Enhanced invasion of hormone refractory prostate cancer cells through hepatocyte growth factor (HGF) induction of urokinase-type plasminogen activator (u-PA)

BACKGROUND: Increased expression of the hepatocyte growth factor (HGF) receptor (MET) is associated with high-grade prostatic adenocarcinoma and metastasis. However, the mechanism through which MET signaling contributes to prostate cancer (CaP) metastasis remains unclear. METHODS: Human PC-3 CaP cells and in vivo selected, isogeneic variant cells of increasing metastatic potential (PC-3M, PC-3M-Pro4, and PC-3M-LN4) were used to investigate the effect of HGF on CaP cell growth, protease production, and invasion. Cell-free urokinase-type plasminogen activator (u-PA) expression and function following HGF treatment were analyzed by Western blot, ELISA, and casein/plasminogen zymography. In vitro invasion stimulated by HGF was measured using Matrigel-coated invasion chambers. RESULTS: Both mRNA and functional protein for MET were detected in each of the CaP cell lines. HGF treatment (0-40 ng/ml) weakly increase proliferation, however, HGF induced soluble u-PA protein and activity 3-fold in the metastatic variant cells. HGF significantly stimulated the invasion of highly metastatic PC-3M-LN4 cells through Matrigel and treatment with specific urokinase receptor inhibitors diminished the HGF-stimulated invasion in a dose-dependent manner. CONCLUSIONS: These results demonstrate the biological significance of u-PA up-regulation in response to HGF in highly metastatic hormone refractory CaP cells.     

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.     

Serum hepatocyte growth factor activator (HGFA) in benign prostatic hyperplasia and prostate cancer

OBJECTIVES: Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor (HGF). We attempted to clarify whether serum levels of HGFA could be used as a marker for prostate cancer. MATERIAL AND METHODS: Serum levels of total HGF and HGFA were measured by enzyme-linked immunosorbent assay in 99 healthy controls, 27 patients with benign prostatic hyperplasia (BPH) and 119 patients with prostate cancer. RESULTS:: The mean+/-S.D. serum levels of HGFA in untreated prostate cancer and BPH cases were 0.42+/-0.24 and 0.50+/-0.26 ng/ml, respectively (no significant difference). Serum HGFA was significantly elevated in hormone-refractory prostate cancer (stage D3) compared to other stages, while HGF did not significantly differ with regard to clinical stage. CONCLUSIONS: Serum HGFA tends was elevated in patients with advanced stage prostate cancer. Further studies in large groups of patients are needed to clarify the clinical value of HGFA.     

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.     

Effects of hepatocyte growth factor on E-cadherin-mediated cell-cell adhesion in DU145 prostate cancer cells

Objectives. To examine how hepatocyte growth factor (HGF) affects cell-cell adhesion junctions on scattering in prostate cancer cells. HGF is known to induce scattering (dispersion of clustered cells into single cells) in various epithelial cells, including prostate cancer cells, but the mechanisms surrounding this action are not fully understood. Cell-cell adhesion junctions are composed of E-cadherin and its associated intracellular catenins and play important roles in the maintenance of cell integrity.Methods. The human prostate cancer cell line DU145 was used in this study. The associations and changes of various adhesion molecules with HGF treatment were investigated by inhibition assays, Western blot analysis, and immunofluorescence staining.Results. In the inhibition assay, anti-E-cadherin neutralizing monoclonal antibody caused the dissociation of DU145 cells similar to the scattering with HGF treatment. The expression of E-cadherin decreased with HGF, and the expression of alpha-catenin and beta-catenin did not change by Western blot analysis. In immunofluorescence staining, HGF caused the translocation of E-cadherin from cell-cell adhesion junctions to the cytoplasm.Conclusions. These results indicate that HGF induces scattering by decreasing the expression of E-cadherin and causes its translocation to the cytoplasm of DU145 cells.     

Monocyte chemotactic protein-1 (MCP-1) acts as a paracrine and autocrine factor for prostate cancer growth and invasion

BACKGROUND: Monocyte chemotactic protein-1 (MCP-1) plays a key role in the recruitment and activation of monocytes during inflammation. Increased MCP-1 serum levels in patients with various cancers were correlated with advanced stage. Here, we evaluated the role of MCP-1 on prostate cancer (CaP) cell proliferation and invasion. METHODS: Expression of MCP-1 in tissue specimens was analyzed by immunohistochemical staining. MCP-1 production was determined by ELISA in conditioned media collected from primary prostate epithelia (PrEC), LNCaP, C4-2B, PC3 cells, and hFOB. Cell proliferation and invasion were assayed by MTS assay and invasion chambers. RESULTS: All CaP cells, as well as hFOB, produced high amount of MCP-1 compared to PrEC cells. MCP-1 expression levels were associated with advanced pathologic stage. MCP-1 induced proliferation and invasion of CaP cells and this was abolished partially either by CCR2 antagonist or PI3 Kinase inhibitor. CONCLUSION: MCP-1 acts as a paracrine and autocrine factor for CaP growth and invasion.     

【原创论文】组蛋白甲基转移化酶基因SETDB1促进前列腺癌细胞的增殖、迁徙和侵袭

SETDB1已在部分人类肿瘤中被确立为癌基因。本研究的目的是检测艇珏坫J基因及蛋白在人前列腺癌组织、细胞系、前列腺增生组织、前列腺上皮细胞中的表达。并研究SETDB1基因对前列腺癌细胞在体外的增殖、迁徙、侵袭的作用。方法：应用实时定量聚合酶链反应（Real-timeqPCR）免疫组化检测SETDB1基因及蛋白在人前列腺癌组织、细胞系、前列腺增生组织、前列腺上皮细胞中的表达。借助siRNA下调SETDB1在前列腺癌细胞系的表达,分别应用细胞计数实验、细胞克隆形成实验、流式细胞技术;细胞划痕实验、细胞小室侵袭实验对SETDB1下调后的细胞进行检测。结果：SETDB1在人前列腺组织、细胞中高表达,下调SETDB1在前列腺癌细胞系的表达后,前列癌细胞的增殖、迁徙、侵袭能力降低。结论：SETDB1可以作为前列腺癌的癌基因进一步研究。     

Prosaptide TX14A stimulates growth, migration, and invasion and activates the Raf-MEK-ERK-RSK-Elk-1 signaling pathway in prostate cancer cells

BACKGROUND: Prosaposin is a neurotrophic factor. Prosaposin knock-out mice have been reported to develop a number of abnormalities, including atrophy of the prostate gland and mitogen-activated protein kinase (MAPK)-inactivation in prostate epithelial cells. These abnormalities underscore a potential fundamental role in prostate development. The trophic factor activity of prosaposin has been localized at a specific amino terminal portion of the molecule that has been the source for a number of biologically active peptides called prosaptides (e.g., TX14A). The expression and function of prosaposin in prostate cancer is not known. METHODS: Using conventional protein expression analysis, immunohistochemical staining, cell proliferation assays, and in vitro invasion assays, we determined the expression of prosaposin and the effect of prosaptide TX14A on cell growth/death protection, motility, invasion, and MAPK signal transduction pathway in prostate cancer cells. RESULTS: We found a higher expression of prosaposin in androgen-independent (AI) prostate cancer cells (PC-3 and DU-145) than in androgen-dependent (AD) LNCaP or normal prostate epithelial cells. Immunohistochemical staining on benign and malignant prostate tissues revealed an intense cytoplasmic anti-prosaposin immunoreactivity in tumor cells, as well as stromal, endothelial, and inflammatory mononuclear cells. The intensity of staining was proportional to the overall Gleason's score. In addition, we demonstrated that TX14A stimulates cell proliferation/survival, migration, and invasion, and activates the Raf-MEK-ERK-RSK-Elk-1 signaling cascade of the MAPK pathway. CONCLUSIONS: These results are suggestive of a potential pleuripotent regulatory function for prosaposin in prostate cancer.     

Serum hepatocyte growth factor activator inhibitor type I (HAI-I) and type 2 (HAI-2) in prostate cancer

BACKGROUND: Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are Kunitz-type serine protease inhibitors for hepatocyte growth factor activator (HGFA). We attempted to clarify whether serum levels of HAI-1 and HAI-2 could be a useful marker in patients with prostate cancer. METHODS: Serum levels of HAI-1 and HAI-2 were measured by enzyme-linked immunosorbent assay in 27 patients with benign prostatic hyperplasia (BPH) and 118 patients with prostate cancer. RESULTS: The mean serum levels of HAI-1 in patients with prostate cancer were significantly higher than those in patients with BPH. Furthermore, the serum HAI-1 levels in patients with distant metastasis and hormone resistant prostate cancer were significantly elevated compared with those in patients with organ-confined diseases. There were no significant differences in serum HAI-2 levels among prostate cancer subgroups according to clinical stage. Significantly elevated levels of HAI-1 were detected in 38 patients with prostate cancer before any treatment. CONCLUSIONS: HAI-1 may be a potential tumor marker for prostate cancer. Further studies in large groups of patients are needed to define the clinical value of HAI-1.     

Pentosan polysulfate (Elmiron): in vitro effects on prostate cancer cells regarding cell growth and vascular endothelial growth factor production

BACKGROUND: Elmiron (ALZA Corp, Mountain View, CA) is the only Food and Drug Administration-approved oral therapy for interstitial cystitis. We hypothesized that Elmiron would affect the growth of prostate cancer in vitro. METHODS: Prostate cancer cell lines (LnCaP, PC3, and DU145) were treated with Elmiron. Cell viability was measured by MTT (3-4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), whereas vascular endothelial growth factor (VEGF) was measured by a commercial enzyme-linked immunosorbent assay. RESULTS: Inhibition of cell growth was observed in all cell lines tested. LnCaP exhibited a mean inhibition of 12% +/- 7% at 24 hours (P = .025) and 20% +/- 15% at 72 hours (P < .001). PC3 exhibited a mean inhibition of 26% +/- 13% at 24 hours (P < .001) and 44% +/- 5% at 72 hours (P < .001). DU145 exhibited a mean inhibition of 9% +/- 6% at 24 hours (P < .015) and 30% +/- 5% at 72 hours (P < .001). PC3 cells exhibited a significant reduction in VEGF levels (P < .001). CONCLUSIONS: The reductions in cell growth and VEGF indicate that Elmiron may act as an antiangiogenic agent and may have application in the treatment of prostate cancer.     

Influence of hepatocyte growth factor secreted from fibroblasts on the growth and invasion of scirrhous gastric cancer

The objective of this study was to show that hepatocyte growth factor (HGF) and HGF receptor (c-met protein) play an important role in the cancer growth and infiltration in scirrhous gastric cancer. The expression level of c-met protein was examined in 90 cases of advanced gastric cancer using anti-c-met antibody. Co-cultivation of each of four gastric cancer cell lines with gastric fibroblasts was performed using a double chamber method. The expression rate of c-met was 79.5% in type 4 tumors, significantly higher than in other types. The expression rates were 63.6% in undifferentiated-type cancer and 36.3% in differentiated-type cancer. Co-cultivation of undifferentiated-type cancer with fibroblasts showed a significantly higher HGF concentration than fibroblasts cultured alone. The growth in three undifferentiated-type cancers was accelerated by an addition of rhHGF and by co-cultivation with fibroblasts and was inhibited by anti-HGF antibody. Moreover rhHGF stimulated the invasion activity of undifferentiated cancer cell lines. These findings suggested that gastric fibroblasts in scirrhous cancer stimulate tumor growth and invasion through activation of the HGF/c-met system.     

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.     

硼替佐米体外对前列腺癌细胞迁移和侵袭的影响及其机制

目的：探讨硼替佐米体外对前列腺癌细胞迁移和侵袭的影响及其机制。方法：选择对数生长的LNCAP前列腺癌细胞株,分别选择不同浓度的硼替佐米处理,在处理24h后进行前列腺癌细胞迁移和侵袭的检测,同时收集细胞进行炎症因子表达实验和Western blot检测分析。结果：LNCAP+硼替佐米(10nmol/L)组、LNCAP+硼替佐米(20nmol/L)组细胞的迁移指数与侵袭指数都明显低于对单独LNCAP组,并且IL-12及TNF-α表达量明显低于单独LNCAP组,对比差异都有统计学意义(P<0.05)不同浓度的硼替佐米处理LNCAP细胞24h后,FAK总蛋白无明显变化,对比差异都无统计学意义(P>0.05)。结论：替佐米能够有效的抑制前列腺癌LNCAP细胞的迁移和侵袭,其作用的发挥与抑制IL-12及TNF-α的表达分泌有关,但对FAK总蛋白的表达无影响。     

GLI-1参与表皮生长因子介导的前列腺癌ARCaP_E细胞体外侵袭活性强化的实验研究

目的:探讨Hedgehog(HH)信号通路效应蛋白GLI-1在表皮生长因子(EGF)介导的人前列腺癌AR-CaPE细胞系体外侵袭活性增强中的作用。方法:以人前列腺癌ARCaP细胞系作为研究模型,免疫荧光技术鉴定ARCaPE内EGF受体(EGFR)和GLI-1的表达情况;100 ng/ml EGF体外作用于ARCaPE后,观察细胞的形态及体外侵袭能力的变化,采用Western印迹检测细胞内ERK信号通路成分和GLI-1蛋白的表达变化情况;Transwell侵袭实验检测EGF(100 ng/ml)与GLI-1拮抗剂GANT61(10μmol/L)单独或联合作用对ARCaPE细胞体外侵袭能力的影响。结果:ARCaPE细胞同时表达EGFR与GLI-1蛋白;EGF诱导上皮样外观的ARCaPE细胞向间质样外观的AR-CaPM转化,增强ARCaPE细胞的体外侵袭能力并显著上调细胞内p-ERK和GLI-1蛋白的表达水平(P<0.05);GANT61明显抑制ARCaPE细胞的体外侵袭能力且减弱EGF对细胞侵袭能力的增强效应(P<0.05)。结论:HH信号通路和EGF/ERK信号通路之间存在一定的相互作用,GLI-1可能在EGF介导的人前列腺癌ARCaPE细胞体外侵袭活性增强过程中发挥着重要作用。     

Inhibition of cortactin and SIRT1 expression attenuates migration and invasion of prostate cancer DU145 cells

Objectives: Cortactin is overexpressed in various types of cancer and enhances cell motility. It has been recently reported that silent mating type information regulation 2 homolog 1 interacts with cortactin and promotes cell migration. Here, we examined the role of cortactin and silent mating type information regulation 2 homolog 1 in migration and invasion of prostate cancer cells. Methods: The cortactin expression levels in DU145, LNCaP and PC3 prostate cancer cells, and in PrEC normal human prostate epithelial cells were evaluated by western blot analysis. In DU145 cells, the expression of cortactin or silent mating type information regulation 2 homolog 1 was inhibited by small interfering RNA, and the effects of their knockdown on migration and invasion were examined by cell migration and invasion assays. To determine the localization of cortactin and silent mating type information regulation 2 homolog 1, western blot and immunofluorescence microscopic analyses were carried out. The functional interaction between silent mating type information regulation 2 homolog 1 and cortactin was also studied by in vivo acetylation assay. Results: The protein expression of cortactin was significantly higher in DU145 cells than in other cell lines. Knockdown of cortactin or silent mating type information regulation 2 homolog 1 expression inhibited both migration and invasion of DU145 cells. Similarly to cortactin, silent mating type information regulation 2 homolog 1 was found to be predominantly expressed in the cytoplasm. Finally, the knockdown of silent mating type information regulation 2 homolog 1 expression increased the acetylation level of cortactin. Conclusions: Our findings suggest that inhibition of cortactin or silent mating type information regulation 2 homolog 1 expression attenuates migration and invasion of DU145 cells and this could represent a promising strategy to regulate metastasis of prostate cancer.