Androgen-independent Prostate Cancer PC-3 Cells Research Articles

The antiandrogen bicalutamide activates the androgen receptor (AR) with a mutation in codon 741 through the mitogen activated protein kinase (MARK) pathway in human prostate cancer PC3 cells

The objective of this study was to assess the effect of antiandrogen on the activation of mutated androgen receptor (AR) and its signaling pathway in prostate cancer. We transfected the AR gene with a point mutation at codon 741 (tryptophan to leucine; W741L) into human androgen-independent prostate cancer PC3 cells lacking the expression of AR, and established PC3 cells overexpressing mutant type AR (PC3/W741L). Changes in the phenotype in these cells were compared to those in PC3 cells transfected with wild- type AR (PC3/Wild) and control vector alone (PC3/Co). There was no significant differences in the growth among PC3/Co, PC3/Wild and PC3/W741L cells. A transactivation assay using these cells showed that bicalutamide activated W741L mutant type AR, but not wild-type AR, while hydroxyflutamide failed to activate either type of ARs. Treatment with specific inhibitors of the MAPK or STST3 pathway (UO126 or AG490, respectively), in contrast to treatment with the Akt pathway inhibitor LY294002, significantly inhibited the dihydrotestosterone-induced activation of both wild-type and mutant ARs; however, activation of W741L mutant AR by bicalutamide was significantly inhibited by treatment with UO126, in contrast to treatment with AG490 or LY294002. Furthermore, treatment of PC3/W741L with bicalutamide, in contrast to treatment with hydroxyflutamide, resulted in significant upregulation of phosphorylated p44/42 MAPK. These findings suggest that the MAPK pathway might be involved in the activation of the AR with a point mutation at codon 741 induced by treatment with the antiandrogen bicalutamide.

NFκB-dependent regulation of urokinase plasminogen activator by proanthocyanidin-rich grape seed extract: effect on invasion by prostate cancer cells

Tumor invasion and metastasis present major obstacles to successful control of androgen-independent prostate cancer. Cell migration is a fundamental aspect of cancer cell metastasis. Urokinase plasminogen activator (uPA) system is implicated in cell migration and cancer metastasis and has potential to be developed as therapeutic target. In recent years, efficacy of dietary nutrients in preventing and curing cancer has gained increasing attention. One such promising candidate is proanthocyanidin-rich grape seed extract (GSE). We investigated the efficacy of GSE in regulating uPA expression and cell migration using highly metastatic androgen-independent PC3 prostate cancer cells as a model. GSE down-regulated uPA as a function of concentration. Additional studies showed that GSE inhibited DNA-binding activity of the transcription factor nuclear factor kappa B (NFkappaB), which in turn decreased NFkappaB-dependent uPA transcription. Invasion assays revealed the inhibitory effect of GSE on PC3 cell migration. These in-vitro experiments demonstrate the therapeutic property of GSE as an antimetastatic agent by targeting uPA.

Involvement of microRNA-21 in mediating chemo-resistance to docetaxel in androgen-independent prostate cancer PC3 cells

To investigate whether microRNA-21 was involved in mediating the chemoresistance of prostate cancer cells to docetaxel. A microarray technique was used to determine the miRNA profile in docetaxel-resistant PC3 cells. Real-time PCR was used to confirm the array results. miR-21 mimics and inhibitors were synthesized and introduced to cells using Lipofectamine 2000. Cell proliferation was examined with the CCK-8 assay. Luciferase reporter containing PDCD 3'UTR was constructed and the activity was detected by a dual luciferase assay. PDCD4 protein expression was evaluated using Western blot. A docetaxel-resistant prostate cancer PC3 cell line (PC3R) was established . Using microarrays, miR-21 was found to be up-regulated in PC3R cells. Ectopic expression of miR-21 increased the resistance to docetaxel in PC3 wild type cells. In contrast, silencing of miR-21 in PC3R cells sensitized the cells to docetaxel. The IC(50) values for miR-21-silencing cells and control cells were 28.31 and 35.89 nmol/L, respectively. PDCD4, a direct target gene of miR-21, could mediate chemoresistance to docetaxel in PC3 cells. Our findings suggest that miR-21 contributed to the resistance of PC3 cells to docetaxel, and that targeting miR-21 may offer a promising therapeutic approach in sensitizing prostate cancer to docetaxel treatment.

Effects of the ErbB1/ErbB2 kinase inhibitor GW2974 on androgen-independent prostate cancer PC-3 cell line growth and NSE, chromogranin A and osteopontin content

Prostate cancer is one of the most frequently diagnosed cancer in men. Treatment by radical prostatectomy, radiotherapy and anti-androgen drugs is successful in patients with localized cancer. However, prolonged androgen deprivation often leads to hormone refractory condition, associated with disease relapse. ErbB1 and ErbB2 activity has been correlated with androgen-independence. We determined the effects of GW2974, a dual inhibitor of ErbB-1 and ErbB-2 tyrosine kinase activity, on growth, NSE, chromogranin A and osteopontin cytosol content in the androgen-independent prostate cancer cell line PC-3. We found that PC-3 cell growth was inhibited by GW2974, whereas NSE and chromogranin A cell contents were stimulated and osteopontin cytosol level was not affected. The present data may have clinical implications for the treatment of advanced prostate cancer.

The multikinase inhibitor sorafenib induces caspase-dependent apoptosis in PC-3 prostate cancer cells

The present study investigated the effects of the multikinase inhibitor sorafenib on androgen-independent cancer cells viability and intracellular signaling. Human androgen-independent PC-3 prostate cancer cells were treated with sorafenib. At concentration that suppresses extracellular signal-regulated kinase phosphorylation, sorafenib treatment reduced the mitochondrial transmembrane potential. Sorafenib also down-modulated the levels of myeloid cell leukemia 1, survivin and cellular inhibitor of apoptosis protein 2. Sorafenib induced caspase-3 cleavage and the mitochondrial release of cytochrome c. However, no nuclear translocation of apoptosis inducing factor was detected after treatment and the pan-caspase inhibitor Z-VAD-FMK had an obvious protective effect against the drug. In conclusion, sorafenib induces apoptosis through a caspase-dependent mechanism with down-regulated anti-apoptotic proteins in androgen-independent prostate cancer cells in vitro.

225 CHEMOSENSITIZATION AND INHIBITION OF TUMOR GROWTH BY SHORT HAIRPIN RNA TARGETING INTERLEUKIN-6 IN ANDROGEN-INDEPENDENT HUMAN PROSTATE CANCER PC3 CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010225 CHEMOSENSITIZATION AND INHIBITION OF TUMOR GROWTH BY SHORT HAIRPIN RNA TARGETING INTERLEUKIN-6 IN ANDROGEN-INDEPENDENT HUMAN PROSTATE CANCER PC3 CELLS Iori Sakai, Hideaki Miyake, Tomoaki Terakawa, and Masato Fujisawa Iori SakaiIori Sakai More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , Tomoaki TerakawaTomoaki Terakawa More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.283AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Interleukin-6 (IL-6), a pleiotropic cytokine involved in the regulation of multiple pathophysiological functions, has been shown to be implicated in the development and progression of several types of malignant tumors, including prostate cancer. The objective of this study was to analyze the inhibitory effects of IL-6 production in androgen-independent prostate cancer PC3 cells on their growth and chemosensitivity. METHODS We established PC3 cells in which the expression vector containing short hairpin RNA (shRNA) targeting IL-6 was introduced (PC3/sh-IL-6). The growth and the sensitivity to docetaxel in PC3/si-IL-6 cells were compared with those in PC3 cells transfected with control vector alone (PC3/Co). RESULTS Measurement of IL-6 concentration in culture supernatant from each cell line showed that expression level of IL-6 protein in PC3/sh-IL-6 cells was approximately 20% of that in PC3/Co cells. Both in vitro and in vivo, the growth of PC3/sh-IL-6 cells was significantly inferior to that of PC3/Co cells. Expression levels of Bcl-2, Bcl-xL, phosphorylated p44/42 mitogen activated protein kinase (MAPK) and Akt in PC3/sh-IL-6 cells appeared to be markedly downregulated compared with those in LNCaP/Co cells. Treatment of both PC3/sh-IL-6 and PC3/Co cells with docetaxel induced the increase in IL-6 production through the upregulation of nuclear factor-κB; however, IL-6 production in PC3/Co cells following docetaxel treatment was approximately twice as much as that in PC3/sh-IL-6 cells. The IC50 of docetaxel in PC3/sh-IL-6 cells decreased by 67% compared with that in PC3/Co cells. Furthermore, pretreatment with the proteasome inhibitor bortezomib, which completely inhibited the docetaxel-induced production of IL-6 in both PC3/sh-IL-6 and PC3/Co cells, resulted in the marked sensitization of both cell lines to docetaxel. CONCLUSIONS IL-6 production by human prostate cancer PC3 cells may play crucial roles in their growth through the modulation of molecules involved in apoptotic cell death and signal transduction pathways. Moreover, IL-6 production by PC3 cells is suggested to strictly regulate the sensitivity of PC3 cells to docetaxel; therefore, the remarkable sensitization of PC3 cells to docetaxel could be achived in PC3 cells through the complete blockade of IL-6 production by the additional treatment with bortezomib. Collectively, the suppression of IL-6 production using shRNA could be a useful therapeutic strategy against androgen-independent prostate cancer. Kobe, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e88 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Iori Sakai More articles by this author Hideaki Miyake More articles by this author Tomoaki Terakawa More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

383 CHARACTERIZATION OF β4-INTEGRIN-MEDIATED CYTOTOXIC EFFECT OF AEXU, A TYPE THREE SECRETION SYSTEM EFFECTOR, ON HUMAN PROSTATE CANCER PC3 CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2010383 CHARACTERIZATION OF β4-INTEGRIN-MEDIATED CYTOTOXIC EFFECT OF AEXU, A TYPE THREE SECRETION SYSTEM EFFECTOR, ON HUMAN PROSTATE CANCER PC3 CELLS Masafumi Kumano, Hideaki Miyake, Takeshi Honda, and Masato Fujisawa Masafumi KumanoMasafumi Kumano Kobe, Japan More articles by this author , Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , Takeshi HondaTakeshi Honda Osaka, Japan More articles by this author , and Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.451AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Type three secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the host cells. AexU, one of TTSS effectors, has been shown to play an important role during A. hydrophila SSU strain pathogenesis through the ribosylation of ADP. Recently, several investigators have suggested the cytotoxic activities of AexU against a wide variety of tumor cells through the modulation of β4-integrin function. The objectives of this study were to evaluate the cytotoxic effect of AexU on human androgen-independent prostate cancer PC3 cells, and to investigate the mechanism of cytotoxicity induced by AexU focusing on the significance of β4-integrin expression in PC3 cells. METHODS We established pGEX vector encoding GST-AexU-HA fusion protein, and purified AexU protein by affinity chromatography. The cytotoxic effects of AexU either alone or in combination with chemotherapeutic agents on PC3 cells were evaluated. We then established PC3 cells in which the expression vector containing short hairpin RNA (shRNA) targeting β4-integrin was introduced (PC3/sh-In). The sensitivity to AexU in PC3/sh-In cells was compared with that in PC3 cells transfected with control vector alone (PC3/Co). RESULTS Expression level of β4-integrin in PC3 cells was significantly greater than those in other human prostate cancer LNCaP and DU145 cells. The cytotoxic effect of AexU on PC3 cells was more significant than those in the remaining two cell lines. Treatment of PC3 cells with AexU resulted in the upregulation of Bax as well as the downregulation of phosphorylated Akt. The IC50 of docetaxel and cisplatin in PC3 cells were decreased by approximately 90% and 80%, respectively, by the combined treatment with sublethal dose of AexU. The cytotoxic effect of AexU on PC3/Co cells was significantly greater than that on PC3/sh-In cells, accompanied by the upregulation of phosphorylated Akt in PC3/sh-In cells compared with that in PC3/Co cells. CONCLUSIONS These findings suggest that treatment with AexU could be a useful therapeutic option against androgen-independent prostate cancer, particularly that overexpressing β4-integrin, through the growth inhibition and the enhanced chemosensitivity. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e151-e152 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masafumi Kumano Kobe, Japan More articles by this author Hideaki Miyake Kobe, Japan More articles by this author Takeshi Honda Osaka, Japan More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

N-cadherin increases after androgen deprivation and is associated with metastasis in prostate cancer

Androgen-deprivation therapy (ADT) is the standard treatment for metastatic prostate cancer. One factor that has been implicated in the metastatic process is the cell adhesion molecule N-cadherin. In this study, we investigated if the expression of N-cadherin was influenced by androgen deprivation and was associated with metastasis in prostate cancer. The effect of androgen deprivation on N-cadherin expression was initially studied in androgen-dependent (AD) LNCaP and androgen-independent (AI) LNCaP-19 and PC-3 prostate cancer cell lines. Expression of N-cadherin increased in the absence of androgens in AI LNCaP-19 primary tumors and metastases and also in vitro, but not in AI PC-3 tumors, indicating a possible involvement of the androgen receptor in the regulation of N-cadherin. N-cadherin was absent in AD LNCaP tumors. No clear associations between N-cadherin and factors related with epithelial-mesenchymal transition or neuroendocrine differentiation could be established. In addition, N-cadherin was evaluated by immunohistochemistry in human prostate tumors. Expression of N-cadherin was more frequently found in tumors from patients treated with ADT than in tumors from patients with no prior hormonal treatment. N-cadherin expression was also associated with metastasis and Gleason score. Furthermore, increased N-cadherin was detected in prostate cancer biopsies already 3 months after initiation of ADT when tumors were in a regressed state. In summary the results indicate that androgen deprivation induces N-cadherin in prostate tumors. Moreover, N-cadherin was increased in castration-resistant tumors in patients with established metastases. This might indicate that castration induces molecular alterations in the tumor cells, resulting in a more invasive and metastatic phenotype.

Chloride Ion Modulates Cell Proliferation of Human Androgen-independent Prostatic Cancer Cell

In the present study, we investigated if the intracellular Cl^- affects cell growth and cell cycle progression of androgen-independent prostate cancer PC3 cells. PC3 cells cultured in a medium containing 113 mM Cl^- for 96 h grew up 9-fold in cell number, while PC3 cells cultured in an 8 mM-Cl^--containing culture medium showed complete arrest of cell growth even after culture for 96 h. Exposure of cells to the 8 mM-Cl^- culture medium diminished phosphorylation levels of Rb and cdc2, which are respectively key accelerators of transition from G₁ to S phase and G₂ to M phase in cell cycle progression. Culturing cells in the 8 mM-Cl^--containing culture medium upregulated the protein expression level of p21 (a CDK inhibitor) inhibiting transition of G₁ to S phase, and diminished the incorporation of 5-ethynyl-2′-deoxyuridine (EdU; a thymidine analogue) into DNA. These results suggest that cells cultured in the low Cl^- medium prolonged the duration of all phases of the cell cycle (G₁, S, and G₂/M), thereby abolishing overall cell cycle progression. Effects of culturing cells in the low Cl^- culture medium on cell cycle progression would be mediated via a change in the intracellular Cl^- concentration ([Cl^-]_i), since [Cl^-]_i was decreased under a low Cl^- culture medium. To clarify this possibility, we studied effects of furosemide and bumetanide, Na⁺/K⁺/2Cl^- cotransporter (NKCC) inhibitors, on proliferation of PC3 cells. Furosemide and bumetanide decreased [Cl^-]_i and cell growth of PC3 cells. These results suggest that a change in [Cl^-]_i would play a critical role in this growth mechanism.

Hesperidin suppressed proliferations of both Human breast cancer and androgen‐dependent prostate cancer cells

Hesperidin, a flavonoid derived from citrus fruits, has been reported to show various biological effects including anticancer activity. This study investigated whether hesperidin affected the proliferation of MCF-7 human breast cancer cells transfected with green fluorescent protein (GFP)/alpha-tubulin (MCF-7-GFP-Tubulin cells), androgen-independent PC-3 and DU-145 prostate cancer cells, and androgen-dependent LNCaP prostate cancer cells. The results were as follows. (1) Hesperidin inhibited the proliferation of MCF-7-GFP-Tubulin cells, probably not through an antimitotic mechanism. (2) Hesperidin also inhibited both basal and testosterone-induced proliferation of LNCaP cells. (3) However, hesperidin did not significantly affect the cell proliferation of two hormone-independent prostate cancer cells, PC-3 and DU-145. It is concluded that hesperidin can inhibit the proliferation of breast cancer cells through mechanisms other than antimitosis and it is suggested that hesperidin be further investigated for the possible interaction with androgenic receptors and involvement in signaling pathway after receptor binding in prostate cancer cells through future research.

Cytoprotective Mitochondrial Chaperone TRAP-1 As a Novel Molecular Target in Localized and Metastatic Prostate Cancer

Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Pten(pc-/-)), also exhibit high levels of TRAP-1. Expression of TRAP-1 in nontransformed prostatic epithelial BPH-1 cells inhibited cell death, whereas silencing of TRAP-1 in androgen-independent PC3 or DU145 prostate cancer cells by small interfering RNA enhanced apoptosis. Targeting TRAP-1 with a novel class of mitochondria-directed Hsp90 inhibitors, ie, Gamitrinibs, caused rapid and complete killing of androgen-dependent or -independent prostate cancer, but not BPH-1 cells, whereas reintroduction of TRAP-1 in BPH-1 cells conferred sensitivity to Gamitrinib-induced cell death. These data identify TRAP-1 as a novel mitochondrial survival factor differentially expressed in localized and metastatic prostate cancer compared with normal prostate. Targeting this pathway with Gamitrinibs could be explored as novel molecular therapy in patients with advanced prostate cancer.

Exogenous p27KIP1 expression induces anti-tumour effects and inhibits the EGFR/PI3K/Akt signalling pathway in PC3 cells

p27 is a cyclin-dependent kinase inhibitor that regulates the progression of cells from G(1) to S phase of the cell cycle. Loss of p27 has been associated with disease progression and with an unfavourable outcome in prostate cancer. In this study, we investigated whether exogenous p27 expression in the human androgen-independent prostate cancer PC3 cell line had any effect on cell growth, and we studied the molecular mechanisms involved. p27 expression was restored in PC3 cells by plasmid delivery. Cell proliferation and apoptosis were assessed in PC3 cells transfected with p27. We also investigated the effects of p27 on the epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway in PC3 cells. By restoring p27 expression in PC3 cells, we observed that p27 reduced proliferation and induced arrest in G(0)/G(1) phase. Moreover, p27-transfected PC3 cells underwent apoptosis, as shown by flow cytometric analysis and western blotting analysis of Bcl-2, Bax, Bad, caspase-3 and poly(ADP-ribose)polymerase expression. Furthermore, the p27-induced anti-tumour action correlated with inhibition of the EGFR/PI3K/Akt signalling pathway, as confirmed by western blotting analysis and densitometry of EGFR, PI3K (p85), Akt and p-Akt(S473) expression. Our results suggest that exogenous expression of p27 inhibits the proliferation of PC3 cells through induction of G(1) arrest and apoptosis, and this process correlates with inhibition of the EGFR/PI3K/Akt signalling pathway.

Neuroendocrine differentiation is involved in chemoresistance induced by EGF in prostate cancer cells

Aims Neuroendocrine (NE) cells were thought to be post-mitotic and non-proliferative. But it was recently reported that NE cells express, and induce surrounding cells to express potent antiapoptotic proteins. We hypothesize that neuroendocrine differentiation (NED), a common phenomenon in prostate cancer, is related to chemoresistance in prostate cancer. Main methods Androgen-independent human prostate cancer DU145 and PC-3 cells were exposed to epidermal growth factor (EGF). MTT assays evaluated changes in chemoresistance after EGF treatment, and flow cytometry examined EGF-induced cell cycle changes in DU145 cells. Western blotting, real-time RT-PCR and transmission electron microscopy were utilized to confirm NED. Key findings After stimulation with EGF, DU145 and PC-3 cells exhibited stronger resistance to cisplatin. Flow cytometry showed that EGF stimulation substantially decreased the proportion of DU145 cells in G 1 phase. EGF treatment increased the expression of neuron-specific enolase, a marker of NED induction. Significance NED in prostate cancer is involved in the chemoresistance induced by EGF. EGF and/or the EGF receptor may be potential targets for medical intervention in chemo-resistant prostate cancer.

Penta-O-galloyl-β-d-glucose Suppresses Prostate Cancer Bone Metastasis by Transcriptionally Repressing EGF-Induced MMP-9 Expression

Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of cancer-related death of men in the United States. Epidermal growth factor (EGF) generated from bone tissue contributes to prostate cancer metastasis through stimulating matrix metalloproteinase (MMP) secretions from prostate cancer cells. In this study, in vitro invasion assay was performed by incubating penta-O-galloyl-beta-D-glucose (5GG) at various concentrations with 2 x 10(4) PC-3 cells for 48 h. The anti-invasive and cytotoxic effects of 5GG were found and evaluated on the human androgen-independent prostate cancer PC-3 cell line by MTT assays and Western blot analyses. 5GG inhibited the EGF-induced cell invasiveness and MMP-9 expression in a dose- and time-dependent manner by reducing the MMP-9 transcriptional activity. To explore the mechanisms for the 5GG-mediated regulation of MMP-9, we further examined the effects of 5GG on transcription factors, including NF-kappaB, AP-1, and mitogen-activated protein kinase (MAPK) activities. The results showed that 5GG suppressed the EGF-induced NF-kappaB nuclear translocation and also abrogated the EGF-induced activation of c-jun N-terminal kinase (JNK), an upstream modulator of NF-kappaB. Moreover, we showed that 5GG reduced EGFR expression through the proteasome pathway. These results suggest that 5GG may exert at least part of its anti-invasive effect in androgen-independent prostate cancer by controlling MMP-9 expression through the suppression of the EGFR/JNK pathway. Finally, 5GG suppresses invasion and tumorigenesis in nude mice treatment with intratibia injection of PC-3 cells. These in vitro and in vivo results suggest that 5GG may be a therapeutic candidate for the treatment of advanced prostate cancer.

Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells

We investigated the effects of transient receptor potential M8 (TRPM8) channel on the proliferation and motility of androgen-independent prostate cancer PC-3 cells. After being permanently transfected with an empty vector and cDNA encoding the TRPM8 protein, cells were analysed for cell cycle distribution and motility using flow cytometry and scratch assay. Immunocytochemistry and Ca2+ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells. Cell cycle distribution and scratch assay analysis revealed that TRPM8 induced cell cycle arrest at the G0/G1 stage (P < 0.05) and facilitated the cell apoptosis induced by starvation (P < 0.05). Furthermore, TRPM8 inhibited the migration of PC-3-TRPM8 cells (P < 0.01) through the inactivation of focal-adhesion kinase. It appears that TRPM8 was not essential for the survival of PC-3 cells; however, the overexpression of TRPM8 had negative effects on the proliferation and migration of PC-3 cells. Thus, TRPM8 and its agonists may serve as important targets for the treatment of prostate cancer.

Differential Effects of Selenium on Benign and Malignant Prostate Epithelial Cells: Stimulation of LNCaP Cell Growth by Noncytotoxic, Low Selenite Concentrations

We examined the hypothesis that nontoxic concentrations of selenium induce apoptosis and growth inhibition selectively in prostate cancer cells but not in benign prostate cells. Nontumorigenic BPH-1 prostate epithelial cells, androgen-sensitive LNCaP, and androgen-independent PC-3 prostate cancer cells were exposed to sodium selenite at 1 to 10 μ mol/l for 24 to 72 h. Cell proliferation, viability, and apoptosis were assessed by MTT assay, trypan blue exclusion, flow cytometry, DNA laddering, and caspase activation. BPH-1 cells were more sensitive for cytotoxic selenium effects than malignant prostate cells, whereas LNCaP cells were more sensitive than PC-3 cells. At noncytotoxic selenium concentrations, there was no apoptosis in BPH-1 and PC-3 cells and no growth inhibition of LNCaP and BPH-1 cells. PC-3 cells were refractory to apoptosis induction but were growth inhibited at noncytotoxic concentrations. LNCaP cells were growth stimulated at 1 μ mol/l and sensitive to apoptosis induction at higher noncytotoxic concentrations. Thus, noncytotoxic selenite concentrations did not induce growth inhibition or apoptosis selectively in prostate cancer cells. Growth stimulation of LNCaP cells by low concentrations suggests the possibility of adverse effects of selenium supplementation on hormone sensitive prostate cancer, whereas inhibition of PC-3 cell proliferation at noncytotoxic concentrations suggests potential benefit of selenium in advanced prostate cancer.

Anti-androgen-independent prostate cancer effects of ginsenoside metabolites In Vitro: Mechanism and possible structure-activity relationship investigation

Treatment of androgen-independent prostate cancer (AIPC) remains unsatisfactory. In our present experiment, natural occurring ginsenosides (NOGs) and intestinal bacterial metabolites (IBMs) were employed to investigate their anti-AIPC cell growth activity using PC-3 cells. Our results showed that the IBMs exerted more portent anti-AIPC activity than NOGs, by decreasing survival rate, inhibiting proliferation, inducing apoptosis, and leading to cell cycle arrest in AIPC PC-3 cells. The increase of LogP and decrease of C-6 steric hindrance, which were caused by deglycosylation by intestinal bacteria, may be the reason for the higher anti-AIPC activity of IBMs.

Nkx3.1 and p27KIP1 Cooperate in Proliferation Inhibition and Apoptosis Induction in Human Androgen–Independent Prostate Cancer Cells

Prostate cancer (PC), which responds well to androgen ablation initially, invariably progresses to treatment resistance. The so-called androgen-independent PC is also a concern, since there is no effective therapy so far. Nkx3.1 is a putative prostate tumor suppressor that is expressed exclusively in the prostate under the regulation of androgen, and p27KIP1 functions as a cell proliferation inhibitor and apoptosis trigger by disrupting the cyclin-dependent kinase (CDK)-cyclin complex. Lack of expressions of Nkx3.1 and/or p27KIP1 have been detected in most advanced PC and is associated with poor clinical progression. Here, we show that endogenous expressions of both Nkx3.1 and p27KIP1 are lost in the androgen-independent PC3 PC cells, while remaining intact in LNCaP PC cells, which contain functional androgen receptor (AR) and are hormone-responsive. Ectopic restoration of either Nkx3.1 or p27KIP1 in PC3 cells results in reduced cell proliferation, and increased cell death. Both effects are synergistically enhanced when the two molecules are coexpressed. p27KIP1 overexpression in PC3 results in increased cell population ceased at the G0/G1 phase, and this cell-cycle-arresting effect is significantly enhanced by the coexpression of Nkx3.1. Flow cytometry further revealed that Nkx3.1 and p27KIP1 also cooperatively render more PC3 cells undergoing apoptosis. Consistently, the coexpression of Nkx3.1 and p27KIP1 leads to the decreased expression of Bcl-2 oncogene and a concomitantly upregulated Bax expression. It also activates caspase 3 and leads to increased cleavage of PARP. Our findings thus reveal the crucial relevance of the combined antiproliferative and proapoptotic activities of Nkx3.1 and p27KIP1 in androgen-independent PC cells, and further suggest that a combined, rather than single gene manipulation may be of clinical value for hormone-refractory PC.

Lipido‐sterolic extract of Serenoa repens (LSESr, Permixon®) treatment affects human prostate cancer cell membrane organization

The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 microg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in omega 6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in omega 6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2-3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status.

Suppression of Wnt/β-catenin signaling inhibits prostate cancer cell proliferation

Although mounting evidence has demonstrated an important role of Wnt/β-catenin signaling in the development and progression of cancer, the therapeutic potential of small molecules that target this pathway for prostate cancer remains largely unknown. We reported herein that the highly invasive androgen-independent PC-3 and DU145 human prostate cancer cells exhibited higher levels of Wnt/β-catenin signaling than the androgen-dependent LNCaP prostate cancer cells and non-cancerous PZ-HPV-7 and PWR-1E prostate cells, and that exogenous Wnt3A treatment exaggerated the difference of the Wnt/β-catenin signaling levels among these prostate cells. Furthermore, we demonstrated that the non-steroidal anti-inflammatory drug, sulindac sulfide, the cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, and the nitric oxide-donating aspirin derivative, NO-ASA, blocked Wnt/β-catenin signaling in PC-3 and DU145 cells. These effects occurred at concentrations comparable to those required to inhibit cell proliferation, indicating that the inhibitory effect of these drugs on prostate cancer cell proliferation may involve the suppression of Wnt/β-catenin signaling. Finally, we showed that a novel small molecule inhibitor of Wnt/β-catenin signaling, PKF118- 310, inhibited Wnt/β-catenin signaling and proliferation in prostate cancer cells within the same concentration range. Together, these results suggest that small molecules that inhibit Wnt/β-catenin signaling have therapeutic potential for the prevention or treatment of prostate cancer.