Babao Dan Reverses Multi-drug Resistance Of Gastric Cancer Via PI3K/AKT/mTOR Signaling Pathways

Objective: To study the effect of BBD on overcoming multi-drug resistance of gastric cancer and regulating PI3K/AKT/m TOR signaling pathway.To elucidate the mechanism of BBD reversal of multi-drug resistance of gastric cancer on the aspects of drug efflux,proliferation,apoptosis and autophagy through in vitro experiments.To provide experimental basis for further expanding the development and application of BBD in clinical practice.Methods: Gastric cancer cell SGC7901 and SGC7901/DDP cell were cultured in vitro;MTT assays were used to detect the effects of Cisplatin(DDP),Doxorubicin(DOX),5-fluorouracil(5-FU)on cell viability,and drug resistance index(RI)was calculated.MTT assay was used to detect the effects of BBD on SGC7901 and SGC7901/DDP cells.SGC7901/DDP cells were treated with 0.25 mg/m L and 0.5 mg/m L BBD combined with different concentrations of DDP,DOX and 5-FU for 48 h,and their IC50 and reversal fold(RF)were calculated.The effects of BBD on DOX and Rhodamine(Rho123)accumulation were observed by fluorescence microscopy in SGC7901/DDP cells.The effect of BBD on the proliferation and cycle distribution of SGC7901/DDP cells were detected by colony formation and flow cytometry.DAPI and Annxin V/PI were used to detect the effect of BBD on apoptosis of SGC7901/DDP cells.Fluorescence microscope and flow cytometry were used to detect the effect of BBD on autophagy in SGC7901/DDP cells,and 3-MA(early autophagy inhibitors)and chloroquine(CQ late autophagy inhibitors)were used for further verification autophagy.The expression of drug efflux-related factors(ABCB1,ABCC1,ABCG2),cell proliferation regulatory factors(Survivin,Cyclin D1,CDK4,p21),apoptosis regulatory factors(Bcl-2,Bax,caspase-3,cleave-caspase-3),autophagy related factors(LC3Ⅱ/Ⅰ,p62,Beclin 1)were detected by Western Blot in SGC7901/DDP cells.Western Blot was used to detect the effects of BBD and 740Y-P on PI3K/ AKT /m TOR signaling pathway activation in SGC7901/DDP cells.Results: SGC7901/DDP cells were multidrug resistant,and the drug resistance index(RI)to DDP,DOX and 5-FU were 1.86,1.501 and 47.70(RI > 1.5).BBD could reverse multi-drug resistance of SGC7901/DDP cells.The reversal fold(RF)of 0.25 mg/ m L and 0.5mg/m L BBD on DDP,DOX and 5-FU were 1.51,4.34,3.14 and 1.55,4.68,3.56(RF > 1.5).BBD can increase the accumulation of DOX and Rho123,reduce drug efflux,and down-regulate the protein expressions of ABCB1,ABCC1 and ABCG2 in SGC7901/DDP cells.BBD inhibits the proliferation and the formation of cell colonies of SGC7901/DDP cells,reduces the proportion of S phase cells,blocks cells in G0/G1 phase,down-regulates the protein expression of Cyclin D1 and CDK4 and up-regulates the protein expression of p21.BBD induced apoptosis of SGC7901/DDP cells,and up-regulated protein expression ratios of cleaved caspase-3/caspase-3 and Bax/Bcl-2.BBD promoted autophagy in SGC7901/DDP cells,down-regulated p62 expression and up-regulated LC3Ⅱ/Ⅰ and Beclin1 expression;Meanwhile,3-MA(early autophagy inhibitors)and CQ(late autophagy inhibitors)also further confirmed that BBD could promote autophagy in SGC7901/DDP cells.BBD down-regulated the proteins expression of p-PI3 K,p-AKT and p-m TOR,indicating that BBD inhibits the activation of PI3K/AKT/m TOR pathway.Meanwhile,PI3 K agonist(740Y-P)also further revealed that BBD inhibited the activation of PI3K/AKT/m TOR signaling pathway.Conclusion: BBD can reverse multi-drug resistance of gastric cancer cells and inhibit drug efflux,proliferation,induction of apoptosis and promotion of autophagy in drug-resistant gastric cancer cells.BBD inhibits activation of PI3K/AKT/m TOR signaling pathway in multi-drug resistant gastric cancer cells,which is one of its important mechanisms.