Inhibitory Effects And Mechanism Of Simvastatin And Nitidine Chloride On Renal Cancer

BackgroundRenal cell carcinoma (RCC) is the most common type of renal cancer, accounting for approximately90-95%kidney neoplasms. Worldwide, mortality due to RCC has exceeded100,000patients each year. About25-30%of patients develop metastases at diagnosis of RCC, with surviva1≥5years ranging from5%to10%, and overall median survival of less than one year. Surgical intervention is the primary treatment for localized RCC, but alone it has limited benefit in patients with aggressive disease. In addition, traditional cytotoxic chemotherapy and immunotherapy have failed to demonstrate a benefit in patients in the adjuvant setting.Recent years have seen a rapid development of molecular targeted therapy. Amongst the first-line targeted therapies, sunitinib and temsirolimus are the most representative, which can block the signaling pathway of multiple receptor tyrosine kinases (RTKs) and mammalian target of rapamycin (mTOR) respectively. Nevertheless, none of the interventions would be considered cost-effective at a willingness-to-pay threshold of30,000pounds per quality-adjusted life-year. Thus, there is a great demand for treatments that can prolong survival without greatly increasing costs or eroding the quality of patients’ lives.Simvastatin (or3-hydroxy-3-methylglutaryl coenzyme A [HMG-CoA] reductase inhibitors), which is a structural analogue of HMG-CoA that inhibit conversion of HMG-CoA to mevalonate. Beyond the cholesterol-reducing properties, simvastatin exhibit numerous pleiotropic effects, including anti-inflammation and immunomodulation, reduction in the risk for various forms of dementia, and a decrease in proteinuria and the progression of kidney disease. Of significance, emerging evidence revealed that simvastatin could exhibit antineoplastic effects in a variety of cancer cells, including prostate cancer, breast cancer, hepatic cancer and colon cancer. However, the precise effect of simvastatin against RCC cells and the underlying molecular mechanisms have not been well established.Nitidine is derived from the root of Zanthoxylum nitidum. Previous studies reported that Nitidine has anti-inflammatory, anti-fungal, anti-oxidant and even anti-HIV functions. Nitidine chloride(NC), a chloride of Nitidine, has been identified as a potential anti-tumor drug because it can inhibit proliferation and induce cell apoptosis in several tumor types including osteosarcoma, gastric cancer and liver cancer. However, the function of anti-metastasis activity in renal cancer cells and the underlying molecular mechanisms have not been well established.Given current distress on treatment of renal cancer, study the effect of simvastatin and nitidine chloride on renal cancer cells is very mingful.ObjectiveIn this study, we investigate the effect of simvastatin and nitidine chloride on cell viability, apoptosis, migration and invasion of786-0and A498cells. Further investigation of the underlying mechanisms indicates AKT/mTOR, ERK and JAK2/SAT3pathways play key roles in these actions.MethodsUsing two different renal cancer cell lines, A498and786-O, the cytotoxicity of simvastatin were determined by MTT assay and colony formation assay in vitro. The apoptosis induced by simvastatin was detected by cytometric apoptosis assays. Scratch migration assay and transwell invasion and migration assay were used to detecte anti-metastasis effect of simvastatin. A xenograft model was used to determine whether simvastatin inhibits tumor growth in vivo. In addition, the protein level of AKT/mTOR, ERK, JAK2/STAT3and apoptosis-associated proteins were examined by Western blot analysis. Using two different renal cancer cell lines, A498and786-0, the cytotoxicity of nitidine chloride were determined by MTT assay in vitro. Scratch migration assay and transwell invasion and migration assay were used to detecte anti-metastasis effect of nitidine chloride. A xenograft model was used to determine whether nitidine chloride inhibits tumor growth in vivo. The apoptosis induced by nitidine chloride was detected by cytometric apoptosis assays. In addition, the protein level of ERK, AKT, MMPs and apoptosis-associated proteins were examined by Western blot analysis.ResultsWith cell viability, colony formation and flow cytometric apoptosis assays, we found that simvastatin potently suppressed cell growth of A498and786-0cells in a time-and dose-dependent manner. Consistently, the xenograft model performed in nude mice exhibited reduced tumor growth with simvastatin treatment. In addition, the inhibitory effects of simvastatin on migration and invasion were also observed in vitro.Furthermore, we examined the apoptotic markers after exposure of different concentrations of simvastatin. The results indicated that simvastatin could induce cleavage of caspase-3and PARP, down-regulate the pro-apoptotic survivin and as well as up-regulate the anti-apoptotic Bax Bcl-2. Cell cycle arrest was reported to contribute to the growth suppression of statins in cancer cells; however, we find no significant effect of simvastatin on cell cycle distribution in RCC cells (data not shown).We examined the change of ERK phosphorylation caused by the administration of simvastatin. We found that the levels of phosphorylated ERK were remarkably decreased both in vitro and in vivo. To determine whether simvastatin played its anti-tumor effects via ERK pathway, we applied siRNA to knockdown ERK expression. After transfected with siRNA of ERK, the cell viability and motility were significantly decreased. These results demonstrated that knockdown of ERK could sensitize renal cancer cells to simvastatin-induced anti-cancer effect. We also found that simvastatin could significantly suppress the phosphorylation/activation of AKT, as well as inhibited its downstream effectors, mTOR. To further investigate whether the pro-apoptotic and anti-metastatic effects are mediated by targeting AKT/mTOR pathway, we applied siRNA to knockdown the expression of AKT. We found that inhibition of AKT activation by knockdown of AKT expression sensitized renal cancer cells to simvastatin-induced anti-cancer effect. To date, there is no data on the effects of simvastatin on regulating IL-6-induced JAK2/STAT3signaling in renal cancer cells. For the first time, we found that simvastatin could significantly inhibit IL-6induced proliferation and metastasis of RCC cells, attenuate IL-6-induced JAK2activation, and subsequently decrease the phosphorylation of STAT3both in vitro and in vivo. Further knockdown of STAT3with siRNA showed significantly decreased cell proliferation and mobility. All these findings suggest JAK2/STAT3pathway may play a crucial role in simvastatin-induced anti-tumor effects in RCC cells. In conclusion, these findings suggested that simvastatin-induced apoptosis and its anti-metastasis activity in RCC cells were accompanied by inhibition of AKT/mTOR, ERK and JAK2/STAT3pathways, which imply that simvastatin may be a potential therapeutic agent for the treatment of RCC patients.With cell viability and flow cytometric apoptosis assays, we found that NC potently suppressed the growth of786-0and A498cells in a time-and dose-dependent manner. Consistently, the xenograft model performed in nude mice exhibited reduced tumor growth with NC treatment. Mechanically, we presented that NC significantly decreased phosphorylation of ERK and Akt, accompanied by up-regulation of P53, Bax, cleavage caspase-3and cleavage PARP, down-regulation of Bcl-2, caspase-3and PARP. Furthermore, a specific MEK inhibitor, PD98059, could potentiate the pro-apoptotic effects of NC, which indicated that NC might trigger apoptosis in renal cancer cells partly via inhibition of ERK activity. Taken together, our results imply that NC could be developed as a potential anticancer agent to renal cancer and worthy of further studies.With scratch assay and transwell assays, we found that NC potently suppressed the migration and invasion of786-0and A498cells. Mechanistically, we presented that NC significantly decreased phosphorylation of AKT, accompanied by down-regulation of MMP-2and MMP-9. Furthermore, a specific AKT inhibitor, LY294002, could enhance the anti-metastasis effects of NC, which indicated that NC suppressed metastasis of renal cancer cells partly via inhibition of AKT activity. Taken together, our results imply that NC can be developed as a potential anti-metastasis agent to renal cancer.ConclusionsTaken altogether, our study demonstrates that simvastatin can markedly exhibit pro-apoptotic and metastasis inhibitory effect on RCC cells. Mechanically, we found AKT/mTOR, ERK, as well as JAK2/STAT3pathways account for the anti-tumor effects of simvastatin. All these results imply that simvastatin may be a promising therapeutic drug for renal cancer patients. Our data, for the first time, demonstrated that NC inhibits the proliferation of786-0and A498renal cancer cells both in vitro and in vivo. Furthermore, our data documented that suppression of ERK pathway contributes to NC-induced apoptosis in renal cancer cells. Thus, this study provides evidence for the therapeutic potential of NC as renal cancer treatment. Our results imply that AKT signaling pathway plays a key role in NC induced anti-metastasis effect in renal cancer cells. All these results suggest a protective activity of NC in renal cell carcinoma development and metastasis.