木榄胚轴总黄酮通过调节PI3K/Akt信号通路诱导人结肠癌HT-29细胞凋亡

Total flavoniods from Bruguiera gymnorrhiza hypocotyls induces apoptosis in human colon cancer HT-29 cells by regulating of PI3K/Akt signaling pathway

目的 研究木榄胚轴总黄酮(TFBH)对人结肠癌HT-29细胞增殖和迁移的影响及其诱导细胞凋亡的作用机制。方法 MTT法检测TFBH对细胞增殖的影响。划痕实验观察TFBH对细胞迁移的影响。流式细胞术检测TFBH对细胞周期和细胞凋亡的影响。Hoechst 33258荧光染色观察TFBH对细胞核荧光强度的影响。Western blot法检测TFBH处理细胞后,凋亡相关蛋白以及PI3K/Akt信号通路蛋白表达情况。结果 不同浓度的TFBH处理HT-29细胞后,细胞活力随着其浓度的增加而降低。与对照组比较,50、62.5 μg·mL^(-1)TFBH组能够抑制HT-29细胞发生迁移。进一步的流式细胞术和Hoechst 33258检测表明TFBH可以诱导细胞周期阻滞在G_(0)/G_(1)期和G_(2)/M期,并诱导细胞凋亡,凋亡细胞核的荧光强度与TFBH给药浓度成正相关。Western blot结果显示,TFBH能够使凋亡相关蛋白Bax/Bcl-2、cleaved caspase-3/caspase-3以及cleaved caspase-9/caspase-9的相对蛋白表达水平增加,并抑制PI3K/Akt信号通路蛋白p-PI3K、p-Akt的表达。结论 TFBH可以在体外抑制人结肠癌HT-29细胞的增殖和迁移,其诱导细胞凋亡机制与抑制PI3K/Akt信号通路、调控Bcl-2凋亡蛋白家族并激活caspase级联反应有关。

Objective To determine the effect of total flavoniods from Bruguiera gymnorrhiza hypocotyls (TFBH) on the proliferation and migration of human colon cancer HT-29 cells and its apoptosis mechanism.Methods HT-29 cells were treated with TFBH and at various concentrations.The proliferation of cells treated with TFBH were checked by MTT assay.Scratch assay was used to observe the effect of TFBH on cell migration.Flow cytometry and Hoechst 33258 fluorescence staining were used to detect the effect on the cell cycle and apoptosis.Western blot was used to evaluate the expression level of apoptosis-related proteins as well as PI3K/Akt signaling pathway proteins.Results After the treatment with different concentrations of TFBH in HT-29 cells,the cell viability decreased in a dose-dependent manner.Compared with the control group,the 50 and 62.5 μg·mL^(-1)TFBH groups inhibited the migration of HT-29 cells.Further evaluation with flow cytometry and Hoechst 33258 confirmed that TFBH induced the cell cycle arrest at G/Gand G/M phase and apoptosis,and the fluorescence intensity of apoptotic cell nucleus was positively correlated with the TFBH administration concentration.Western blot showed that TFBH up-regulated the expression of apoptosis-related proteins Bax/Bcl-2,cleaved caspase-3/caspase-3 and cleaved caspase-9/caspase-9,and significantly inhibited the expression of p-PI3K and p-Akt.Conclusion TFBH can inhibit the proliferation and migration of human colon cancer HT-29 cells in vitro.TFBH-induced apoptosis is related to the suppression of PI3K/Akt signaling pathway and thereafter activation of Bcl-2 apoptosis protein family and the caspase cascade reactions.