The Mechanisms Study Of The Antitumor Actions Of Cordycepin During Induction Of Apoptotic Cell Death And Inhibition Of Cell Migration In Clear Cell Renal Cell Carcinoma(ccRCC)

Background Renal cell carcinoma(RCC)denotes cancer originated from epithelial cells in kidney and accounts for more than 90% of kidney cancers.Renal cell carcinoma encompasses more than 10 kinds of histopathological and molecular subtypes,of which clear cell renal cell carcinoma is the most common one.Approximately 75% of renal cell carcinoma are clear cell renal cell carcinoma(cc RCC).Epidemiological survey shows that over the past 20 years,the incidence of renal cell carcinoma worldwide has increased by 2% every year.Worldwide,the age-standardized incidence and mortality of renal cell carcinoma were 6 per 100,000 and 2.1 per 100,000 respectively.Although the diagnosis and treatment technologies of renal cell carcinoma have made great progress,up to 20%~30% of patients with renal cell carcinoma have distant metastasis.The prognosis of patients with metastatic renal cell carcinoma has historically been poor with the 5-year survival rate of about 8%.In recent years novel immune-based and targeted treatments have been developed with the understanding of the pathogenesis of renal cell carcinoma has deepened.However,so far,no one drug obtains overall response rate of 100% and durable complete responses,and at present neo-adjuvant therapy is still worthy of research and development for renal cell carcinoma.Chinese traditional medicine(CTM),especially Chinese herbal medicine,provides an alternative for the development of modern Western medicine.In recent years herbal medicine and the extracted active composition attract more and more attention for treatment of many chronic diseases including cancers.Cordycepin is the main active ingredient extracted from traditional Chinese medicine Cordyceps sinensis.Previous studies in lung cancer,breast cancer,ovarian cancer and leukemia showed that cordycepin harbored broad-spectrum anti-tumor activity.Nonetheless,the action of cordycepin and the underlying molecular mechanisms in renal cell carcinoma are still unclear and warrant further investigation.Objective To investigate the effect of cordycepin on the survival and migration of clear cell renal cell carcinoma;To preliminarily elucidate the molecular mechanisms of cordycepin actions in clear cell renal cell carcinoma;To lay an experimental and theoretical foundation for the future potential clinical application of cordycepin in the treatment of renal cell carcinoma.Methods1.Clear cell renal cell carcinoma cell line Caki-1 was treated with different concentrations(10 μg/ml,20 μg/ml,30 μg/ml,40 μg/ml,50 μg/ml)of cordycepin for 48 hours and then cell viabilities were determined using CCK-8(cell counting kit-8);Caspase-3 activities were also measured biochemically after treatment.2.Clear cell renal cell carcinoma cell line Caki-1 was treated with 30 μg/ml cordycepin in the presence or absence of 50μmol/L pan-caspase inhibitor z-VADfmk for 48.Then the cell viability was determined by CCK-8 cell viability assay kit;Cleavage of apoptosis marker protein PARP(poly ADP-ribose polymerase)and caspase-3 were detected by Western Blot assays.3.Transwell cell migration experiments were conducted to analyze the effect of 10 μg/ml of cordycepin on the migration ability of Caki-1 clear cell renal cell carcinoma cells for 24 hours and 48 hours.4.Clear cell renal cell carcinoma Caki-1 cells were treated with cordycepin at the concentration of 10 μg/ml,20 μg/ml,30 μg/ml for 48 hours.Then Western Blot assays were performed to detect the changes of PTEN(phosphatase and tensin homologue phosphatase deleted on chromosome 10)expression and the serine / threonine protein kinase Akt phosphorylation levels;real-time fluorescence quantitative PCR(Q-PCR)were performed to determine the effects of cordycepin treatment on micor RNA-21(mi R-21)expression in clear cell renal cell carcinoma Caki-1 cells.5.PTEN expression in clear cell renal cell carcinoma was knockdown by transfection of mi R-21 mimic or PTEN si RNA(small interfering small interfering RNA).Western Blot experiments were performed to verified the PTEN knockdown effect;Effects of knockdown of PTEN on cordycepin-induced apoptotic cell death of clear cell renal cell carcinoma Caki-1 cells were determined by CCK-8 cell viability assay kit;Transwell cell migration assays were done to detect the influence of knockdown of PTEN on cordycepin-induced cell migration inhibition in clear cell renal cell carcinoma Caki-1 cells.Results1.Results from cell viability assays by CCK-8 cell counting assay kit showed that,compared with control,forty-eight hours treatment with cordycepin(10 μg/ml,20 μg/ml,30 μg/ml,40 μg/ml and 50 μg/ml)induced significant decrease in cell viability of clear cell renal cell carcinoma Caki-1 cells in dose-dependent manner(P<0.05 vs.control);With the gradual increase of cordycepin concentration,cell apoptosis marker,caspase-3 activities increased gradually correspondingly in clear cell renal cell carcinoma Caki-1 cells(P<0.05 vs.control).Cordycepin(30 μg/ml)–induced cell death in renal cell carcinoma Caki-1 cells was significantly inhibited by 50 μmol/L pan-caspase inhibitor z-VAD-fmk treatment(P<0.05);Western Blot detection results showed that 30 μg/ml cordycepin induced cleavage of PARP and caspase-3 in renal cell carcinoma Caki-1cells were completely inhibited by 50 μmol/L pan-caspase inhibitor z-VAD-fmk treatment.2.Transwell cell migration assays showed that,compared with control,10 μg/ml cordycepin treatment significantly inhibited the migration of clear cell renal cell carcinoma Caki-1 cells(P<0.05 vs.control).3.Western Blot detections results showed that compared with the control group,cordycepin treatment(10 μg/ml,20 μg/ml and 30 μg/ml)in clear cell renal cell carcinoma Caki-1 cell significantly increased the PTEN expression level(P<0.05 vs.control)and decreased protein kinase Akt phosphorylation level(P<0.05 vs.control)in concentration-dependent form.Real-time fluorescence quantitative PCR results showed that compared with the control,cordycepin treatment(10 μg/ml,20 μg/ml,30 μg/ml)dose-dependently increased the expression level of mi R-21 in clear cell renal clear cell carcinoma Caki-1cells(P<0.05 vs.control).4.Western Blot detection results demonstrated that transfection with mi R-21 mimic or PTEN si RNA reduced the expression level of PTEN protein in clear cell renal cell carcinoma Caki-1 cells;CCK-8 cell viability assay results showed that transfection of clear cell renal cell carcinoma Caki-1 cell with mi R-21 mimic or PTEN si RNA could significantly suppress cordycepin treatment(30 μg/ml)induced cell death(P<0.05);Transwell cell migration assay results illustrated that transfection of clear cell renal cell carcinoma Caki-1 cell with mi R-21 mimic or PTEN si RNA could also significantly attenuate the inhibitory effects of cordycepin treatment(10 μg/ml)on cell migration(P<0.05).Conclusions Cordycepin treatment can increase mi R-21 expression and decrease PTEN phosphatase expression in clear cell renal cell carcinoma cell line Caki-1 cells;Cordycepin treatment can induce apoptotic cell deth and inhibit cell migration of clear cell renal cell carcinoma cell line Caki-1 cells via modulation of expressions of mi R-21 and PTEN;Cordycepin shows the potential clinical application value in the treatment of renal cell carcinoma.