Custódio J B, Cardoso C M, Madeira V M, Almeida L M
Laboratório de Bioquimíca, Faculdade de Farmácia, Universidade de Coimbra, Coimbra, Portugal.
Toxicol In Vitro. 2001 Aug-Oct;15(4-5):265-70. doi: 10.1016/s0887-2333(01)00019-4.
Etoposide (VP-16) is widely used for the treatment of several forms of cancer. The cytotoxicity of VP-16 has been assigned to the induction of apoptotic cell death but the signaling pathway for VP-16-induced apoptosis is essentially unknown. There is some evidence that this process depends on events associated with the loss of mitochondrial membrane potential (Delta Psi) and/or release of apoptogenic factors, putatively as a consequence of mitochondrial permeability transition (MPT) induction. This work evaluates the interference of VP-16 with MPT in vitro, which is characterized by the Ca(2+)-dependent depolarization of Delta Psi, the release of matrix Ca(2+) and by extensive swelling of mitochondria. Delta Psi depolarization and Ca(2+) release were measured with ion-selective electrodes, and mitochondrial swelling was monitored spectrophotometrically. Incubation of rat liver mitochondria with VP-16 results in a concentration-dependent induction of MPT, evidenced by an increased sensitivity to Ca(2+)-induced swelling, depolarization of Delta Psi, Ca(2+) release by mitochondria and stimulation of state 4 oxygen consumption. All of these effects are prevented by preincubating the mitochondria with cyclosporine A, a potent and specific inhibitor of the MPT. Therefore, VP-16 increases the sensitivity of isolated mitochondria to the Ca(2+)-dependent induction of the MPT. Together, these data provide a possible mechanistic explanation for the previously reported effects of VP-16 on apoptosis induction.
依托泊苷（VP - 16）被广泛用于治疗多种癌症。VP - 16的细胞毒性已归因于诱导凋亡性细胞死亡，但其诱导凋亡的信号通路基本未知。有证据表明，该过程取决于与线粒体膜电位（ΔΨ）丧失和/或凋亡因子释放相关的事件，推测这是线粒体通透性转换（MPT）诱导的结果。本研究评估了VP - 16在体外对MPT的干扰作用，MPT的特征是ΔΨ的Ca²⁺依赖性去极化、基质Ca²⁺的释放以及线粒体的广泛肿胀。用离子选择性电极测量ΔΨ去极化和Ca²⁺释放，并用分光光度法监测线粒体肿胀。用VP - 16孵育大鼠肝线粒体导致MPT呈浓度依赖性诱导，表现为对Ca²⁺诱导的肿胀敏感性增加、ΔΨ去极化、线粒体Ca²⁺释放以及对状态4氧消耗的刺激。预先用环孢素A（一种有效的MPT特异性抑制剂）孵育线粒体可防止所有这些效应。因此，VP - 16增加了分离线粒体对Ca²⁺依赖性MPT诱导的敏感性。总之，这些数据为先前报道的VP - 16对凋亡诱导的作用提供了一种可能的机制解释。
