title: Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells.
abstract: Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.
authors.affiliation: a Department of Clinical and Biological Sciences ; University of Turin ; Turin , Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Ullio
authors.affiliation: b Division of Pharmacology; Faculty of Health Sciences; Linköping University ; Linköping , Sweden.
authors.forename: Ulf T
authors.initials: UT
authors.lastname: Brunk
authors.affiliation: c Department of Earth and Environmental Sciences ; University of Milan-Bicocca ; Milan , Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Urani
authors.affiliation: c Department of Earth and Environmental Sciences ; University of Milan-Bicocca ; Milan , Italy.
authors.forename: Pasquale
authors.initials: P
authors.lastname: Melchioretto
authors.affiliation: a Department of Clinical and Biological Sciences ; University of Turin ; Turin , Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Bonelli
authors.affiliation: a Department of Clinical and Biological Sciences ; University of Turin ; Turin , Italy.
authors.forename: Francesco M
authors.initials: FM
authors.lastname: Baccino
authors.affiliation: a Department of Clinical and Biological Sciences ; University of Turin ; Turin , Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Autelli
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects* / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D007501:Iron / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D008668:Metallothionein / Q000187:drug effects*; D009336:Necrosis / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D014409:Tumor Necrosis Factor-alpha; D008668:Metallothionein; D007501:Iron
keywords: TNF; autophagy; cell death; hepatoma cells; iron; lysosomes; oxidative stress