Effects of hepatocyte growth factor on urokinase-type plasminogen activator (uPA) and uPA receptor in DU145 prostate cancer cells

Urokinase-type plasminogen activator (uPA) and the uPA receptor (uPAR) are involved in a proteolytic cascade resulting of extracellular matrix degradation. Upstream, uPA and uPAR are regulated by various factors including hepatocyte growth factor (HGF), which stimulates the uPA/uPAR proteolytic system and increases invasion of cancers. We recently demonstrated that HGF induces invasion of DU145 prostate cancer cells into collagen gel matrix. We therefore examined effects of HGF on uPA and uPAR expression in DU145 cells. Effects of HGF on uPA expression in culture medium were determined by Western blotting and fibrin zymography, effects on uPAR expression in cell-associated protein were examined by Western blotting. HGF increased uPA and uPAR production in a dose-dependent manner up to 10 ng/mL, while effects of 20 ng/mL were approximately equal to those of 10 ng/mL. HGF stimulated uPA production beyond that in control cultures from 8 h until 48 h after HGF addition. HGF stimulated a uPA/uPAR proteolytic network in DU145 cells, which may be important for acquisition invasive potential by prostate cancer.     

Density-dependent regulation of epidermal growth factor receptor expression in DU 145 human prostate cancer cells

Androgen-independent prostate cancer cells may rely on an autocrine loop for growth stimulation, and have been shown to express both the epidermal growth factor receptor (EGFR) and its stimulatory ligands. We have shown here that DU 145 prostate cancer cells have a decreased amount of EGFR in confluent cultures when compared to levels seen in subconfluent cultures. This down-regulation of EGFR numbers is not due to cell proliferation or nutrient depletion, but can be correlated only with whether cell-cell contact exists throughout the culture. This is reminiscent of the situation existing in some tumors whereby EGFR expression is higher in cells at the invading margins of the tumor.     

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.     

iNOS基因转染对雄激素非依赖性前列腺癌细胞DU145生长的影响

目的:观察诱导型一氧化氮合酶(iNOS)基因转染对雄激素非依赖性前列腺癌细胞DU145生物学行为的影响。方法:将iNOS基因转染到DU145细胞并筛选出阳性细胞进行扩增,并设空载体组和对照组。观察细胞的形态变化,MTT法绘制生长曲线;流式细胞术检测细胞凋亡率;了解NOS抑制剂对转染细胞的影响。结果:转染iNOS后,DU145细胞分泌的NO[(272.50±15.82)μmol/L]显著高于空载体组[(122.00±18.93)μmol/L]和对照组[(121.00±6.98)μmol/L](P<0.05)。流式细胞术检测结果提示转染iNOS组细胞凋亡率[(42.78±2.01)%]明显高于空载体组[(30.65±1.46)%]和对照组[(28.96±1.50)%](P<0.05)。MTT测定结果提示转染组细胞生长较空载体组和对照组减慢(P<0.05),NOS抑制剂可以加快其生长,但无显著性差异(P>0.05)。结论:iNOS基因转染可以使DU145细胞分泌较高浓度的NO,诱导细胞凋亡,抑制细胞生长,为晚期雄激素非依赖性前列腺癌的基因治疗提供一个有效的靶点。     

Proliferation of DU145 prostate cancer cells is inhibited by suppressing insulin-like growth factor binding protein-2

BACKGROUND: Insulin-like growth factor binding protein-2 (IGFBP-2) is expressed by all human prostate cancer cell lines and dramatically increases in the serum of prostate cancer patients. However, the role of IGFBP-2 in prostatic tumorigenesis is not known. The aim of the present study was to investigate the effects of IGFBP-2 on the proliferation of DU145 human prostate cancer cells in culture. METHODS: Using cell proliferation assays, we examined the effects of exogenously administered and endogenously modulated levels of IGFBP-2 on the proliferation of DU145 cells. RESULT: Cell growth was stimulated by exogenously administered IGFBP-2, but significantly retarded (P < 0.05) by its neutralizing antibody. Overexpression of IGFBP-2 by transfection also stimulated cell growth, which was significantly (P < 0.05) inhibited in transfectants expressing antisense mRNA to IGFBP-2. Furthermore, the proliferation of IGFBP-2 overexpressing cells was significantly dampened by exogenously administered IGFBP-2 antibody. CONCLUSIONS: IGFBP-2 is an autocrine growth factor for DU145 human prostate cancer cells and cell proliferation can be significantly retarded by neutralizing or inhibiting its synthesis. These findings provide a strong rationale for targeting IGFBP-2 in the testing of novel strategies to treat prostate cancer.     

Adenovirus-mediated expression of a soluble fibroblast growth factor receptor inhibits in vitro growth of prostate DU145 cells

BACKGROUND: Fibroblast growth factor (FGF) family plays a key role in prostate cancer. The soluble FGF receptor (sFGFR) has been studied with regards to inhibiting cancer growth and was shown to have a dominant negative effect on cellular signaling and function. Using replication deficient adenovirus-mediated gene transfer, we tested if sFGFR expression may have a suppressive effect on in vitro growth of prostate cancer cells. METHODS: Western analysis was used to verify expression of sFGFR1 and to examine the effect of sFGFR1 on MAP kinase phosphorylation. The effect on proliferation and invasiveness of DU145 cells was examined using the WST-1 and Matrigel Invasion assay, respectively. RESULTS: Activation of MAP kinase (pERK1 and 2) by exogenous FGF1, 2, and 7 was suppressed to baseline levels by sFGFR, which was not seen with EGF. Proliferation and invasion of DU145 cells were significantly suppressed by sFGFR. CONCLUSIONS: A replication deficient adenoviral vector system reproducibly expresses sFGFR in prostate cells. Suppression of in vitro growth in DU145 cells by sFGFR provides the basis of a novel therapeutic approach.     

Growth-inhibitory activity of melatonin on human androgen-independent DU 145 prostate cancer cells

BACKGROUND: The pineal hormone melatonin has been shown to exert a direct oncostatic activity on neoplastic cells, particularly from breast cancer. In the present study, we evaluated the effects of melatonin on the proliferation and on the cell cycle distribution of human androgen-independent DU 145 prostate cancer cells. Experiments were also performed to gain insights into the possible mechanism of action of the hormone. METHODS: The effects of melatonin on DU 145 cell proliferation was analyzed by counting the cells by hemocytometer at the end of treatment. The effects of the pineal hormone on cell cycle distribution were evaluated by FACS analysis. RT-PCR studies were performed to detect Mel(1a) and Mel(1b) expression in DU 145 cells. The cellular localization of (125)I-melatonin binding sites was investigated by radioreceptor assay. A commercially available binding-protein assay kit was utilized to evaluate intracellular cAMP levels. RESULTS: Melatonin, in physiological doses, significantly inhibited DU 145 cell proliferation and induced cell cycle withdrawal by accumulating cells in G0/G1 phase. The mRNA for Mel(1a) receptors was found to be expressed in DU 145 cells; however, by radioreceptor assay, no binding sites for (125)I-melatonin could be detected in membrane preparations, suggesting that, in these cells, the level of translation of this mRNA is too low to possibly mediate the antiproliferative action of the hormone. In agreement with this hypothesis, melatonin did not affect forskolin-induced intracellular cAMP accumulation. Binding sites for (125)I-melatonin were found in nuclear extracts of DU 145 cells. CONCLUSIONS: Melatonin exerts a direct oncostatic activity on human androgen-independent prostate cancer cells, by affecting cell cycle progression. This activity seems to be mediated by nuclear, but not by membrane, receptors.     

茶多酚对前列腺癌细胞DU145生长的影响

目的:探讨茶多酚对前列腺癌细胞生长的影响及其作用机制。方法:选取激素非依赖性前列腺癌细胞系DU145为研究对象,在药物组的培养基中加入茶多酚使其终浓度分别为50、100、250、500μg/ml,对照组加入正常细胞培养基。各组细胞培养48 h后,采用MTT比色法检测细胞生存率,然后通过Western印迹分析和荧光定量RT-PCR法检测各组细胞survivin基因表达情况。结果:加入茶多酚溶液48 h后,各药物组细胞存活率分别为0.97±0.12、0.71±0.07、0.20±0.03和0.08±0.01,与对照组相比,除50μg/ml组(P=0.42)外,其余3个药物组细胞存活率均明显低于对照组(P﹤0.01)。随茶多酚作用时间增加,各组细胞存活率呈现下降趋势,到96 h时,除50μg/ml组,其余3组细胞存活率均在5%以下。加药48 h后,对照组、100、250、500μg/ml药物组的sur-vivin表达条带灰度值分别为15 075±48、13 425±31、2 017±24、1 274±22,与对照组相比,3个药物组survivin蛋白表达均明显减少(P均﹤0.01)。另外,50、100、250、500μg/ml药物组给药48 h时的survivin mRNA量分别为0.74±0.03、0.64±0.02、0.52±0.01、0.21±0.02,均明显少于对照组(P均﹤0.01)。结论:茶多酚可以抑制前列腺癌DU145细胞的生长,且这种作用可能与survivin基因表达减少有关。     

隐丹参酮对前列腺癌DU145细胞中异黏蛋白表达的影响

目的:探讨隐丹参酮对前列腺癌DU145细胞增殖及细胞凋亡的作用,并初步探讨隐丹参酮对DU145细胞中异黏蛋白(MTDH)表达及下游PI3K/AKT信号通路的影响。方法:四氮唑蓝(MTT)比色法检测不同浓度隐丹参酮分别作用DU145细胞24、48、72 h后对细胞的生长抑制作用;原位末端转移酶标记(TUNEL)法检测细胞凋亡情况;Western印迹检测不同浓度隐丹参酮及作用不同时间对DU145细胞中MTDH蛋白的表达影响;RT-PCR技术检测隐丹参酮分别作用DU145细胞12、24、48 h后细胞中MTDH mRNA的表达情况;Western印迹检测隐丹参酮作用DU145细胞48 h后细胞中MTDH、AKT、p-AKT、Bcl-2蛋白的表达情况。结果:隐丹参酮能够明显抑制DU145细胞增殖,且抑制效应呈剂量和时间依赖性(P0.05);以10μmol/L隐丹参酮作用DU145细胞24、48、72 h后细胞凋亡率分别为(29.42±4.51)%、(55.07±5.67)%和(70.84±4.66)%,明显高于对照组(3.1±2.48)%(P0.05)。Western印迹和RT-PCR结果显示,隐丹参酮可在转录和翻译水平下调MTDH的表达(P0.05),抑制AKT信号通路和抗凋亡蛋白Bcl-2的表达(P0.05)。结论:隐丹参酮可抑制前列腺癌DU145细胞的增殖,促进细胞凋亡,其机制可能是通过下调MTDH表达,抑制其下游PI3K/AKT信号通路。     

Secretion of epidermal growth factor and related polypeptides by the DU 145 human prostate cancer cell line

Epidermal growth factor (EGF)-like proteins may be important in regulating the growth of human prostate cancer cells and in the development of hormone independence. In the present study, we demonstrated that the DU 145 human prostate cancer cell line secretes EGF-like polypeptides into serum-free culture medium. The production of both authentic EGF and transforming growth factor-alpha was demonstrated by specific radioimmunoassays. In addition to 6-7k monomers, immunoreactive higher molecular weight species were isolated by gel chromatography. The DU 145 cells also specifically bound human EGF, with both high- (Kd 1.8 x 10(-10) M) and low- (Kd 1.1 x 10(-9) M) affinity binding sites. Exogenous EGF stimulated 3H-thymidine incorporation when cells were plated at low density in serum-free culture medium, while at higher cell density neither cell division nor 3H-thymidine incorporation was significantly altered. We postulate that DU 145 cells may be autologously stimulated by endogenously produced EGF and related polypeptides, which, under normal culture conditions, precludes any further effect of exogenous EGF.     

薄荷醇抑制前列腺癌DU145细胞的增殖和迁移

目的探讨薄荷醇对雄激素非依赖性前列腺癌DU145细胞增殖和迁移能力的影响。方法通过RT-PCR、免疫组织化学和Western blot方法检测TRPM8和TRPA1的表达;MTT和划痕试验检测薄荷醇对DU145细胞的增殖和迁移能力的影响;流式细胞术检测TRPM8对DU145细胞周期和凋亡的影响。结果 RT-PCR、免疫组织化学和Western blot提示TRPM8在DU145细胞中高表达而TRPA1在DU145细胞中不表达;薄荷醇能诱导细胞周期阻滞于G0/G1期,与未经薄荷醇处理的细胞相比,经100μmol/L薄荷醇处理的细胞培养24、48和72h后,G0/G1期细胞显著增加(49.12%±1.92%vs61.71%±2.70%、77.65%±1.63%、71.81%±2.46%,P<0.05,P<0.01),进而抑制细胞增殖(P<0.05),并抑制细胞迁移(P<0.05),流式细胞术检测显示薄荷醇并不引起细胞凋亡。结论 TRPM8可能成为前列腺癌治疗的一个新靶点,对于高表达TRPM8的雄激素非依赖性前列腺癌针对TRPM8通道的药物治疗可能比TRPM8基因治疗更为实用,因此,薄荷醇作为一个潜在的抗肿瘤药物也拥有很大的发展前景。     

Autocrine regulation of DU145 human prostate cancer cell growth by epidermal growth factor-related polypeptides

The DU145 human prostate cancer cell line possesses epidermal growth factor (EGF) receptors and synthesizes both EGF and the related polypeptide transforming growth factor-alpha (TGF-alpha). A monoclonal antibody to the EGF receptor was used to determine whether these characteristics were indicative of a functional autocrine regulatory system. This antibody competed effectively with [125I]EGF for binding to DU145 cell binding sites over a 1 x 10(-11) to 1 x 10(-7) M concentration range, and did so with a capability similar to that of the two natural ligands. It inhibited growth of these cells in both 3% fetal bovine serum-supplemented and serum-free medium; in experiments with incubation times of 3-5 days there was a 45-50% reduction in cell number. Growth suppression by the EGF receptor blockade of cells plated at a density of 1.5 x 10(4) cells/ml/well was reversed competitively by the addition of EGF to the medium; 0.3 nM completely eliminated the inhibitory effect of a 1 x 10(-9) M antibody concentration. It is concluded that DU145 cell growth is regulated by an EGF-mediated autocrine loop.     

表皮生长因子信号通路对前列腺癌细胞DU145表面整合素α5、β1的调控

目的 探讨表皮生长因子（EGF）信号通路对雄激素非依赖型前列腺癌细胞系DU145表面整合素α5、β1的调控.方法 应用流式细胞术、逆转录聚合酶链反应（RT-PCR）和Western蛋白印迹（Western blot）法测定EGF、丝裂原活化蛋白激酶（MAPK）信号通路抑制剂PD98059对DU145细胞整合素α5和β1表达的影响;采用细胞粘附能力测定和Transwell小室法检测EGF及PD98059对DU145细胞粘附能力和迁移能力的影响.结果 EGF上调DU145细胞表面整合素β1亚基的蛋白和mRNA的表达,分别为对照组的231%和248%（方差值为9.0和24.6,P均＜0.01）,并可促进DU145细胞对纤维连接蛋白（Fn）的粘附和迁移能力.而PD98059则具有相反的作用,下调DU145细胞表面整合素β1亚基的蛋白和mRNA的表达,分别为对照组的60%和63%（方差值为6.3和15.3,P均＜0.01）,并可抑制DU145细胞对Fn的粘附和迁移能力.EGF和PD98059对整合素α1的表达无明显影响.结论 EGF可能通过MAPK信号通路上调前列腺癌DU145细胞表面整合素β1亚基的表达,从而促进DU145细胞对Fn的粘附能力和迁移能力;此调节作用主要发生在mRNA转录水平.     

环巴胺对DU145细胞增殖和细胞周期的影响

目的:研究Hedgehog信号通路阻断剂(环巴胺)对DU145细胞增殖的抑制作用。方法:不同浓度(1、10、50、100μmol/L)环巴胺干预DU145细胞,分别在24、48、72h后采用噻唑蓝比色法检测其对细胞增殖的抑制作用;流式细胞术检测环巴胺对细胞周期的影响;RT-PCR检测50μmol/L环巴胺作用48h后实验组和对照组细胞周期蛋白E(cyclinE)mRNA表达水平的差异。结果:环巴胺对DU145细胞的抑制作用呈时效和量效依赖关系,当浓度>10μmol/L作用24h后会显著抑制细胞增殖,10、50、100μmol/L浓度组对细胞的抑制率分别为7.42%、12.70%和59.15%,与空白对照组相比,差异有统计学意义(P<0.05)。流式细胞术检测发现,当环巴胺浓度达10μmol/L以上,干预48h后G1期细胞比例明显升高。对照组、10、50μmol/L浓度组的G1期细胞百分比分别为:(52.17±2.21)%、(60.13±2.75)%和(74.30±3.52)%,差异有统计学意义(P<0.01);凋亡峰也随环巴胺浓度增加而逐渐增高。50μmol/L环巴胺作用48h后DU145细胞cyclinEmRNA表达显著降低,与空白对照组相比降低61.90%(P<0.01)。结论:环巴胺可以抑制DU145细胞的增殖能力,其机制可能与下调DU145细胞cyc-linEmRNA表达水平,从而将DU145细胞阻滞于G1期有关。环巴胺亦可以诱导DU145细胞凋亡。     

表皮生长因子信号通路对前列腺癌细胞DU145黏附和迁移能力的影响

目的 探讨表皮生长因子（EGF）信号通路对雄激素非依赖型前列腺癌细胞株DU145黏附和迁移能力的影响。方法 应用细胞黏附能力测定和划痕实验测定EGF对DU145黏附和迁移能力的作用，应用流式细胞术测定EGF对DU145细胞表面整联蛋白α5和β1表达的影响，应用逆转录-聚合酶链反应（RT-PCR）和Western blot测定EGF对DUl45细胞整联蛋白α5和β1亚基总蛋白和mRNA表达的影响。结果 EGF可促进DUl45对纤连蛋白（Fn）的黏附和迁移作用，同时，Fn的受体a5β1的表达发生了变化，EGF上调细胞表面β1亚基的表达，而丝裂源激活蛋白激酶（MAPK）信号通路抑制剂PD98059则具有相反的作用，这种调节作用主要发生在mRNA水平上。结论 EGF可能通过MAPK信号通路，上调β1的表达，从而促进DU145细胞的侵袭能力。     

Stable lower PAR expression decreased DU145 prostate cancer cell growth in SCID mice.

BACKGROUND: PAR is a novel gene ubiquitously expressed in normal and malignant tissues with a trend towards higher expression in tumor cells. PAR biological function is unknown. Here we report the effect of lowering PAR expression on in vitro and in vivo proliferation of DU145 cells. METHODS: Decreased PAR expression was achieved by stable transfection of DU145 cells with antisense PAR cDNA cloned in pCMV-Script expression vector. The proliferative potential of DU145 transfectants was studied by cell counts, colony formation in soft agar, flow cytometry, and growth in severe combined immunodeficient (SCID) mice. RESULTS: DU145 transfectants exhibited a decreased cell proliferation in tissue culture and a low efficiency of colony formation in soft agar. Flow cytometry revealed an arrest of these cells in G2-M phase of mitotic cycle. A dramatic decrease of tumor growth was observed when DU145 transfectant cells were inoculated in SCID mice, compared with controls. Histological examination of these tumors showed a marked decrease in cell density and in number of mitoses while control tumors showed a high cell density and numerous mitoses. CONCLUSIONS: The data presented here provide the first evidence for PAR gene cellular function and its possible implication in malignant transformation.     

Inactivation of the NF2 tumor suppressor protein merlin in DU145 prostate cancer cells

BACKGROUND: The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact-dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21-activated kinase (PAK) inactivates merlin's growth suppressing function, and is regulated by cell-culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells. METHODS: Phosphorylation of merlin in five prostate cancer cell lines (LNCaP, DU145, PC3, 22RV1, and LAPC-4) was examined by Western blotting using anti-phospho-merlin (Ser518) antibody. The activity of PAK, an upstream regulator of merlin phosphorylation, was measured by Western blotting using phospho-PAK (Ser141) antibody. The effects of various cell-culture conditions on the phosphorylation levels of merlin and PAK were analyzed. RESULTS: Both merlin expression and phosphorylation were low in LNCaP, PC3, 22RV1, and LAPC-4 prostate cancer cells. In DU145 cells, total and phosphorylated merlin were abundant, but phosphorylation was not inhibited by high cell density, serum withdrawal, the addition of hyaluronic acid or inhibition of CD44 expression, all of which are reported to inhibit merlin phosphorylation in non-neoplastic cells. PAK activation was elevated in DU145 cells and the addition of a PAK-specific inhibitor peptide but not the Rac1-specific inhibitor NSC23766 inhibited both PAK and merlin phosphorylation. CONCLUSIONS: Merlin is inactivated in DU145 prostate cancer cells by PAK-mediated constitutive phosphorylation, identifying a novel mechanism of merlin inactivation in neoplastic cells.     

NK4基因转染对前列腺癌DU145细胞增殖、迁移、侵袭和凋亡的影响

研究HGF拮抗剂NK4在前列腺癌中的作用。将包含NK4cDNA的表达载体pBudCE4．1-EGFP-NK4转染到DU145细胞中。体外实验检测白分泌的NK4对肿瘤细胞增殖、迁移、转移及凋亡的影响。体内实验裸鼠分为三组,分别皮下种植DUl45、空质粒转染的Dul45和NK4转染的DUl45细胞,检测皮下肿瘤的大小、细胞凋亡及细胞增殖情况。体外实验结果显示转染NK4的DUl45细胞可以分泌NK4蛋白。自分泌的NK4抑制HGF诱导的肿瘤细胞增殖、迁移及转移,促进凋亡（P〈0．01）。NK4可以调节HGF受体c－Met及其下游ERKl与Akt1／2蛋白的活性。体内实验显示,NK4转染DUl45细胞组的肿瘤生长及细胞增殖受到抑制,同时肿瘤细胞凋亡增加。本实验显示包含NK4cDNA的表达载体转染前列腺癌细胞可以有效地调节肿瘤细胞的增殖、迁移、侵袭及凋亡。NK4作用于HGF／c－Met可以作为前列腺癌治疗的一个有效靶点。     

DU145 human prostate cancer cells express functional receptor activator of NFkappaB: new insights in the prostate cancer bone metastasis process

Prostate cancer metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer, frequently resulting in significant morbidity and mortality. As the underlying mechanisms are not fully characterized, understanding the biological mechanisms that govern prostate cancer metastases to bone at the molecular level should lead to the determination of new potential therapeutic targets. Receptor activator of NFkappaB ligand (RANKL)/RANK/Osteoprotegerin (OPG) are the key regulators of bone metabolism both in normal and pathological condition, including prostate cancer bone metastases. In the present study, we demonstrated that human prostate cancer cell lines, DU145 and PC3 express biologically functional RANK. Indeed, soluble human RANKL (shRANKL, 100 ng/ml) treatment induced ERK 1/2, p38 and IkappaB phosphorylations in these cells. shRANKL administration also promoted DU145 and PC3 prostate cancer cell invasion in vitro. Whereas human OPG (hOPG) administration alone (100 ng/ml) had no marked effect, combined association of both agents abolished the RANKL-induced DU145 cell invasion. As RANKL had no direct effect on DU145 cell proliferation, the observed effects were indeed related to RANKL-induced cell migration. DU145 human prostate cancer cells promoted osteoclastogenesis of osteoclast precursors generated from mouse bone marrow. Moreover, DU145 cells produced soluble factor(s) that up-regulate the proliferation of MC3T3-E1 pre-osteoblasts through the activation of the ERK 1/2 and STAT3 signal transduction pathways. This stimulation of pre-osteoblast proliferation resulted in an increased local RANKL expression that can activate both osteoclasts/osteoclast precursors and prostate cancer cells, thus facilitating prostate cancer metastasis development in bone. We confirm that RANKL is a factor that facilitates metastasis to bone by acting as an activator of both osteoclasts and RANK-positive prostate cancer cells in our model. Furthermore, the present study provides the evidence that blocking RANKL-RANK interaction offer new therapeutic approach not only at the level of bone resorbing cells, but also by interfering with RANK-positive prostate cancer cells in the prostate cancer bone metastasis development.