角蛋白18磷酸化与奥沙利铂作用下结肠癌HCT116细胞凋亡的关系

The study on the correlationship between keratin phosphorylation with oxaliplatin induced apoptosis of colon cancer cell HCT116

目的探讨在化疗药物作用下角蛋白18(K18)磷酸化与结肠癌HCT116细胞凋亡的关系。方法以不同浓度的奥沙利铂作用于HCT116细胞,Annexin V-FITC/PI双标记流式细胞术和钙黄绿素-AM/碘化丙啶(Calcein-AM/PI)法检测细胞凋亡情况,免疫印迹(western blotting)方法检测K18磷酸化表达水平;HCT116细胞分别转染绿色荧光蛋白(GFP)、野生型K18和33、52丝氨酸(Ser33A、Ser52A)磷酸化位点突变的K18质粒后,用奥沙利铂处理,An-nexin V-FITC/PI和Calcein-AM/PI法分析K18及其突变对细胞凋亡的影响。结果奥沙利铂可以使HCT116细胞发生明显的凋亡,而且凋亡比例随着药物浓度的增加而升高,经20、60、100μmol/L奥沙利铂处理后,细胞较对照组均出现凋亡增加,依次为(15.6±3.1)%、(30.1±4.9)%和(62.6±6.8)%,两两比较差异均有统计学意义(P均<0.05);K18的Ser33、Ser52磷酸化水平也随着药物浓度的增加而增加;单纯质粒转染的各细胞组之间的凋亡率差异均无统计学意义(P>0.05),但经过奥沙利铂处理后,Ser33A和Ser52A质粒转染的HCT116细胞的凋亡率[(27.3±6.7)%和(31.2±8.1)%]明显低于GFP和K18质粒转染的细胞凋亡率[(40.5±4.8)%和(50.9±6.3)%,P<0.05],转染K18质粒组的HCT116细胞的凋亡率高于GFP转染对照组,差异有统计学意义(P<0.05);Ser33A和Ser52A质粒转染组之间差异无统计学意义(P>0.05)。结论 K18 Ser33和Ser52磷酸化水平随着细胞凋亡水平的增加而增加;K18过表达提高了HCT116细胞对奥沙利铂的敏感性,而抑制K18 Ser33和Ser52的磷酸化可以减少化疗药物作用下结肠癌细胞的凋亡。

Objective To study the correlationship between phosphorylation of K18 with apoptosis of human colorectal cancer HCT 116 cells in the effect of oxaliplatin. Methods HCT 116 cells were treated with different concentra- tions of oxaliplatin （OXA）, then FITC-conjugated annexin V and PI double stained flow cytometry, Calcein-AM/PI stain- ing and western blotting（WB） were used to analyze cell apoptosis and phosphorylation of K18. HCT 116 cells were trans- fected with green fluorescent protein（GFP） vector, wild type K18 expression vector and Ser33A, Ser52A phosphorylation site mutant K18 expression vector, then treated with OXA. FITC-conjugated annexin V and PI double stained flow cytom- etry, Calcein-AM/PI staining were used to analyze the effect of K18 and its phosphorylation to cell apoptosis. Results OXA could induced obvious apoptosis of I-ICT116 cells, the apoptosis rate of HCT1l6 cells increased along with the in- crease of OXA concentration,and after 20, 60, 100μmol/L OXA treatment, comparing with the control group, the apopto- sis of cells was found increasing, followed by （15.6±3.1）%, （30.1±4.9）% and （62.6±6.8）%, there was significant difference between each group. The phosphorylation of Ser33 and Ser52 K18 increased along with the increase of OXA concentra- tion. There was no significant difference among different plasmid transfected groups （P 〉 0.05）. While after treated with OXA, the apoptosis rates of Ser33A and Ser52A transfected HCT116 cells[（27.3±6.7）%, （31.2±8.1 ）%] were much lower than GFP and K18 transfected cells[（40.5±4.8）%, （50.9±6.3）%, P 〈 0.05], obviously, the apoptosis of K18 transfected group was higher than GFP transfected group, and the difference was statistically significant（P 〈 0.05）. There was no sig-nificant difference between Ser33A and Ser52A transfected groups （P 〉 0.05）. Conclusion The phosphorylation of KI 8 （Ser33, Ser52）increases along with the increase of cell apoptosis. Overexpression of K18 increases the chemotherapeutic drug sensitivity of HCTll6 cells, while reduction of phosphorylation of K18 （Ser33, Ser52） decreases chemotherapeutic drug sensitivity of HCT 116 cells.