没食子酸诱导非小细胞肺癌A549 细胞凋亡的机制研究

Gallic acid induces apoptosis of non-small cell lung cancer A549 cells via JAK/STAT3 pathway

摘要:

目的 探讨没食子酸（GA）对非小细胞肺癌A549 细胞凋亡的影响及其机制。方法 将A549 细 胞随机分为对照组和不同浓度GA 组，分别给予正常培养基及不同浓度GA 培养6 ～ 48 h。噻唑蓝比色法（MTT） 检测细胞活力，流式细胞仪检测细胞凋亡，实时荧光定量聚合酶链反应（qRT-PCR）测定细胞中酪氨酸激酶1 （JAK1）、信号转导及转录激活因子3（STAT3）、B 淋巴细胞瘤2 基因（Bcl -2）及Bcl-2 相关X 蛋白（Bax） mRNA 表达；Western blot 检测细胞中Bcl-2、Bax、JAK1、STAT3、磷酸化酪氨酸激酶1（p-JAK1）、磷酸化信 号转导及转录激活因子3（p-STAT3）的蛋白表达。结果 12 ～ 28μg/ml GA 作用A549 细胞不同时间后，与 对照组相比，GA 组细胞生存率降低（P <0.05），细胞凋亡率增加（P <0.05），并且随GA 浓度增加，干预时间 延长，其对细胞的生长抑制及凋亡诱导作用逐渐增强。同时，与对照组比较，GA 组细胞中Bax mRNA 及蛋白 表达逐渐增高（P <0.05），p-JAK1、p-STAT3、Bcl-2 的蛋白表达及Bcl-2 mRNA 表达逐渐降低（P <0.05）； 并且随GA 浓度增加，其对细胞中Bax、Bcl-2、p-JAK1、p-STAT3 蛋白表达的调节作用逐渐增强，但对细 胞中JAK1、STAT3 mRNA 及蛋白表达水平无影响（P >0.05）。结论 GA 呈浓度、时间依赖性诱导A549 细 胞凋亡，抑制其生长，作用机制可能与抑制细胞中JAK1、STAT3 的磷酸化激活，进而调节Bax、Bcl-2 的表 达有关。

Abstract:

Objective To investigate the effect of gallic acid (GA) on proliferation and apoptosis of non-small cell lung cancer A549 cells and the underlying mechanisms. Methods A549 cells were randomly divided into a normal control group and three GA groups. The normal control group was regularly cultured while the GA groups were cultured with different concentrations of GA (12, 20 and 28 μg/ml) for 6-48 h, respectively. The viability of A549 cells was analyzed by MTT assay. Apoptosis induction was detected by flow cytometry. qRT-PCR was used to examine the mRNA expressions of Bax, Bcl-2, JAK1 and STAT3. The protein expressions of Bcl-2, Bax, JAK1, STAT3, p-JAK1 and p-STAT3 were determined by Western blot. Results The viability of the cells in the GA groups was significantly lower than that in the normal control group after 6-48 h culture (P < 0.05), additionally, the percentages of the apoptotic cells in the GA groups were significantly higher than that in the control group after 24 h (P < 0.05). Furthermore, the growth inhibitory effect and the apoptosis induction effect of GA became more and more dramatic with the prolongation of incubation time and increase of GA concentration. It was also found that GA up-regulated Bax mRNA and protein expressions and down-regulated Bcl-2, p-JAK1 and p-STAT3 protein expressions as well as Bcl-2 mRNA level compared with the control group (P < 0.05), however, the mRNA or protein expressions of JAK1 and STAT3 showed no significant differences (P > 0.05). Conclusions GA inhibits viability and induces apoptosis of A549 cells in a dose- and time-dependent manner via blocking the phosphorylation of JAK1 and STAT3 followed by decreased Bcl-2 and increased Bax expressions.