吴茱萸碱对人结肠癌细胞周期阻滞及凋亡的影响

摘要

OBJECTlVE To investigate the effect of evodiamine on proliferation of human colorectal cancer cells and to explore its mechanism of apoptosis in human colorectal cancer cells. METHODS Human colorectal cancer cell lines ( HCT-116 ) were cultured with evodiamine at the concentrations of 1.5, 3.0 and 6.0 μmol·L-1 for 24, 48 and 72 h,respectively. The proliferation of cells was detected by CCK-8, the cell cycle and apoptosis of cells treated were measured by flow cytometry, changes in cell morphology were observed under an inverted microscope and morphological changes in apoptotic cells were observed under a fluorescence microscope after Hoehst staining. The protein expression of P53, Bax, Bcl-2, cleaved caspase 3 and cyclin A1 was examined by Western blot. RESULTS The proliferation of cells was significantly inhibited by evodiamine in a time and concentration-dependent manner( rTime=0.744, P=0.05;rConcentration=0.953, P<0.01). The percentage of cells in S phase increased from (13.9± 7.0)% to (39.7±11.7)%(P<0.05) and the rate of apoptosis cells up-regulated from (4.2±3.0)% to (14.9±5.8)%(P<0.05) when HCT-116 cells were treated with different concentrations of evodiamine for 48 h. Plasmatorrhexis and distortion occurred in a concentration-dependent manner while a large amount of cell debris and a large number of floating cells were observed under the microscope. Hoechst staining indicated cell shinkage, nuclear condensation and appare pale. The expression of P53, Bax and cleaved caspase 3 was obviously increased while the Bcl-2 and cyclin A1 significantly decreased. CONCLUSlON Evodiamine can induce the apoptosis of human colorectal cancer cells mainly by down-regulating the expression of cyclin A1 and activation of P53 signal pathway, breaking the balance of Bcl-2/Bax, and ultimately activating caspase 3.%目的：探讨吴茱萸碱对人结肠癌细胞周期阻滞及凋亡的影响及其作用机制。方法体外培养人结肠癌HCT-116细胞分别加入吴茱萸碱1.5,3.0,6.0和12μmol·L－1继续培养24,48和72 h,CCK-8法检测吴茱萸碱对HCT-116细胞存活的影响；用流式细胞术检测吴茱萸碱对HCT-116细胞凋亡和细胞周期的影响；倒置显微镜镜下观察细胞形态变化；荧光显微镜观察 Hoechst 染色后细胞凋亡的形态学改变；Western蛋白质印迹法检测细胞周期蛋白A1的表达及凋亡相关蛋白P53, Bax ,Bcl-2,激活型胱天蛋白酶3的表达。结果吴茱萸碱1.5,3.0和6.0μmol·L－1作用24,48和72 h后,HCT-116细胞增殖受到抑制( P<0.05),且呈时间( r时间＝0.744, P<0.05)和浓度( r浓度＝0.953, P<0.01)依赖性。吴茱萸碱作用HCT-116细胞48 h后, S期细胞由正常对照组的(13.9±7.0)％增加至(39.7±11.7)％(P<0.05)；细胞凋亡率由正常对照组的(4.2±3.0)％增加至(14.9±5.8)％(P<0.05)。光学显微镜下可见,随着药物浓度的增加,细胞的形态发生改变,胞膜破裂,产生细胞碎片,并出现大量漂浮细胞；Hoechst染色可见部分细胞出现细胞核固缩、核发白、染色质凝集等凋亡变化；P53、Bax和激活型胱天蛋白酶3蛋白表达增加, Bcl-2、细胞周期蛋白A1蛋白表达下降。结论吴茱萸碱能够通过下调细胞周期蛋白 A1的表达；激活 P53信号通路,下调Bcl-2、上调Bax,破坏Bcl-2/Bax的比例；激活胱天蛋白酶3,共同促进人结肠癌HCT-116细胞的凋亡。