阿西替尼通过调控自噬对结肠癌HCT-116细胞增殖、凋亡的影响研究

目的研究阿西替尼(AXI)对结肠癌HCT 116细胞增殖、凋亡及自噬的影响。方法体外培养结肠癌HCT 116细胞,分为AXI 1组(150 μmol／L)、AXI 2组(300 μmol／L)、AXI 3组(600 μmol／L)及无任何添加的正常对照组(NC组)。四甲基偶氮唑蓝法(MTT)检测各组结肠癌HCT 116细胞增殖情况;AnnexinV FITC／PI法检测各组结肠癌HCT 116细胞凋亡情况;Western Blot检测结肠癌HCT 116细胞自噬相关蛋白LC3 Ⅱ、beclin1,雷帕霉素靶蛋白(mTOR)信号通路中p mTOR、p P70S6K蛋白水平、增殖相关蛋白CyclinD1、c Myc,以及凋亡相关cleaved caspase3、B细胞淋巴瘤 2(Bcl 2)、Bax蛋白表达水平。结果与NC组比较,AXI 1组和AXI 2组结肠癌HCT 116细胞增殖抑制率、凋亡率、LC3 Ⅱ、beclin1、Bax、cleaved Caspase3蛋白表达水平依次增加(P<005),p mTOR、p P70S6K、Cyclin D1、c Myc、Bcl 2蛋白表达水平依次减少(P<005)。AXI 2组和AXI 3组结肠癌HCT 116细胞增殖抑制率、凋亡率、LC3 Ⅱ、beclin1、p mTOR、p P70S6K、CyclinD1、c Myc、cleaved caspase3、Bcl 2、Bax蛋白表达水平比较,差异无统计学意义(P>005)。结论AXI可通过抑制mTOR通路激活,诱导结肠癌HCT 116细胞自噬,抑制其增殖,促进其凋亡,初步揭示了AXI对结肠癌细胞的作用机制,为结肠癌的靶向治疗提供了新思路。

Effects of acetinib on proliferation and apoptosis of colon cancer HCT-116 cells by regulating autophagy

Abstract] Objective: To study the effects of AXI on proliferation and apoptosis of human colon cancer HCT-116 cells by regulating autophagy. Methods: Colon cancer HCT-116 cells were cultured in vitro and the colon cancer HCT-116 cells in logarithmic phase were treated with different concentrations of AXI (1 mol/L, 7.5 mol/L, 15 mol/L, 30 mol/L, 60 mol/L). Normal growth colon cancer HCT-116 cells were set as control group (NC group). The proliferation of HCT-116 cells was detected by MTT assay, flow cytometry and Annexin V-FITC/PI were used to detect the apoptosis of HCT-116 cells in colon cancer, and Western Blot was used to detect the expressions of autophagy-related proteins LC3 II, beclin1, proliferation-related proteins CyclinD1, c-Myc, apoptosis-related cleaved-caspase3, B-cell lymphoma-2 (Bcl-2) and Bax in colon cancer cells. Results: Compared with NC group, AXI 1 mol/L, 7.5 mol/L, 15 mol/L, 30 mol/L inhibited the proliferation of HCT-116 cells in a concentration-dependent and time-dependent manner (P < 0.05). The inhibitory rate of 30 mol/L for 48 hours on the proliferation of HCT-116 cells was 50.21%, and close to half suppression dose IC50, which was the best concentration and effect time. There was no significant difference in the expressions of proliferation, apoptotic rate of colon cancer HCT-116 cells, LC3 II, beclin1, CyclinD1, c-Myc, cleaved-caspase 3, Bcl-2, Bax protein between AXI 30 mol/L and 60 mol/L (P > 0.05). Compared with NC group, the apoptotic rate of HCT-116 cells, LC3 II, beclin1, Bax, cleaved-Caspase3 protein expression levels of colon cancer HCT-116 cells in AXI 15 mol/L and 30 mol/L treatment groups increased in turn (P < 0.05), while the expression levels of Cyclin D1, c-Myc and Bcl-2 protein decreased in turn (P < 0.05). Conclusions: AXI may inhibit the proliferation of HCT-116 cells, induce autophagy and promote apoptosis by up-regulating the expressions of LC3 II, beclin1, Bax, cleaved-Caspase3 and down-regulating the expressions of Cyclin D1, c-Myc, Bcl-2.