Melatonin and prostate cancer cell proliferation: interplay with castration,epidermal growth factor,and androgen sensitivity

BACKGROUND: Potential modulatory effects of melatonin on the proliferation of androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells were reported recently. In this study, we investigated the effects of combined melatonin and castration on LNCaP tumor growth in vivo, the interactions between melatonin and epidermal growth factor (EGF) on LNCaP cell proliferation, and melatonin actions on the proliferation of PC-3 and DU 145 cells. METHODS: Tumor development and growth in castrated nude mice inoculated with LNCaP cells or in intact animals inoculated with DU 145 cells, with or without daily melatonin treatment, were monitored by observation and caliper measurement. MT(1) receptor expression in native or transfected prostate cancer cell lines was examined by immunocytochemistry or 2-[(125)I]iodomelatonin binding. Cyclin D1 expression in LNCaP cells was assessed by Western blotting, and cell proliferation was measured by thymidine incorporation and/or cell count. RESULTS: Melatonin treatment was associated with further decreases in LNCaP tumor incidence and growth rate in castrated nude mice. Melatonin and 2-iodomelatonin (a melatonin receptor agonist) attenuated EGF-stimulated increases in LNCaP cell proliferation and cyclin D1 levels. Melatonin had no effect on the proliferation or growth of MT(1) receptor-expressing DU 145 cells, and of PC-3 cells in which MT(1) receptor protein was undetectable. The proliferation of transfected PC-3 cells expressing MT(1) receptor was unaffected by 2-iodomelatonin. CONCLUSION: Together with previous data, the present results indicate synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor. Androgen-sensitive prostate cancer cell proliferation may be modulated by opposite changes in cyclin D1 levels induced by activated MT(1) and EGF receptors. In androgen-insensitive prostate cancer cells, MT(1) receptor-mediated signal transduction may become defective not only through changes in membrane receptor protein expression and/or functions, but also by means of alterations in downstream postreceptor signaling events.     

Long-term androgen-ablation causes increased resistance to PI3K/Akt pathway inhibition in prostate cancer cells

BACKGROUND: In advanced stages of prostate cancer, the phosphatidylinositol-3' kinase (PI3K)/Akt signaling cascade, one of the major survival pathways in the cell, is frequently constitutively activated due to mutation or loss of the tumor suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Using cell culture models representing different tumor stages, we explored the effect of inhibition of this survival pathway on the induction of apoptosis. METHODS: Inhibition of the survival kinase Akt and induction of apoptosis was analyzed in androgen-insensitive DU145 and PC-3 cells, in androgen-responsive LNCaP, and in androgen-independent long-term androgen-ablated LNCaP-abl cells representing therapy-resistant prostate cancer cells. Activated Akt was determined by immunoblotting using a phospho-Akt specific antibody. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry and measurement of cleavage of the caspases substrate poly-ADP-ribose polymerase (PARP). RESULTS: IGF-1, EGF, and heregulin but not PDGF or activators of protein kinase A induced phosphorylation of Akt in DU145 cells and activation was completely blocked by the PI3K inhibitor LY294002. In the hormone-responsive prostate cancer cell line LNCaP that has a constitutively switched-on Akt kinase, LY294002 caused a dose- and time-dependent Akt inhibition, which was absent in long-term androgen-ablated LNCaP sublines. In agreement with the resistance to inhibition of the PI3K/Akt pathway, long-term androgen-ablated LNCaP sublines remained relatively resistant to induction of cell death by LY294002 or the cytotoxic drug etoposide. Inhibition of the PI3K/Akt pathway restored the sensitivity of long-term androgen-ablated cells to induction of apoptosis by a cytotoxic drug almost completely. CONCLUSION: These results suggest that long-term androgen ablation therapy for prostate cancer reinforces the PI3K/Akt pathway and impedes its inhibition thus contributing to increased resistance of tumor cells to induction of apoptosis. With regard to treatment of therapy-refractory prostate cancer, these findings suggest effectiveness of a combination of cytotoxic treatment and inhibition of the PI3K-Akt survival pathway in tumor cells after failure of androgen-ablation therapy.     

Growth inhibition of androgen-insensitive human prostate carcinoma cells by a 19-norsteroid derivative agent,mifepristone

Mifepristone, also known as RU 486, is a 19-norsteroid derivative. Currently, mifepristone is being tested in clinical trials on meningioma and breast cancer. In this study we analyzed whether mifepristone could inhibit the growth of human prostate cancer cells including androgen-insensitive (PC-3 and DU145) and androgen-sensitive (LNCaP) cell lines. At 1-nM concentration, mifepristone exhibited a marginal stimulatory action on LNCaP and PC-3 cells. Nevertheless, a dose-dependent growth inhibition on those same cell lines was observed at concentrations of 1 μM and 10 μM. Twenty-day exposure to the clinically achievable concentration of 1 μM mifepristone resulted in consistent inhibition of all three cell lines studied. Furthermore, this in vitro growth inhibition was reflected in an in vivo nude mouse system. Mifepristone at the dosage of 4 mg/100 g body weight completely suppressed the growth of PC-3 tumors for 21 days, although this was followed by a growth rate similar to that of the control tumor. To understand the possible mechanism of mifepristone inhibition, PC-3 cells were exposed to mifepristone in comparison with dexamethasone (Dex), progesterone, and 5 alpha-dihydrotestosterone (DHT), each at 1-μM concentration. The results demonstrated that while both DHT and Dex alone had essentially no effect on cell growth, progesterone alone resulted in a 20% growth inhibition, while mifepristone had more than 60% inhibition with a 16-day exposure. At an equal concentration, the degree of growth inhibition of PC-3 cells by mifepristone or progesterone was partially diminished by simultaneous exposure to Dex. In conclusion, our results demonstrated that the growth of androgen-insensitive prostate cancer cells can be directly inhibited by mifepristone in cultures. This in vitro inhibition is reflected in xenografted tumors.     

EGFR and Her-2 regulate the constitutive activation of NF-kappaB in PC-3 prostate cancer cells

BACKGROUND: The mechanism through which NF-kappaB (NF-kappaB) is constitutively activated in prostate cancer cells remains unclear. We investigated whether members of the ErbB family of epidermal growth factor receptors (EGFR) are involved in the constitutive activation of NF-kappaB in prostate cancer cell lines. METHODS AND RESULTS: EGFR, Her-2, and ErbB3 are expressed and constitutively activated in PC-3, DU145, and LNCaP prostate cancer cells lines. Using several pharmacological ErbB inhibitors, we demonstrate that EGFR and Her-2 are involved in the constitutive activation of NF-kappaB in PC-3 cells through two different mechanisms. EGFR activates NF-kappaB through the phosphorylation of IkappaBalpha on serines 32/36 thereby influencing the nuclear translocation of the p65 subunit. In contrast, Her-2 activates NF-kappaB independently of IkappaBalpha phosphorylation on serines 32/36. CONCLUSION: This study directly implicates ErbB receptors in the activation of NF-kappaB in PC-3 prostate cancer cells.     

Downregulation of androgen receptor expression by luteolin causes inhibition of cell proliferation and induction of apoptosis in human prostate cancer cells and xenografts

BACKGROUND: Androgen receptor (ARs) play a crucial role in the development and progression of prostate cancer. Recent studies have suggested that prostate cancer cell proliferation is inhibited by AR downregulation. Our aim was to investigate how luteolin, a natural flavonoid, affects cell growth and AR expression in prostate cancer cells and xenografts. METHODS: We assessed prostate cancer cell (LNCaP, DU145, and PC-3) proliferation and apoptosis by MTT assay, flow cytometric analysis, and Western analysis. AR function was measured by evaluating the AR target molecule, prostate-specific antigen (PSA), by RT-PCR, Western blotting, and enzyme-linked immunosorbent assay. We determined the mechanism of AR downregulation with cycloheximide chase assays, proteasome inhibitor, and coimmunoprecipitation experiments. The effects of luteolin on growth inhibition in vivo were examined by LNCaP xenografts in SCID mice. RESULTS: Luteolin significantly repressed prostate cancer cell proliferation and induced apoptosis in LNCaP cells. PC-3 and DU145 cells were less susceptible to luteolin-mediated growth inhibition. Luteolin simultaneously suppressed intracellular and secreted PSA levels and repressed AR mRNA and protein expression in a dose- and time-dependent manner. Luteolin reduced the association between AR and heat-shock protein 90, causing AR degradation through a proteasome-mediated pathway in a ligand-independent manner. Luteolin also suppressed LNCaP xenograft tumor growth in SCID mice. CONCLUSION: Luteolin-mediated AR downregulation contributes to the inhibition of cell proliferation and the induction of apoptosis in LNCaP human prostate cancer cells, suggesting that AR is a molecular target for luteolin-mediated anticancer activity. Luteolin may act as a chemopreventive or chemotherapeutic agent for prostate cancer.     

FOXN2在前列腺癌组织的表达及对前列腺癌细胞生物学的影响

探讨双头框蛋白N2（FOXN2）在前列腺癌（PCa）组织中的表达以及对PCa细胞生物学的影响。方法 选取宜宾市第二人民医院50例接受手术治疗的PCa患者的标本及癌旁组织,通过RT-qPCR实验和Western blot实验分别检测PCa组织和癌旁组织中的FOXN2 mRNA及蛋白的表达水平,并分析PCa组织中FOXN2与患者临床病理特征的相关性。通过RT-qPCR实验检测正常前列腺细胞RWPE-1、PCa细胞PC-3、DU145、LNCaP中FOXN2 mRNA的表达水平。选取PC-3细胞为研究对象,分为FOXN2过表达组、空载体对照组以及对照组,分别通过MTT实验、流式细胞实验、Transwell实验以及Western blot实验检测各组细胞增殖、凋亡、迁移和侵袭情况以及相关蛋白Bax、CyclinD1、MMP-2的表达量。结果 与癌旁组织相比,FOXN2的mRNA和蛋白表达水平显著降低（P<0.05）,与TCGA数据库中结果一致。FOXN2低表达与PCa患者淋巴转移、TNM分期以及Gleason评分有关（P=0.003、0.005、0.002）。与人正常前列腺细胞RWPE-1相比,FOXN2在PCa细胞PC-3、DU145、LNCaP中均呈现低表达（P<0.05）,其中PC-3细胞中表达量最低。与对照组和空载体对照组相比,FOXN2过表达组的PC-3细胞在作用24 、48 、72 h后增殖能力显著下降（F=290.400、57.735、113.014,P<0.05）,CyclinD1蛋白表达水平显著下降（P<0.05）;PC-3细胞的凋亡率显著升高（P<0.05）,Bax蛋白表达水平显著升高（P<0.05）;PC-3细胞的迁移和侵袭能力显著下降（P<0.05）,MMP-2蛋白表达水平显著下降（P<0.05）。结论 FOXN2在PCa组织及细胞中低表达,过表达FOXN2可抑制PCa细胞的增殖、迁移和侵袭,并促进细胞凋亡。     

Prostate stromal cell-derived hepatocyte growth factor induces invasion of prostate cancer cell line DU145 through tumor-stromal interaction.

BACKGROUND: In prostate cancer, several growth factors derived from stromal cells regulate tumor cell growth. Hepatocyte growth factor (HGF) possesses biological activities that promote cancer proliferation and invasion through tumor-stromal interaction. We examined how prostate stromal cell-derived HGF affects invasion of prostate cancer cells through this interaction. METHODS: The effects of HGF, various growth factors (transforming growth factor (TGF)-alpha, TGF-beta1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor), and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3, and DU145) were determined by collagen gel invasion assay. DU145 cells and PrSC were cocultured for Matrigel invasion chamber assay. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by the ELISA method and Western blotting. RESULTS: LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or cocultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta1. Native-type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. CONCLUSIONS: PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction, wherein DU145 cells secreted some HGF-inducer(s) for PrSC.     

Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells

BACKGROUND: Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro. METHODS: We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively. RESULTS: Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin. CONCLUSIONS: Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells.     

Interleukin-6 protects LNCaP cells from apoptosis induced by androgen deprivation through the Stat3 pathway

BACKGROUND: Elevated expression of interleukin-6 (IL-6) is implicated in the progression of hormone refractory prostate cancer. Previous studies demonstrated that IL-6 promotes androgen-independent growth of prostate cancer cells. In this study, the effect of IL-6 on apoptosis induced by androgen deprivation was investigated. METHODS: The effect of IL-6 on apoptosis induced by androgen deprivation in LNCaP cells was examined by cell death ELISA and Western blot using cleaved poly (ADP-ribose) polymerase (PARP) and caspase-9, as well as Bcl-xL and phosphorylated Bad. The Stat3 in IL-6-mediated anti-apoptosis in prostate cancer cells was examined using either dominant-negative or constitutively activated Stat3 mutants. RESULTS: Overexpression of IL-6 renders androgen sensitive LNCaP human prostate cancer cells more resistant to apoptosis induced by androgen deprivation. LNCaP cells undergo apoptosis after 72 hr of androgen deprivation, an outcome is largely absent in clones overexpressing IL-6 as measured by cell death ELISA and chromatin degradation assays. IL-6 over-expressing cells resulted in a significant decrease in the expression of cleaved PARP and cleaved caspase-9 as well as an increase in the expression of Bcl-xL and phosphorylated Bad. Addition of IL-6 antibody completely abolished the anti-apoptotic activity of IL-6. This protective effect of IL-6 was reversed by the expression of a dominant-negative Stat3 mutant, Stat3F. Furthermore, ectopic expression of a constitutively active Stat3 antagonized androgen deprivation-induced cell death of LNCaP cells. CONCLUSION: These results indicate that IL-6 protects androgen sensitive LNCaP cells from apoptosis induced by androgen deprivation, and Stat3 activation play an important role in IL-6-mediated anti-apoptosis in prostate cancer cells.     

PC-SPES: a unique inhibitor of proliferation of prostate cancer cells in vitro and in vivo

BACKGROUND: Management of prostate cancer that has spread beyond the capsule is a difficult problem. Innovative and nontoxic approaches to the disease are urgently required. Recently, a commercially available herbal mixture called PC-SPES showed potent antitumor activities on a variety of malignant cells in vitro. METHODS: PC-SPES was evaluated for its ability to inhibit clonal growth, and to induce cell cycle arrest of three human prostate cancer cell lines (LNCaP, PC-3, and DU 145). Western blot analysis examined the effect of PC-SPES on levels of p21(waf1), p27(kip1), Bcl-2, and E-cadherin in the three cell lines; and telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay. Furthermore, the effect of oral PC-SPES (250 mg/kg/day) on growth of PC-3 and DU 145 tumors present in male BNX nu/nu triple immunodeficient mice was studied. LNCaP cells were not analyzed in mice because they grow only with difficulty in these immunodeficient mice. RESULTS: PC-SPES markedly inhibited clonal growth of LNCaP, PC-3, and DU 145 prostate cancer cells, with a 50% inhibition (ED50) at approximately 2 microl/ml. Pulse-exposure studies showed that a 5-day pulse-exposure to PC-SPES (2 microl/ml) in liquid culture achieved a 50% inhibition of PC-3 clonal growth in soft agar, suggesting that the growth inhibition mediated by the extracts remained after removal of PC-SPES. Cell cycle analysis using the prostate cancer cell lines found that PC-SPES induced a significant increase in the number of cells in G0-G1 and G2/M, with a concomitant decrease in the number of cells in S phase. PC-SPES (2 microl/ml, 4 days) increased slightly the levels of p21(waf1) in the three cell lines, decreased by 40% the levels of Bcl-2 in PC-3, and the levels of p27(kip1) and E-cadherin and telomerase were unchanged in each of the lines. In vivo treatment with oral PC-SPES of male BNX mice having DU 145 tumors produced significant inhibition of their growth (P < 0.001), with no objective side effects including blood chemistries, weights, or autopsy analysis. The PC-SPES showed no statistical effect on the in vivo growth of PC-3 cells. CONCLUSIONS: PC-SPES inhibits clonal proliferation of human prostate cancer cells both in vitro and in vivo, using a murine model.     

Differential retention of alpha-vitamin E is correlated with its transporter gene expression and growth inhibition efficacy in prostate cancer cells

BACKGROUND: Epidemiological studies showed Vit E has protective effects against prostate cancer (PCa). Interestingly, different prostate cancer cells have different sensitivity to alpha-Vit E or VES treatment. The goal of this study is to determine whether cellular Vit E bioavailability and its transport proteins are important contributing factors. METHODS: alpha-Vit E and its ester form, VES, were used to treat prostate cancer LNCaP, PC3, and DU145 cells, and their growth rates were determined by MTT assay. Cellular levels of Vit E were quantified using HPLC as the index of bioavailability. The expression levels of Vit E transport proteins were determined by real-time PCR. RESULTS: Among these PCa cells, only LNCaP cells were sensitive to 20 microM alpha-Vit E treatment, while both LNCaP and PC3 cells were sensitive to 20 microM VES treatment. Coordinately, cellular levels of alpha-Vit E and VES positively correlated to their inhibitory effects. Further study found expression levels of Vit E transport proteins, including tocopherol associated protein (TAP), scavenger receptor class B type I (SR-BI), alpha-tocopherol transfer protein (TTP), and ATP binding cassette transporter A1 (ABCA1), were different in various PCa cells, which may contribute to cellular Vit E bioavailability. This notion is further supported by the findings that overexpression or knockdown of TTP could coordinately alter cellular alpha-Vit E levels in PCa cells. CONCLUSION: Antiproliferative efficacy of alpha-Vit E is correlated with its cellular bioavailability in PCa cells. Modulating the expression of the efflux or influx transporters could sensitize the growth inhibition efficacy of Vit E in prostate cancer cells.     

C16 ceramide accumulates following androgen ablation in LNCaP prostate cancer cells

BACKGROUND: Adenocarcinoma of the prostate is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related deaths among men in the United States. The most successful therapies to date for this tumor have involved some form of androgen ablation. However, these therapies become ineffective as the tumor evolves to an androgen-insensitive state. Ceramide is a lipid second messenger that has been shown to mediate growth arrest or cell death when added exogenously to prostate cancer cells. As a first step toward understanding the events that lead to the transition of prostate cancer cells to an androgen-independent state, we considered investigating the effect of androgen ablation on endogenous ceramide levels in androgen-sensitive and androgen-insensitive prostate cancer cells. METHODS: To investigate the mechanisms of growth arrest/apoptosis in androgen-sensitive (LNCaP) and insensitive (DU-145, PC-3) cells, we used various methods including nonyl acridine orange (NAO) staining, propidium iodide (PI) staining/cell-cycle analysis, lipid analysis, and Western blotting assays. RESULTS: In this study, we demonstrate that androgen ablation drives G(0)/G(1)-phase cell-cycle arrest followed by progressive apoptosis in vitro, in LNCaP cells. Lipid analysis indicated an increase in C16 ceramide, which was generated via the de novo pathway as revealed by blockade of ceramide synthase by fumonisin B1. The addition of 5alpha-dihydrotestosterone (DHT) or fumonisin B1 rescued LNCaP cells from apoptosis induced by androgen ablation, and decreased levels of intracellular C16 ceramide. Neither apoptosis nor an increase in C16 ceramide was observed in androgen-independent cell lines following androgen ablation.     

Role of hepatocyte growth factor in invasion of prostate cancer cell lines through tumor-stromal interaction

We examined how prostate stromal cell-derived hepatocyte growth factor (HGF) affects invasion of prostate cancer cells through tumor-stromal interaction. The effects of HGF, various growth factors [transforming growth factor (TGF)-alpha, TGF-beta 1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor], and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3 and DU145) were determined by collagen gel invesion assay. DU145 cells and PrSC were co-cultured for matrigel invasion chamber assay. LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta 1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or co-cultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta 1. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by ELISA method and Western blotting. Native type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. In summary, PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction wherein DU145 cells secreted some HGF-inducer(s) for PrSC.     

Effect of the neuropeptides bombesin and calcitonin on the growth of prostate cell lines, PC-3, DU 145, and LNCaP

Neuroendocrine cells are present in normal and tumoral prostate tissue, the neuropeptides secreted by this cells have a biological functions that have not been fully elucidated. The presence of neuroendocrine cells in prostatic carcinoma have been shown to increase tumor progression. We characterized the in vitro proliferative influence of bombesin and calcitonin in androgen-insensitive, PC-3 and DU-145, and androgen-sensitive, LNCaP, cell lines of human prostate cancers. The influence of these neuropeptides on proliferation were assessed using the colorimetric XTT assay and by cells counts with a hemocytometer. The growth of PC-3 and DU-145 cell lines is stimulated by bombesin and calcitonin but exerted any stimulatory effect on the proliferation of the LNCaP cell line. This indicate that bombesin and calcitonin can modulate proliferation of androgen-insensitive human prostate cell lines "in vitro" and may be potential paracrine growth promoters in stablished androgen irresponsive human prostatic carcinoma cells.     

靶向沉默核干因子对前列腺癌PC-3细胞增殖能力的影响

目的:检测前列腺癌PC-3、LNCaP及DU145细胞中核干因子(Nucleostemin,NS)基因的表达,研究NS基因沉默后对PC-3细胞增殖能力的影响。方法:采用免疫细胞化学法及逆转录-聚合酶链反应(RT-PCR)分别检测NS蛋白及mRNA在3种前列腺癌细胞中的表达。用NS特异性小发夹RNA表达质粒转染PC-3细胞,分别用RT-PCR及Western印迹方法检测转染后细胞(简称NS-shRNA-PC-3)中NSmRNA及蛋白的变化。比较NS基因沉默前后PC-3细胞体外、裸鼠体内增殖能力及凋亡情况的变化。结果:3种细胞中均显示NS基因高表达。转染后NS-shRNA-PC-3细胞中NS表达显著降低,细胞增殖速度减慢,G0/G1期细胞百分率显著升高,早期凋亡细胞增多。体内致瘤实验显示,NS基因沉默后,PC-3细胞在裸鼠体内增殖能力显著降低。结论:NS在前列腺癌细胞系中呈高表达,RNA干扰沉默NS基因后PC-3细胞增殖能力显著降低,凋亡细胞增多。     

Human prostate cancer cells express neuroendocrine cell markers PGP 9.5 and chromogranin A

BACKGROUND: A proportion of men with prostate cancer will progress to develop metastatic disease involving the lymph-nodes and bone. To identify novel candidates associated with metastatic progression, we compared the proteomic profiles of LNCaP (lymph-node metastatic, androgen-dependant) and PC-3 (bone metastatic, androgen-independent), human prostate cancer cells. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), was used to identify differentially expressed proteins. Western blotting was used to validate the identity of any candidates. Immunohistochemistry was used to assess tissue expression. RESULTS: 2D-PAGE followed by ESI-MS/MS analyses identified the expression of glutathione S-transferase-pi (GST-pi) and protein gene product 9.5 (PGP 9.5) in PC-3 cells, but absent expression in LNCaP cells. PGP 9.5 expression in PC-3 cells was confirmed by Western blotting, in addition to expression in DU145 cells. Analysis of cell conditioned media showed that PGP 9.5 was secreted. Sequencing of the PGP 9.5 gene promoter region in bisulfite modified DNA, suggested that the regulation of expression involves promoter hypermethylation. RT-PCR analysis for Chromogranin A (ChA) mRNA (a marker of neuroendocrine cells), showed expression in PC-3 and DU145 cells but was undetectable in LNCaP cells. Immunohistochemistry localised PGP 9.5 expression exclusively within neuroendocrine cells and nerve fibres. CONCLUSIONS: Our unexpected finding that the neuroendocrine cell markers PGP 9.5 and ChA are expressed by PC-3 and DU145 cells, suggests that these cells may have been derived from metastatic adenocarcinomas which had undergone neuroendocrine differentiation or alternatively the expression occurred ectopically as a result of cell culture.     

mRNA expression of the five membrane-type matrix metalloproteinases MT1-MT5 in human prostatic cell lines and their down-regulation in human malignant prostatic tissue

BACKGROUND: The aim of this study was to assess the expression of membrane-type matrix metalloproteinases (MT-MMPs) 1-5 in the human prostatic cell lines BPH-1, LNCaP, DU 145, PC-3, in malignant and non-malignant prostatic tissue samples, and in epithelial cells cultured from these tissue samples. METHODS: Matched malignant and non-malignant tissue specimens were obtained from 12 men with untreated prostate carcinoma after radical prostatectomy. Expression of mRNA for the five MT-MMPs was quantified by real-time PCR technique and normalized to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The expression of the five MT-MMPs was distinctly different not only between the prostate cell lines but also varied in the same cell line. There was a general higher expression of all MT-MMPs except for MT3-MMP in the androgen-insensitive cells DU 145 and PC-3 compared with that in the androgen-sensitive LNCaP cells. Their relatively high expression in the benign prostatic cell line BPH-1 and also in the primary cell cultures from malignant and non-malignant tissue samples argues against a simple association between MT-MMP expression and invasiveness. In malignant tissue samples and their corresponding cell cultures, the expression of most MT-MMPs was down-regulated in comparison to the normal counterparts. There was no correlation between tumor classification data and the MT-MMP expression results. CONCLUSIONS: In contrast to other carcinoma, the down-regulation of most MT-MMPs is typical for prostate carcinoma. It seems to occur mainly in epithelial cells and has to be examined as special characteristic of this tumor entity in further studies.     

Regulation of lnvasive Potential of Human Prostate Cancer Cell Lines by Hepatocyte Growth Factor

Background: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro.
Methods: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgenindependent), and LNCaP (androgen-dependent). We studied the expression of the HCF receptor c-met proto-oncogene (c-met) by Western blotanalysis, and alsodetermined theeffectsof HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay.
Results: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells.
Conclusion: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.