Arrest In PC-3 Cells Research Articles

Synergistic effects of bloom helicase (BLM) inhibitor AO/854 with cisplatin in prostate cancer

To determine the synergistic effect and mechanism of AO/854, a new Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, cell viability assays, neutral comet assays, and Western blotting (WB) were performed on prostate cancer (PCa) cells. According to our findings, combining AO/854 and CDDP enhanced the antiproliferative capabilities of PC3 cell lines. As evidenced by the upregulation of γH2AX, cleaved caspase-3/caspase-3, and BAX/Bcl-2, AO/854 dramatically increased PC3 apoptosis and DNA damage induced by CDDP. Furthermore, combining AO/854 and CDDP synergistically inhibited PC3 cell migration and invasion. In addition, AO/854 inhibited CDDP-induced S-phase cell-cycle arrest in PC3 cells while enhancing G2/M-phase cell-cycle arrest. In vivo, the antitumor efficacy of the combination therapy group was greater than that of the groups treated with AO/854 or CDDP alone. Our findings indicate that synergistic chemotherapy with AO/854 and CDDP may be a novel anticancer strategy for PCa.

Flubendazole suppresses VEGF-induced angiogenesis in HUVECs and exerts antitumor effects in PC-3 cells.

Flubendazole, an FDA-approved anthelmintic, has been predicted to show strong VEGFR2 inhibitory activity in silico screening combined with invitro experimental validation, and it has shown anti-cancer effects on some human cancer cell lines, but little is known about the anti-angiogenesis effects and anti-prostate cancer effects. In this study, we analyzed the binding modes and kinetic analysis of flubendazole with VEGFR2 and first demonstrated that flubendazole suppressed VEGF-stimulated cell proliferation, wound-healing migration, cell invasion and tube formation of HUVEC cells, and decreased the phosphorylation of extracellular signal-regulated kinase and serine/threonine kinase Akt, which are the downstream proteins of VEGFR2 that are important for cell growth. What's more, our results showed that flubendazole decreased PC-3 cell viability and proliferation ability, and suppressed PC-3 cell wound healing migration and invasion across a Matrigel-coated Transwell membrane in a concentration-dependent manner. The antiproliferative effects of flubendazole were due to induction of G2-M phase cell cycle arrest in PC-3 cells with decreasing expression of the Cyclin D1 and induction of cell apoptosis with the number of apoptotic cells increased after flubendazole treatment. These results indicated that flubendazole could exert anti-angiogenic and anticancer effects by inhibiting cell cycle and inducing cell apoptosis.

Theaflavin-3,3'-di-gallate represses prostate cancer by activating the PKCδ/aSMase signaling pathway through a 67 kDa laminin receptor.

Prostate cancer is a major cause of morbidity and mortality in men. Theaflavin-3,3'-digallate (TF-3) is an important functional ingredient of black tea. We aimed to evaluate the cytotoxic effects of TF-3 on prostate cancer and to identify the underlying molecular mechanism. In this study, we explored the effects of TF-3 on prostate cancer in PC-3 cells and in NOD/SCID mice with prostate cancer. The results demonstrated that TF-3 inhibited prostate cancer cell proliferation by regulating the PKCδ/aSMase signaling pathway. The anti-prostate cancer effect of TF-3 was attributed to the expression of the 67 kDa laminin receptor (67LR), which is overexpressed in various cancers, playing a vital role in the growth and metastasis of tumor cells. Stable knockdown of 67LR could efficiently inhibit TF-3 induced apoptosis and cell cycle arrest in PC-3 cells, through interacting with the PKCδ/aSMase signaling pathway. In vivo studies also confirmed the above findings that TF-3 effectively inhibited tumor growth in terms of tumor volume. TF-3 treatment can significantly inhibit tumor growth and up-regulate the phosphorylation of PKCδ and the expression of aSMase in tumor xenografts developed by subcutaneously implanting PC-3 cells and 67LR-overexpressing PC-3 cells in mice. However, in tumor xenografts formed by subcutaneously implanting 67LR-knockdown PC-3 cells, TF-3 has no significant effect on PKCδ/aSMase pathway regulation and tumor growth inhibition.

Combined Anti-Cancer Effects of Platycodin D and Sorafenib on Androgen-Independent and PTEN-Deficient Prostate Cancer

Castration-resistant (androgen-independent) and PTEN-deficient prostate cancer is a challenge in clinical practice. Sorafenib has been recommended for the treatment of this type of cancer, but is associated with several adverse effects. Platycodin D (PD) is a triterpene saponin with demonstrated anti-cancer effects and a good safety profile. Previous studies have indicated that PC3 cells (PTEN -/-, AR -/-) are sensitive to PD, suggesting that it may also be a useful treatment for castration-resistance prostate cancer. We herein investigated the effects of combining PD with sorafenib to treat PTEN-deficient prostate cancer cells. Our data show that PD promotes sorafenib-induced apoptosis and cell cycle arrest in PC3 cells. Of interest, PD only promoted the anti-cancer effects of sorafenib in Akt-positive and PTEN-negative prostate cancer cells. Mechanistic studies revealed that PD promoted p-Akt ubiquitination by increasing the p-Akt level. PD also increased the protein and mRNA expression of FOXO3a, the downstream target of Akt. Meanwhile, PD promoted the activity of FOXO3a and increased the protein expression of Fasl, Bim and TRAIL. Interestingly, when FOXO3a expression was inhibited, the antitumor effects of both PD and sorafenib were individually inhibited, and the more potent effects of the combination treatment were inhibited. Thus, the combination of PD and sorafenib may exert potent anti-cancer effects specifically via FOXO3a. The use of Akt inhibitors or FOXO3a agonists, such as PD, may represent a promising approach for the treatment of androgen-independent and PTEN-deficient prostate cancer.

Gephyromycin C, a novel small-molecule inhibitor of heat shock protein Hsp90, induces G2/M cell cycle arrest and apoptosis in PC3 cells in vitro

Gephyromycin C (GC), a natural compound isolated from a marine-derived actinomycete Streptomyces sp. SS13I, which exerts anti-proliferative effect on PC3 cells. However, its underlying mechanism of the anti-cancer effect remains unknown. The results of SRB assays showed that GC inhibited the proliferation of PC3 cells with an IC50 value of 1.79 ± 0.28 μM. GC also induced G2/M cell cycle arrest which was accompanied by declining levels of cyclin proteins. Possible mechanisms were investigated and it was found that GC bound to Hsp90 and caused the degradation of Hsp90 client proteins (AKT, CHK1, P53, CDK4, Raf-b, and Raf-1). The fluorescent polarization assay with FITC-labeled geldanamycin (FITC-GA) demonstrated that GC was able to compete with FITC-GA in binding to wild type Hsp90 with an IC50 of 2.15 μM. Results of a docking study also suggested that GC interacted with the N-terminal domain of Hsp90. Our results showed that GC could bind to Hsp90, which resulted in down-regulation of Hsp90 client proteins and G2/M arrest in PC3 cells. Since the antitumor effects of this kind of angucycline via targeting Hsp90 has not been reported before, our results indicate that GC is a novel inhibitor of Hsp90 from marine resources and worthy of further study.

Drug repurposing: Discovery of troxipide analogs as potent antitumor agents

Drug repurposing plays a vital role in the discovery of undescribed bioactivities in clinical drugs. Based on drug repurposing strategy, we for the first time reported a novel series of troxipide analogs and then evaluated their antiproliferative activity against MCF-7, PC3, MGC-803, and PC9 cancer cell lines and WPMY-1, most of which showed obvious selectivity toward PC-3 over the other three cancer cell lines and WPMY-1. Compound 5q, especially, could effectively inhibit PC3 with an IC50 value of 0.91 μM, which exhibited around 53-fold selectivity toward WPMY-1. Data indicated that 5q effectively inhibited the colony formation, suppressed the cell migration, and induced G1/S phase arrest in PC3 cells. Also, compound 5q induced cell apoptosis by activating the two apoptotic signaling pathways in PC3 cells: death receptor-mediated extrinsic pathway and mitochondria-mediated intrinsic pathway. Compound 5q up-regulated the expression of both pro-apoptotic Bax and P53, while down-regulated anti-apoptotic Bcl-2 expression. Besides, compound 5q significantly increased the expression of cleaved caspase 3/9 and cleaved PARP. Therefore, the successful discovery of compound 5q may further validate the feasibility of this theory, which will encourage researchers to reveal undescribed bioactivities in traditional drugs.

ANNONA MURICATA NON‐SELECTIVELY SUPPRESS PC3 CELLS by INHIBITNG CELL PROLIFERATION and INDUCING CELL CYCLE ARREST

Annona muricata (AM) also known as graviola or soursop is a tropical evergreen tree that belongs to family Annonaceae. The leaves of the tree have various therapeutic benefits against numerous diseases including various multi drug resistant cancers. The major phytochemical constituents associated with its anticancer activity are annonaceous acetogenis (AAGs). It was hypothesized that AAGs exhibit their anticancer activity by possibly inhibiting mitochondrial NADH‐ubiquinone complex which limits the ATP supply to the cancer cells. However, the exact mechanism is unknown. The purpose of the study was to determine whether ethyl acetate fraction (EAF) is selectively kills prostate cancer cells and the mechanism by which the EAF induces cell death or reduces cell proliferation.3‐(4,5‐dimethylthiazol‐2‐y1)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfopheny1)‐2H‐tetrazolium (MTS) cell viability assay was performed on both cancer and healthy cells and it was observed that EAF non‐selectively reduces cell viability of PC3 cells and non‐cancer cells. To further examine the effect of EAF on PC3 cells, cell proliferation assay was performed, and it was observed that EAF inhibits cell proliferation in PC3 cells. Western blot analysis for cyclin D1 expression level was performed and EAF exposed cells showed reduced cyclin D1 levels suggesting that AM induces cell cycle arrest in PC3 cells. Immunoblotting analysis suggested that there was no significant effect on poly (ADP ribose) polymerase 1 (PARP‐1) expression levels however EAF significantly increases c‐PARP expression levels indicating that EAF does not induce apoptosis in prostate cancer cells. These results suggest that EAF reduces cell viability of PC3 cells by inhibiting cell proliferation via inducing cell cycle arrest.

Amide-tethered quinoline-resorcinol conjugates as a new class of HSP90 inhibitors suppressing the growth of prostate cancer cells

The study is focused on the design and synthesis of amide tethered quinoline-resorcinol hybrid constructs as a new class of HSP90 inhibitor. In-vitro studies of the synthetic compounds led to the identification of compound 11, which possesses potent cell growth inhibitory effects against HCT116, Hep3B and PC-3 cell lines, exerted through HSP90 inhibition. Compound 11 triggers degradation of HSP90 client proteins along with concomitant induction of HSP70, demonstrates apoptosis inducing ability and causes G2M phase cell cycle arrest in PC-3 cells. Molecular modeling was used to dock compound 11 into the HSP90 active site and key interactions with the amino acid residues of the HSP90 chaperone protein were determined.

Abstract 2960: NCX4040, a nitric oxide (NO) donating form of aspirin as a potential treatment for prostate cancer

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to be beneficial in preventing various types of cancers including prostate cancer (PCa). Of all the NSAIDs in use, Aspirin appears to be one of the promising anticancer agents for PCa management. However, experimental and epidemiological studies indicated that larger doses of Aspirin and long term supplementation is required to achieve anticancer effect with accompanied side effects. Therefore, our study focused on evaluating Aspirin derivative, namely NCX4040 which is a NO releasing form of Aspirin as a potent alternative for PCa treatment while minimizing the side effects. In the present study, we assessed the anticancer effects of NCX4040 on PC-3, a bone metastatic PCa cell line employing various in-vitro cellular assays. In our preliminary studies, we compared the effects of NCX4040 with that of Aspirin and NO releasing compound DETA/NO on PC-3 cells using MTT as read out. These results showed that NCX4040 is highly potent in inhibiting the viability of PC-3 cells when compared to Aspirin or DETA/NO treatments. Hence, further studies were focused on characterizing the anticancer properties as well as the underlying anticancer mechanism of NCX4040. Of note, our results showed that NCX4040 robustly inhibited the colony forming ability of PC-3 cells in-vitro implicating its antitumorigenic potential. In addition to inhibiting colony formation of PC-3 cells, NCX4040 also suppressed the Transwell matrigel invasion of PC-3 cells suggesting the antimetastatic effects of NCX4040. The observed antimetastatic effect of NCX4040 in PC-3 PCa cells appears to be via targeting actin cytoskeleton as confirmed by confocal microscopic analysis using phalloidin stain. Following these findings, we performed cell cycle analysis to determine whether NCX4040 enforces cell cycle arrest in PC-3 cells. These results showed that NCX4040 induced robust G0 cell cycle arrest in a dose dependent manner. Moreover, flow cytometry analysis using Annexin V and PI staining revealed that NCX4040 induced potent apoptotic cell death in PC-3 cells. To delineate the underlying anticancer mechanism of NCX4040, PC-3 cells treated with NCX4040 was subjected to apoptotic protein array analysis. Results of this study inferred that NCX4040 down regulated the expression of key anti-apoptotic proteins such as Survivin, XIAP and IAPs. In conclusion, our study suggests that NCX4040 is a promising alternative agent for PCa treatment. Citation Format: SOMAIAH CHINNAPAKA, Gnanasekar Munirathinam. NCX4040, a nitric oxide (NO) donating form of aspirin as a potential treatment for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2960.

Lipophilic Grape Seed Proanthocyanidin Exerts Anti-Proliferative and Pro-Apoptotic Effects on PC3 Human Prostate Cancer Cells and Suppresses PC3 Xenograft Tumor Growth in Vivo.

The in vitro antiprostate cancer activity of lipophilic grape seed proanthocyanidin (LGSP) against the PC3 cell line was evaluated by MTT assay, flow cytometry, and immunoblot analysis, and the in vivo antiprostate cancer effect was evaluated by a PC3-derived mouse xenograft model via oral gavage LGSP. Ki67 and cleaved caspase 3 immunostaining experiments were performed in tumor tissues. LGSP exhibited a strong inhibitory effect on PC3 cell proliferation by inducing apoptosis. Treatment with LGSP resulted in a G1 phase cell cycle arrest in PC3 cells, which was further confirmed by decreasing the expression of cyclin D1 and CDK 4 and increasing the expression of the tumor suppressors p21 and p27. Furthermore, activation of cleaved fragments of caspases 3, caspases 9, and PARP indicated that LGSP-induced apoptosis is caspase-dependent. Upstream of caspase cascade, LGSP increased the cytochrome c release in cytoplasm. After treatment with LGSP, the Bcl-2/Bax ratio also decreased in PC3 cells. In tumor studies, LGSP inhibited the growth of PC3-derived mouse xenografts by inhibiting tumor cell proliferation and inducing apoptosis. Our findings suggest that LGSP is an effective antiprostate cancer component and deserves further study.

Effect of dihydroartemisinin on UHRF1 gene expression in human prostate cancer PC-3 cells.

As the second most common cancer in men around the world, prostate cancer is increasingly gaining more attention. Dihydroartemisinin (DHA) has been proven to be a promising anticancer agent in vitro as well as in vivo in accumulating data. However, the detailed mechanisms of how DHA action in human prostate cancer PC-3 cells remain elusive. This study aimed to investigate the effects of DHA, a novel anticancer agent, by inhibiting the expression of ubiquitin like containing PHD and ring finger 1 (UHRF1) in PC-3 cells. The apoptosis and cell-cycle distribution were detected by flow cytometry. Quantitative real-time PCR was performed to examine both UHRF1 and DNA methyltransferase 1 (DNMT1) expressions at mRNA levels, whereas the expressions of UHRF1, DNMT1, and p16 proteins at protein levels were detected by Western blotting. Methylation levels of p16 CpG islands were determined by bisulfite genomic sequencing. We showed that DHA induced the downregulation of UHRF1 and DNMT1, accompanied by an upregulation of p16 in PC-3 cells. Decreased p16 promoter methylation levels in DHA-treated groups were also observed in PC-3 cells. Furthermore, DHA significantly induced apoptosis and G1/S cell-cycle arrest in PC-3 cells. Our results suggested that downregulation of UHRF1/DNMT1 is upstream to many cellular events, including G1 cell arrest, demethylation of p16, and apoptosis. Together, our study provides new evidence that DHA may serve as a potential therapeutic agent in the treatment of prostate cancer.

8-Farnesyloxycoumarin induces apoptosis in PC-3 prostate cancer cells by inhibition of 15-lipoxygenase-1 enzymatic activity.

Prostate cancer is the second most common cancer in men worldwide. Overexpression of 15-lipoxygenase-1 (15-LOX-1) has been reported in prostate cancer patients. This study aimed to investigate the cytotoxic and anticancer effects of 8-farnesyloxycoumarin (8f), a prenylated coumarin, by inhibition of 15-LOX-1 activity, in prostate cancer cells. The activity of 15-LOX-1 and the inhibitory effects of 8f on this enzyme were first assessed in PC-3 and DU145 prostate cancer cells. The MTT assay was used to examine the cytotoxicity effects of 8f on PC-3 cells following 15-LOX-1 inhibition. To determine the type of cell death, chromatin condensation and DNA damage were examined by DAPI staining and comet assay, respectively. Furthermore, the effects of 8f on the cell cycle were evaluated by PI staining and flow cytometry. The activity of 15-LOX-1 was determined to be higher in PC-3 compared with DU145 cells; thus, this cell line was selected for further experiments. 8f induced cell death in PC-3 cells in a dose-dependent and time-dependent manner, with IC50 values similar to cisplatin, which was used as a control. However, 8f did not significantly affect the viability of HFF3, human foreskin fibroblast cells, under identical conditions. The appearance of apoptotic cells after 8f treatment was confirmed by the presence of PC-3 cells containing condensed chromatin as shown by DAPI staining. The comet assay indicated the induction of DNA damage in cancerous cells compared with normal cells. In addition, 8f induced a potent G1 cell-cycle arrest in PC-3 cells. Our results showed that the antitumor effects of 8f on PC-3 cells were promoted by apoptosis induction, probably via inhibition of 15-LOX-1 activity, thus suggesting that 8f may have therapeutic value in prostate cancer treatment.

Abstract 4576: Hepatocyte nuclear factor 4 alpha suppresses cell proliferation and induces cellular senescence and apoptosis in prostate cancer cells

Abstract Hepatocyte nuclear factor 4α (HNF4α, NR2A1) is a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors. Recent advances show that it is a key transcriptional regulator of many genes, involved in xenobiotic and drug metabolism and also cancers of gastrointestinal tract. However, the exact functional roles of HNF4α gene in prostate cancer progression are still not fully understood. In this study we used the overexpression (gain-of-function) and knockdown (loss-of-function) approaches to determine the functional roles of HNF4α in prostate cancer. Our results showed that HNF4α exhibited a reduced expression pattern in many prostate cancer cells compared to immortalized prostatic cell lines. Its overexpression could significantly inhibit the cell proliferation of prostate cancer cells, and trigger the cellular senescence and apoptosis by activation of p21 signal pathway in a p53-independent manner. We showed that stable overexpression of HNF4α could significantly induce G2/M arrest in PC3 cells, while shRNA-mediated knockdown of HNF4α could induce G1 growth arrest, suggesting that HNF4α might play a negative role in the cell cycle regulation of PC3 cells. Moreover, stable HNF4α knockdown could confer resistance to paclitaxel treatment and enhance colony formation capacity in prostate cancer cells. Together, our results suggest that HNF4α may play a tumor suppressor function in prostate cancer progression.This work was supported by a Direct Grant for Research 2015-2016 (2015. 1. 064), CUHK. Citation Format: Zhu WANG, Franky Leung Chan. Hepatocyte nuclear factor 4 alpha suppresses cell proliferation and induces cellular senescence and apoptosis in prostate cancer cells. [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 4576.

Abstract 46: The impact of monocarboxylate transporter expression on metabolic function in prostate cancer cells

Abstract INTRODUCTION The tumor microenvironment is subjected to variations in oxygen tension which results in hypoxic regions. Hypoxia in tumors is associated with radioresistance and a metastatic phenotype. Hypoxic conditions cause tumor cells to favor anaerobic metabolism, however this switch to glycolysis is also found in tumor cells under aerobic conditions, known as the ‘Warburg effect’. An increased reliance on glycolysis results in large quantities of lactate, which needs to be transported out of tumor cells to maintain the intracellular pH. The monocarboxylate transporters, MCT1 and MCT4, regulate the transport of lactate in tumor cells and here we report the effect of overexpression or knockdown of these transporters on metabolism, growth and response to therapy in prostate cancer cell lines. METHODS Human prostate cancer cell lines were stably transfected, using lentiviral vectors, to show overexpression of MCT1 (DU145, LNCaP and PC3), overexpression of MCT4 (LNCaP), or knockdown of MCT4 (DU145 and PC3). Using these stable cell lines, the effect of overexpression or silencing of MCT1 or MCT4 was assessed in vitro. A lactate assay was used to investigate the effect on intra- and extracellular lactate under varying oxygen tensions. Effect on cell cycle progression was assessed using flow cytometry and toxicity of docetaxel was determined using MTT and SRB assays. Finally, a metabolome siRNA screen was carried out to determine if MCT4 expression was related to sensitivity to toxicity resulting from knockdown of a range of metabolic proteins. RESULTS Expression levels of MCT1 and MCT4 were confirmed by western blot and immunofluorescence. Silencing of MCT4 in DU145 and PC3 cell lines increased intracellular lactate under hypoxic conditions. No effect on intra- or extracellular lactate was observed in cell lines with overexpression of MCT1. Changes in expression of MCT1 or MCT4 did not appear to result in sensitization or resistance to docetaxel treatment under either normoxic or hypoxic conditions. Overexpression of MCT1 in PC3 cells caused cell cycle arrest in the G2/M phase in both normoxia and hypoxia. The metabolome screen indicated that, in wild type LNCaP cells, down-regulation of the protein product of the genes encoding 2,4-Dehydrocholesterol Reductase or Pyruvate Dehydrogenase Phosphatase Catalytic Subunit 2 was synthetically lethal. Overexpression of MCT4 resulted in protection against these lethal effects. CONCLUSIONS Altering the expression of MCT1 and MCT4 in human prostate cancer cells had a variety of effects on cell function. Overexpression of MCT1 caused cell cycle arrest in PC3 cells which may impact on radiosensitivity as cells are most vulnerable to radiation treatment when in the G2/M phase. The identification of two proteins that cause toxicity when silenced in LNCaP wild-type cells, but not LNCaP cells showing overexpression of MCT4, is an interesting lead for further investigation. Citation Format: Laura Hutchinson, Amy Boyers, Amy Chadwick, Ian Stratford. The impact of monocarboxylate transporter expression on metabolic function in prostate cancer cells. [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 46.

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.

Higher Initial DNA Damage and Persistent Cell Cycle Arrest after Carbon Ion Irradiation Compared to X-irradiation in Prostate and Colon Cancer Cells.

The use of charged-particle beams, such as carbon ions, is becoming a more and more attractive treatment option for cancer therapy. Given the precise absorbed dose-localization and an increased biological effectiveness, this form of therapy is much more advantageous compared to conventional radiotherapy, and is currently being used for treatment of specific cancer types. The high ballistic accuracy of particle beams deposits the maximal dose to the tumor, while damage to the surrounding healthy tissue is limited. In order to better understand the underlying mechanisms responsible for the increased biological effectiveness, we investigated the DNA damage and repair kinetics and cell cycle progression in two p53 mutant cell lines, more specifically a prostate (PC3) and colon (Caco-2) cancer cell line, after exposure to different radiation qualities. Cells were irradiated with various absorbed doses (0, 0.5, and 2 Gy) of accelerated 13C-ions at the Grand Accélérateur National d’Ions Lourds facility (Caen, France) or with X-rays (0, 0.1, 0.5, 1, 2, and 5 Gy). Microscopic analysis of DNA double-strand breaks showed dose-dependent increases in γ-H2AX foci numbers and foci occupancy after exposure to both types of irradiation, in both cell lines. However, 24 h after exposure, residual damage was more pronounced after lower doses of carbon ion irradiation compared to X-irradiation. Flow cytometric analysis showed that carbon ion irradiation induced a permanent G2/M arrest in PC3 cells at lower doses (2 Gy) compared to X-rays (5 Gy), while in Caco-2 cells the G2/M arrest was transient after irradiation with X-rays (2 and 5 Gy) but persistent after exposure to carbon ions (2 Gy).

Abstract B51: The tyrphostin, NT157, suppresses insulin receptor substrates and augments therapeutic response of prostate cancer

Abstract Insulin-like growth factor (IGF) signaling is associated with castrate-resistant prostate cancer (CRPC) progression. Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and anti-apoptotic signaling from IGF-1 receptor (IGF-1R), insulin receptor and other oncoproteins. This study demonstrates that IRS1/2 expression is increased in PCa and, persists in CRPC. Furthermore, this study assesses the anti-cancer activity of NT157, a small-molecule tyrphostin targeting IRS proteins, using androgen-responsive (LNCaP) and -independent (PC3) prostate cancer cells in vitro and in vivo. NT157 treatment resulted in dose-dependent inhibition of IGF1R activation, suppression of IRS protein expression, inhibition of IGF-1-induced AKT activation, but increased ERK activation in NT157-treated cells in vitro. These effects were correlated with decreased proliferation and increasing apoptosis of LNCaP cells and increasing G2M arrest in PC3 cells. NT157 also suppressed androgen-responsive growth, delayed CRPC progression of LNCaP xenografts and suppressed PC3 tumor growth alone and in combination with docetaxel. This study reports the first preclinical proof-of-principle data that this novel small-molecule tyrosine kinase inhibitor targeting human IRS suppresses IRS1/2 expression, delays CRPC progression, and suppresses growth of CRPC tumors in vitro and in vivo. Demonstration that IRS expression can be increased in response to a variety of stressors that may lead to resistance or reduced effect of the therapies indicate that NT157-mediated IRS1/2 down-regulation is a novel therapeutic approach for management of advanced prostate cancer. Citation Format: Naokazu Ibuki, Mazyar Ghaffari, Hadas Reuveni, Mitali Pandey, Ladan Fazli, Haruhito Azuma, Martin E. Gleave, Alexander Levitzki, Michael E. Cox. The tyrphostin, NT157, suppresses insulin receptor substrates and augments therapeutic response of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B51.

Assessment of the anticancer mechanism of ferulic acid via cell cycle and apoptotic pathways in human prostate cancer cell lines.

Studies on genetic changes underlying prostate cancer and the possible signaling pathways are getting increased day by day, and new treatment methods are being searched for. The aim of the present study is to investigate the effects of ferulic acid (FA), a phenolic compound, on cell cycle, apoptosis, invasion, and colony formation in the PC-3 and LNCaP prostate cancer cells. The effect of FA on cell viability was determined via a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) method. Total RNA was isolated with Tri Reagent. Expression of 84 genes for both cell cycle and apoptosis separately was evaluated by reverse transcriptase PCR (RT-PCR). Protein expressions were evaluated by Western blot analysis. Furthermore, apoptotic effects of FA were observed with terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Effects of FA on cell invasion and colony formation were determined using Matrigel chamber and colony assay, respectively. The half maximal inhibitory concentration (IC50) dose of FA was found to be 300 μM in PC-3 cells and 500 μM in LNCaP cells. According to RT-PCR results, it was observed that FA inhibited cell proliferation by increasing the gene expressions of ATR, ATM, CDKN1A, CDKN1B, E2F4, RB1, and TP53 and decreasing the gene expressions of CCND1, CCND2, CCND3, CDK2, CDK4, and CDK6 in PC-3 cells. On the other hand, it was seen that FA suppressed cell proliferation by increasing in the gene expressions of CASP1, CASP2, CASP8, CYCS, FAS, FASLG, and TRADD and decreasing in the gene expressions of BCL2 and XIAP in LNCaP cells. In this study, protein expression of CDK4 and BCL2 genes significantly decreased in these cells. It could induce apoptosis in PC-3 and LNCaP cells. Also, it was observed that FA suppressed the invasion in PC-3 and LNCaP cells. Moreover, it suppressed the colony formation. In conclusion, it has been observed that FA may lead to cell cycle arrest in PC-3 cells while it may cause apoptosis in LNCaP cells.

Tumor-suppressive microRNA-145 induces growth arrest by targeting SENP1 in human prostate cancer cells.

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer-related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin-like modifier (SUMO)-specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA-145 (miRNA-145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3′-untranslated region of SENP1 mRNA is a potential target of miR-145. Here we found that low expression of miR-145 was correlated with high expression of SENP1 in PCa cell line PC-3. The transient introduction of miR-145 caused cell cycle arrest in PC-3 cells, and the opposite effect was observed when miR-145 inhibitor was transfected. Further studies revealed that the SENP1 3′-untranslated region was a regulative target of miR-145 in vitro. MicroRNA-145 also suppressed tumor formation in vivo in nude mice. Taken together, miR-145 plays an important role in tumorigenesis of PCa through interfering SENP1.

The tyrphostin NT157 suppresses insulin receptor substrates and augments therapeutic response of prostate cancer.

Insulin-like growth factor (IGF) signaling is associated with castrate-resistant prostate cancer (CRPC) progression. Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and antiapoptotic signaling from IGF1 receptor (IGF1R), insulin receptor, and other oncoproteins. This study demonstrates that IRS1/2 expression is increased in prostate cancer, and persists in CRPC. Furthermore, this study assesses the anticancer activity of NT157, a small molecule tyrphostin targeting IRS proteins, using androgen-responsive (LNCaP) and -independent (PC3) prostate cancer cells in vitro and in vivo. NT157 treatment resulted in dose-dependent inhibition of IGF1R activation, suppression of IRS protein expression, inhibition of IGF1-induced AKT activation, but increased ERK activation in NT157-treated cells in vitro. These effects were correlated with decreased proliferation and increasing apoptosis of LNCaP cells and increasing G2-M arrest in PC3 cells. NT157 also suppressed androgen-responsive growth, delayed CRPC progression of LNCaP xenografts, and suppressed PC3 tumor growth alone and in combination with docetaxel. This study reports the first preclinical proof-of-principle data that this novel small molecule tyrosine kinase inhibitor suppresses IRS1/2 expression, delays CRPC progression, and suppresses growth of CRPC tumors in vitro and in vivo. Demonstration that IRS expression can be increased in response to a variety of stressors that may lead to resistance or reduced effect of the therapies indicate that NT157-mediated IRS1/2 downregulation is a novel therapeutic approach for management of advanced prostate cancer.