黄芩素诱导乳腺癌细胞自噬

Baicalein induces autophagy in breast cancer cells

目的:考察黄芩素诱导乳腺癌细胞的自噬作用,并初步探讨其机制。方法:采用MTT实验考察黄芩素对乳腺癌MCF-7细胞和4T1细胞活力的影响,确定给药剂量。Western blot检测黄芩素(25、50和100μmol/L)及联合自噬抑制剂3-MA作用下,MCF-7细胞和4T1细胞中自噬特征蛋白LC3-Ⅱ和LC3-Ⅰ的表达水平,流式细胞术Annexin V/PI双染法观察3-MA对黄芩素诱导MCF-7细胞和4T1细胞凋亡的影响,确认黄芩素诱导自噬的作用。通过Western blot考察自噬信号通路相关蛋白p-mTOR、mTOR、p-AKT和AKT的蛋白水平,结合AKT-mTOR激活剂EGF明确AKT-mTOR通路在黄芩素诱导乳腺癌自噬中的作用。结果:50μmol/L及其以上剂量的黄芩素可显著抑制乳腺癌MCF-7细胞和4T1细胞的活力,其作用具有显著的时效和量效性。Western blot结果表明,50和100μmol/L黄芩素作用下MCF-7细胞和4T1细胞的LC3-Ⅱ/LC3-Ⅰ比例显著增强,3-MA加入后又明显降低。流式细胞术结果显示,相比黄芩素单用组,联合自噬抑制剂可促进MCF-7细胞的坏死和凋亡。通路蛋白研究表明mTOR和AKT总量不变,其活化蛋白水平在黄芩素作用下显著降低,而加入EGF后又再次增加。结论:黄芩素可通过抑制AKT-mTOR通路诱导乳腺癌MCF-7细胞和4T1细胞自噬。

AIM ： To investigate the autophagy of breast cancer cells induced by baicalein and to explore its mechanism. METHODS： The effects of baicalein on the viability of MCF-7 cells and 4T1 cells were investigated by MTT assay, and the dosage of the drug was determined. The expression levels of microtubule-associated protein 1 light chain 3- II （LC3-II） and LC3-I in the MCF-7 cells and 4T1 cells treated with baicalein at doses of 25, 50 and 100 （jimol/L, or combined with autophagy inhibitor 3-methyladenine （3-MA） were determined by Western blot. In order to confirm the role of baicalein in autophagy, the effect of 3-MA on the apoptosis of both MCF-7 cells and 4T1 cells induced by baicalein was analyzed by flow cytometry. The protein levels of p-mTOR, mTOR, p-AKT and AKT were examined by Western blot and the role of AKT-mTOR pathway in the induction of autophagy in breast cancer induced by baicalein was determined by the combination of activators. RESULTS ： Baicalein at 50 jjimol/L and above doses significantly inhibited the viability of breast cancer cells in a dose- and time-dependent manner. The expression of LC3-II/LC3-I in both MCF-7 cells and 4T1 cells was significantly enhanced after the action of baicalein, and the ratio of LC3-II/LC3-I was significantly decreased after 3-MA addition. The results of flow cytometry showed that, compared with baicalein group, the combination of baicalein and 3-MA promoted the levels of necrosis and apoptosis. Moreover, the protein levels of p-mTOR and p-AKT were significantly de-creased and were rescued by EGF, while their total protein levels were not changed. CONCLUSION： Baicalein induces autophagy through AKT-mTOR pathway both in MCF-7 cells and 4T1 cells.