益气除痰方抑制p-AKT活性诱导A549细胞失巢凋亡的研究

Yi-Qi-Chu-Tan Formula Induces Anoikis by Inhibition of p-AKT in Human A549 Cancer Cell

目的:观察益气除痰方含药血清对肺腺癌A549细胞失巢凋亡的诱导作用以及对P-akt、Pp38蛋白以及下游凋亡相关基因表达的影响。方法:采用软琼脂集落培养实验和poly-HEMA培养检测含药血清对锚定不依赖性增殖和失巢凋亡影响,流式细胞仪检测细胞凋亡率;Western blot检测含药血清对Pakt、P-p38蛋白的影响,RT-PCR测定用药前后调亡相关基因mRNA表达的变化。结果:培养10 d左右,A549细胞在软琼脂可形成明显集落,低、中、高剂量含药血清及阳性组细胞集落形成数较阴性组明显减少(P<0.05);流式细胞仪检测显示,阴性对照组,低、中、高剂量药物组,阳性对照组凋亡率分别为0.57±0.40%,10.23±1.46%、16.30±0.96%、27.10±1.22%,45.80±1.45%,差异有统计学意义(P<0.05);Western blot结果显示:与阴性组相比较,低、中、高剂量组,P-p38蛋白相对表达量分别为1.04、1.11、1.09,未见明显变化;而P-akt蛋白相对表达量分别为0.81、0.58、0.53,明显降低;RTPCR显示益气除痰方可显著上调p53 mRNA、bax mRNA表达,下调bcl-2 mRNA表达。结论:益气除痰方含药血清可诱导A549失巢凋亡,其作用机制可能与降低A549细胞P-akt蛋白表达,进而调控其信号传导通路下游的凋亡相关基因p53、bcl-2、bax的表达有关,而与P-p38蛋白的调控无明显相关。

Objective： To investigate the impacts of the serum of Yi-qi-chu-tan formula on anoikis in human lung cancer cell line A549 and explore its effect on the expression of proteins P-akt, P-p38 as well as of the anoikis-related genes p53, bax and bcl- 2. Methods： Detecting the effect of different concentrations herbs serum on anchorage independent proliferation and anoikis of A549 cells was assessed by soft agar colony experiment and poly HEMA cultivation. Apoptosis was measured by flow c, ytometry. The protein expression of P-akt and P-p38 was detected by Western blot. The related mRNA expression were examined by RT-PCR. Results： A549 cells can form clear colonies in 10 days. In the number of colonies, the negative group was more than the herb serum groups and posi- tive group significantly （P 〈 0. 05 ）, and the herb serum group showed a dose-dependent, there were significant difference between the four groups （ P 〈 0.05 ） . The apoptotic rate of the negative group, low, medium, high dose group and the positive group respec- tively were0.57±0.40%, 10.23±1.46%, 16. 30±0.96%, 27.10±_1.22%, 45.80±1.45% （P〈0.05） .Inthelow, medi-um and high dose group the expression of P-akt respectively were 0. 81, 0. 58, 0. 53, while the P-p38 were 1.04, 1.11, 1. 09. Yi- qi-chu-tan formula can increase tile mRNA expression of p53 and bax and decrease the mRNA expression of bcl-2 and had no influence on the expression of P38. Conclusion： Yi-qi-chu-tan Formula can induce anoikis by reducing P-Akt to regulate the expression of anoikis-related genes in the downstream of PI3K/akt signaling pathway in A549 cell and which presents a dose-dependent.