ABT-263诱导A431细胞凋亡及其对AKT下游信号通路影响

ABT-263 induced A431 cell apoptosis and its effect on AKT downstream signaling pathway

目的观察抗凋亡蛋白抑制剂ABT-263对人皮肤鳞状细胞癌细胞系A431细胞功能的影响,并探讨相关分子机制。方法 (1)取对数生长期的人永生化表皮细胞(Ha Ca T细胞)与A431细胞提取总蛋白,采用蛋白印迹法检测B淋巴细胞瘤-2(BCL-2)和BCL-XL的蛋白表达水平。(2)分别以浓度为50μmol/L的ABT-263(抑制剂组)和二甲基亚砜(对照组)处理A431细胞,培养4和9 h后,采用透射扫描电子显微镜(TEM)检测各组细胞的形态变化。(3)以剂量为0、10、25、40和50μmol/L的ABT-263处理A431细胞24 h后,采用CCK-8法检测细胞活力。(4)以不同剂量的ABT-263处理A431细胞,采用蛋白印迹法检测活化半胱氨酸天冬氨酸蛋白酶(Caspase-3)、活化聚腺苷酸二磷酸核糖转移酶-1(PARP-1)、磷酸化蛋白激酶B(p AKT)ser473、磷酸化糖原合成酶激酶-3β(p GSK3β)和磷酸化组蛋白H2AX(γH2AX)的蛋白相对表达水平。结果 A431细胞中的BCL-2和BCL-XL蛋白相对表达水平均高于Ha Ca T细胞(P<0.01)。TEM检查结果显示,抑制剂组A431细胞从正常的形态逐渐变化为凋亡的形态,表现为微绒毛消失,核染色质密度增高并凝聚在核膜周边,核仁裂解。10、25、40和50μmol/L剂量组A431细胞的细胞活力均低于对照组(P<0.05)。10、30和50μmol/L剂量组A431细胞的活化Caspase-3和活化PARP-1蛋白的相对表达水平均高于对照组(P<0.05)。10、25、40、50μmol/L剂量组A431细胞的p AKT(ser473)和p GSK3β的蛋白相对表达水平均低于对照组(P<0.05),γH2AX蛋白相对表达水平高于对照组(P<0.05)。A431细胞活力和p GSK3β蛋白相对表达水平均随抑制剂剂量的增加而减少(P<0.01),活化Caspase-3和γH2AX的蛋白相对表达水平均随抑制剂剂量的增加而增加(P<0.01),均呈剂量-效应关系。结论 ABT-263可诱导A431细胞发生线粒体途径的凋亡,并可诱导AKT/GSK3β信号通路失活,从而促进A431细胞凋亡,呈一定程度的剂量-效应关系。

Objective To investigate the effect of ABT-263, an anti-apoptotic protein inhibitor, on human cutaneous squamous cell carcinoma A431 cells, and to explore its molecular mechanisms. Methods i）Total protein was extracted from human immortalized epidermal cells （HaCaT cells ） and A431 cells in logarithmic growth phase. The protein expression of B-cell lymphoma-2 （BCL-2） and BCL2-1ike 1 （BCL-XL） was detected by Western blotting, ii） The A431 cells were treated with ABT-263 （inhibitor group） and dimethyl sulfoxide （control group） at a concentration of 50 μmol/L for 4 and 9 hours. The morphological changes of the cells were examined by transmission electron microscopy, iii ） The A431 cells were treated with 0, 10, 25, 40, and 50 μmo]/L of ABT-263 for 24 hours, and the cell viability was determined by CCK-8 assay, iv） The A431 cells were treated with different doses of ABT-263, and the expression of cleaved Caspase-3, cleaved poly （ ADP-fibose ） polymerase-1 （ PARP-1 ） , phosphorylated protein kinase B [ pAKT （ser473） ], phosphorylated glycogen synthase kinase-313 （pGSK3） and phosphorylated histone H2AX （7H2AX） was detected by Western blot. Results The relative expression of BCL-2 and BCL-XL in A431 cells were higher than those in HaCaT cells （P 〈 0.01 ）. Transmission electron microscopy results showed that A431 cells in inhibitor group gradually nuclear chromatin de, nsitvand aggregation around the nuclear membrane, and nuclear fragmentation. The cell viability of A431 cells in 10, 25, 40 and 50 μmol/L groups were lower than those in control group （ P 〈 0. 05 ）. The relative expression of cleaved Caspase-3 and cleaved PARP-1 in A431 cells in 10, 30 and 50μmol/L groups were higher than those in control group （P 〈0. 05）. The relative expression of pAKT （ser473） and pGSK3β in A431 cells in 10, 25, 40 and 50 μmol/L groups were lower than those of the control group （P 〈0. 05） and YH2AX protein expression was higher than that of the control group（P 〈 0. 05）. A431 cell viability and pGSK3β protein expression decreased with the increase of inhibitor dosage （P 〈0. 01 ）. The relative expression of cleaved Caspase-3 and yH2AX protein increased with the increase of inhibitor dosage （P 〈 0. 01 ） , showing dose-effect relationship. Conclusion ABT-263 can induce apoptosis of A431 cells through mitochondria pathway and induce the inactivation of AKT/GSK3[3 pathway, which can promote the apoptosis of A431 cells with a dose- effect relationship.