Akt和核因子KB信号通路在胃癌细胞化学治疗耐药中的作用

The impact of Akt and nuclear factor kB pathway on chemoresistance of gastric cancer cell

目的 探讨Akt、核因子（NF）-kB信号通路在胃癌细胞化学治疗耐药中的作用，以及Akt、NF-kB信号通路的相互作用关系。方法采用化学治疗药物阿霉素、足叶乙甙及两药联合应用Akt抑制剂（Wortmannin）或NF-kB抑制剂（MG-132）分别作用于胃癌细胞（SGC-7901）。用四甲基偶氮唑盐比色法（MTT法）检测SGC-7901细胞增长率；TUNEL法和膜联蛋白V／碘化丙啶（PI）双标法检测肿瘤细胞凋亡；免疫细胞化学法检测NF-kB／P65蛋白表达；电泳迁移率实验（EMSA）法检测NF-kB-DNA结合活性的变化；Western印迹法检测磷酸化Akt或磷酸化NF—KB抑制因子（IxB）a蛋白的表达。结果①阿霉素和足叶乙甙均能明显抑制SGC-7901细胞的生长，并呈时间、剂量依赖性；分别联合应用Wortmannin或MG-132后能进一步抑制其生长。②化学治疗药物剂量依赖性地诱导SGC-7901细胞凋亡和NF—xB或Akt活化，联合应用MG-132或Wortmannin均能增强化学治疗药物的诱导凋亡作用和抑制NF-kB或Akt活化作用。③Wortmannin可明显抑制NF-kB的活化，而MG-132对Akt的活化无明显影响。结论化学治疗药物诱导SGC7901细胞凋亡的同时诱导Akt、NF-kB活化，抑制Akt、NF-kB的活化可增加化学治疗药物的疗效。

Objective To evaluate the role of Akt and nuclear factor （NF）-kB pathway in the development of chemoresistance in gastric cancer and the relation between Akt and NF-kB. Methods SGC-7901 cells were exposed to chemotherapeutic drugs （ doxorubicin and etoposide ） or chemotherapeutic drugs combined with Wortmannin or MCr132. The cell growth was detected using MTT method. The apoptosis of SGC-7901 cells was measured by TUNEL and Annexin V/PI methods. The protein level of NF-kB was analyzed by immunocytochemical staining. Electrophoretic mobility shift assay （EMSA） was used to confirm the increased nuclear translocation of NF-kB/P65. The protein levels of p-Akt and p-IkBa were determined by Western blotting. Results① Both chemotherapeutic drugs could obviously inhibit the growth of SGC-7901 cells in time-dose-dependent manner. Pretreatment of SGC-7901 cells with Wortmannin or MG-132 could promote this inhibitory effect. ② Both chemotherapeutic drugs could induce apoptosis of SGC-7901 and activate Akt and NF-kB in a close-dependent manner. Wortmannin or MG-132 pretreatment could enhance the apoptosis of SGC 7901 cells and bloke NF-kB activation induce by doxorubicin or etoposide.③ The activation of NF-kB was found in SGC-7901 cells stimulated with Wortmannin,but no activation of Akt was noted in those treated with MG-132. Conclusions The chemotherapeutic drugs can both induce apoptosis and activate Akt and NF-kB in SGC-7901 cells. The efficacy of chemotherapeutic drugs can be increased via inhibiting activation of Akt or NF-kB.