益气除痰方通过抑制Akt信号通路诱导顺铂耐药肺癌细胞凋亡的作用及分子机制研究

Effects and Mechanism of Yiqi Chutan Formula on Inducing Apoptosis of Cisplatin-resistance Lung Cancer Cells by Inhibiting Akt Signaling Pathway

目的探讨益气除痰方诱导顺铂耐药肺癌细胞凋亡的作用及其分子机制。方法制备益气除痰方含药血清,采用Annexin V/PI双染结合流式细胞术检测益气除痰方对人肺腺癌顺铂耐药细胞株A549/DDP1细胞凋亡的影响。采用Caspase分光光度法检测试剂盒检测益气除痰方对A549/DDP细胞内Caspase-8和Caspase-3激酶活性的影响。采用Western blotting检测益气除痰方对A549/DDP细胞Akt/FoxO信号通道相关蛋白的影响。结果与空白血清对照组比较,A549/DDP细胞经不同浓度益气除痰方作用后,凋亡相关蛋白Caspase-8和Caspase-3激酶活性增强(P<0.05),呈剂量依赖性效应。益气除痰方能明显抑制Akt的磷酸化(P<0.05),随着剂量的增加,其抑制作用增强,而Akt总蛋白的水平基本未受影响;相对应FoxO3a蛋白磷酸化水平逐渐降低(P<0.05),而Fox O3a总蛋白的水平基本未受影响;并且经益气除痰方作用后,FoxO3a下游的Bim的表达显著升高(P<0.05),均呈现明显的剂量依赖性效用。结论益气除痰方可能通过抑制Akt/FoxO信号转导从而激活内源性凋亡通路,诱导肿瘤细胞凋亡,进而逆转肺癌细胞顺铂耐药。

Objective To observe the effect of Yiqi Chutan formula（YQCTF） on the apoptosis of cisplatin- resistant human lung cancer cells and to explore the apoptotic pathways involved in YQCTF- induced apoptosis. Methods Serum of YQCTF was made. The Annexin V/Propidium iodide（PI） double labeling method combined with flow cytometry was employed to detect the early apoptotic cells, and the effect of YQCTF on the enzymatic activities of Caspase- 8 and Caspase- 3 in Cisplatin- resistant A549/DDP cells were assessed with Caspase spectrophotometry kit. Meanwhile, the effect of YQCTF on the phosphorylation of Akt and its crucial downstream mediator of Fox O3 a,and the expression of Bim（a target gene of Fox O3a）,was examined by Western blotting. Results Compared with the blank serum group,the activities of Caspase- 8 and Caspase- 3 were obviously dose- dependently enhanced after the A549/DDP cells were treated with differenct concentrations of YQCTF. YQCTF could obviously inhibit the phosphorylation of Akt and Fox O3 a, and the effect on Akt became stronger with the increase of dose and the prolongation of time. However,YQCTF had no effect on Akt and Fox O3 a total protein level. The expression of Bim（the downstream target gene of Fox O3a） was increased in dose- dependent manner after treatment with YQCTF. Conclusion YQCTF suppressed the Akt pathway and triggered the apoptosis of A549/DDP cells which were resistant to cisplatin treatment, thereby mediating the anti- cancer and anti- chemoresistance activity of YQCTF.