Dept. of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
Am J Physiol Lung Cell Mol Physiol. 2010 May;298(5):L696-703. doi: 10.1152/ajplung.00322.2009. Epub 2010 Feb 12.
Bleomycin, a chemotherapeutic agent, can cause pulmonary fibrosis in humans and is commonly used to induce experimental pulmonary fibrosis in rodents. In cell culture, bleomycin causes single- and double-stranded DNA breaks and produces reactive oxidative species, both of which require iron (Fe(2+)) and O(2). The mechanism of bleomycin-induced apoptosis is controversial due to its complexity. We investigated bleomycin apoptotic signaling events in primary pulmonary endothelial cells. Time course experiments revealed that bleomycin induced apoptosis within 4 h. Caspase-8, the initiator caspase for the extrinsic pathway, was activated within 2 h and preceded activation of the effector caspases-3 and -6 (4 h). Caspase-9, the initiator of the intrinsic pathway and release of cytochrome c from the mitochondria were not detected at these time points. Bleomycin induced the expression of Bcl-2 and Bcl-x(L), Bcl-2 family member proteins that protect cells from the mitochondria-dependent intrinsic apoptosis. Real-time quantitative RT-PCR results demonstrated that, at 4-8 h, bleomycin induced expression of TNF and TNF receptor family genes known to induce the extrinsic apoptotic pathway. Silencing of the death receptor adaptor protein Fas-associated death domain by short interfering RNA significantly reduced bleomycin-induced apoptosis. Apoptosis was also abrogated by caspase-8 inhibition, but only slightly reduced by caspase-3 inhibition. Together, these data suggest that bleomycin initiates apoptosis via the extrinsic pathway.
博莱霉素是一种化疗药物，可导致人类肺纤维化，常用于诱导啮齿动物实验性肺纤维化。在细胞培养中，博莱霉素可导致单链和双链 DNA 断裂，并产生活性氧，这两者都需要铁（Fe(2+)）和 O(2)。博莱霉素诱导细胞凋亡的机制因复杂性而存在争议。我们研究了原代肺内皮细胞中博莱霉素诱导的凋亡信号事件。时间进程实验表明，博莱霉素在 4 小时内诱导细胞凋亡。起始外源性途径的效应半胱天冬酶 caspase-8 在 2 小时内被激活，并早于效应半胱天冬酶 caspase-3 和 caspase-6 的激活（4 小时）。在这些时间点未检测到起始内源性途径的 caspase-9 和细胞色素 c 从线粒体中的释放。博莱霉素诱导 Bcl-2 和 Bcl-x(L)的表达，Bcl-2 家族蛋白可保护细胞免受线粒体依赖性内在凋亡的影响。实时定量 RT-PCR 结果表明，在 4-8 小时，博莱霉素诱导已知诱导外源性凋亡途径的 TNF 和 TNF 受体家族基因的表达。短干扰 RNA 沉默死亡受体衔接蛋白 Fas 相关死亡结构域显著减少博莱霉素诱导的凋亡。凋亡也被 caspase-8 抑制剂阻断，但 caspase-3 抑制剂仅轻度减少。总之，这些数据表明博莱霉素通过外源性途径起始细胞凋亡。
