P53-mediated cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells
P53-mediated cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells
复制标题
DOI：
10.1111/j.1745-7254.2007.00588.x
复制
发表时间：
2007-07-01
影响因子：
8.2
通讯作者：
Ikejima, Takashi
中科院分区：
文献类型：
--
作者：
Cui, Qiao;Yu, Jing-hua;Ikejima, Takashi
目的：研究冬凌草甲素体外诱导MCF-7人乳腺癌细胞凋亡的非caspase-3机制。方法：采用MTT（噻唑蓝）法检测冬凌草甲素处理的MCF-7细胞的活力。通过相差显微镜观察具有浓缩细胞核的凋亡细胞。通过琼脂糖凝胶电泳测定核小体DNA片段化。通过乳酸脱氢酶测定测定细胞凋亡比率。通过流式细胞术分析测量细胞周期交替和线粒体膜电位。 Western blot检测Bax、Bcl-2、caspase-3、caspase-9、热休克蛋白（Hsp）90、p53、p-p53、p21、聚ADP核糖聚合酶（PARP）和半胱天冬酶激活DNase抑制剂（ICAD）蛋白的表达。结果：冬凌草甲素以时间和剂量依赖性方式抑制细胞生长。通过上调 p53 和 p21 蛋白表达来改变细胞周期。 Pancaspase 抑制剂 Z-VAD-fmk 和 calpain 抑制剂 II 均降低细胞死亡率。在冬凌草甲素诱导的 MCF-7 细胞凋亡中检测到核小体 DNA 断裂和 Delta Psi(mit) 下调，这涉及线粒体后 caspase-9 依赖性途径。 Bcl-2和Hsp90表达水平的降低以及Bax和p21表达水平的增加与磷酸化p53磷酸化水平的升高呈正相关。此外，PARP 部分被钙蛋白酶而不是被 capase-3 裂解。结论：DNA 损伤引起线粒体和 caspase-9 通路的改变以及 p53 介导的细胞周期停滞，但与冬凌草甲素诱导的 MCF-7 细胞中的 caspase-3 活性无关。
Aim: To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro.Methods: The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis.Results: Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of Delta Psi(mit) were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3.Conclusion: DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.