Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells
Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells
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DOI：
10.1074/jbc.m302559200
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发表时间：
2003-09-05
影响因子：
4.8
通讯作者：
Perez-Soler, R
中科院分区：
文献类型：
--
作者：
Ling, YH;Liebes, L;Perez-Soler, R
硼替佐米是一种蛋白酶体抑制剂，在多种肿瘤细胞系中显示出显著的抗肿瘤活性，处于I、II和III期临床试验中，最近已被批准用于治疗多发性骨髓瘤患者。硼替佐米抑制蛋白酶体后导致细胞凋亡的事件顺序尚不清楚。检测硼替佐米对线粒体凋亡途径组分的影响：活性氧（ROS）的产生、线粒体膜电位（Deltapsi（m））的改变和线粒体释放细胞色素c。对于人H460肺癌细胞，0.1 μ M硼替佐米暴露显示在24小时开始诱导凋亡性细胞死亡，在处理48 - 72小时后效果增加。在3 - 6小时后，以时间依赖性的方式观察到ROS产生的升高、Deltapsi（m）的增加以及细胞色素c释放到胞质溶胶中。与鱼藤酮和抗霉素A（线粒体电子传递链复合物I和III的抑制剂）或环孢霉素A（线粒体通透性转换孔的抑制剂）共孵育，可抑制硼替佐米诱导的ROS生成，增加Deltapsi（m）和细胞色素c释放。抗氧化剂Tiron阻断硼替佐米诱导的ROS产生、Deltapsi（m）增加和细胞色素c释放。Tiron处理还防止硼替佐米诱导的PARP蛋白裂解和细胞死亡。苄氧基羰基-VAD-氟甲基酮（一种泛半胱天冬酶抑制剂）虽然阻止了硼替佐米诱导的聚（ADP-核糖）聚合酶裂解和凋亡性死亡，但并未改变硼替佐米诱导的ROS生成和Deltapsi（m）增加。在PC-3前列腺癌细胞（Bcl-2过表达）中，硼替佐米诱导的ROS生成减少，Deltapsi（m）增加与细胞对硼替佐米的耐药性和药物诱导的细胞凋亡减弱相关。在p53缺失的H358细胞中瞬时转染野生型p53引起硼替佐米诱导的细胞凋亡的刺激，但未能增强ROS的产生和Deltapsi（m）的增加。因此，ROS的产生通过介导Deltapsi（m）的破坏和细胞色素c从线粒体的释放在硼替佐米诱导的凋亡级联的启动中起关键作用。
Bortezomib, a proteasome inhibitor, shows substantial anti-tumor activity in a variety of tumor cell lines, is in phase I, II, and III clinical trials and has recently been approved for the treatment of patients with multiple myeloma. The sequence of events leading to apoptosis following proteasome inhibition by bortezomib is unclear. Bortezomib effects on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration in the mitochondrial membrane potential (Deltapsi(m)), and release of cytochrome c from mitochondria. With human H460 lung cancer cells, bortezomib exposure at 0.1 muM showed induction of apoptotic cell death starting at 24 h, with increasing effects after 48 - 72 h of treatment. After 3 - 6 h, an elevation in ROS generation, an increase in Deltapsi(m), and the release of cytochrome c into the cytosol, were observed in a time-dependent manner. Co-incubation with rotenone and antimycin A, inhibitors of mitochondrial electron transport chain complexes I and III, or with cyclosporine A, an inhibitor of mitochondrial permeability transition pore, resulted in inhibition of bortezomib-induced ROS generation, increase in Deltapsi(m), and cytochrome c release. Tiron, an antioxidant agent, blocked the bortezomib-induced ROS production, Deltapsi(m) increase, and cytochrome c release. Tiron treatment also protected against the bortezomib-induced PARP protein cleavage and cell death. Benzyloxycarbonyl-VAD-fluoromethyl ketone, an inhibitor of pan-caspase, did not alter the bortezomib-induced ROS generation and increase in Deltapsi(m), although it prevented bortezomib-induced poly( ADP-ribose) polymerase cleavage and apoptotic death. In PC-3 prostate carcinoma cells ( with overexpression of Bcl-2), a reduction of bortezomib-induced ROS generation, Deltapsi(m) increase was correlated with cellular resistance to bortezomib and the attenuation of drug-induced apoptosis. The transient transfection of wild type p53 in p53 null H358 cells caused stimulation of the bortezomib-induced apoptosis but failed to enhance ROS generation and Deltapsi(m) increase. Thus ROS generation plays a critical role in the initiation of the bortezomib-induced apoptotic cascade by mediation of the disruption of Deltapsi(m) and the release of cytochrome c from mitochondria.