The Mechanism Of Dioscin Promotes The Degradation And Decreases The Expression Of The P-gp To Reverse Cancer Drug Resistance And The EMT Process

Background and ObjectiveOvercoming drug-resistance is one of the most vital challenge in cancer therapy.Tumor drug-resistance can trigger metastasis,cancer recurrence and poor prognosis,ect.Lots of studies reported that drug resistance is link to the expression and function of ATP-binding cassette（ABC）transporter proteins,especially,the high-expression of P-glycoprotein（P-gp/MDR1/ABCB1）enable to inhibit the antitumor effect of the antitumor drug through removing chemotherapy drugs from cancer cells and decreasing the concentration of drugs.Especially,paclitaxel（PTX）is a first-line chemotherapy in the clinic,and as a substrate of the P-gp protein,which made cancer crlls obtain the acquired drug-resistance to PTX.Acquired drug-resistance can elevate Epithelial Mesenchymal Transformation（EMT）,that is associated with tumor migration and invasion,accelerating tumorigenesis and development.Dioscin,as an effective element,deprives from Dioscorea oppositifolia,whose antitumor effect has been attention recent years.The aim of study to explore dioscin exerts the effect and mechanism of reversing drug resistance through mediating degradation and transcription of the P-gp and blocks metastasis and invasion,and providing a promising method to deal with tumor drug-resistance for clinic cancer therapy.Method1.SRB assay was used to detect the cell viability of the drug-resistant cancer cells TE-1/PTX,Hela/PTX treated with paclitaxel,cisplatin and 5-Fu and IC₅₀,meanwhile,calculating the resistant index respectively.Western blotting was used to detect the expression of P-gp between cancer parental cells and PTX-resistant cancer cells to evaluate the drug resistant level.2.The cytotoxic effects of dioscin on parental celld TE-1,Hela and drug-resistant cells TE-1/PTX and Hela/PTX were detected.The plate colony formation used to detect to examine the differences between parental cancer cells and resistant cancer cells and the effect of dioscin on colony formation in resistant cancer cells.3.SRB assay was performed to detect the sensitive change and reversing drug resistant standard of drug-resistant cells TE-1/PTX and Hela/PTX to PTX in the presence of dioscin,and cell morphology changes.The effect of dioscin on Rh123accumulation in P-gp overexpressing drug-resistant cells and parental cells were detected by flow cytometry to test P-gp function.Western blotting was used to test the effect of different concentrations and treatment time of the dioscin on P-gp protein of drug-resistant cells.4.Western blotting was used to detect the effect of different concentration dioscin to the related protein of the AKT/MAPKs/GSK3βpathway,which can regulate the transcription of P-gp in drug-resistant cells.Co-Immunoprecipitation assay was used to analyze the interaction of P-gp and ubiquitin to value the influence of dioscin on P-gp degradation in TE-1/PTX and Hela/PTX.5.Wound healing assay and Transwell assay were used to detect the effect of dioscin on the cell migration ability.Western blotting was used to detect the effect of dioscin on EMT-related proteins in resistant cancer cells.6.The Hela/PTX cell xenograft models were established and evaluated the effect of dioscin on drug-resistant cell cancer tissue growth ability through the changes of tumor volume,tumor weight and HE staining.Western blotting assay was used to detect the effect of dioscin on.Result1.Compared to parental cells,the resistant indexs of Hela/PTX to the PTX,cispltain,5-Fu were respectively 7.89,5.33 and 5.77;the resistant indexs of TE-1/PTX to the PTX and cisplatin were respectively 4.89 and 3.86;Drug-resistant cancer cells possessed resistance to the antitumor drug in a degree.There was more expression P-gp in PTX-resistant cells and showed that TE-1/PTX and Hela/PTX as P-gp overexpression cell model could devote to the follow experiments.2.Dioscin decreased the cancer cells viability in dose-and time-dependent manners.Furthermore,dioscin could significantly curb the colony formation of PTX-resistant cancer cells.3.Dioscin enhanced significantly the pharmaceutical sensitivity of PTX to the multi-drug resistant（MDR）cells,indicated that dioscin exerted the function of reversing drug-resistance.Dioscin could attenuate obviously the expression of P-gp of PTX-resistant cancer cells in dose-and time-dependent manner.Therefore,dioscin remarkably increased the intracellular accumulation of rhodamine 123（Rh123）and decreased the function of P-gp efflux.4.Dioscin inhibited expression of the related proteins in AKT/MAPKs/GSK3βpathway in dose-dependent manner,including p-AKT,p-p38,p-JNK,p-Erk and p-GSK3β.Which affects the transcription and expression of the P-gp protein.5.Co-IP assay displayed that dioscin could activate the ubiquitin-proteasome system and promote the degradation of the P-gp ubiquitination in drug-resistant cancer cells.6.Compared with parental cells,MDR cells possessed the strong migration.After treatment with dioscin,the expression levels of Slug,Snail,N-cadherin,FAK and Vimentin were down-regulated,while the expression levels of E-cadherin,Claudin-1,and ZO-1 were up-regulated,hence,dioscin could attenuate metastasis and invasion through abolishing EMT process.7.In xenograft tumor model,dioscin could effectively inhibit the growth of drug-resistant tumor.Dioscin also had no obvious effect in body weight and no obvious toxicity to main organs of nude mice.Dioscin inhibit EMT-related proteins and AKT/MAPKs/GSK3βpathway to decrease drug-resistance and inhibit tumor growth.Conclusion1.There are overexpression of the P-gp protein in multi-drug resistant cancer cells that can enhance migration and activate EMT process.2.Dioscin can effectively elevate the cytotoxic effects of the PTX to the multi-drug resistant cells and reverse drug-resistance through promoting the ubitiqutin degradation and inhibiting the AKT/MAPKs/GSK3βpathway to attemuate the expression of the P-gp.Meanwhile,dioscin is able to inhibit the metastasis and invasion that is link to blocking EMT process.3.Dioscin can inhibit the growth of drug-resistant cancer cells Hela/PTX in the axillary xenograft model of nude mice,and the maximum dose of dioscin has no obvious toxicity to nude mice.The mechanism of diioscin in vivo was basically consistent with the results in vitro.