Androgen-dependent LNCaP Prostate Cancer Research Articles

Aspirin Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Hormone-Refractory Prostate Cancer Cells through Survivin Down-Regulation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether acetylsalicylic acid (ASA), so-called aspirin, enhances TRAIL-induced apoptosis in androgen-dependent LNCaP and androgen-independent LNCaP-derived prostate cancer cells. To evaluate the cell death effects of TRAIL in combination with ASA on tumor cells, we performed DNA fragmentation assay and immunoblot analysis for poly(ADP-ribose) polymerase-1, caspases, and anti-apoptotic proteins. We observed that ASA promoted TRAIL-induced apoptotic death in both LNCaP and its derived cells (C4, C4-2, and C4-2B). These enhancements of TRAIL's effect were related to the decrease in survivin protein expression by pretreatment with ASA. We also confirmed that knockdown in survivin expression by transfecting survivin small interfering RNA increased TRAIL-induced apoptosis. To study the mechanism of survivin down-regulation, we determined the levels of mRNA and the activities of survivin promoter in the ASA-treated and untreated cells. Reduction of the intracellular levels of survivin protein was due to a decrease in transcriptional activity. Data from electrophoretic mobility shift assay and chromatin immunoprecipitation analyses revealed that ASA inhibited the transcription factor E2F-1 binding activity to the survivin promoter region, which is known to regulate survivin gene transcription. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated by down-regulating survivin, and the down-regulation of survivin is due to inhibition of E2F-1 binding activity to the survivin promoter region.

Saw Palmetto induces growth arrest and apoptosis of androgen-dependent prostate cancer LNCaP cells via inactivation of STAT 3 and androgen receptor signaling

PC-SPES is an eight-herb mixture that has an activity against prostate cancer. Recently, we purified Saw Palmetto (Serenoa repens) from PC-SPES and found that Saw Palmetto induced growth arrest of prostate cancer LNCaP, DU145, and PC3 cells with ED50s of approximately 2.0, 2.6, and 3.3 microl/ml, respectively, as measured by mitochondrial-dependent conversion of the the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Saw Palmetto induced apoptosis of LNCaP cells in a time- and dose-dependent manner as measured by TUNEL assays. Also, Saw Palmetto increased the expression of p21waf1 and p53 protein in LNCaP cells. In addition, we found that Saw Palmetto down-regulated DHT- or IL-6-induced expression of prostate specific antigen in conjunction with down-regulation of the level of androgen receptor in the nucleus as measured by Western blot analysis. Moreover, Saw Palmetto down-regulated the IL-6-induced level of the phosphorylated form of STAT 3 in LNCaP cells. Furthermore, Saw Palmetto inhibited the growth of LNCaP cells present as tumor xenografts in BALB/c nude mice without adverse effect. These results indicate that Saw Palmetto might be useful for the treatment of individuals with prostate cancer.

Rap2 regulates androgen sensitivity in human prostate cancer cells

Progression of prostate cancer to a fatal androgen-independent disease is associated with activation of MAP kinase, consistent with chronic stimulation of the Ras-signaling pathway. We have previously shown that Ras activation is sufficient to induce androgen-independent growth of prostate cancer cells. One mechanism of MAP kinase regulation is modulation of Ras signaling by other Ras family members, the Rap gene paralogs Rap1a/b and Rap2a/b. Here we ask if Rap proteins play a role in determining androgen sensitivity of human prostate cancer cells either alone or in the context of an activated Ras. To evaluate the role of Rap proteins in androgen responsiveness we use Rap over-expression with or without mutated Ras co-transfection and Rap siRNA knockdown to evaluate androgen-dependent prostate-specific antigen (PSA) promoter reporter expression and cell growth in androgen-dependent LNCaP and independent C4-2 human prostate cancer cells. Rap1 is equally expressed between LNCaP and C4-2 cells and thus we focused on Rap2 which is minimally expressed in C4-2. Rap2a affects androgen-dependent PSA reporter expression in a dose-dependent manner in LNCaP and C4-2 cells. Low levels of Rap2a enhance PSA reporter expression, whereas higher concentrations inhibit expression. We show that Rap2a antagonizes the enhanced PSA reporter expression conferred by an active RasV12 gene in prostate cancer cells. siRNA knockdown data indicate that Rap2 has a greater effect on androgen-stimulated growth in LNCaP than in C4-2 cells. We show that Rap2 is involved in androgen-mediated transcriptional and growth responses of human prostate cancer cells.

A 90 kDa fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

Highly tumorigenic human androgen receptor‐positive prostate cancer cells overexpress angiogenin

We have recently established a highly tumorigenic cell line, LNCaP-CR, derived from human androgen-dependent prostate cancer LNCaP cells. In the present study, we examined the genes responsible for the high tumorigenicity of LNCaP-CR cells. The cDNA microarray analysis and protein array of secreted factors indicated angiogenin (ANG), an angiogenic factor, as a candidate gene. Reverse transcription-polymerase chain reaction and immunoassay confirmed that LNCaP-CR cells expressed high levels of ANG but not vascular endothelial growth factor (VEGF), compared with the parental LNCaP cells. We also proved that another tumorigenic androgen receptor-positive prostate cancer cell line, 22Rv1, secretes higher levels of ANG than VEGF. To assess the contribution of ANG to the highly tumorigenic phenotype, we transfected the ANG gene into LNCaP cells in order to overexpress ANG, and also transfected ANG small interfering RNA-expressing constructs into LNCaP-CR cells to downregulate ANG. Overexpression of ANG in LNCaP cells did not affect their growth in vitro, but it significantly enhanced tumorigenicity and angiogenesis in vivo. In contrast, knockdown of ANG expression in LNCaP-CR cells also did not affect the growth in vitro, but it led to a significant decrease in tumorigenicity and angiogenesis. Taken together, ANG is one of the genes responsible for the high tumorigenicity of LNCaP-CR cells. Thus, our results support the idea that ANG is an attractive target for cancer therapy and show that LNCaP-CR cells are useful for studying ANG action and experimental therapeutic approaches targeting ANG.

Differential efficacy of 3-hydroxy-3-methylglutaryl CoA reductase inhibitors on the cell cycle of prostate cancer cells

Members of the statin family of 3-hydroxy-3-methylglutaryl CoA reductase inhibitors are being investigated for the therapy and prevention of cancers because of their growth-inhibitory effects on epithelial cells. Some epidemiologic studies show that patients taking statins show a lower incidence of cancer compared with those taking other cholesterol-lowering medication. In contrast, other studies show that statin use does not correlate with cancer risk. To address this discrepancy, we investigated the efficacy of different statins on the PC-3 prostate cancer cell line and the androgen-dependent LNCaP prostate cancer cell line. Clinically used statins, lovastatin, fluvastatin, and simvastatin inhibit proliferation of the two prostate cancer cells by inducing a G1 arrest. Lovastatin induced the arrest at 0.5 micromol/L, a concentration easily reached in the serum after oral administration. Pravastatin, however, was less effective at inhibiting 3-hydroxy-3-methylglutaryl CoA reductase in PC-3 cells and had to be present at 200 times higher concentrations to effect a cell cycle arrest. Another potential source of variability is the different levels of the cyclin-dependent kinase (cdk) inhibitor p27 noted in prostate cancers particularly because statins have been suggested to act through the induction of cdk inhibitors. All three statins (lovastatin, fluvastatin, and simvastatin) inhibited cyclin E/cdk2 kinase leading to hypophosphorylation of Rb, but this inhibition was correlated with a loss of the activating phosphorylation on Thr160 of cyclin E-associated cdk2 and not dependent on the cdk inhibitors p21 and p27. Therefore, p27 status is unlikely to confound the epidemiologic data on the efficacy of statins in prostate cancer. To make definitive conclusions about the efficacy of statins on cancer prevention, however, the epidemiologic studies should take into account the type of statin used and the serum concentrations achieved and ensure that the tested statin inhibits the specific type of cancer in vitro at those concentrations.

Zanthoxyli Fructus induces growth arrest and apoptosis of LNCaP human prostate cancer cells in vitro and in vivo in association with blockade of the AKT and AR signal pathways

Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR-target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types.

Hypoxia regulation of expression and angiogenic effects of vasoactive intestinal peptide (VIP) and VIP receptors in LNCaP prostate cancer cells

Vascular endothelial growth factor (VEGF) is a main factor promoting neovascularization (angiogenesis) of solid tumours as prostate carcinoma. Hypoxia stimulates VEGF gene expression by activating the hypoxia-inducible factor-1 (HIF-1α). In the present study, the hypoxia-mimicking agent Ni2+ induced vasoactive intestinal peptide (VIP) expression at both mRNA and peptide levels but it did not modify the expression of VIP receptors (VPAC1, VPAC2 and PAC1 receptors) in androgen-dependent human LNCaP prostate cancer cells. VIP increased the mRNA levels of VPAC1 and PAC1 receptors whereas it decreased VPAC2 receptor mRNA level. These features support that hypoxia up-regulation of VIP gene expression in prostatic carcinoma may lead to VIP regulation of the expression of its receptors by means of autocrine/paracrine mechanisms. Either VIP or hypoxia mimetics with Ni2+ increased VEGF expression whereas both conditions together resulted in an additive response. It suggests two independent mechanisms for the observed pro-angiogenic activities of VIP and hypoxia. VIP did not stimulate HIF-1α mRNA expression but increased the translocation of HIF-1α from the cytosolic compartment to the cell nucleus. Moreover, VIP was unable to modify the expression of the HIF-1α inhibitor FIH-1 discarding the possibility of an indirect effect of VIP on HIF-1 transactivation.

Differential regulation of IGFBP-3 by the androgen receptor in the lineage-related androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer models

Despite evidence implicating insulin-like growth factor binding protein-3 (IGFBP-3) as a growth inhibitor of prostate cancer (CaP), little is known about changes in its regulation and function during progression to androgen independence. The expression levels of IGFBP-3 were determined by cDNA microarray analysis and tissue microarrays (TMAs) after androgen ablations. LNCaP (LN-BP3) and C4-2 (C4-2-BP3) sublines were used to compare the apoptotic effects of IGFBP-3 in LNCaP (androgen-dependent) and C4-2 (androgen-independent) cells. After androgen deprivation, IGFBP-3 mRNA levels increased more in C4-2 compared to LNCaP cells. Androgens suppressed IGFBP-3 levels in a dose-dependent manner in LNCaP and C4-2 cell. IGFBP-3 expression was increased after NHT in human CaP tissues. Apoptotic rates increased in LN-BP3, but not C4-2-BP3 cells, following doxycycline-mediated IGFBP-3 induction. C4-2 cell survival in an androgen-depleted environment may be facilitated through differential resistance to the apoptotic effects elicited by IGFBP-3.

Differential effects of whole soy extract and soy isoflavones on apoptosis in prostate cancer cells

Previous studies have suggested that soy isoflavones, especially genistein and diadzein, have chemoprotective effects in prostate cancer, however, the precise mechanism are unclear. Soy contains a mixture of soy isoflavones and other bioactive components, thus a whole food approach may be more effective than using individual soy isoflavones for chemoprevention. This study sought to compare the effects of whole soy extracts with genistein and diadzein on prostate cancer cell proliferation, apoptosis and changes in NFκB activation. Androgen-dependent LnCap and androgen-independent PC3 prostate cancer cells were treated with soy extract, genistein or diadzein for 48h and analyzed for cell cycle alterations, Annexin V staining and multicaspase activity. The soy extract induced an increase in the subG1 peaks, Annexin V and multicaspase staining at concentrations as low as 25μM in PC3 and LnCap cells, whereas genistein and diadzein did not induce apoptosis until concentrations >100μM. Furthermore, addition of 25μM soy extract induced a higher percentage of apoptotic cells than equal concentrations of genistein or diadzein (p<0.001). Preliminary data did not show suppression of NFκB activation with treatments. Soy extracts also had no effect on apoptosis in the benign prostate cells, suggesting that the effects of soy are cancer-cell specific. These results suggest that a whole food approach using foods that bear a combination of active compounds may be more efficacious for chemoprevention compared to individual compounds. In vivo studies exploring the effect of soy and individual soy isoflavones on hormone-induced prostate cancer Noble rat model is underway. This work was supported by CA107693.

Alteration of gene expression in response to bone morphogenetic protein-2 in androgen-dependent human prostate cancer LNCaP cells

Bone morphogenetic protein (BMP)-2, a multifunctional member of the transforming growth factor (TGF)-beta superfamily with powerful osteoinductive effects, has various biological activities in a variety of cells. We observed that BMP-2 inhibits cell proliferation in the androgen-dependent human prostate cancer cell line, LNCaP. To investigate the mechanism of inhibition of androgen-dependent growth by BMP-2, we compared the gene expression in LNCaP cells treated with dihydrotestosterone (DHT) to that of LNCaP cells treated with DHT and BMP-2, using DNA microarray analysis. Of 8,400 human genes on the gene chip, 38 genes were up-regulated by >2.0-fold and 48 genes were down-regulated by <0.5-fold by treatment with BMP-2. These genes were involved in a variety of cellular functions, including signal transduction, transcription regulation, enzymes, transporters, structural molecules and translation. RT-PCR analysis showed that CH1CL and BMX were up-regulated and DACH1 and WNT5A were down-regulated by treatment with BMP-2. Furthermore, we detected an increase of WNT5A protein in the medium by DHT and inhibition of the increase by BMP-2. In the present study, we identified several BMP-2-responsive genes in LNCaP cells. Further studies of the roles of these genes may clarify the mechanisms underlying the inhibition of cell proliferation by BMP-2 and identify better approaches for the prevention and treatment of prostate cancer.

Establishment of a highly tumorigenic LNCaP cell line having inflammatory cytokine resistance

Human androgen-dependent prostate cancer LNCaP cells are low tumorigenic even in immunodeficient mice and were killed by the synergistic effect of inflammatory cytokines, IL-β and IL-6. To establish a highly tumorigenic LNCaP cell line, we isolated the cytokine-resistant LNCaP-CR cell line and examined the phenotypes. The parental LNCaP cells were induced to commit apoptosis by the addition of IL-1β and IL-6, but LNCaP-CR cells showed strong resistance against the cytokine action. However, LNCaP-CR cells did not exhibit any resistance to various antitumor drugs investigated. While LNCaP cells formed only palpable tumors in SCID mice, LNCaP-CR cells readily made tumors and their growth was significantly higher than that of LNCaP cells. Moreover, LNCaP tumor-bearing mice gained the weight gradually, but LNCaP-CR tumor-bearing mice significantly lost their body weight. LNCaP-CR cells still responded to androgen action and expressed AR, erbB2, IL-1R, IL-6R, gp130, STAT3, p21, Bcl-2 and caspase-3 as well as LNCaP cells. These results indicate that LNCaP-CR cell line is a new type of tumorigenic LNCaP cell lines and should be useful for identifying responsible genes of tumorigenicity, cytokine resistance, and also cachexia.

Novel Role of Cold/Menthol-sensitive Transient Receptor Potential Melastatine Family Member 8 (TRPM8) in the Activation of Store-operated Channels in LNCaP Human Prostate Cancer Epithelial Cells

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I(cold/menthol)) that shows inward rectification and high Ca(2+) selectivity, which are dramatically different properties from "classical" TRPM8-mediated I(cold/menthol). Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I(cold/menthol) and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca(2+) release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca(2+) release channel, potentially involved in a number of Ca(2+)- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

Role of SRC-1 in the Promotion of Prostate Cancer Cell Growth and Tumor Progression

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor-negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.

Vitamin D receptor agonists induce prostatic acid phosphatase to reduce cell growth and HER-2 signaling in LNCaP-derived human prostate cancer cells

We have previously shown that concentrations of 1α,25-dihydroxyvitamin D 3 (1,25D) that induce G 0/G 1 cell cycle arrest in androgen-dependent LNCaP prostate cancer cells also decrease expression of c-Myc, a proto-oncogene that stimulates progression from G 1 to S phase of the cell cycle. Since both c-Myc expression and cell cycle progression are regulated by tyrosine kinase activation, we examined the ability of 1,25D to alter tyrosine kinase signaling in LNCaP cells and the androgen-independent LNCaP C81 (C81 LN) cell line. 1,25D selectively reduced protein tyrosine phosphorylation within both the LNCaP and C81 LN cells. This reduction in tyrosine kinase signaling appears to result from elevated levels of cellular prostatic acid phosphatase (PAcP). Western blots and biochemical assays revealed 1,25D increases the level of active PAcP in both cell lines. In addition, 1,25D decreased tyrosine phosphorylation of HER-2, an EGFR family member inactivated by PAcP, and the HER-2 downstream adaptor protein p52 Shc in C81 LN cells. Inhibition of HER-2 signaling by AG825 reduces growth of C81 LN cells and the parental LNCaP cells. These data therefore suggest that 1,25D-mediated decreases in LNCaP and C81 LN cell growth are in part due to decreases in tyrosine kinase signaling that result from up-regulation of PAcP.

The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells

TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1-4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.

Methylseleninic acid sensitizes prostate cancer cells to TRAIL-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Unfortunately, some tumor cells remain resistant to TRAIL. Therefore, agents that sensitize malignant cells to TRAIL-mediated cell death might be of particular importance for the development of novel antitumor therapeutic regimens. Recent studies establish a critical role of selenium in prostate cancer prevention in vitro and in vivo. Here, we demonstrate that concomitant administration of TRAIL and methylseleninic acid (MSA) produces synergistic effects on the induction of apoptosis in androgen-dependent LNCaP and androgen-independent DU-145 prostate cancer cells. MSA rapidly and specifically downregulates expression of the cellular FLICE inhibitory protein, a negative regulator of death receptor signaling. In addition, we demonstrate that the synergistic effects of MSA and TRAIL result from the activation of the mitochondrial pathway-mediated amplification loop. Addition of MSA effectively blocked TRAIL-mediated BAD phosphorylation at Ser112 and Ser136 in DU-145 cells and was accompanied by induction of the mitochondrial permeability transition and release of apoptogenic cytochrome c and Smac/DIABLO proteins from the mitochondria and into the cytosol. These results suggest that selenium-based dietary compounds may help to overcome resistance to TRAIL-mediated apoptosis in prostate cancer cells.

Equiguard™ suppresses androgen-dependent LNCaP prostate cancer cell proliferation by targeting cell cycle control via down regulation of the retinoblastoma protein Rb and induction of apoptosis via the release of cytochrome c

Lack of life-prolonging therapies has provided much of the impetus for seeking complementary and alternative management/treatment options by prostate cancer (CaP) patients. Among these, the use of dietary supplements and botanical products has been showing a sustained increase in recent years, owing in part to some encouraging pre-clinical and clinical data shown in a limited number of herbal products. Notably, however, the majority of herbal and dietary supplement products have not been rigorously studied with regard to their efficacy. In vitro mechanistic experiments are considered essential preludes and requisites to more lengthy and costly animal and human studies, in that they may provide relevant insights and scientific basis for effects some of these products purportedly might demonstrate. In vitro studies in our laboratory have shown that a polyherbal supplement, Equiguard, exhibits anti-tumor activity against hormone dependent LNCaP cells cultured in both androgen-proficient (FBS) and -deficient (CS-FBS) conditions. Clinically relevant anti-prostate cancer effects of Equiguard are vividly illustrated by growth suppression and down regulated expression of prostate specific genes, respectively, androgen receptor (AR) and prostate specific antigen (PSA). However, the mechanistic bases contributing to these effects have not been well characterized. This communication describes experiments aimed at further understanding growth arrest elicited by Equiguard in LNCaP cells cultured in FBS and CS-FBS conditions. We have focused on aspects of cell cycle control and induction of apoptosis. Regulation of cell cycle progression by Equiguard was analyzed by examining changes in the expression of Rb and cyclins D/E. Using Western blot analysis, we showed that treatment caused inhibition of Rb phosphorylation, which was accompanied by the reduction of cyclins D/E expression, in both culture conditions. Moreover, cells treated with Equiguard and cultured with FBS-supplemented media showed up-regulation of cyclin-dependent kinase inhibitor Kip1/p27. These results support the interpretation that suppression of Rb phosphorylation mediated the observed growth arrest induced by Equiguard under androgen-proficient condition. In contrast, Equiguard-treated cells cultured in CS-FBS had lowered expression of the Kip1/p27, suggesting that different control mechanisms, possibly evoked by changes in cellular microenvironments, contributed to growth suppression by Equiguard. The growth suppressive effects of Equiguard in both culture conditions were also evaluated with respect to induction of apoptosis. While Equiguard elicited apoptosis was accompanied by an increase in the level of cytosolic cytochrome c, the relative accumulation of cytochrome c in the cytosol was unaffected by culture conditions. These results suggest that the ability to trigger apoptosis as one aspect of the control of cell growth by Equiguard is integrally linked to the release of cytochrome c, by a mechanism largely independent of the presence of androgens.

Protection of androgen-dependent human prostate cancer cells from oxidative stress-induced DNA damage by overexpression of clusterin and its modulation by androgen.

Recent studies reported that oxidative stress is one of the major factors associated with the progression of prostate cancer through the accumulation of DNA damage. In the present study, we investigated the effect of oxidative stress on cell injury using androgen-dependent human prostate cancer LNCaP cells overexpressing clusterin, which has been shown to play crucial roles in the acquisition of resistance to several apoptotic stimuli. We introduced clusterin cDNA into LNCaP cells which do not express a detectable level of clusterin expression, and generated a clusterin-overexpressing cell line (LNCaP/Cl) and a control vector only-transfected cell line (LNCaP/Co). The effects of hydrogen peroxide (H2O2) treatment on the LNCaP sublines with and without the addition of dihydrotestosterone (DHT) were analyzed using the in vitro mitogenic assay and lipid peroxidation assay, and morphological changes in the LNCaP sublines after H2O2 treatment were examined by staining with Hoechst 33258. The degrees of DNA damage induced by H2O2 into the LNCaP sublines were evaluated by the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. H2O2-induced apoptosis in LNCaP/Cl was significantly suppressed compared with that in LNCaP/Co through the inhibition of membrane damage; however, the measurement of 8-OHdG level demonstrated that DNA damage was more intensively accumulated in LNCaP/Cl cells than LNCaP/Co cells. Furthermore, DHT suppressed the incidence of apoptotic cell death and enhanced the formation of 8-OHdG in both LNCaP/Cl and LNCaP/Co cells after H2O2 treatment in a dose-dependent manner. These findings suggest that clusterin may contribute to conferring resistance to oxidative stress-mediated cellular injury on prostate cancer cells, especially in the presence of androgen.

Proteasome inhibitor PS-341 down-regulates prostate-specific antigen (PSA) and induces growth arrest and apoptosis of androgen-dependent human prostate cancer LNCaP cells.

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of nuclear factor kappaB (NF-kappaB) in vitro. In addition, recent clinical studies of PS-341 have demonstrated some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of androgen-dependent human prostate cancer LNCaP cells in conjunction with markedly up-regulated levels of p21(waf1) and p53. In addition, we found that PS-341 down-regulated both 5alpha-dihydrotestosterone (DHT)- and interleukin-6 (IL-6)-induced expression of prostate-specific antigen (PSA) as measured by western blot analysis. PS-341 down-regulated basal levels of the androgen receptor (AR) in the nucleus; however, it did not affect DHT-induced nuclear translocation of AR in these cells. Reporter assays using a series of promoters of the PSA gene showed that down-regulation of PSA by PS-341 was caused by inhibition of the transcriptional activity of the androgen receptor response element (ARE) in these cells. Taken together, the results indicate that PS-341 induced growth arrest and apoptosis of LNCaP cells by blockade of the AR signaling pathway. The proteasome may be a molecular target for treatment of a variety of cancers including prostate cancer.