Cytoprotective autophagy induction by withaferin A in prostate cancer cells involves GABARAPL1

Withaferin A (WA) is a naturally occurring steroidal lactone with proven cancer chemopreventive activity in preclinical models of different cancers including prostate adenocarcinoma. Previously we compared the RNA-seq data from control and WA-treated 22Rv1 human prostate cancer cells to identify mechanistic targets of this phytochemical. The Gene Ontology pathway analysis of the RNA-seq data revealed significant upregulation of genes associated with autophagy upon WA treatment in 22Rv1 cells. In this study, we extended these findings to investigate the mechanism underlying WA-induced autophagy. Initially, we confirmed autophagy induction by WA treatment by transmission electron microscopy using three prostate cancer cell lines (LNCaP, 22Rv1, and PC-3). Fourteen common genes altered by 8- and 16-hour exposure to WA were identified from human autophagy PCR array and these results were consistent with the RNA-seq data. Two key autophagy markers (LC3BII and SQSTM1) were robustly increased in WA-exposed LNCaP, 22Rv1, and PC-3 cells as determined by immunoblotting, and this effect was elevated in the presence of autophagy inhibitor bafilomycin A1 (BafA1). BafA1 treatment augmented WA's cytotoxicity and subsequently its proapoptotic potential. WA treatment induced GABARAPL1 (ATG8L) protein expression in all three cell lines and its knockdown by RNA interference attenuated WA-mediated apoptosis. WA-induced autophagy was not affected in the presence of an antioxidant (EUK134). Taken together, the present study reveals that WA-mediated autophagy is cytoprotective and mediated by GABARAPL1.     

α-Viniferin activates autophagic apoptosis and cell death by reducing glucocorticoid receptor expression in castration-resistant prostate cancer cells

Prostate cancer (PCa) is one of the most commonly diagnosed urological malignancies. However, there are limited therapies for PCa patients who develop biochemical recurrence after androgen deprivation therapy (ADT). In the present study, we investigated the therapeutic efficacy and mechanism of α-Viniferin (KCV), an oligostilbene of trimeric resveratrol, against human PCa cells and found that it markedly inhibited the proliferation of LNCaP, DU145, and PC-3 cancer cells in a time- and dose-dependent manner, and had a strong cytotoxicity in non-androgen-dependent PCa cells. In addition, KCV inhibited AR downstream expression in LNCaP cells, and inhibited activation of GR signaling pathway in DU145 and PC-3. Further investigation indicated that KCV could induce cancer cell apoptosis through AMPK-mediated activation of autophagy, and inhibited GR expression in castration-resistant prostate cancer(CRPC). These findings suggest that KCV may prove to be a novel and effective therapeutic agent for the treatment of CRPC.     

Antiproliferative effects of AVN944, a novel inosine 5-monophosphate dehydrogenase inhibitor, in prostate cancer cells

Inosine 5-monophosphate dehydrogenase II, a key enzyme in the de novo synthesis of purine nucleotides, is expressed in prostate tumors and prostate cancer cells. AVN944 is a new, specific, noncompetitive IMPDH inhibitor. In this study, we investigated the effects of IMPDH inhibitor AVN944 on LNCaP, CWR22Rv1, DU145 and PC-3 human prostate cancer cells. AVN944 inhibited proliferation of these 4 prostate cancer cell lines and was associated with cell cycle G1 arrest of LNCaP cells and S-phase block of androgen-independent CWR22Rv1, DU145 and PC-3 cells. AVN944 induced caspase-dependentand caspase-independent cell death in LNCaP, CWR22Rv1, and DU145 cells. AVN944 induced expression of p53-target proteins Bok, Bax and Noxa in androgen-responsive cell lines and suppressed expression of survivin in prostate cancer cells regardless of their androgen sensitivity. AVN944 also induced differentiation of androgen-independent prostate cancer cells as indicated by morphological changes and increased expression of genes coding for prostasomal proteins, keratins and other proteins, including tumor suppressor genes MIG-6 and NDRG1. AVN944-differentiated androgen-independent DU145 and PC-3 cells are sensitized to TRAIL-induced apoptosis as demonstrated by induction of caspases and PARP cleavage. In summary, AVN944 inhibited the growth of human prostate cancer cells by inducing cell cycle arrest, cell death as well as differentiation. AVN944 is a novel, promising therapeutic agent that might be combined with other agents for treatment of human prostate cancer.     

Growth of human prostate cancer cells is significantly suppressed in vitro with sodium butyrate through apoptosis

Histone deacetylase inhibitors (HDACis) have shown significant antiproliferative and apoptotic properties in various types of cancer cells, including prostate cancer cells, and are therefore being evaluated as a treatment modality. However, the mechanism by which sodium butyrate (SB) induces apoptosis is not completely understood. We focused on SB which exists in the intestine and is therefore expected to have less adverse effects. In this study, three prostate cancer cell lines (LNCaP, DU145 and PC-3) were treated in vitro with different concentrations of SB. Cell proliferation was studied by the XTT assay; cell cycle analysis and induction of apoptosis were studied by laser scanning cytometry. Western blot analysis was used to study p21, p27, CDK2, CDK4, CDK6, caspase-3, caspase-7, Fas, FADD, TRADD, Bcl-2 and Bax protein expression. SB inhibited cell growth and induced apoptosis in a concentration-dependent manner in human prostate cancer cells (LNCaP, DU145 and PC-3). Western blot analysis showed dose-dependent increases of p21 levels in DU145 and PC-3 cells, and dose-dependent decreases of CDK2, CDK4, CDK6 and procaspase-3 protein levels in all three prostate cancer cell lines. Bcl-xL was significantly down-regulated in DU145 cells, and Bcl-2 was significantly down-regulated in PC-3 and LNCaP cells. No significant changes were observed in procaspase-7, TRADD and Bax expression, although slight decreases in Fas and FADD expression were seen in all three prostate cancer cell lines. Analysis of cell morphology using laser scanning microscopy detected condensed and fragmented nuclei. In conclusion, SB induces G1 and G2 arrest by increasing p21 expression resulting in CDK2, CDK4 and CDK6 down-regulation. SB potently induced apoptosis, which was accompanied by DNA fragmentation, down-regulated Bcl-2 in LNCaP and PC-3 cells, Bcl-xL in DU145 cells, and down-regulated procaspase-3, but not procaspase-7, in these human prostate cancer cell lines. These results suggest that SB may serve as a new modality for the treatment of hormone refractory prostate cancer.     

Metastatic model for human prostate cancer using orthotopic implantation in nude mice.

BACKGROUND: Understanding the mechanism of prostate cancer metastasis is essential to the design of a more effective therapy. An effective therapy for this disease will depend on the development of a clinically relevant in vivo model. PURPOSE: We describe the development of such a model by using orthotopic implantation of human prostate cells in BALB/c nude mice. METHOD: We compared the tumorigenicity of and the incidence of metastasis of human prostate cancer PC-3M and LNCaP-FGC (LNCaP) cell lines subsequent to prostatic (orthotopic) or subcutaneous (ectopic) implantations in male nude mice. RESULTS: LNCaP cells produced tumors only in the prostate. Enhanced tumorigenicity at the orthotopic site was found for PC-3M cells. Lymph node metastases were observed in practically all mice given an injection of PC-3M cells in the prostate, but they were uncommon with subcutaneous injection of these cells. Bilateral orchiectomy did not alter the tumorigenicity of either PC-3M or LNCaP cells or the incidence of lymph node metastasis by PC-3M cells. LNCaP tumors in the mouse prostate (but not PC-3M tumors) elaborated detectable levels of human prostate-specific antigen (PSA) in the serum, even when tumors were small (1.5 mm in diameter). Immunohistochemistry analysis revealed the presence of the PSA marker in tissue sections of LNCaP but not of PC-3M tumors. CONCLUSIONS: The implantation of human prostate cancer cells in an ectopic environment does not permit expression of metastatic potential. In contrast, intraprostatic implantation does. IMPLICATIONS: These data suggest that the orthotopic injection of human prostate cancer cells into the nude mouse may provide a valuable model to study the biology and therapy of human prostate cancer.     

The Chinese medicinal herbal formula ZYD88 inhibits cell growth and promotes cell apoptosis in prostatic tumor cells

Prostate cancer is a leading cause of cancer death in American males. Currently, there is no curative therapy available once prostate cancer has metastasized. A major systemic therapy for metastatic prostate cancer is anti-androgen therapy. Unfortunately this therapy is only palliative and rarely curative, and eventually the tumor cells develop resistance to further hormone manipulation. It is therefore imperative to develop alternative effective therapies. In the present study, the effect of a Chinese herbal formula, ZYD88, on regulation of cell growth and cell apoptosis was examined in prostatic tumor cells. ZYD88 decreased cell viability of multiple prostatic tumor cell lines, DU-145, PC-3, MDA-PCa 2b and LNCaP in a time- and dose-dependent manner. It also produced a rapid and dose-dependent increase in caspase 3 activity in LNCaP and PC-3 cells, and induced DNA fragmentation in LNCaP cells, indicating cell apoptosis. In cotransfection assays, ZYD88 inhibited androgen-induced prostate specific antigen (PSA) gene promoter activity, and induced estrogen-target gene promoter activity. These data suggest that ZYD88 is a potential agent for prostate cancer therapy, and deserves further study.     

Tributyrin induces differentiation, growth arrest and apoptosis in androgen-sensitive and androgen-resistant human prostate cancer cell lines

Progression to androgen independence remains the main problem that impacts on survival and quality of life in prostate cancer patients. We have investigated the potency of tributyrin, an orally available prodrug of butyrate, to induce growth arrest, differentiation and apoptosis in LNCaP, PC-3 and TSU-PR1 human prostate cancer cell lines. Cells were treated with 0.1 to 5 mM tributyrin or sodium butyrate. Growth inhibition, cell cycle arrest and apoptosis induction was assessed using standard methods. Both agents induced a more differentiated, fibroblast-like phenotype in androgen-sensitive as well as androgen-resistant cell lines. Expression of prostate-specific antigen was increased in LNCaP cells by tributyrin as a indicator of differentiation. The IC(50) for sodium butyrate was 2.5 mM in PC-3 and TSU-PR1 cells. LNCaP cells exhibited <50% growth inhibition at 5 mM sodium butyrate. However, the IC(50) for tributyrin was 0.8 mM in PC-3 cells, 1.2 mM in TSU-PR1 cells and 3.1 mM in LNCaP cells. Flow cytometry revealed a strong G1-arrest after exposure to tributyrin or sodium butyrate. Both agents resulted in a strong increase of apoptosis rates compared with mock-treated cells. Overall, tributyrin had a 2.5- to 3-fold growth inhibitory and apoptosis-inducing potency compared with equimolar concentrations of sodium butyrate. Our results demonstrate that tributyrin is more potent than butyrate in regard to cell growth inhibition and apoptosis induction at pharmacologically relevant concentrations. Hence, tributyrin may be a promising candidate for clinical protocols in prostate cancer.     

Sorafenib-induced defective autophagy promotes cell death by necroptosis

Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5−/− cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.     

Allyl isothiocyanate,a constituent of cruciferous vegetables,inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosis

Dietary isothiocyanates (ITCs) are highly effective in affording protection against chemically induced cancers in laboratory animals. In the present study, we demonstrate that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits proliferation of cultured PC-3 (androgen-independent) and LNCaP (androgen-dependent) human prostate cancer cells in a dose-dependent manner with an IC(50) of approximately 15-17 micro M. On the other hand, survival of a normal prostate epithelial cell line (PrEC) was minimally affected by AITC even at concentrations that were highly cytotoxic to the prostate cancer cells. Reduced proliferation of PC-3 as well as LNCaP cells in the presence of AITC correlated with accumulation of cells in G(2)/M phase and induction of apoptosis. In contrast, AITC treatment failed to induce apoptosis or cause G(2)/M phase arrest in PrEC cells. A 24 h treatment of PC-3 and LNCaP cells with 20 micro M AITC caused a significant decrease in the levels of proteins that regulate G(2)/M progression, including Cdk1 (32-50% reduction), Cdc25B (44-48% reduction) and Cdc25C (>90% reduction). A significant reduction in the expression of cyclin B1 protein (approximately 45%) was observed only in LNCaP cells. A 24 h exposure of PC-3 and LNCaP cells to an apoptosis-inducing concentration of AITC (20 micro M) resulted in a significant decrease (31-68%) in the levels of anti-apoptotic protein Bcl-2 in both cell lines, and approximately 58% reduction in Bcl-X(L) protein expression in LNCaP cells. In conclusion, it seems reasonable to hypothesize that AITC, and possibly other ITCs, may find use in the treatment of human prostate cancers.     

Pancratistatin induces apoptosis and autophagy in metastatic prostate cancer cells

The Amaryllidaceae alkaloid pancratistatin has been proven to selectively induce apoptotic cell death in a variety of human cancer cells with an insignificant effect on non-cancerous cells. In this study we report, for the first time, the effects of pancratistatin (PST) on models of metastatic prostate cancer. The effects of pancratistatin on prostate cancer DU145 and LNCaP cell lines was assessed by microscopy, enzymatic activity assays and Western blotting. Apoptosis was determined by nuclear condensation and caspase activation, and autophagy was observed by MDC staining and LC3 expression levels. Human prostate xenografts were used to test the potential therapeutic efficacy of intra-tumor administration of pancratistatin in vivo. Pancratistatin treatment reduced cell viability and induced apoptosis in androgen-responsive (LNCaP) and androgen-refractory (DU145) prostate cancer cell lines in a dose- and time-dependent manner, but with an insignificant effect on normal human fibroblast (NHF) cells at the effective dose. Increased reactive oxygen species production and collapse of mitochondrial membrane potential resulted from treatment with pancratistatin in both cancer cell lines. This study presents the novel finding that pancratistatin treatment caused decreased migration capacity and increased autophagy levels in metastatic prostate cancer cells. Importantly, in this proof-of-concept study, pancratistatin reduced the volume of xenograft tumors compared to control-treated animals, and was well-tolerated. Our results highlight the potential of pancratistatin for clinical development as a selective therapeutic for treatment of metastatic prostate cancer.     

A concentrated aglycone isoflavone preparation (GCP) that demonstrates potent anti-prostate cancer activity in vitro and in vivo.

PURPOSE: Isoflavones have anticancer activities, but naturally occurring isoflavones are predominantly glycosylated and poorly absorbed. Genistein combined polysaccharide (GCP; Amino Up Chemical Co., Sapporo, Japan), is a fermentation product of soy extract and basidiomycetes mycillae that is enriched in biologically active aglycone isoflavones. This study analyzes GCP in vitro and in vivo for potential utility as a prostate cancer chemopreventative agent. EXPERIMENTAL DESIGN: Androgen-sensitive LNCaP and androgen-independent PC-3 cells were grown with various concentrations of GCP. In vitro cell growth was analyzed by the WST-1 assay, and apoptosis was assessed by fluorescence-activated cell sorting and detection of poly(ADP-ribose) polymerase cleavage using Western blot techniques. Effects of GCP on expression of cell cycle-regulatory proteins p53 (LNCaP only), p21, and p27 and the protein kinase Akt were considered using Western blot techniques. An in vivo LNCaP xenograft model was used to study the effects of a 2% GCP-supplemented diet on tumor growth in comparison with a control diet. RESULTS: GCP significantly suppressed LNCaP and PC-3 cell growth over 72 h (89% and 78% in LNCaP and PC-3, respectively, at 10 microg/ml; P < 0.0001). This reduction was associated with apoptosis in LNCaP cells, but not in PC-3 cells. GCP induced p27 and p53 (LNCaP only) protein expression within 6 h and suppressed phosphorylated Akt in both cell lines. The 2% GCP-supplemented diet significantly slowed LNCaP tumor growth, increasing apoptosis (P < 0.001), and decreasing proliferation (P < 0.001) over 4 weeks. CONCLUSIONS: GCP has potent growth-inhibitory effects against prostate cancer cell lines in vitro and in vivo. These data suggest GCP has potential as an effective chemopreventive agent against prostate cancer cell growth.     

Guggulsterone-induced apoptosis in human prostate cancer cells is caused by reactive oxygen intermediate dependent activation of c-Jun NH2-terminal kinase

Guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, causes apoptosis in cancer cells but the sequence of events leading to cell death is poorly understood. We now show that guggulsterone-induced cell death in human prostate cancer cells is caused by reactive oxygen intermediate (ROI)-dependent activation of c-Jun NH(2)-terminal kinase (JNK). Exposure of PC-3 and LNCaP cells to apoptosis inducing concentrations of guggulsterone resulted in activation of JNK and p38 mitogen-activated protein kinase (p38 MAPK) in both cell lines and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LNCaP cells. The guggulsterone-induced apoptosis in PC-3/LNCaP cells was partially but statistically significantly attenuated by pharmacologic inhibition (SP600125) as well as genetic suppression of JNK activation. On the other hand, pharmacologic inhibition of p38 MAPK activation in PC-3 or LNCaP cells (SB202190) and ERK1/2 activation in LNCaP cells (PD98059) did not protect against guggulsterone-induced cell death. The guggulsterone treatment caused generation of ROI in prostate cancer cells but not in a normal prostate epithelial cell line (PrEC), which was also resistant to guggulsterone-mediated JNK activation. The guggulsterone-induced JNK activation as well as cell death in prostate cancer cells was significantly attenuated by overexpression of catalase and superoxide dismutase. In addition, guggulsterone treatment resulted in a decrease in protein level and promoter activity of androgen receptor in LNCaP cells. In conclusion, the present study reveals that the guggulsterone-induced cell death in human prostate cancer cells is regulated by ROI-dependent activation of JNK and guggulsterone inhibits promoter activity of androgen receptor.     

Regulation of macroautophagy in ovarian cancer cells in vitro and in vivo by controlling glucose regulatory protein 78 and AMPK

In this study we show that diindolylmethane (DIM) induces autophagy in ovarian cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations of DIM for 24 hours resulted in a concentration dependent induction of autophagy as measured by flowcytometry. Electron microscopy confirmed the presence of autophagosomes in DIM treated cells. Western blot analysis showed that DIM treatment increased the expression of LC3B, a hall mark of autophagy as well as p62 and Atg 12 proteins that are accumulated during autophagy. Autophagy inhibitors bafilomycin or chloroquine inhibited DIM induced autophagy. Furthermore, DIM treatment significantly increased the expression of ER stress regulators such as Grp78, IRE1 and GADD153. Cycloheximide or ER stress inhibitor mithramycin not only blocked ER stress proteins that were activated by DIM but also autophagy. Silencing Grp78 or GADD 153 significantly blocked the expression of LC3B and p62 indicating that autophagy in our model is mediated by ER stress. Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the cytosolic calcium levels which lead to the activation of AMPK in our model. Chelating cytosolic calcium with BAPT-AM abrogated not only the phosphorylation of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical inhibitor or siRNA blocked the induction of LC3B or p62, indicating that DIM mediated autophagy requires activation of AMPK. Oral administration of DIM significantly suppressed SKOV-3 tumor xenografts in nude mice. Activation of ER stress and autophagy were observed in the tumors of DIM treated mice. Taken together, these results suggest that induction of autophagy by DIM in ovarian cancer cells was associated with ER stress and AMPK activation.     

Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathway

Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1-4). In this study, we investigated the role of EP receptors in PGE2-induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM-10 microM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2-induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2'5'-dideoxyadenosine, at concentrations that inhibited PGE2-induced cAMP, significantly blocked PGE2-induced VEGF secretion in PC-3 cells. We conclude that PGE2-induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways.     

eNOS protects prostate cancer cells from TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anti-cancer agent because it induces apoptosis of most tumor cells with little or no effect on normal cells. In this study, we investigated the effect of TRAIL on human prostate normal and cancer cell lines, and found that the prostate cancer cell lines PC-3, ALVA-31, DU 145 and TSU-Pr1 were sensitive to TRAIL-induced apoptosis, while normal PrEC cells and cancer cell line LNCaP were resistant. No correlation was found between the sensitivity of cells to TRAIL and the expression of TRAIL receptors DR4 and DR5, and pro-apoptotic proteins Bax and Bak. However, LNCaP cells displayed a high Akt activity. Furthermore, we found that endothelial nitric oxide synthase (eNOS), one of the Akt substrates, was highly expressed in LNCaP but not in other cells. Inhibition of eNOS activity by NOS inhibitor sensitized LNCaP cells to TRAIL. Moreover, PC-3 cell clones stably expressing eNOS were resistant to TRAIL-induced apoptosis. Taken together, these results indicate that eNOS can regulate the sensitivity of prostate cancer cells to TRAIL, and down-regulation of eNOS activity may sensitize prostate cancer cells to TRAIL-based therapy.     

PC-1/PrLZ contributes to malignant progression in prostate cancer

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.     

NCL1, a highly selective lysine-specific demethylase 1 inhibitor,suppresses prostate cancer without adverse effect

Herein, we investigated therapeutic potential of a novel histone lysine demethylase 1 (LSD1) inhibitor, NCL1, in prostate cancer. Hormone-sensitive prostate cancer cells, (LNCaP) and castration resistant cancer cells (PC3 and PCai1) were treated with NCL1, and LSD1 expression and cell viability were assessed. Prostate cancer cells showed strong LSD1 expression, and cell viability was decreased by NCL1. ChIP analysis showed that NCL1 induced H3K9me2 accumulation at the promoters of androgen-responsive genes. NCL1 also induced G1 cell cycle arrest and apoptosis. In addition, autophagosomes and autolysosomes were induced by NCL1 treatment in LNCaP. Furthermore, LC3-II expression was significantly increased by NCL1 and chloroquine. In mice injected subcutaneously with PCai1 and intraperitoneally with NCL1, tumor volume was reduced with no adverse effects in NCL1-treated mice. Finally, LSD1 expression in human cancer specimens was significantly higher than that in normal prostate glands. In conclusion, NCL1 effectively suppressed prostate cancer growth without adverse events. We suggest that NCL1 is a potential therapeutic agent for hormone-resistant prostate cancer.