Keratin 18 phosphorylation increases autophagy of colorectal cancer HCT116 cells and enhanced its sensitivity to oxaliplatin
YAN Xiaodong;SHI Ying;KOU Buxin;ZHU Zhenyu;CHAI Jie;CHEN Dexi;GUO Hongliang;Department of General Surgery,Shandong Cancer Hospital;School of Medicine and Life Sciences,Shandong Academy of Medical Sciences,University of Jinan;Department of Infectious Diseases,Beijing Youan Hospital,Capital Medical University;
Objective To study the correlation between the phosphorylation of keratin 18( K18) and the autophagy and apoptosis of HCT116 cells under the effect of oxaliplatin( OXA) and investigate its possible mechanism. Methods HCT116 cells were transfected with empty plasmid,wild-type K18 expression plasmid and 33,52 phosphorylation site mutated K18( Ser33 /52A) expression plasmid separately,and all cells were then treated with 60 μmol/L OXA,followed by supplementation of autophagy inhibitor 3-methyladenine( 3-MA) or autophagy inducer rapamycin. FITC-conjugated annexin V and propidium iodide( PI) double staining combined with flow cytometry,calcein-AM / PI staining were used to analyze the effects of K18 and its mutants on cell apoptosis; Western blotting was performed to detect the expressions of K18 phosphorylation,autophagy related proteins microtubule associated protein 1 light chain 3( LC3) and beclin-1. Results Transfection of Ser33 /52 A plasmid significantly reduced the level of K18 phosphorylation. After treated with OXA,the apoptosis rate of K18 plasmid transfected group was significantly higher than that of empty plasmid transfected group,while the apoptosis rate of Ser33 /52 A plasmid transfected HCT116 cells was significantly lower than that of empty plasmid or K18 plasmid transfected group. Compared with empty plasmid group,the autophagy of K18 plasmid transfected group was significantly promoted,while the autophagy in Ser33 /52 A plasmid transfected group was significantly inhibited. Conclusion K18 overexpression enhanced the autophagy in HCT116 cells and increased its sensitivity to OXA. The decrease of K18 ser33 and ser52 phosphorylation inhibited autophagy and decreased apoptosis of HCT116 cells.