Mechanism Study Of Combined Treatment Of Telomerase And Autophagy Inhibitor On The Survival Of Malignant Melanoma Cell Line

Background: Malignant melanoma(MM)is the most aggressive and invasive skin malignant tumor and accounts for 20,000 of new cancer diagnoses in China each year.For the majority of patients with MM,the disease is locally advanced when diagnosed and the therapeutic efficacy remains unfavorable.Presently,the treatment of advanced MM is mainly dacarbazine-based combination therapy and progress has been made in immunotherapy represented by PD-1 and CTLA-4 antibody in recent years.However,the benefits in an unselected patient population are modest.The prevention and treatment of MM must aim at its pathogenesis.Telomerase is a reverse transcriptase,which maintains telomere length by synthesizing repetitive sequences of telomere.Over 90% of tumor cells express highly active telomerase,which is closely related to the tumorigenesis and progression of MM.Autophagy is a cell lysosomal dependent degradation pathway and plays an important role in the tumorigenesis and progression of MM.Further,it has the function of anti apoptosis and resistance to chemotherapeutic drugs.It is a trend to explore the combination of different drugs in the treatment of MM in clinical trials.The aim of this study is to investigate the role of telomerase and autophagy in the development of MM,and to explore the potential mechanism of combined inhibition of telomerase and autophagy in the treatment of MM.Methods: 1.Telomerase inhibitor BIBR1532 was used to treat MM cell line A375.And then,CCK-8 was used to detect cell viability,Annexin Ⅴ/PI double staining was used to detect cell apoptosis status,to observe the effect of telomerase inhibitor on the survival of MM cells.2.Telomerase inhibitor BIBR1532 was used to treat MM cell line A375.And then,Western Blot was performed to detect the expression of LC3 II,p62 protein,m RFP-GFP-LC3 transfected MM cell line A375 was used to detect the status of autophagy flux,to explore the activation of autophagy using telomerase inhibitor on MM cells.3.Combination of telomerase inhibitors BIBR1532 and autophagy inhibitor CQ were used to treat MM cell line A375.And then,CCK-8 was used to detect cell viability,Annexin Ⅴ/PI double staining was used to detect cell apoptosis status,JC-1 staining was used to detect the changes of mitochondrial membrane potential,PI staining was used to detect cell cycle conditions,and colony formation experiment was performed to detect cell proliferation ability,to investigate the possibility of combined inhibition of telomerase and autophagy in the treatment of MM.Results: 1.Telomerase inhibitor BIBR1532(50umol/L)inhibited the growth and induced apoptosis of MM cell line A375.2.Telomerase inhibitor BIBR1532(50umol/L)up-regulated LC3 II expression and down-regulated p62 expression in MM cell line A375.Using m RFP-GFP-LC3 transfected MM cell line A375 showed that after BIBR1532(50umol/L)treatment,the autophagy flux increased significantly.3.Telomerase inhibitor BIBR1532(50umol/L)combined with autophagy inhibitor CQ(20umol/L)significantly promoted cell death,induced cell apoptosis and mitochondrial membrane potential disruption,blocked the cell cycle at G2/M phase,and inhibited the clone formation of MM cell line A375.Conclusion: Telomerase inhibitors have inhibitory effect on the survival of MM cells and induce the activation of autophagy in MM cells.Inhibition of autophagy improves the sensitivity of MM cells to telomerase inhibitors.