Yan Xiaodong, Shi Ying, Kou Buxin, Zhu Zhenyu, Chai Jie, Chen Dexi, Guo Hongliang
Department of General Surgery, Shandong Cancer Hospital, Jinan 250117; School of Medicine and Life Sciences, Shandong Academy of Medical Sciences, University of Jinan, Jinan 250022, China.
Department of Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Jan;32(1):34-8.
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.
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.
Transfection of Ser33/52A 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 Ser33/52A 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 Ser33/52A plasmid transfected group was significantly inhibited.
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.
研究奥沙利铂（OXA）作用下，角蛋白18（K18）磷酸化与HCT116细胞自噬及凋亡的相关性，并探讨其可能机制。
分别将空载质粒、野生型K18表达质粒及33、52位磷酸化位点突变的K18（Ser33/52A）表达质粒转染HCT116细胞，随后用60 μmol/L OXA处理所有细胞，再添加自噬抑制剂3-甲基腺嘌呤（3-MA）或自噬诱导剂雷帕霉素。采用异硫氰酸荧光素（FITC）标记的膜联蛋白V与碘化丙啶（PI）双染结合流式细胞术、钙黄绿素-AM/PI染色分析K18及其突变体对细胞凋亡的影响；采用蛋白质免疫印迹法检测K18磷酸化、自噬相关蛋白微管相关蛋白1轻链3（LC3）和贝林1的表达。
转染Ser33/52A质粒显著降低了K18磷酸化水平。经OXA处理后，K18质粒转染组的凋亡率显著高于空载质粒转染组，而Ser33/52A质粒转染的HCT116细胞凋亡率显著低于空载质粒或K18质粒转染组。与空载质粒组相比，K18质粒转染组的自噬显著增强，而Ser33/52A质粒转染组的自噬显著受抑制。
K18过表达增强了HCT116细胞的自噬并增加了其对OXA的敏感性。K18第33位和第52位丝氨酸磷酸化水平降低抑制了HCT116细胞的自噬并减少了其凋亡。
