression or treat mPA. Our group is actively involved in the study of selected OE miRNAs for their potential as circulating markers for mPA. [Table: see text]

Benzyl isothiocyanate induces protective autophagy in human prostate cancer cells via inhibition of mTOR signaling

Benzyl isothiocyanate (BITC) is a dietary chemopreventive agent that inhibits the growth of various human cancer cells by causing apoptotic cell death. In this study, we demonstrate that BITC not only induces apoptosis but also induces autophagy in human hormone-sensitive (Rv1) and -refractory (PC3) prostate cancer cells. In BITC-treated cells, the induction of autophagy was detected by monitoring the processing of an autophagy marker protein, microtubule-associated protein 1 light chain 3 (LC3), the aggregation of LC3 into granular structures and the formation of acidic organelles. Inhibition of autophagy using 3-methyladenine increased BITC-induced apoptosis, whereas the administration of caspase inhibitor suppressed BITC-induced cell death. Our data also showed that BITC inhibits mammalian target of rapamycin (mTOR) kinase activity in a dose-dependent manner. The expression of phospho-mTOR (Ser2481), an indicator of mTOR intrinsic catalytic activity, and phospho-UNC-51-like kinase 1 (Ser757), a direct substrate of mTOR, were decreased in BITC-treated cells. However, the increased expression of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and antiapoptotic Bcl-2 were detected only in PC3 cells at later stages of BITC treatment. Collectively, our results show that BITC induces a protective autophagy response in Rv1 and PC3 cells through inhibition of the mTOR signaling pathway. Activation of the AKT survival pathway was only observed in PC3 cells, representing a resistance mechanism of advanced prostate cancer upon BITC treatment. These findings could potentially contribute to the beneficial effect of BITC in prostate cancer treatments.