Fulda S, Susin S A, Kroemer G, Debatin K M
University Children's Hospital, Ulm, Germany.
Cancer Res. 1998 Oct 1;58(19):4453-60.
Apoptosis mediated by anticancer drugs may involve activation of death-inducing ligand/receptor systems such as CD95 (APO-1/Fas), cleavage of caspases, and perturbance of mitochondrial functions. We investigated the sequence of these events in SHEP neuroblastoma cells transfected with Bcl-2 or Bcl-X(L) using two different drugs, namely, doxorubicin (Doxo), which activates the CD95/CD95 ligand (CD95-L) system, and betulinic acid (Bet A), which does not enhance the expression of CD95 or CD95-L and which, as shown here, directly targets mitochondria. Apoptosis induced by both drugs was inhibited by Bcl-2 or Bcl-X(L) overexpression or by bongkrekic acid, an agent that stabilizes mitochondrial membrane barrier function, suggesting a critical role for mitochondria. After Doxo treatment, enhanced CD95/CD95-L expression and caspase-8 activation were not blocked by Bcl-2 or Bcl-X(L) and were found in cells with a mitochondrial transmembrane potential (delta psi(m)) that was still normal (delta psi(m)high cells). In marked contrast, after Bet A treatment, caspase-8 activation occurred in a Bcl-2- or Bcl-X(L)-inhibitable fashion and was confined to cells that had lost their delta psi(m) (delta psi(m)low cells). Mitochondria from cells treated with either Doxo or Bet A induced cleavage of both caspase-8 and caspase-3 in cytosolic extracts. Thus, caspase-8 activation may occur upstream or downstream of mitochondria, depending on the apoptosis-initiating stimulus. In contrast to caspase-8, cleavage of caspase-3 or poly(ADP-ribose)polymerase was always restricted to delta psi(m)low cells, downstream of the Bcl-2- or Bcl-X(L)-controlled checkpoint of apoptosis. Cytochrome c, released from mitochondria undergoing permeability transition, activated caspase-3 but not caspase-8 in a cell-free system. However, both caspases were activated by apoptosis-inducing factor, indicating that the mechanism of caspase-8 activation differed from that of caspase-3 activation. Taken together, our findings demonstrate that perturbance of mitochondrial function constitutes a central coordinating event in drug-induced cell death.
抗癌药物介导的细胞凋亡可能涉及死亡诱导配体/受体系统的激活，如CD95（APO-1/Fas）、半胱天冬酶的切割以及线粒体功能的紊乱。我们使用两种不同的药物，即阿霉素（Doxo）和桦木酸（Bet A），研究了转染了Bcl-2或Bcl-X(L)的SHEP神经母细胞瘤细胞中这些事件的顺序。阿霉素激活CD95/CD95配体（CD95-L）系统，而桦木酸不增强CD95或CD95-L的表达，并且如本文所示，它直接靶向线粒体。两种药物诱导的细胞凋亡均被Bcl-2或Bcl-X(L)的过表达或被一种稳定线粒体膜屏障功能的药剂——邦克雷酸所抑制，这表明线粒体起着关键作用。在阿霉素处理后，增强的CD95/CD95-L表达和半胱天冬酶-8的激活未被Bcl-2或Bcl-X(L)阻断，并且在具有仍正常的线粒体跨膜电位（Δψm）的细胞（Δψm高细胞）中发现。与之形成显著对比的是，在桦木酸处理后，半胱天冬酶-8的激活以一种可被Bcl-2或Bcl-X(L)抑制的方式发生，并且局限于已经失去其Δψm的细胞（Δψm低细胞）。用阿霉素或桦木酸处理的细胞的线粒体在细胞溶质提取物中诱导了半胱天冬酶-8和半胱天冬酶-3的切割。因此，半胱天冬酶-8的激活可能发生在线粒体的上游或下游，这取决于凋亡起始刺激。与半胱天冬酶-8不同，半胱天冬酶-3或聚（ADP-核糖）聚合酶的切割总是局限于Δψm低细胞，位于Bcl-2或Bcl-X(L)控制的细胞凋亡检查点的下游。从经历通透性转变的线粒体释放的细胞色素c在无细胞系统中激活了半胱天冬酶-3但未激活半胱天冬酶-8。然而，两种半胱天冬酶均被凋亡诱导因子激活，这表明半胱天冬酶-8激活的机制不同于半胱天冬酶-3激活的机制。综上所述，我们的研究结果表明线粒体功能的紊乱构成了药物诱导的细胞死亡中的一个核心协调事件。
