野马追总黄酮的体内外抗乳腺癌活性及机制研究

Study on Anti-breast Cancer Activity and Mechanism of Total Flavonoids from Eupatorium Lindleyanum DC.

关键词:  野马追总黄酮  乳腺癌  细胞周期  细胞凋亡  自噬

Key words:  total flavonoids from Eupatorium lindleyanum DC.  breast cancer  cell cycle  apoptosis  autophagy

摘要:

目的 探究野马追总黄酮的体内外抗乳腺癌活性及相关的作用机制。方法 MTT法检测野马追总黄酮对乳腺癌细胞（MCF-7、T47D、MDA-MB-231、MDA-MB-468）和人正常乳腺上皮细胞MCF-10A增殖的影响；流式细胞术检测野马追总黄酮对乳腺癌细胞周期和凋亡的影响；吖啶橙（AO）染色法检测野马追总黄酮对细胞自噬的影响；Western blotting检测细胞周期、凋亡、自噬相关蛋白的变化，以及PI3K/Akt信号通路相关蛋白的变化；建立MDA-MB-231细胞的裸鼠移植瘤模型，观察野马追总黄酮的体内抗肿瘤活性。结果 野马追总黄酮能明显抑制乳腺癌细胞增殖，并呈剂量依赖性，而对人正常乳腺上皮细胞无显著的抑制作用；野马追总黄酮能增加G₂/M期细胞比例，上调p-cdc-2蛋白表达，下调cdc-2和Cyclin B1蛋白表达；野马追总黄酮能显著增加乳腺癌细胞凋亡比例，上调促凋亡蛋白Bad和Bax表达，下调抗凋亡蛋白Bcl-2表达；野马追总黄酮能够诱导乳腺癌细胞产生自噬，自噬相关蛋白LC3-Ⅱ表达上升，p62表达降低；野马追总黄酮能够显著抑制PI3K和Akt的磷酸化水平；野马追总黄酮能够在体内抑制裸鼠肿瘤的体积。结论 野马追总黄酮通过抑制PI3K/Akt信号通路，阻滞细胞周期、诱导凋亡和自噬，进而在体内外抑制乳腺癌细胞的生长。

Abstract:

OBJECTIVE To investigate the anti-breast cancer activity and related mechanism of total flavone from Eupatorium lindleyanum DC.(TFE). METHODS MTT assay was used to detect the effect of TFE on the proliferation of breast cancer cells (MCF-7, T47D, MDA-MB-231, MDA-MB-468) and human normal mammary cells(MCF-10A). Flow cytometry was used to determine the effect of TFE on cell cycle and apoptosis in breast cancer. Acridine orange(AO) staining was used to detect the effect of TFE on autophagy. The changes of cell cycle, apoptosis, autophagy related proteins, as well as the changes of PI3K/Akt signaling pathway related proteins were detected by Western blotting assay. Xenograft model in nude mice of MDA-MB-231 cells was established to observe the antitumor activity in vivo of TFE. RESULTS TFE could significantly inhibit the proliferation of breast cancer cells in a dose-dependent manner, but had no significant inhibitory effect on human normal mammary cells. TFE could induce cell cycle arrest at G₂/M phase, and the expression of cell cycle related proteins Cyclin B1 and cdc2 were decreased, while p-cdc2 was increased. TFE could significantly increase the apoptosis rate of breast cancer cells, and the expression of apoptotic protein Bad and Bax were increased, while the anti-apoptotic protein Bcl-2 was decreased. TFV could induce autophagy in breast cancer cells, and the expression of autophagy related protein LC3-Ⅱ increases, while the expression of p62 decreases. TFE significantly inhibited the phosphorylation of PI3K and Akt. TFE inhibited tumor volume in nude mice in vivo. CONCLUSION TFE inhibits the growth of breast cancer cells in vivo and in vitro by inhibiting the PI3K/Akt signaling pathway, cell cycle arrest, inducing apoptosis and autophagy.