Growth Of Human Prostate Cancer Cells Research Articles

Mechanistic Insights into Harmine-Mediated Inhibition of Human DNA Methyltransferases and Prostate Cancer Cell Growth.

Mammalian DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, are key DNA methylation enzymes and play important roles in gene expression regulation. Dysregulation of DNMTs is linked to various diseases and carcinogenesis, and therefore except for the two approved anticancer azanucleoside drugs, various non-nucleoside DNMT inhibitors have been identified and reported. However, the underlying mechanisms for the inhibitory activity of these non-nucleoside inhibitors still remain largely unknown. Here, we systematically tested and compared the inhibition activities of five non-nucleoside inhibitors toward the three human DNMTs. We found that harmine and nanaomycin A blocked the methyltransferase activity of DNMT3A and DNMT3B more efficiently than resveratrol, EGCG, and RG108. We further determined the crystal structure of harmine in complex with the catalytic domain of the DNMT3B-DNMT3L tetramer revealing that harmine binds at the adenine cavity of the SAM-binding pocket in DNMT3B. Our kinetics assays confirm that harmine competes with SAM to competitively inhibit DNMT3B-3L activity with a Ki of 6.6 μM. Cell-based studies further show that harmine treatment inhibits castration-resistant prostate cancer cell (CRPC) proliferation with an IC50 of ∼14 μM. The CPRC cells treated with harmine resulted in reactivating silenced hypermethylated genes compared to the untreated cells, and harmine cooperated with an androgen antagonist, bicalutamide, to effectively inhibit the proliferation of CRPC cells. Our study thus reveals, for the first time, the inhibitory mechanism of harmine on DNMTs and highlights new strategies for developing novel DNMT inhibitors for cancer treatment.

TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated human prostate cancer cell growth.

Androgen receptor (AR) inhibitors represent the mainstay of prostate cancer treatment. In a genome-wide CRISPR-Cas9 screen using LNCaP prostate cancer cells, loss of co-repressor TLE3 conferred resistance to AR antagonists apalutamide and enzalutamide. Genes differentially expressed upon TLE3 loss share AR as the top transcriptional regulator, and TLE3 loss rescued the expression of a subset of androgen-responsive genes upon enzalutamide treatment. GR expression was strongly upregulated upon AR inhibition in a TLE3-negative background. This was consistent with binding of TLE3 and AR at the GR locus. Furthermore, GR binding was observed proximal to TLE3/AR-shared genes. GR inhibition resensitized TLE3KO cells to enzalutamide. Analyses of patient samples revealed an association between TLE3 and GR levels that reflected our findings in LNCaP cells, of which the clinical relevance is yet to be determined. Together, our findings reveal a mechanistic link between TLE3 and GR-mediated resistance to AR inhibitors in human prostate cancer.

Effects of Black Raspberries and Their Constituents on Rat Prostate Carcinogenesis and Human Prostate Cancer Cell Growth In Vitro

Prostate cancer patients often use dietary supplements, such as black raspberries, which are a rich source of compounds with antioxidant and anticancer activity, particularly on gastrointestinal cancers. Feeding black raspberries inhibited mammary cancer induction in rats and growth of cancer cells in nude mice, indicating systemic bioavailability of bioactive compounds. We tested whether feeding black raspberries and its constituents would inhibit prostate cancer development. However, we did not find preventive effects in two rat prostate carcinogenesis models, even though the berry anthocyanin metabolite protocatechuic acid was detectable in their prostates. Black raspberry extract, the anthocyanin cyanidin-3-rutinoside and protocatechuic acid did not inhibit prostate cancer cell growth in vitro, but ellagic acid and its urolithin A metabolite did at high concentrations. Prostate cancer cell migration was not affected by these agents nor was growth in soft agar, except that ellagic acid reduced colony formation at physiological concentrations and protocatechuic acid at high concentrations. Low bioavailability of bioactive berry compounds and metabolites may limit exposure of tissues such as the prostate, since we found that cyanidin-3-rutinoside was not bioavailable to prostate cancer cells, but its aglycone cyanidin was and inhibited their growth. Thus, black raspberries are unlikely to prevent prostate cancer.

Morphological and molecular effects of phenolic extract from coconut kernel on human prostate cancer cell growth in vitro

Coconut is an indispensable ingredient in the diet and traditional medicine of individuals belonging to the Indian subcontinent. Coconut is of high nutritional value owing to the presence of all essential dietary components, viz, saturated fatty acids, arginine rich proteins, fibre and minor components like vitamin E, phytosterols, polyphenols and flavonoids. The polyphenolic content present in coconut kernel is of particular interest due to their numerous reported beneficial effects such as reduction of oxidative stress, combating cancer and in modulating anti-inflammatory pathways. Therefore, in the present study the cytotoxic effect of the polyphenol rich fraction from coconut kernel (CKf) was evaluated in human prostate cancer (DU-145) cells. Individual components present in CKf was determined by LC-MS analysis. It showed that CKf contained several bioactive molecules which have potential anticancer activity viz, coumaric acid, myristin, chlorogenic acid and triterpenoid methyl esters. The cytotoxic effect of CKf at various concentrations (2.5–20 μg/ml) on DU-145 was assessed using MTT assay, AO/EB staining, mitochondrial superoxide/ROS production and changes in intracellular calcium levels, 24 hrs post treatment. Changes in the cell morphology and nucleus were observed using Scanning Electron Microscopy and Confocal microscopy. ROS and mitochondrial superoxide levels was evaluated using DCHF-DA and MitoSOX staining respectively. The impact of ROS on changes in cellular calcium levels was also studied using Fura-2-AM. LDH leakage from C K f treated and control cells were observed colorimetrically. Further, PCR analysis was done to detect changes in mitochondria associated apoptotic gene expression. It was also observed that C K f treatment increased the expression of pro-apoptotic genes - Bax, Bid, Bak and p53 in a dose-dependent manner. Based on the above results, it can be concluded that C K f may be used as a part of a dietary regime for controlling the progression of prostate cancer.

PARP1-siRNA suppresses human prostate cancer cell growth and progression.

Poly (ADP-ribose) polymerase (PARP) inhibitors, such as olaparib or rucaparib, have shown treatment efficacy in BRCA1/2-deficient tumors. However, since PARP inhibitors (PARPi) mainly modulate the activation of PARP but not its expression, whether small interfering RNA (siRNA) specific to PARP has the same function as PARPi has not been well defined. In the present study it was demonstrated that PARP1-siRNA could reduce prostate cancer (PCa) cell progression regardless of the BRCA1/2 mutation. PARP1 silencing could significantly inhibit PC3 cell migration and invasion. Additionally, PARP1-siRNA also inhibited the proliferation of PC3 and Du145 cells. After the induction of apoptosis by docetaxel, cleaved-caspase3 of DU145 and C4-2 cells increased significantly in the PARP1-siRNA group. In the xenograft nude mouse model, PARP1-siRNA could suppress xenograft tumor size of PC3 cells and produce a more regular morphology. Invitro and invivo, PARP1 silencing significantly downregulated vimentin expression and upregulated E-cadherin expression, both of which are epithelial-mesenchymal transition (EMT) markers. It has been revealed that PI3K inhibition could sensitize the effect of PARPi. Notably, PARP1-siRNA could suppress the expression of EGFR and p-GSK3β (Ser9) in PCa cells, which was different from PARPi. Our results indicated that PARP1-siRNA can suppress the growth and invasion capacity of PCa cells, thereby suggesting that PARP1-siRNA, which is different from PARPi, may provide a potential treatment method for PCa.

Dual inhibition of BRD4 and PI3K by SF2523 suppresses human prostate cancer cell growth in vitro and in vivo

Bromodomain-containing protein 4 (BRD4) and phosphatidylinositol 3-kinase (PI3K) are both key oncogenic proteins in human prostate cancer. In the current study, we examined the anti-prostate cancer cell activity by SF2523, a BRD4 and PI3K dual inhibitor. We showed that SF2523 potently inhibited survival and proliferation of the primary human prostate cancer cells. SF2523 induced profound apoptosis activation in prostate cancer cells. The dual inhibitor was yet non-cytotoxic to the prostate epithelial cells. At the molecular level, SF2523 downregulated BRD4-regulated genes (cyclin D1, c-Myc and androgen receptor) and almost blocked AKT-S6K1 activation in prostate cancer cells. In vivo, SF2523 intraperitoneal administration at the well-tolerated dose inhibited human prostate cancer xenograft growth in severe combined immunodeficient (SCID) mice. BRD4-regulated genes (cyclin D1, c-Myc and androgen receptor) and AKT-S6K1 activation were inhibited in SF2523-treated tumors. Together, dual inhibition of BRD4 and PI3K by SF2523 suppresses human prostate cancer cell growth in vitro and in vivo.

α-Pinene Inhibits Human Prostate Cancer Growth in a Mouse Xenograft Model

Background: α-Pinene is one of the most widely found terpenoids in nature. Substantial evidence shows that α-pinene has cancer prevention properties. In this study, the PC-3 cell line was used to establish subcutaneous xenograft tumors in nude mice. Methods: Cytotoxicity was measured with the MTT assay, and apoptosis and cell cycle analyses were conducted using flow cytometry in vitro. The PC-3 cell line was used to establish subcutaneous xenograft tumors in nude mice. Results: We found that treatment with α-pinene significantly inhibited human prostate cancer cell growth and induced apoptosis and cell cycle arrest in the cell line-based model. Furthermore, tumor progression was inhibited more in mice treated with α-pinene than in control mice. We detected less Ki67 and proliferation cell nuclear antigen in paraffin sections from xenograft tumor specimens taken from α-pinene-treated mice than in those from the control group. Meanwhile, α-pinene treatment induced apoptosis in xenograft tumors as determined by the TUNEL assay. Conclusions: These data strongly suggest that α-pinene inhibits prostate cancer growth in a xenograft model and may be an effective therapeutic agent for prostate cancer treatment.

MicroRNA-488 mediates the growth and glycolysis of human prostate cancer cells by targeting 6-phosphofructo-2-kinase 3 gene

Objective To investigate the effect of micrioRNA (miRNA, miR) -488 on glycolysis and proliferation of prostate cancer cells and the underlying mechanism. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to examine the expression of miR-488 in prostate cancer cells. Bioinformatics prediction and luciferase reporter gene assay were utilized for the identification of the target genes of miR-488. The expression levels of 6-phosphofructo-2-kinase 3 (PFKFB3) mRNA and protein were detected by Real-time PCR and Western blotting after the miR-488 mimic was transfected 24 h later. After miR-488 mimics was transfected, or co-transfected with PFKFB3 over-expression plasmid for 24 h, the capacity of proliferation and the glycolysis of prostate cancer cells (PC-3 and DU145) were determined by cell counting kit-8 (CCK-8), glucose uptake and lactate assay, respectively. Results Compared to the normal prostate epithelium cell RWPE-1, the expression of miR-488 in PC-3 and DU145 cell lines was 0.26±0.03 (P=0.031) and 0.19±0.04 (P=0.021), but there was no statistically significant difference in LNCaP and 22RV1 cells. The glucose uptake rate and the lactate secretion rate were 0.54±0.03 (P=0.042) and 0.52±0.01 (P=0.032), and 0.55±0.02 (P=0.037) and 0.61±0.17 (P=0.048) after up-regulating the expression of miR-488, which indicating the decline of glycolysis ability and the proliferation ability. Bioinformatics prediction and luciferase reporter gene assay showed that PFKFB3 was the target gene of miR-488. After the expression of miR-488 was up-regulated, the expression levels of PFKFB3 mRNA and protein were down-regulated. When miR-488 and PFKFB3 over-expression plasmids were co-transfected into the cells, the proliferation was elevated and the glucose uptake rate and lactate secretion rate were 0.79±0.12 and 0.82±0.11, and 0.77±0.07 (P=0.015) and 0.83±0.04 (P=0.026) respectively, indicating a higher glycolysis rate than the control. Conclusion MiR-488 can regulate the ability of proliferation and glycolsis of prostate cancer cells by targeting PFKFB3 gene. Key words: MicroRNA-488; 6-phosphofructo-2-kinase 3; Prostate cancer; Proliferation; Glycolysis

Nintedanib antiangiogenic inhibitor effectiveness in delaying adenocarcinoma progression in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP)

BackgroundIn recent times, anti-cancer treatments have focused on Fibroblast Growth Factor (FGF) and Vascular-Endothelial Growth Factor (VEGF) pathway inhibitors so as to target tumor angiogenesis and cellular proliferation. One such drug is Nintedanib; the present study evaluated the effectiveness of Nintedanib treatment against in vitro proliferation of human prostate cancer (PCa) cell lines, and growth and progression of different grades of PCa lesions in pre-clinical PCa transgenic adenocarcinoma for the mouse prostate (TRAMP) model.MethodsBoth androgen-independent (LNCaP) and androgen-dependent (PC3) PCa cell lines were treated with a range of Nintedanib doses for 72 h, and effect on cell growth and expression of angiogenesis associated VEGF receptors was analyzed. In pre-clinical efficacy evaluation, male TRAMP mice starting at 8 and 12 weeks of age were orally-fed with vehicle control (10% Tween 20) or Nintedanib (10 mg/Kg/day in vehicle control) for 4 weeks, and sacrificed immediately after 4 weeks of drug treatment or sacrificed 6–10 weeks after stopping drug treatments. At the end of treatment schedule, mice were sacrificed and ventral lobe of prostate was excised along with essential metabolic organ liver, and subjected to histopathological and extensive molecular evaluations.ResultsThe total cell number decreased by 56–80% in LNCaP and 45–93% in PC3 cells after 72 h of Nintedanib treatment at 2.5–25 μM concentrations. In pre-clinical TRAMP studies, Nintedanib led to a delay in tumor progression in all treatment groups; the effect was more pronounced when treatment was given at the beginning of the glandular lesion development and continued till study end. A decreased microvessel density and VEGF immunolocalization was observed, besides decreased expression of Androgen Receptor (AR), VEGFR-1 and FGFR-3 in some of the treated groups. No changes were observed in the histological liver analysis.ConclusionsNintedanib treatment was able to significantly decrease the growth of PCa cell lines and also delay growth and progression of PCa lesions to higher grades of malignancy (without inducing any hepatotoxic effects) in TRAMP mice. Furthermore, it was observed that Nintedanib intervention is more effective when administered during the early stages of neoplastic development, although the drug is capable of reducing cell proliferation even after treatment interruption.

The Downregulation of the Expression of CD147 by Tumor Suppressor REIC/Dkk-3, and Its Implication in Human Prostate Cancer Cell Growth Inhibition.

The cluster of differentiation 147 (CD147), also known as EMMPRIN, is a key molecule that promotes cancer progression. We previously developed an adenoviral vector encoding a tumor suppressor REIC/Dkk-3 gene (Ad-REIC) for cancer gene therapy. The therapeutic effects are based on suppressing the growth of cancer cells, but, the underlying molecular mechanism has not been fully clarified. To elucidate this mechanism, we investigated the effects of Ad-REIC on the expression of CD147 in LNCaP prostate cancer cells. Western blotting revealed that the expression of CD147 was significantly suppressed by Ad-REIC. Ad-REIC also suppressed the cell growth of LNCaP cells. Since other researchers have demonstrated that phosphorylated mitogen-activated protein kinases (MAPKs) and c-Myc protein positively regulate the expression of CD147, we investigated the correlation between the CD147 level and the activation of MAPK and c-Myc expression. Unexpectedly, no positive correlation was observed between CD147 and its possible regulators, suggesting that another signaling pathway was involved in the downregulation of CD147. This is the first study to show the downregulation of CD147 by Ad-REIC in prostate cancer cells. At least some of the therapeutic effects of Ad-REIC may be due to the downregulation of the cancer-progression factor, CD147.

Ecotropic viral integration site 1, a novel oncogene in prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men in the western world. Mutations in tumor suppressor genes and in oncogenes are important for PCa progression, whereas the role of stem cell proteins in prostate carcinogenesis is insufficiently examined. This study investigates the role of the transcriptional regulator Ecotropic Viral Integration site 1 (EVI1), known as an essential modulator of hematopoietic and leukemic stem cell biology, in prostate carcinogenesis. We show that in healthy prostatic tissue, EVI1 expression is confined to the prostate stem cell compartment located at the basal layer, as identified by the stem cell marker CD44. Instead, in a PCa progression cohort comprising 219 samples from patients with primary PCa, lymph node and distant metastases, EVI1 protein was heterogeneously distributed within samples and high expression is associated with tumor progression (P<0.001), suggesting EVI1 induction as a driver event. Functionally, short hairpin RNA-mediated knockdown of EVI1 inhibited proliferation, cell cycle progression, migratory capacity and anchorage-independent growth of human PCa cells, while enhancing their apoptosis sensitivity. Interestingly, modulation of EVI1 expression also strongly regulated stem cell properties (including expression of the stem cell marker SOX2) and in vivo tumor initiation capacity. Further emphasizing a functional correlation between EVI1 induction and tumor progression, upregulation of EVI1 expression was noted in experimentally derived docetaxel-resistant PCa cells. Importantly, knockdown of EVI1 in these cells restored sensitivity to docetaxel, in part by downregulating anti-apoptotic BCL2. Together, these data indicate EVI1 as a novel molecular regulator of PCa progression and therapy resistance that may control prostate carcinogenesis at the stem cell level.

Abstract 831: c-Myc is a novel target of prostate cancer cell growth inhibition by honokiol

Abstract Honokiol (HNK) has been shown to inhibit human prostate cancer cell growth in vitro and in vivo, but the underlying mechanism is not fully understood. The present study demonstrates that c-Myc, a key mediator of prostate cancer growth, is a novel target of HNK. Exposure of prostate cancer cells to plasma achievable concentrations of HNK resulted in a marked decrease in levels of total and/or phosphorylated c-Myc. HNK-mediated suppression of c-Myc protein level was due in part to repression of its transcription or protein degradation. We also observed down-regulation of c-Myc in tumors from PC-3 xenografted mice upon oral administration of HNK when compared with control; although the difference was not significant. Stable overexpression of c-Myc conferred protection against HNK-mediated inhibition of colony formation and G0-G1 cell cycle arrest in PC-3 cells. Moreover, HNK suppressed the expression of c-Myc downstream target genes (Cyclin D1 and EZH2) and these effects were restored in Myc-overexpressing PC-3 cells. Consistent with these results, cells with stable knockdown of EZH2 were more sensitive to growth inhibition by HNK compared with control cells. Because androgen receptor (AR) is implicated in regulation of c-Myc expression, we explored the possibility whether AR could affect the expression of c-Myc and its target genes. Indeed, AR overexpression in PC-3 cells enhanced the expression of both c-Myc and its target genes. In conclusion, it is reasonable to postulate that HNK may be effective in retarding growth of c-Myc-driven prostate cancer. This study was supported by the grants CA101753 and CA115498 awarded by the National Cancer Institute. Citation Format: Krishna B. Singh, Eun-Ryeong Hahm, Shivendra V. Singh. c-Myc is a novel target of prostate cancer cell growth inhibition by honokiol. [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 831.

Pyridine analogues of curcumin exhibit high activity for inhibiting CWR-22Rv1 human prostate cancer cell growth and androgen receptor activation.

The concentrations required for curcumin to exert its anticancer activity (IC50, 20 µM) are difficult to achieve in the blood plasma of patients, due to the low bioavailability of the compound. Therefore, much effort has been devoted to the development of curcumin analogues that exhibit stronger anticancer activity and a lower IC50 than curcumin. The present study investigated twelve pyridine analogues of curcumin, labeled as groups AN, BN, EN and FN, to determine their effects in CWR-22Rv1 human prostate cancer cells. The inhibitory effects of these compounds on testosterone (TT)-induced androgen receptor (AR) activity was determined by performing an AR-linked luciferase assay and by TT-induced expression of prostate-specific antigen. The results of the current study suggested that the FN group of analogues had the strongest inhibitory effect of growth on CWR-22Rv1 cultured cells, and were the most potent inhibitor of AR activity compared with curcumin, and the AN, BN and EN analogues. Thus, the results of the present study indicate the inhibition of the AR pathways as a potential mechanism for the anticancer effect of curcumin analogues in human prostate cancer cells. Furthermore, curcumin analogues with pyridine as a distal ring and tetrahydrothiopyran-4-one as a linker may be good candidates for the development of novel drugs for the treatment of prostate cancer, by targeting the AR signaling pathway.

2',4'-dihydroxychalcone, a flavonoid isolated from Herba oxytropis, suppresses PC-3 human prostate cancer cell growth by induction of apoptosis.

Natural products are a promising source for the development of novel cancer therapies, due to their potential effectiveness and low toxicity profiles. As a main component of Herba oxytropis, 2',4'-dihydroxychalcone (TFC) is known to demonstrate anti-tumor activity in vitro. In the present study, TFC was found to potently inhibit proliferation and induce apoptosis in PC-3 human prostate cancer cells in a dose-dependent manner. The results demonstrated that the induction of apoptosis is associated with cell cycle arrest at the G0/G1 phase and activation of caspase-3/-7. Additional mechanistic studies of two biomarkers, phosphatase and tensin homolog (PTEN) and cyclin-dependent kinase inhibitor 1B (p27Kip1), in prostate cancer revealed that TFC treatment significantly upregulated the expression of PTEN and p27Kip1. The findings of the present study indicate that TFC-induced apoptosis in PC-3 cells via upregulation of PTEN and p27Kip1, which results in cell cycle arrest in G0/G1 phase, activation of caspase-3/-7 and induction of apoptosis. Therefore, TFC may be a potential compound for human prostate cancer therapy.

The inhibitory effects of AR/miR-190a/YB-1 negative feedback loop on prostate cancer and underlying mechanism.

Prostate cancer at advanced stages including metastatic and castration-resistant cancer remains incurable due to the lack of effective therapies. MiR-190a belongs to the small noncoding RNA family and has an important role in breast cancer metastasis. However, it is still unknown whether miR-190a plays a role in prostate cancer development. Herein, we first observed AR/miR-190a/YB-1 forms an auto-regulatory negative feedback loop in prostate cancer: miR-190a expression was down-regulated by AR activation; YB-1 functions are as an AR activator; miR-190a inhibited AR expression and transactivation through direct binding to 3′UTR of YB-1 gene. MiR-190a contributes the human prostate cancer cell growth through AR-dependent signaling. Moreover, we examined the expression of miR-190a and observed a significant decrease in human prostate cancers. Reduced expression of miR-190a was inversely correlated to AR levels of prostate cancer patients, and patients with higher miR-190a expression in their tumor have improved tumor-free survival. Taken together, our findings identified a biochemical and functional link between miR-190a with reduced expression in advanced prostate cancer, YB-1 and AR signaling in prostate cancer.

Vitamin D3 potentiates the growth inhibitory effects of metformin in DU145 human prostate cancer cells mediated by AMPK/mTOR signalling pathway

Metformin and vitamin D₃ both exhibit a strong antiproliferative action in numerous cancer cell lines, including in human prostate cancer cells. Here we showed that the combination of the two drugs had a much stronger effect on DU145 human prostate cancer cell growth than either drug alone. In this research, cell proliferation was measured by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was determined with Hoechst 33342 staining. Western blotting and cell cycle analyses were used to elucidate potential mechanisms of interaction between the drugs. It is shown that in cultured DU145 cells, vitamin D₃ combined with metformin exhibits synergistic effects on cell proliferation and apoptosis. The underlying antitumor mechanisms may involve altered cycle distribution with a G1/S cell cycle arrest, activation of phospho-AMPK with subsequent inhibition of downstream mTOR signalling pathway, down-regulate c-Myc expression, and reducing the level of anti-apoptotic protein p-Bcl-2. In conclusion, metformin and vitamin D₃ synergistically inhibit DU145 cell growth, indicating a promising clinical therapeutic strategy for the treatment of androgen-independent prostate cancer.

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.

Glucose-Based Regulation of miR-451/AMPK Signaling Depends on the OCT1 Transcription Factor

In aggressive, rapidly growing solid tumors such as glioblastoma multiforme (GBM), cancer cells face frequent dynamic changes in their microenvironment, including the availability of glucose and other nutrients. These challenges require that tumor cells have the ability to adapt in order to survive periods of nutrient/energy starvation. We have identified a reciprocal negative feedback loop mechanism in which the levels of microRNA-451 (miR-451) are negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator OCT1 by 5' AMP-activated protein kinase (AMPK), which is activated by glucose depletion-induced metabolic stress. Conversely, in a glucose-rich environment, unrestrained expression of miR-451 suppresses AMPK pathway activity. These findings uncover miR-451 as a major effector of glucose-regulated AMPK signaling, allowing tumor cell adaptation to variations in nutrient availability in the tumor microenvironment.

Ursolic Acid Mediated Inhibition of Human Prostate Cancer Cell Growth in vitro Involves Alterations in Cell Cycle Related Protein Expression

Ursolic acid is a constituent of the American cranberry (Vaccinium macrocarpon) and other Vaccinium fruit found primarily in the peel. In this study, the effects of ursolic acid (UA) on the cell cycle behaviour of DU145 human prostate cancer cells in vitro was examined. Following treatment of cells with UA (50 μM) for 6 hours, the cellular viability of the cells decreased. UA treatment increased the proportion of cells in the G1 phase of the cell cycle. The relative proportion of cells in G2-M phase was similar in both control and UA-treated cells. UA treatment also caused a decrease in the proportion of cells in S-phase relative to untreated cells. These cell cycle alterations were associated with changes in expression of cell cycle associated regulatory proteins. UA decreased the protein expression levels of cyclin A, cyclin D, p16 , pRBp107 and increased the expression of CDK2, CDK4, cyclin E, p21, p27 and pRBp130. CyclinB1 protein expression levels were apparently unaffected by UA. These findings demonstrate that ursolic acid can affect the cell growth behaviour of human prostate cancer cells in vitro. [Cranberry Institute (Wisconsin Board) & Telus MRFD Prostate Cancer Research Fund (PEI Division) funded]

Abstract 2131: Effects of black raspberries and their constituents on rat prostate carcinogenesis and human prostate cancer cell growth

Abstract Black raspberries (BRBs) and their constituents may inhibit human GI tract cancers and prevent breast cancer in a rat model. Anthocyanins and polyphenols in BRBs are responsible for their anti-cancer effects. Berries and other fruits are used as dietary supplements by cancer patients. As prostate cancer remains a major health problem, it is thus important to determine whether BRBs or BRB constituents may prevent or treat this malignancy. We previously reported (AACR Ann Mtg 2013) that the BRB anthocyanin metabolite protocatechuic acid (PCA) was detectable in the prostate of rats fed BRB, indicating that it reaches this tissue, but BRB consumption by Wistar rats did not prevent prostate cancer induced by MNU and chronic testosterone treatment. We now studied the effect of feeding AIN-93M diet containing 5% or 10% lyophilized BRB powder (exchanged for starch) on induction of prostate cancer in NBL rats by chronic testosterone &amp; 17β-estradiol treatment via SC Silastic implants. Incidence of adenocarcinomas arising from periurethral prostatic ducts was similar in rats fed control diet and those fed berry-containing diets (73-90%), as was tumor multiplicity (1.7-1.8 tumors per prostate). In contrast, we previously found that cyanidin-3-rutinoside, the most abundant anthocyanin in BRBs, inhibits growth of LAPC-4 human prostate cancer cells. We now used PC-3 and LNCaP prostate cancer cells to study effects of ethanolic extract of lyophilized BRB powder and major BRB constituents: the polyphenol ellagic acid (EA), cyanidin-3-rutinoside, and PCA. Growth rates were measured by hemocytometer counting with trypan blue exclusion. BRB extract (1 µg/ml to 1 mg/ml) inhibited viability of LNCaP cells dose-dependently, but linear trend was not significant (p=0.09). Similar, but also not quite significant, decreases in LNCaP cell numbers were caused by PCA and EA, while EA increased the number of dead cells (ANOVA p=0.0471, linear trend p=0.0385). No significant changes occurred in PC-3 cell viability with any of the treatments. Effects on anchorage independent growth of PC-3 cells were assessed by soft agar colony forming assays. A dose-dependent reduction in the number of colonies occurred after EA treatment (ANOVA p=0.0065, linear trend p=0.0007) and BRB extract (ANOVA p&amp;lt;0.0001, linear trend p&amp;lt;0.0001), but no change in colony formation of cells treated with cyanidin-3-rutinoside or PCA. Compared to vehicle, BRB at 1 mg/ml maximally reduced colonies by 50%, and 3.0 µg/ml EA reduced colonies by 39%. No effects on cell migration of PC-3 cells, assessed by wound healing assays, were found for any of the treatments. Thus, BRBs may be beneficial for treatment of prostate cancer and EA is at least partially responsible for the anti-cancer activity of BRBs, but, surprisingly, cyanidin-3-rutinoside and protocatechuic acid did not have significant effects. (Supported by NIH Grant R21 CA152879). Citation Format: Jillian N. Eskra, Michael J. Schlicht, Maarten C. Bosland. Effects of black raspberries and their constituents on rat prostate carcinogenesis and human prostate cancer cell growth. [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 2131. doi:10.1158/1538-7445.AM2014-2131