Tian Xin, Luo Ying, Liu Yun-peng, Hou Ke-zuo, Jin Bo, Zhang Jing-dong, Wang Su
Department of Medical Oncology, The First Affiliated Hospital, China Medical University, Shenyang 110001, China.
Zhonghua Xue Ye Xue Za Zhi. 2006 Jan;27(1):21-4.
To investigate the expression of mitochondria-mediated apoptosis pathway related genes bcl-2, Bax, survivin and Smac/DIABLO in bufalin induced HL-60 cell apoptosis.
Cell viability was determined by trypan blue exclusion, apoptosis by morphology and flow cytometry, expressions of Bcl-2, Bax, Survivin, Smac/DIABLO and caspase-3 protein by Western blot, and expression of survivin mRNA by RT-PCR.
Proliferation of HL-60 cells was inhibited by bufalin and the IC(50) at 24, 48 and 72 h were 25.8, 8.0 and 2.1 nmol/L, respectively. Apoptosis was induced when the cells were treated with bufalin at concentrations of 50.0 nmol/L and higher. Compared with the control, HL-60 cells treated with bufalin at 50.0 nmol/L for 6, 12, 24 and 48 h showed decrease of Bcl-2 protein expression to 88.6%, 53.3%, 19.2% and 9.5%, Bcl-2/Bax ratio (control 2.0) to 1.7, 1.1, 0.4 and 0.2, Survivin protein expression to 75.2%, 54.8%, 37.5% and 20.3%, and survivin mRNA to 85.7%, 39.4%, 12.5%and 0%, respectively. The expression of Smac/DIABLO protein was downregulated to 77.5% (12 h), 21.2% (24 h) and 15.3% (48 h) in mitochondrial fraction and upregulated to 1.4-(12h), 2.0-(24 h) and 3.5- folds (48 h) in cytosolic fraction, respectively. The active subunits of caspase-3 were displayed after treatment for 8 h till 48 h.
Apoptosis induced by bufalin is related to downregulation of expressions of bcl-2 and survivin, decrease of Bcl-2/Bax ratio, mitochondrial release of Smac/DIABLO, and activation of caspase-3. The mitochondria-mediated apoptotic pathway may be involved in the apoptosis induced by bufalin in HL-60 cells.
研究线粒体介导的凋亡途径相关基因bcl-2、Bax、survivin和Smac/DIABLO在蟾毒灵诱导HL-60细胞凋亡中的表达情况。
采用台盼蓝拒染法检测细胞活力，通过形态学和流式细胞术检测细胞凋亡，采用蛋白质印迹法检测Bcl-2、Bax、Survivin、Smac/DIABLO和caspase-3蛋白的表达，采用逆转录聚合酶链反应（RT-PCR）检测survivin mRNA的表达。
蟾毒灵可抑制HL-60细胞增殖，24、48和72 h的半数抑制浓度（IC50）分别为25.8、8.0和2.1 nmol/L。当细胞用50.0 nmol/L及以上浓度的蟾毒灵处理时可诱导凋亡。与对照组相比，用50.0 nmol/L蟾毒灵处理6、12、24和48 h的HL-60细胞，Bcl-2蛋白表达分别降至88.6%、53.3%、19.2%和9.5%，Bcl-2/Bax比值（对照组为2.0）分别降至1.7、1.1、0.4和0.2，Survivin蛋白表达分别降至75.2%、54.8%、37.5%和20.3%，survivin mRNA分别降至85.7%、39.4%、12.5%和0%。Smac/DIABLO蛋白在线粒体部分的表达在12 h时下调至77.5%，24 h时下调至21.2%，48 h时下调至15.3%；在胞质部分的表达在12 h时上调至1.4倍，24 h时上调至2.0倍，48 h时上调至3.5倍。处理8 h至48 h后显示出caspase-3的活性亚基。
蟾毒灵诱导的凋亡与bcl-2和survivin表达下调、Bcl-2/Bax比值降低、Smac/DIABLO从线粒体释放以及caspase-3激活有关。线粒体介导的凋亡途径可能参与了蟾毒灵诱导的HL-60细胞凋亡。
