Establishment Of An Epirubicin-resistant Cell Line Of Human Breast Cancer:Characterization And Mechanism Of MDR

Objective:The objective of this study was to establish a epirubicin-resistant human breast cancer cell line and investigate its drug-resistant mechanism.Methods1.We established a epirubicin-resistant human breast cancer cell line MDA-MB-231/EPI by established by continuous exposure to epirubicin.2.The drug sensitivity and cell growth curve were examined by tetrazolium dye(MTT)test.3.Cell morphology was observed before and after HE staining.4.The ultrastructure was observed using transmission electron microscope(TEM).5.Flow cytometry detected the distribution of cell cycle,the expression of P-gp,the accumulation and discharge of doxorubicin and rhodamine.6.The levels of Mrpl and bcl-2 mRNA were determined by QRT-PCR7.The level of LC3 was detected by western blot.Results1.After 12 months,we established an epirubicin resistant human breast cancer cell line,named MDA-MB-231/EPI.2.Compared with MDA-MB-231,the ratio of nucleus/cytoplasm increased,the growth rate slowed down,and the cell proportion of G0/G1 phase increased in MDA-MB-231/EPI.The resistant index of epirubicin,paclitaxel,etoposide and cisplatin were respectively 26.27,12.261,1.739 and 3.834 times in MDA-MB-231/EPI.3.The MrplmRNA enhanced 53 times(P<0.01)and P-gp expression increased 90 fold(P<0.01)in MDA-MB-231/EPI.The accumulation and efflux of ADM and Rh123 enhanced in MDA-MB-231/EPI.4.The LC3 conversion stably higher than the parental cells(P<0.05)and the autophagosome was significantly increased in MDA-MB-231/EPI.After using the autophagy inhibitor chloroquine,autophagy was inhibited and the sensitivity of epirubicin enhanced(P<0.05).5.Compared with MDA-MB-231 cells,epirubicin induced bcl-2 mRNA increased in MDA-MB-231/EPI cells(P<0.05).After using the autophagy inhibitor chloroquine,bcl-2 mRNA decreased in MDA-MB-231/EPI cells(P ?0.05),but not in MDA-MB-231 cells.Conclusions1.We successfully establish an epirubicin-resistant cell MDA-MB-231/EPI.2.The biological characteristics of MDA-MB-231/EPI cells are more immature than MDA-MB-231 cells.MDA-MB-231/EPI cells possess the feature of multi-drug resistance3.The expression levels and the drug efflux abilities of Mrp 1 mRNA and P-gp are higher in MDA-MB-231/EPI cells than that in MDA-MB-231 cells.4.Autophagy may protect MDA-MB-231/EPI cell by inhibiting drug induced apoptosis.