P16^INK4A Involes Epirubicin Resistance Via Cell Cycle Arrest In Breast Cancer Cells

Object In this research,we have established two kinds of breast cancer resistant cell lines and silenced P16^INK4A expression,indicating that P16^INK4A involves in epirubicin chemoresistance via regulating cell cycle distribution.Methods Breast cancer cell lines MDA-MB-231 and HS-578 T were used as parental cells,MDA-MB-231/EPI and HS-578T/EPI were established as resistant cells.For MDA-MB-231/EPI and HS-578T/EPI cells,there were different tolerant concentration.In MDA-MB-231/EPI cells,epirubicin screen concentration gradient was 0.5,1,2,4,6 and 8mg/L.In HS-578T/EPI cells,its gradient was 0.5,1 and 1.5mg/L.IC50 and RI of parental and resistant cells were measured by MTT assay.Cellular growth curves were built by cell number count and MTT assay.Cell cycle distribution was tested by flow cytometry?FCW?.Expression of P16^INK4A,cell cycle-related protein and multidrug resistant protein were detected by Western-Blot.P16^INK4A expression was silenced by lentivirus-loaded si RNA.Results 1.Chemoresistance test: Cells were treated with epirubicin,paclitaxel and 5-fluorouracil for 24 hours,48 hours and 72 hours.For resistant cells?MDA-MB-231/EPI and HS-578T/EPI?,their IC50 s of three agents were more than that of parental cells?MDA-MB-231 and HS-578T?,relating to screen concentration positively?p<0.01?.In resistant cells,their IC50 and RI were related to epirubicin screen concentration positively?p<0.01?.2.P-glycoprotein?P-gp?,multidrug resistant protein 1?MRP1?and ATP-binding casttel protein 1?ABCG1?,three kinds of resistant-related protein,their expression in resistant cells was more than that of in parental cells?p<0.001?.3.Cell cycle-related phenotype of cells: Growth curves built by cell number count and MTT assay showed that growth speed of resistant cells was slower than that of parental cells?p<0.001?.Results of doubling time in logistic growth phase illustrated that doubling time of resistant cellswas longer than that of parental cells,relating to screen concentration.Percentage of G1 phase cells increased in resistant cells,showed by flow cytometry,relating to screen concentration positively.P16^INK4A expression increased in resistant cells.However,expression of CDK6,Cyclin D1 and Rb protein phosphorylation drooped in resistant cells?p<0.001?.4.Silence of P16^INK4A expression: P16^INK4A expression was downregulated by P16^INK4A si RNA.CDK6,Cyclin D1 expression and Rb protein phosphorylation increased?p<0.01?.For resistant cells,after P16^INK4A silence,IC50 of EPI in 24 hours,48 hours and 72 hours induced?p<0.001?.Growth curves showed growth speed increased and doubling time dropped?p<0.01?.Flow cytometry illustrated that percentage of G1 phase cells induced?p<0.01?and S phase cells increased?p<0.01?.Conclusion P16^INK4A,inhibiting CDK6,Cyclin D1 expression and Rb protein phosphorylation,involves in chemoresistance of EPI via cell cycle arrest in G1 phase.