Mechanism Study Of Inhibitory Effect Of 17-AAG In Huma Renal Cell Carcinoma

PartⅠThe clinicopathologic significance of HSP90,HIF-2α,and VEGF expression in renal cell carcinoma.Objective To investigate the expression of heat shock protein 90(Hsp90), hypoxia-inducible factor 2α(HIF-2α),and Vascular endothelial growth factor (VEGF).Analyze the relationship among these protein and the clinicopathologic features of human renal cell carcinoma.To explore whether Hsp90,HIF-2α,and VEGF play an important role in the outcome of renal cell carcinoma.Methods Measure the expressions of Hsp90,HIF-2α,and VEGF In 60 case of resected specimens of renal cell carcinoma and 25 case their corresponding nomal renal tissues by immunohistochemical S-P method.Analyze the significance of these protein with the clinicopathologic features and outcome in human renal cell carcinoma.RT-PCR and western blot were used to detect the expression levels of HIF-2αand VEGF mRNAs and proteins.Results Increased expressions of Hsp90,HIF-2α,and VEGF were detected in renal cell carcinoma,and significant correlations were observed among them.the expressions of HSP90,HIF-2α,and VEGF were closely related with the stage,LN metastasis and the size of tumor of renal cell carcinoma.The overall survival rate is higher in renal cell carcinoma,which have low expression of HIF-2αand VEGF.The survival rate is slightly higher in renal cell carcinoma,which have low expression of Hsp90.Higher expression of HIF-2αwas detected only at protein level,and for VEGF both at mRNA and protein level via RT-PCR and western blot. Conclusions The expressions of Hsp90,HIF-2α,and VEGF are overexpressed in renal cell carcinoma.It is likely that HIF-2αis involved in the angiogenesis of renal cell carcinoma via upregulation of VEGF expression.Furthermore,Hsp90 and HIF-2αlikely plays an important role in renal cell carcinoma together.PartⅡStudy of the impact of biological behaviors on human renal cell carcinoma cells by 17-AAG in vitro.Objective To explore the effects of 17-AAG on human renal cell carcinoma cells in vitro.Methods human renal cell carcinoma cell line Caki-1 cells were cultured with or without 17-AAG under normoxic and hypoxic conditions,respectively.MTT assay was used to determine the proliferation rate of Caki-1 cells.Apoptosis was detected by the means of Hoechst 33342/PI double staining,AnnexinV-FITC/PI double staining and DNA ladder.Western blot were used to detect the expression of HIF-2αand VEGF protein.Results In experimented group cells,ability of proliferation was inhibited in hypoxic state,meanwhile,apoptotic rate increased.Expressions of HIF-2αand VEGF protein were significantly inhibited in Caki-1 cells of experimental group,compared with in that of negative control group or blank control group.Conclusions 17-AAG can inhibit cell proliferation,and promote apoptosis in human renal cell carcinoma cell of Caki-1 in vitro,and likely due to the inhibition of HIF-2αand VEGF expression.PartⅢResearch for inhibition human renal cell carcinoma by 17-AAG in vivoObjective To research growth of human renal cell carcinoma by 17-AAG in vivo via transplanted subcutaneously in nude mouse.Methods human renal cell carcinoma transplanted subcutaneously in nude mouse. 17AAG,DMSO,and NS were intratumorally injected with 125mg/kg respectively in subcutaneous xenograft model,inhibitory effect of tumor growth was observed,HIF-2αand VEGF protein expressions were detected by immunohistochemical S-P method, TUNEL stain and transmission electron microscopy was used to determine apoptosis.Results Growth rate of subcutaneous xenograft significantly decreased experimental group,the tumors size and weight of experimental group were significantly lesser than those of negative control group and blank control group.For experimental group, expressions of HIF-2αand VEGF proteins were weaker than those of negative control group and blank control group.Conclusions 17-AAG can dramatically inhibit the growth of human renal cell carcinoma,likely due to upregulation the expression of HIF-2αand VEGF.