title: Mediating of caspase-independent apoptosis by cadmium through the mitochondria-ROS pathway in MRC-5 fibroblasts.
abstract: Cadmium (Cd) is an environmental pollutant of global concern with a 10-30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 microM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 microM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd.
authors.affiliation: Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei, Taiwan, ROC. cmshih@tmu.edu.tw
authors.forename: Chwen-Ming
authors.initials: CM
authors.lastname: Shih
authors.forename: Wun-Chang
authors.initials: WC
authors.lastname: Ko
authors.forename: Jui-Sheng
authors.initials: JS
authors.lastname: Wu
authors.forename: Yau-Huei
authors.initials: YH
authors.lastname: Wei
authors.forename: Leng-Fang
authors.initials: LF
authors.lastname: Wang
authors.forename: E-E
authors.initials: EE
authors.lastname: Chang
authors.forename: Tsui-Yun
authors.initials: TY
authors.lastname: Lo
authors.forename: Huey-Hwa
authors.initials: HH
authors.lastname: Cheng
authors.forename: Chien-Tsu
authors.initials: CT
authors.lastname: Chen
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor; D002104:Cadmium / Q000494:pharmacology*; D020169:Caspases / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D004579:Electron Transport; D005347:Fibroblasts / Q000166:cytology*; D005420:Flavoproteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008168:Lung / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: C494829:AIFM1 protein, human; D000590:Amino Acid Chloromethyl Ketones; D000975:Antioxidants; D051033:Apoptosis Inducing Factor; D005420:Flavoproteins; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D002104:Cadmium; D006861:Hydrogen Peroxide; D020169:Caspases