Activity In PC12 Cells Research Articles

Exiguolysin, a Novel Thermolysin (M4) Peptidase from Exiguobacterium oxidotolerans.

This study details a comprehensive biochemical and structural characterization of exiguolysin, a novel thermolysin-like, caseinolytic peptidase secreted by a marine isolate of Exiguobacterium oxidotolerans strain BW26. Exiguolysin demonstrated optimal proteolytic activity at 37 °C and pH 3, retaining 85% activity at 50 °C, highlighting its potential stability under broad reaction conditions. SDS-PAGE and LC-MS analysis identified the enzyme as a 32 kDa M4-family metalloprotease. Exiguolysin activity was inhibited by 1,10-phenanthroline, confirming its dependence on metal ions for activity. Zymographic analysis and substrate specificity assays revealed selective hydrolysis of matrix metalloproteinase (MMP) substrates but no activity against elastase substrates. Analysis of the predicted gene sequence and structural predictions using AlphaFold identified the presence and position of HEXXH and Glu-Xaa-Xaa-Xaa-Asp motifs, crucial for zinc binding and catalytic activity, characteristic of 'Glu-zincins' and members of the M4 peptidase family. High-throughput screening of a 20 × 20 N-alpha mercaptoamide dipeptide inhibitor library against exiguolysin identified SH-CH2-CO-Met-Tyr-NH2 as the most potent inhibitor, with a Ki of 1.95 μM. Notably, exiguolysin selectively inhibited thrombin-induced PAR-1 activation in PC-3 cells, potentially indicating a potential mechanism of virulence in modulating PAR-1 signalling during infection by disarming PARs. This is the first detailed characterization of a peptidase of the M4 (thermolysin) family in the genus Exiguobacterium which may have industrial application potential and relevance as a putative virulence factor.

Combination of navitoclax (Bcl-2 and Bcl-xL inhibitor) and Debio-0932 (Hsp90 inhibitor) suppresses the viability of prostate cancer cells via induction of apoptotic signaling pathway.

Prostate cancer is one of the most common cancers in men. Given the diverse nature of prostate cancer and its tendency to respond differently to various treatments, combination therapies are often employed to enhance outcomes. In this study, the synergetic efficiency of chemotherapeutic drug Navitoclax and heat shock protein 90 (Hsp90) inhibitor Debio-0932 was evaluated in human prostate cancer cell line (PC3). Our results indicated that Navitoclax-Debio-0932 combination exhibited synergistic activity in PC3 cells at concentrations lower than IC50 values. The combination of Navitoclax and Debio-0932 decreased PC3 cell viability in a dose dependent manner at 48h. To investigate the apoptotic potential of the Navitoclax-Debio-0932 combination against prostate cancer cells, the mRNA and protein expression levels of apoptotic and antiapoptotic markers (Bax, Bcl-2, Bcl-xL, Cyt-c, Apaf-1, Casp-3, Casp-7, and Casp-9) were measured using RT-PCR and ELISA assay. Furthermore, the cleavage activity of Casp-3 was determined by colorimetric assay. The results revealed that Navitoclax-Debio-0932 combination potently induced intrinsic apoptotic pathway in PC3 cells rather than using drugs alone. The combined treatment of Navitoclax and Debio-0932 displayed synergistic cytotoxic and apoptotic effects on prostate cancer cells, presenting a promising approach for combination therapy in prostate cancer.

The Effect of Nano-Encapsulated Orlistat on Prostate Cancer: An In vitro Study

Background: The targeted inhibition of fatty acid synthase (FASN) by Orlistat, a potent FASN inhibitor, has been shown to block tumor proliferation and induce apoptosis in cultured tumor cells. Since Orlistat is insoluble, its solubility in blood circulation is limited. Cancer nanotherapeutics are rapidly progressing and are being implemented to solve several limitations of conventional drug delivery systems. Aim: In this in vitro study, we aimed to overcome the limitation of orlistat changes by designing a nanoparticle and investigating its selective cytotoxic effects on cancer cells. Method: To investigate the effects of encapsulated Orlistat on prostate cancer, we measured apoptosis and the distribution of cells via flow cytometry, confocal microscope, and Western blot. Results: There is an improvement in the solubility of Orlistat with the effect of nanoparticles; it was observed that Orlistat decreased the expression of FASN and caused dose-dependent cytotoxicity with Caspase-3, Bax, and PARP activation in PC-3 cells, while it did not have a significant effect on normal prostate epithelial cells. Conclusion: The present results indicate that the limitation of Orlistat can be overcome by assembling a nano-micellar formulation that can induce apoptotic death in the PC-3 cell line but normal prostate epithelial cells.

Interstitial fluid flow contributes to prostate cancer invasion and migration to bone; study conducted using a novel horizontal flow bioreactor

Prostate cancer bone metastasis is the leading cause of cancer-related mortality in men in the United States, causing severe damage to skeletal tissue. The treatment of advanced-stage prostate cancer is always challenging due to limited drug treatment options, resulting in low survival rates. There is a scarcity of knowledge regarding the mechanisms associated with the effects of biomechanical cues by the interstitial fluid flow on prostate cancer cell growth and migration. We have designed a novel bioreactor system to demonstrate the impact of interstitial fluid flow on the migration of prostate cancer cells to the bone during extravasation. First, we demonstrated that a high flow rate induces apoptosis in PC3 cells via TGF-β1 mediated signaling; thus, physiological flow rate conditions are optimum for cell growth. Next, to understand the role of interstitial fluid flow in prostate cancer migration, we evaluated the migration rate of cells under static and dynamic conditions in the presence or absence of bone. We report that CXCR4 levels were not significantly changed under static and dynamic conditions, indicating that CXCR4 activation in PC3 cells is not influenced by flow conditions but by the bone, where CXCR4 levels were upregulated. The bone-upregulated CXCR4 levels led to increased MMP-9 levels resulting in a high migration rate in the presence of bone. In addition, upregulated levels of α v β 3 integrins under fluid flow conditions contributed to an overall increase in the migration rate of PC3 cells. Overall, this study demonstrates the potential role of interstitial fluid flow in prostate cancer invasion. Understanding the critical role of interstitial fluid flow in promoting prostate cancer cell progression will enhance current therapies for advanced-stage prostate cancer and provide improved treatment options for patients.

Effects of brucine on mitochondrial apoptosis and expression of HSP70 in prostate cancer cells.

BackgroundMost prostate cancer patients are already in the middle/advanced stages of the disease at the time of detection. Whether brucine can regulate apoptosis in prostate cancer cells through the expression of heat shock protein 70 (HSP70) has not been reported. We preliminarily investigated the effects of brucine on the mitochondrial apoptosis and HSP70 expression of prostate cancer cells and analyzed brucine’s possible mechanisms in the hope of providing an experimental basis for its clinical application.MethodsThe effect of brucine on the activity of PC-3 cells was determined by the methodology of tetrazolium (MTT) assay method. The effect of brucine on apoptosis was measured by Hoechst 33258 staining and flow cytometry, and western blotting was used to detect the expression levels of the apoptosis-related heat shock protein 70 (HSP70), anti-apoptotic protease activating factor 1 (Apaf-1) and anti-cysteine protease-3 (caspase-3).ResultsAt 24 and 48 h, the activity of human prostate cancer PC-3 cells in the low, medium, and high dose brucine groups was significantly lower than that of the control group, with a dose-dependent difference (P<0.01). The nuclei of the control group fluoresced uniformly with intact nuclei, whereas the nuclei of the brucine group appeared crinkled, and dense granular masses of strong blue fluorescence were visible. The apoptosis rate of human prostate cancer PC-3 cells in the low, medium, and high dose brucine groups was significantly higher than that in the control group (P<0.01) and was dose-dependent. The expression levels of the HSP70 protein in the human prostate cancer PC-3 cells in the low, medium, and high dose brucine groups were significantly lower than those in the control group, while the expression levels of the Apaf-1 and caspase-3 proteins were significantly higher than those in the control group (P<0.01) and showed a dose-dependent relationship.ConclusionsBrucine downregulated HSP70 expression in human prostate cancer PC-3 cells and inhibited the mitochondrial apoptotic signaling pathway, thus acting as an anti-apoptotic agent.

A rice bran phytochemical, cyanidin 3-glucoside, inhibits the progression of PC3 prostate cancer cell.

Prostate cancer is one of the high incidences and the most invasive cancer that is also highly resistant to chemotherapy. Currently, several natural products have been considering using as the supplements for anti-cancer therapy. This study aims to identify the potential active anti-cancer ingredients in the bran extracts of the native Thai rice (Luempua cultivar). Rice bran fraction enriched in anthocyanins was successively isolated and processed until the major purified compound obtained. The sub-fractions and the purified, rice bran, cyanidin 3-glucoside (RBC3G), were studied for biological effects (cell viability, migration, and invasion assays) on human prostatic cancer (PC3) cells using immunohistochemical-staining and immuno-blotting approaches. The sub-fractions and the purified RBC3G inhibited epithelial mesenchymal transition (EMT) characteristics of PC3 cells by blocking the expression of several cytoskeletal associate proteins in a concentration dependent manner, leading to decreasing of the cancer cell motility. RBC3G reduced the expression of Smad/Snail signaling molecules but enhanced the expression of cell surface protein, E-cadherin, leading to a delay tumor transformation. The RBC3G also inhibited matrix metalloproteinase-9 and nuclear factor-kappa B expression levels and the enzymes activity in PC3 cells, leading to a slow cell migration/invasion process. The results suggested that RBC3G blunt and/or delay the progressive cancer cell behaviors by inhibit EMT through Smad signaling pathway(s) mediating Snail/E-cadherin expression.

Induction of proliferative and mutagenic activity by benzo(a)pyrene in PC-3 cells via JAK2/STAT3 pathway

Environmental carcinogen benzo(a)pyrene (BaP) is a representative compound of polycyclic aromatic hydrocarbons (PAHs). BaP is strongly associated with prostate carcinogenesis. However, the molecular mechanism of BaP in development of prostate carcinoma remains largely unknown. The aim of this study was to investigate the effect and mechanism of BaP on the development in prostate cancer. PC-3 cells were exposed to different concentrations of BaP for 24, 48, 72 h, respectively. We analyzed the effect of BaP on PC-3 cell viability, cell cycle, DNA strand breaks, mutagenic activity, and migration. The expression of associated regulatory genes and the effect of JAK2/STAT3 signaling were also measured to explore the relationships among BaP metabolism, the JAK2/STAT3 pathway and proliferative activity in PC-3 cells. We observed significant effects on proliferation, DNA strand breaks and mutagenic activity after BaP exposure in PC-3 cells, and inhibitors of CYP1 and the AhR transcription factor α -naphthoflavone (ANF) and CH223191 treatment clearly reduced both cell survival and mutagenesis associated with BaP exposure. Reduction in G0-G1 phase population and elevation in S phase were observed after BaP exposure. Migratory cells for PC-3 were significantly increased. The results were further confirmed by the expression of mRNA levels in the significant increments of Snail, Slug, MMP-9, CYP1A1, CYP1B1, CycilnD1, CDK4 and significant reduction of E-cadherin. Significant enhancements were found in the expression of JAK2, STAT3 after BaP treatment. Additionally, activator IL-6 significantly enhanced the effect of BaP on cell survival, mutagenic activity, Cyclin D1, CDK4, Snail, and JAK2/STAT3 expression in PC-3 cells. Significant reductions in cell survival, mutagenic activity, Cyclin D1, CDK4, Snail, and JAK2/STAT3 expression were found after inhibitor AG490, ANF and CHJ223191 treatment. These findings reveal that BaP enhances the proliferative and mutagenic activity via JAK2-STAT3 pathway in PC-3 cells, and provide the additional evidence to understand the crucial role of BaP in prostate cancer carcinogenesis.

ESTAT3 Inhibitor AG-490 Inhibits the Growth of Prostate Cancer by miR-503-5p Both In Vivo and In Vitro.

Objective:To explore the effect and the related mechanism of STAT3 inhibitor AG-490 on inhibiting the proliferation of prostate cancer cells.Methods:PC3 cells and DU145 cells were cultured stably and treated with AG-490 to detect the changes in the activity of PC3 cells and DU145 cells. Thirty 6-8 weeks male BALB/c nude mouse were randomly divided into a control group, a DMSO group, and an AG-490 group to detect differences in various indexes .Results:The overexpression of miR-503-5p depends on the activation of STAT3. After treatment with AG-490, The proliferation and invasion of PC3 cells and DU145 cells and the expression of miR-503-5p were all reduced. Luciferase reporter assay demonstrated that the target proteins of miR-503-5p include PDCD4, TIMP-3, and PTEN. After treatment with AG-490, the expression of PDCD4, TIMP-3, and PTEN in cells was significantly up-regulated. IL-6-induced overexpression of miR-503-5p and restored the expression of STAT3, demonstrating the correlation between STAT3 and miR-503-5p. AG-490 can inhibit tumor growth and induce tumor cell apoptosis in the PC3 BALB/c nude mouse xenograft model. Western blotting and immunohistochemical staining showed that the expression levels of STAT3, Ki67, Bcl-2 and MMP-2 in the AG-490 group were significantly reduced, and the expression of PDCD4, TIMP-3 and PTEN increased.Conclusion:AG-490 can inhibit the growth of prostate cancer cells in a miR-503-5p-dependent manner by targeting STAT3. AG-490 is expected to become a new candidate drug for the treatment of prostate cancer.

Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer.

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.

Cytotoxicity of AMANTADIG – a semisynthetic digitoxigenin derivative – alone and in combination with docetaxel in human hormone-refractory prostate cancer cells and its effect on Na+/K+-ATPase inhibition

Cardiac glycosides (CGs) are natural compounds used to treat congestive heart failure. They have garnered attention as a potential cancer treatment option, especially because they bind to Na+/K+-ATPase as a target and activate intracellular signaling pathways leading to a variety of cellular responses. In this study we evaluated AMANTADIG, a semisynthetic cardenolide derivative, for its cytotoxic activity in two human androgen-insensitive prostate carcinoma cell lines, and the potential synergistic effects with docetaxel. AMANTADIG induced cytotoxic effects in both cell lines, and a combination with docetaxel showed a moderate and strong synergism in DU145 and PC-3 cells, respectively, at concentrations considerably lower than their IC50 values. Cell cycle analyses showed that AMANTADIG and its synergistic combination induced G2/M arrest of DU145 and PC-3 cells by modulating Cyclin B1, CDK1, p21 and, mainly, survivin expression, a promising target in cancer therapy. Furthermore, AMANTADIG presented reduced toxicity toward non-cancerous cell type (PBMC), and computational docking studies disclosed high-affinity binding to the Na+/K+-ATPase α subunit, a result that was experimentally confirmed by Na+/K+-ATPase inhibition assays. Hence, AMANTADIG inhibited Na+/K+-ATPase activity in PC-3 cells, as well as in purified pig kidney at nanomolar range. Altogether, these data highlight the potent effects of AMANTADIG in combination with docetaxel and offer important insights for the development of more effective and selective therapies against prostate cancer.

Notch-4 silencing inhibits prostate cancer growth and EMT via the NF-κB pathway.

Epithelial-mesenchymal transition (EMT) is implicated in the metastasis of human prostate cancer (PCa). Notch signaling has been established as a regulator of EMT. Notch-4 has emerged as a mammary proto-oncogene and a target in several cancers. However, the role and the mechanism of action of Notch-4 in PCa are still unclear. In the present study, we first observed a marked increase in Notch-4 expression in the PCa cell lines DU145, PC3 and LnCAP compared with the non-malignant prostate epithelial cell line RWPE1. Knocking down the expression of Notch-4 suppressed the viability and proliferation in the PCa cell lines DU145 and PC3. Also, further study showed that a decline in Notch-4 significantly promoted apoptosis in PC3 cells. Notch-4 silencing also resulted in decreased cell migration and invasion and affected the expression of EMT markers. We hypothesized that Notch-4 ablation suppresses the activity of NF-κB, so we used PMA to stimulate NF-κB p50 and p65 activation in PC3 cells. The results indicate that PMA treatment impaired the action of Notch-4 ablation in the biology of PC3 cells including cell growth, apoptosis, migration, invasion and EMT. The results of the present study show that RNAi targeting against Notch-4 expression suppresses PCa progression.

Effect of Turkish propolis extracts on expression of voltage-gated sodium channel Nav 1.5 and 1.7 α-Isoforms in PC-3 human prostate cancer cells

Purpose: To investigate the effect of dimethyl sulfoxide (DMSO) and water extracts of Turkish propolis (WEP) on mRNA expression of Nav 1.5 and 1.7 α isoforms of Voltage-Gated Sodium Channel (VGSC) proteins in PC-3 human prostate cancer cells. Methods: DMSO and WEP (20 μg/mL each) were incubated for 24 h with PC-3 cells and total RNA was extracted using a commercial kit. Real-time polymerase chain reaction (RTPCR) assay was used to determine mRNA levels of the isoforms of VGSC. Expressions of VGSC were assumed to be 100 % in PC-3 cells incubated without extract. Results: Both extracts decreased the expression of VGSC isoforms to varying extents. Expressions of Nav 1.5 and 1.7 was 61.43 ± 4.92 and 58.17 ± 2.88 %, respectively for DMSO; while for WEP, the values were 83.54 ± 15.96 and 80.40 ± 13.87 20 μg/mL, respectively. Conclusion: This results suggest that DMSO and water extracts of Turkish propolis may have antimetastatic activity in PC-3 cells due to down-regulation of expressions mRNA of VGSC α-isoforms. Keywords: Propolis, Voltage-gated sodium channel (VGSC), PC-3 Human prostate cancer cells

LncRNA GAS5 inhibits proliferation and progression of prostate cancer by targeting miR-103 through AKT/mTOR signaling pathway.

In prior research, evidence has been found for a relation between low exposure of long non-coding RNAs (lncRNAs) and prostate tumor genesis. This study aims to clarify the underlying mechanisms of lncRNA GAS5 in prostate cancer (PCa). In total, 118 pairs of PCa tissues and matched adjacent non-tumor tissues were collected. Additionally, lncRNA GAS5 exposure levels were determined using RT-PCR and in situ hybridization. In addition, dual-luciferase report assay was performed to verify the target effect of lncRNA GAS5 on miR-103. The exposure levels of the proteins related to the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) axis, including AKT, mTOR, and S6K1, were measured by western blot PC3 cells infected with lncRNA GAS5 mimic; lncRNA GAS5 siRNA; or a combination of lncRNA and miR-103. The proliferation, invasion, and migration ability of PC3 cells after being infected were tested by MTT assay, wound healing assay, and transwell assays. Finally, nude mouse xenograft models were used to measure lncRNA GAS5 effects on prostate tumor growth in vivo. The lncRNA GAS5 levels were reduced significantly in the PCa tissues and cell lines (P<0.05). A low exposure of lncRNA GAS5 caused AKT/mTOR signaling pathway activation in PC3 cells (P<0.05). In addition, over-exposure of lncRNA GAS5 was proven to significantly decelerate PCa cell progression in vitro and tumor growth in vivo through inactivating the AKT/mTOR signaling pathway (P<0.05). This study proves that lncRNA GAS5 plays a significant role in the decelerating PCa development via mediating the AKT/mTOR signaling pathway through targeting miR-103.

Abstract 5044: CtBP1 regulates olfactory and adhesion pathways in prostate cancer and metabolic syndrome

Abstract Metabolic syndrome (MeS) is a cluster of pathophysiological disorders that comprise at least three of the following factors: abdominal obesity, elevated triglycerides, dyslipidemia, high blood pressure and impaired glucose tolerance. Several studies associated MeS with increased risk for several cancer types, including prostate cancer (PCa). C-terminal binding protein 1 (CtBP1) is a potent transcriptional co-repressor of tumor suppressor genes. This protein is activated by low NAD+/NADH ratio produced by highly energetic environment such as high fat diet (HFD) intake. Previously, we identified CtBP1 as a novel molecular link between MeS and prostate tumor growth. The aim of this work was to assess the CtBP1 related pathways in PCa and MeS. We developed a MeS in vivo model by chronically feeding male nude mice with HFD. Control diet (CD) fed animals were maintained at the same conditions. These mice were inoculated with PC3 stable CtBP1 depleted or control cells. RNA obtained from xenografts was used for Genome-wide expression profiles (Affymetrix) and Gene Set Enrichment Analysis (GSEA). These analyses yielded several important pathways regulated by CtBP1, such as “Cell Adhesion” (COL17A1, PRRS2, CDH3, ITGB4, LCN2, CDH1, GJB5, TGM2 and SPARC) and “Olfactory” (OR4C45, OR5P2, GUCA1C and CLCA2). CtBP1 significantly diminished the capability of PCa cell lines to adhere to a collagen matrix, repressing the epithelial marker CDH1 and inducing the mesenchymal marker VIM expressions. Interestingly, CtBP1 associated to ITGB4 promoter gene, strongly repressing its expression. CtBP1 depletion increased the plasma membrane of the cell attached to the substrate and the number of filopodia. From olfactory pathway, we particularly focus on Chloride Channel Accessory 2 (CLCA2), a reported breast cancer tumor suppressor gene that function inhibiting epithelial to mesenchymal transition (EMT) and invasion processes. We found that CtBP1 regulates the transcription of CLCA2 in xenografts generated on HFD-fed mice. Using a panel of luciferase reporter plasmids with variable length of the CLCA2 promoter region, we determined that CtBP1 represses CLCA2 promoter activity in PC3 cells. Moreover, we established that CtBP1 associates to the CLCA2 proximal promoter region by chromatin immunoprecipitation (ChIP). Finally, several proteins regulate CLCA2 promoter activity independently (p53, ET2 and BRCA1) or dependently of CtBP1 (p300 and HDAC2). Altogether, these results demonstrated a new role for CtBP1 in the regulation of cellular adhesion, EMT and invasion reinforcing a potential function for this protein in cancer progression. Hence, CtBP1 pathway might help to identify new molecular candidates for better prediction of PCa progression in a subset of patients with MeS Citation Format: Guillermo N. Dalton, Juliana Porretti, Cintia Massillo, Georgina Scalise, Paula Lucía Farré, Cristian P. Moiola, Alejandra Paez, Geraldine Gueron, Paola De Luca, Adriana De Siervi. CtBP1 regulates olfactory and adhesion pathways in prostate cancer and metabolic syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5044.

Lymphatic endothelial cells actively regulate prostate cancer cell invasion.

Lymphatic vessels serve as the primary route for metastatic spread to lymph nodes. However, it is not clear how interactions between cancer cells and lymphatic endothelial cells (LECs), especially within hypoxic microenvironments, affect the invasion of cancer cells. Here, using an MR compatible cell perfusion assay, we investigated the role of LEC-prostate cancer (PCa) cell interaction in the invasion and degradation of the extracellular matrix (ECM) by two human PCa cell lines, PC-3 and DU-145, under normoxia and hypoxia, and determined the metabolic changes that occurred under these conditions. We observed a significant increase in the invasion of ECM by invasive PC-3 cells, but not poorly invasive DU-145 cells when human dermal lymphatic microvascular endothelial cells (HMVEC-dlys) were present. Enhanced degradation of ECM by PC-3 cells in the presence of HMVEC-dlys identified interactions between HMVEC-dlys and PCa cells influencing cancer cell invasion. The enhanced ECM degradation was partly attributed to increased MMP-9 enzymatic activity in PC-3 cells when HMVEC-dlys were in close proximity. Significantly higher uPAR and MMP-9 expression levels observed in PC-3 cells compared to DU-145 cells may be one mechanism for increased invasion and degradation of matrigel by these cells irrespective of the presence of HMVEC-dlys. Hypoxia significantly decreased invasion by PC-3 cells, but this decrease was significantly attenuated when HMVEC-dlys were present. Significantly higher phosphocholine was observed in invasive PC-3 cells, while higher glycerophosphocholine was observed in DU-145 cells. These metabolites were not altered in the presence of HMVEC-dlys. Significantly increased lipid levels and lipid droplets were observed in PC-3 and DU-145 cells under hypoxia reflecting an adaptive survival response to oxidative stress. These results suggest that in vivo, invasive cells in or near lymphatic endothelial cells are likely to be more invasive and degrade the ECM to influence the metastatic cascade. Copyright © 2016 John Wiley & Sons, Ltd.

Coffee inhibits nuclear factor-kappa B in prostate cancer cells and xenografts

Chronic inflammation contributes to prostate cancer and the transcription factor Nuclear Factor-kappa B (NF-κB) is constitutively active in most such cancers. We examine the effects of coffee on NF-κB and on the regulation of selected genes in human-derived prostate cancer cells (PC3) and in PC3 xenografts in athymic nude mice. PC3 cells stably transduced with an NF-κB-luciferase reporter were used both in vitro and for xenografts. NF-κB activity was measured by reporter assays, DNA binding and in vivo imaging. Gene expression was measured in PC3 cells, xenografts and tumor microenvironment by low-density arrays. Western blotting of activated caspases was used to quantify apoptosis.Coffee inhibited TNFα-induced NF-κB activity and DNA-binding in PC3 cells. Furthermore, coffee increased apoptosis and modulated expression of a number of inflammation- and cancer-related genes in TNFα-treated PC3 cells. In vivo imaging revealed a 31% lower NF-κB-luciferase activation in the xenografts of the mice receiving 5% coffee compared to control mice. Interestingly, we observed major changes in gene expression in the PC3 cells in xenografts as compared to PC3 cells in vitro. In PC3 xenografts, genes related to inflammation, apoptosis and cytoprotection were down-regulated in mice receiving coffee, and coffee also affected the gene expression in the xenograft microenvironment. Our data demonstrate that coffee inhibits NF-κB activity in PC3 cells in vitro and in xenografts. Furthermore, coffee modulates transcription of genes related to prostate cancer and inflammation. Our results are the first to suggest mechanistic links between coffee consumption and prostate cancer in an experimental mouse model.

Abstract 1219: ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity

Abstract The physiological mechanisms that can restore biological activity of mutant p53 is an area of high interest given that mutant p53 expression is observed in one third of prostate cancer and more than 50% of all cancers. Here we demonstrate that ID4 (inhibitor of differentiation-4) a dominant negative regulator of bHLH transcription factors can restore mutant p53 transcriptional activity in prostate cancer cells. ID4 is highly expressed in the normal prostate and decreased in prostate cancer due to promoter hypermethylation. Prostate cancer cell lines: DU145 harbors mutant p53 and also lacks ID4 expression; LNCaP cells express wild-type p53 and ID4; whereas PC3 cells are null for p53 and express low levels of ID4. p53 mutants (P223L and V274F) in DU145 cells are within the DNA binding domain and abrogate p53 transcriptional activity due to structural de-stabilization and/or DNA interactions. Ectopic expression of ID4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, transcriptional targets of p53. DNA binding, p53 luciferase reporter studies and ChIP analysis demonstrated that mutant p53 gains ID4 dependent DNA binding and transcriptional activity in part due to CBP/p300 dependent acetylation of p53 at lysine 373. Loss of ID4 in LNCaP cells also abrogated wild-type p53 DNA binding and transcriptional activity with concomitant loss of CBP/p300 requirement and decreased acetylation of p53. To further elucidate ID4 dependent restoration of biological activity of p53 we stably transfected p53-null cell line PC3, which has endogenous ID4 with mutant p53 mimicking DU145 cells. mRNA, protein levels and apoptosis assays were used to determine the effects on cell death and to determine if mutant p53 had the ability to transactivate upstream/downstream targets (MDM2, PUMA, and p21). To further validate our gene expression profile of p53 responsive targets, we performed DNA binding assays as well as p53 luciferase reporter assays to demonstrate wild-type and mutant p53 transcriptional activity in PC3 cells. Also, to elucidate the mechanism by which ID4 promotes apoptosis, we performed acetylation studies in LNCaP and DU145 xenografts (+/-) ID4 as well as p53 co-immunoprecipation studies to demonstrate interaction of both lysine residues 320 and 373. Our results indicate that transfected mutant p53 in PC3 cells were able to induce expression of downstream targets as well as effect biological processes such as apoptosis and cell cycle arrest. Also, post-translational modifications on residues lys320 and lys373 indicated activation of mutant p53. Collectively, based on our studies we demonstrate that ID4 promotes post-translational modifications of p53, which in turn regulates p53 transcriptional activity. NIH grant #R01CA128914 Citation Format: Derrick Jerone Morton, Divya Patel, Jugal Joshi, Pankaj Sharma, Ashley Knowell, Aisha Hunt, Jaideep Chaudhary. ID4 and p53 cross-talk promotes restoration of mutant-p53 transcriptional activity. [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 1219. doi:10.1158/1538-7445.AM2015-1219

Dual targeting of mTORC1 and mTORC2 by INK-128 potently inhibits human prostate cancer cell growth in vitro and in vivo.

Both mammalian target of rapamycin (mTOR) complexes 1 and 2 (mTORC1/2) are often over-activated in prostate cancer cells and are associated with cancer progression. In the current study, we evaluated the potential anti-prostate cancer activity of INK-128, an ATP-competitive mTORC1/2 dual inhibitor, both in vitro and in vivo. Our results showed that INK-128 exerted potent anti-proliferative activity in established (PC-3 and LNCaP lines) and primary (patient-derived) human prostate cancer cells by inducing cell apoptosis. The latter was evidenced by increase of annexin V percentage, formation of cytoplasmic histone-associated DNA fragments, and cleavage of caspase-3. INK-128-induced prostate cancer cell apoptosis and cytotoxicity were alleviated upon pretreatment of cells with the pan-caspase inhibitor z-VAD-FMK or the specific caspase-3 inhibitor z-DVED-FMK. At the molecular level, INK-18 blocked mTORC1/2 activation in PC-3 cells and LNCaP cells and downregulated mTOR-regulated genes including cyclin D1, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α. ERK-MAPK activation and androgen receptor expression were, however, not affected by INK-128 treatment. In vivo, oral administration of INK-128 significantly inhibited growth of PC-3 xenografts in nude mice. The preclinical results of this study suggest that INK-128 could be further investigated as a promising anti-prostate cancer agent.

Novel naftopidil-related derivatives and their biological effects as alpha1-adrenoceptors antagonists and antiproliferative agents

Eleven novel naftopidil-related compounds that contain amide and indole groups were designed and synthesized. The biological effects of these compounds on three α1-adrenoceptor subtypes and cancerous human prostate cell lines (PC-3, DU-145, and LNCaP) were determined. Compounds 2, 3, 5, 11, and 12 exhibited an α1-adrenoceptor antagonistic activity, whereas compounds 9, 10, and 12 displayed moderate antiproliferative activities. Compound 3 exhibited a significant α1D/1A blocking activity in isolated rat tissues (97.7- and 64.6-fold selective for α1D and α1A compared with α1B) but not a relevant cytotoxic activity. Compound 12 demonstrated a potent and selective α1D/1A antagonistic activity (47.9- and 19.1-fold for α1D and α1A compared with α1B) and a potent antiproliferative activity in PC-3 cells (IC50 = 15.70 μM). Further testing confirmed that compound 12 inhibited the growth of PC-3 cells by inducing apoptosis and G0/G1 cell cycle arrest, which was mediated by α1-adrenoceptor. Therefore, compound 12 is a potential multipotent agent that can act as an effective α1-adrenoceptor subtype antagonist for treating benign prostatic hyperplasia and a preventive medication against human prostate cancer.

Identification of a Prostate Cancer Cell Proteinase Activated Receptor/MMP Signaling Axis

HypothesisProteinase Activated Receptors (PARs) are G‐Protein‐Coupled Receptors activated by the proteolytic unmasking of a cryptic tethered ligand. We proposed that prostate cancer cells produce PAR‐regulating enzymes.MethodsWe monitored PC3, DU145 and LNCaP cell supernatant cleavage of PARs in intact cells by fluorescence imaging using N‐ and C‐ terminus dual‐fluorophore‐tagged PARs. Loss of the N‐terminal tag denotes receptor cleavage‐activation. We also used N‐terminal nano‐luciferase‐tagged PARs that, via luciferase release, document PAR‐cleaving proteinase activity in cell‐derived supernatants. MAPKinase and transwell migration assays were used to monitor PAR activation in PC3 cells.ResultsActivation of PARs 1 &amp; 2 triggered MAPKinase signaling and cancer cell migration. Distinct PAR‐cleaving proteinases in conditioned media from all three prostate cancer cell lines were observed with the N‐Luc‐Luciferase assay, with differential inhibition by matrix metalloproteinase inhibitors and soya trypsin inhibitor. Further, when expressed in PC3 cells, the dual‐fluorophore‐tagged PAR1 was seen in a constitutively cleaved‐active state, with an absent N‐terminus.ConclusionOur data show that prostate cancer‐derived cells secrete PAR‐regulating proteinases that, via an autocrine process, can regulate PAR function in the tumour microenvironment. Since PAR activation triggers prostate cell migration and MAPKinase, PAR antagonists and inhibitors of PAR‐activating proteinases represent therapeutic targets for treating prostate cancer.FundingProstate Cancer Canada Movember Discovery grant.