Bleomycin induces the extrinsic apoptotic pathway in pulmonary endothelial cells.
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.
Dept. of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
Ognoon
O
Mungunsukh
Autumn J
AJ
Griffin
Young H
YH
Lee
Regina M
RM
Day
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D001761:Bleomycin / Q000633:toxicity*; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D053200:Fas-Associated Death Domain Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
D061945:Caspase Inhibitors; D017931:DNA Primers; D053200:Fas-Associated Death Domain Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D001761:Bleomycin; D020169:Caspases