NK4 And TRPC6 Cation Channel Operate The Biological Outcome Of HGF In Prostate Cancer

IntroductionProstate cancer (PC) is one of the leading threats to men's health in western societies. Following metastasis to bone and lymph nodes, the primary cause of prostate cancer mortality is the progression from androgen dependent (AD) to andro gen-independent (AI) growth. In the early stages of the disease, androgen-ablation therapy can cause tumor regression, and is currently the most successful treatment option. However, once the tumor achieves androgen independence, there is no effective therapy. Therefore, new prognostic markers and therapeutic strategies may be of great benefit to prostate cancer patients.Hepatocyte growth factor (HGF) is a heterodimers molecule with a number of biological activities, including regulation of migration, invasion and angiogenesis in cancer. HGF is composed of aα-chain, containing the N-terminal hairpin domain and four kringle domains, and a serine protease-likeβ-chain. Over expression of HGF and its receptor, c-Met, in prostate cancer has been reported. Moreover, a higher plasma level of HGF in prostate cancer patients is associated with an advanced stage of malignancy and a poor prognosis.Recently, the HGF antagonist, NK4, was developed. It is a variant form of HGF, comprising the N-terminal and the subsequent four kringle domains of HGF. Since NK4 retains the capacity to bind to the HGF receptor, c-Met, it competes with HGF and inhibits the biological activity of HGF, such as cell proliferation, migration and morphologicchanges. But NK4 itself can't induce c-Met phosphorylation. Competitive inhibitory effects of NK4 on HGF/c-Met have been demonstrated in some types of human cancer cells. In addition, HGF is intimately involved in growth of human prostate cancer and that progression from the androgen-dependent to the androgen-independent state. Using these characteristics of prostate cancer, we have found a new approach for investigating prostate cancer.Cytosolic Ca2+ is an important transducer of HGF signal. HGF combines with c-Met to active phospholipase C (PLC), and then phosphatidylinositol diphosphate (PIP2) is hydrolyzed, generating inositol triphosphate (IP3) and diacylglycerol (DAG). DAG can activate receptor operated calcium channels (ROCC) and caused Ca2+ influx. The increasing of intracellular free Ca2+ active some protein phosphatase, thereby, affected cell DNA replication, leading to cell malignant transformation and tumor cell proliferation and differentiation. Intracellular Ca2+ is related to tumor proliferation, which is involved in the regulation of tumor growth, invasion, metastasis and differentiation. However, the mechanism of Ca2+ entry is unclear.Transient receptor potential (TRP) channels are a large family of nonselective cation channels. TRPC channels, a subfamily of TRP channels in mammalian cells, include 7 members (TRPC1～7), and are mostly permeable to Ca2+. TRPC6, an important way of intracellular calcium signal generation, could regulate intracellular calcium concentration and enzyme activity, which directly or indirectly affect cell biological behaviour. Recently, it has been shown that in Huh-7 cells, the expression of EGF and HGF receptors in hepatocarcinoma probably induces TRPC6 expression, increasing the rate of cell proliferation. Additionally, TRPC6 is critical for HGF-induced growth, migration and morphogenesis in human renal tubular cell line HK2. RNA interference could effectively knock down the expression of target genes. We used TRPC6siRNA to explore the role of TRPC6 in HGF-induced prostate cancer cell proliferation.The above results suggest HGF has a critical role in the growth of PC. To clarify the role of HGF on the growth of PC, We designed the following experiment:①We used the pBudCE4.1-EGFP/NK4 expressing NK4 gene to investigate the anti-tumor effect and the possible mechanism of NK4 on human prostate cancer DU145 cells. This study may supply an experimental basis for the clinical application of NK4 in prostate cancer treatment.②We studied TRPC6 protein expression in benign and malignant human prostate tissues, and correlated TRPC6 protein immunostaining with the stage, grade and androgen responsiveness of the tumors. Furthermore, study was designed to investigate the possible relationship between TRPC6 and HGF and the function of TRPC6 channels in cell proliferation of prostate cancer by using physiological and molecular techniques.Material and methodsⅠ. Material1. Cell linesThe 22Rv1 and DU145 prostate cancer cell lines were obtained from Institute of Basic Medical Sciences Chinese Academy of Medical Sciences.2. SpecimenClinical specimens were collected from the patients registered at Shengjing Hospital of China Medical University (Shenyang, China) between 2004 and 2008.153 prostate cancer samples derived from 142 patients (1 sample in 131 patients,2 samples in 11 patients) and 20 benign prostate tissues derived from BPH patients for TRPC6 protein staining.Ⅱ. Methods1. Plasmid constructionPlasmid pBudCE4.1-EGFP/NK4 was constructed, transformed, amplified and purified. Plasmids were transfected into DU145 cells by Lipofectamine 2000. The cells were selected in the presence of Zeocin, and resistant clones were obtained. Western blot was carried out to detect the expression of NK4 protein.2.MTTAfter transfection, the proliferation of cell was detected by MTT. 3. In vitro migration assayTranswell chamber was used to determine the motility of prostate cancer cells.0.2 ml cells(1×105/ml)was added to the upper well. The chambers were placed in lower wells containing 0.5 ml of RPMI 1640 supplemented with 10% fetal calf serum and 10 ng/ml of HGF for 12 h in 5% CO2 at 37℃. At the end of the incubation period, cells in the upper chamber were removed, and the migrating cells were fixed with cold methanol/acetic acid and then stained with Giemsa. The number of migrating cells was evaluated by random observation of five fields on every section. The average number of cells per field was defined as migration index.4. In vitro invasion assayMatrigel-coated invasion chambers were used to determine the motility of prostate cancer cells.0.2 ml cells (1×105/ml) was added to the upper well. The chambers were placed in lower wells containing 0.5 ml of RPMI 1640 supplemented with 10% fetal calf serum and 10 ng/ml of HGF for 12 h in 5% CO2 at 37℃. At the end of the incubation period, cells in the upper chamber were removed from the top of the filter and the migrating cells were fixed with cold methanol/acetic acid for 30 min, and then stained with Giemsa. The number of migrating cells was evaluated by random observation.5. ImmunohistochemistrySections from paraffin-embedded specimens were deparaffinized and rehydrated, then were incubated at room temperature overnight in an anti-TRPC6 antibody. In control experiments, the primary antibody was replaced with PBS. The TRPC6 in immunohistochemistry score was evaluated by a grading system ranging from 0 to 5:0, 0%～1% of tumor cells are positive; 1,1%～5% of tumor cells positive; 2,5%～10% of tumor cells positive; 3,10%～20% of tumor cells positive; 4,20%～50% of tumor cells positive; 5,> 50% of tumor cells positive.6. RT-PCR analysis Total RNA was isolated from cells using Trizol Reagent. Total RNA was then reverse-transcribed into cDNA. For the PCR reaction, specific sense and antisense primers were selected. The bands were quantified densitometrically.7. Western blotTotal proteins from prostate cancer cells were harvested. Protein samples were separated by electrophoresis on SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed with anti-c-Met, anti-phosphorylation- c-Met, anti-Aktl/2, anti-phosphorylation-Akt1/2, anti-ERK1, anti-phosphorylation- ERK1, anti-HGF-α, anti-TRPC6 and anti-β-actin antibody, then developed with the enhanced chemiluminescence system using specific peroxidase-conjugated anti-IgG secondary antibodies. The bands were quantified densitometrically.8. Silencing of TRPC6Transfection was accomplished using 3 target sequences for TRPC6. FAM-siRNA plasmids and Western blot were used to detect transfection and silence efficiency respectively.9. Intracellular Ca2+ measurementsCells grown on glass bottom cell culture dishes were placed in external solution and loaded with 5μM Fura 2-acetoxy methyl ester (Fura 2-AM) at 37℃. Cells were washed, and dye was allowed to deesterify for 1 h. Fluorescence intensity was recorded over the entire surface of each single cell and intracellular Ca2+ was evaluated from the ratio of the fluorescence emission intensities excited at the two wavelengths.10. Cell cycle analysisCell suspensions were transferred to 95% ethanol while mixing thoroughly and stored at room temperature for 30 min. Cells were washed three times with PBS and then treated with ribonuclease for 15 min. Propidium iodide was added and cells were allowed to incubate for an additional 30 min. Using flow cytometry, cell cycle was measured. 11. Statistical analysisDate analyses were performed with the Statistical Package for Social Sciences software (SPSS11.5). P< 0.05 was regarded as statistically significant.Results1. Construction and identification of eukaryotic expression plasmid. Enzyme analysis and agarose gel electrophoresis showed that there were 1326bp band. The two eukaryotic expression vectors containing pBudCE4.1-EGFP/NK4 were successfully constructed. DU145 cells of transferred NK4 gene expressed NK4 protein.2. MTT showed that HGF-induced cell proliferation was suppressed by autocrine NK4, NK4 alone had no effect on cell proliferation. Cell migration index increased after HGF treatment. Although again, this HGF-induced motility was significantly inhibited by autocrine NK4. Autocrine NK4 suppressed HGF-induced tumor cell invasion.3. Western blot showed that c-Met was detected in DU145 cells. HGF induced phosphorylation of c-Met, ERK1 and Aktl/2. And that was inhibited by autocrine NK4.4. Immunohistochemistry showed that TRPC6 expression in PC was higher than BPH. The increasing staining recorded was associated with the histological grade, Gleason score and extraprostatic extension of prostate cancer. TRPC6 was expressed in the DU145, PC3 and 22Rvl prostate cancer cell lines.5. Western blot revealed a 70%～80% decrease of TRPC6 protein expression in the TRPC6 siRNA1 (siRNA1) and TRPC6 siRNA2 (siRNA2) groups. HGF up-regulated TRPC6 expression. OAG increased [Ca2+]i in the presence of extracellular Ca2+, but not in the absence of extracellular Ca2+. A further experiment showed OAG-induced Ca2+ entry significantly decreased after TRPC6 siRNA. After treatment with HGF, OAG-induced Ca2+ entry increased in DU145 cells. TRPC6 knockdown arrested DU145 cells at G2/M phase. Conclusion1. Genetic modification of DU145 cells with NK4 cDNA yields a significant effect on their proliferation, migration and invasion through inhibiting the HGF-induced phosphorylation of c-Met, ERK1 and Akt1/2. Molecular targeting of HGF/c-Met by NK4 could be applied as a novel therapeutic approach to prostate cancer.2. TRPC6 was expressed in BPH, prostate cancer and, in the DU145, PC3 and 22Rv1 prostate cancer cell lines, and was associated with the cancer's histological grade, Gleason score and extraprostatic extension status.3. TRPC6 could regulate calcium influx in DU145 cells. Knockdown of TRPC6 channels in DU145 cells suppressed the cell proliferation and arrested cell cycle at G2/M phase. HGF increases the expression of TRPC6, which results in an increase of OAG-induced calcium entry. So the Ca2+ inflow regulated by TRPC6 channels is essential for HGF-induced growth proliferation of prostate cancer.