Objectives: To investigate the mechanism of luteolin for inhibiting proliferation of lung cancer A549 cells.
Methods: A549 cells treated with different concentrations of luteolin for 48 h were evaluated for changes in cell viability, proliferation, reactive oxygen species (ROS) production and apoptosis using MTT assay, plate cloning assay, EdU staining, DCFH-DA assay and Hoechst33258 staining. The changes in cell autophagy were examined with MDC staining, and the expressions of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-9), autophagy-related proteins (LC3B, Beclin 1, and P62), AKT/mTOR pathway proteins, and HO-1 protein were detected using Western blotting.
Results: Treatment with luteolin dose-dependently inhibited the viability and proliferation of A549 cells, increased intracellular ROS levels, up-regulated the expressions of Bax, cleaved caspase-9, and Beclin 1, increased the LC3B-II/LC3B-I ratio, down-regulated the expressions of Bcl-2 and P62, and induced cell apoptosis and autophagy. Luteolin also significantly inhibited the phosphorylation of AKT and mTOR and down-regulated the expression of HO-1 protein in the cells.
Conclusions: Luteolin induces apoptosis and autophagy to inhibit proliferation of A549 cells by increasing ROS production, inhibiting the AKT/mTOR pathway and down-regulating HO-1 protein expression.
目的: 探究木犀草素（Lut）对肺癌A549细胞增殖的抑制作用及其内在机制。方法: 用不同浓度的Lut处理A549细胞48 h，通过MTT法检测细胞活性，通过平板克隆和EdU染色检测细胞增殖，通过DCFH-DA法检测细胞活性氧（ROS）水平，通过Hoechst33258 染色法检测细胞凋亡水平，通过MDC染色法检测细胞自噬水平，通过Western blotting实验检测细胞凋亡相关蛋白Bax、Bcl-2、Cleaved caspase-9，自噬相关蛋白LC3B、Beclin1、P62，AKT/mTOR通路蛋白以及HO-1蛋白的表达。结果: Lut剂量依赖性的抑制A549细胞的活力和增殖能力（P<0.05），引发细胞内ROS水平增加（P<0.05），上调凋亡相关蛋白Bax、Cleaved caspase-9和自噬相关蛋白Beclin1的表达，增加LC3B-II/LC3B-I的比值，下调抗凋亡蛋白Bcl-2和自噬相关蛋白P62的表达，诱导细胞凋亡和自噬（P<0.001）。此外，Lut可显著抑制AKT和mTOR的磷酸化，下调HO-1蛋白的表达（P<0.05）。结论: Lut通过增加细胞内ROS的产生，抑制AKT/mTOR通路以及下调HO-1蛋白水平诱导A549细胞的凋亡和自噬。.
Keywords: AKT/mTOR signaling pathway; HO-1; apoptosis; autophagy; luteolin; reactive oxygen species.