title: Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine.
abstract: Hydroxychloroquine (HCQ) is a lysosomotropic amine with cytotoxic properties. Here, we show that HCQ induces signs of lysosomal membrane permeabilization (LMP), such as the decrease in the lysosomal pH gradient and the release of cathepsin B from the lysosomal lumen, followed by signs of apoptosis including caspase activation, phosphatidylserine exposure, and chromatin condensation with DNA loss. HCQ also induces mitochondrial membrane permeabilization (MMP), as indicated by the insertion of Bax into mitochondrial membranes, the conformational activation of Bax within mitochondria, the release of cytochrome c from mitochondria, and the loss of the mitochondrial transmembrane potential. To determine the molecular order among these events, we introduced inhibitors of LMP (bafilomycin A(1)), MMP (Bcl-X(L), wild-type Bcl-2, mitochondrion-targeted Bcl-2, or viral mitochondrial inhibitor of apoptosis from cytomegalovirus), and caspases (Z-VAD.fmk) into the system. Our data indicate that caspase-independent MMP is rate-limiting for LMP-mediated caspase activation. Mouse embryonic fibroblasts lacking the expression of both Bax and Bak are resistant against hydroxychloroquine-induced apoptosis. Such Bax(-/-) Bak(-/-) cells manifest normal LMP, yet fail to undergo MMP and subsequent cell death. The data reported herein indicate that LMP does not suffice to trigger caspase activation and that Bax/Bak-dependent MMP is a critical step of LMP-induced cell death.
authors.affiliation: Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Boya
authors.forename: Rosa-Ana
authors.initials: RA
authors.lastname: Gonzalez-Polo
authors.forename: Delphine
authors.initials: D
authors.lastname: Poncet
authors.forename: Karine
authors.initials: K
authors.lastname: Andreau
authors.forename: Helena L A
authors.initials: HL
authors.lastname: Vieira
authors.forename: Thomas
authors.initials: T
authors.lastname: Roumier
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Perfettini
authors.forename: Guido
authors.initials: G
authors.lastname: Kroemer
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000502:physiology; D002460:Cell Line / Q000166:cytology / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019254:Genes, bcl-2; D006367:HeLa Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D006886:Hydroxychloroquine / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects*; D019169:Jurkat Cells / Q000166:cytology / Q000187:drug effects; D008247:Lysosomes / Q000502:physiology*; D018942:Macrolides; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects*; D010539:Permeability / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction; D014162:Transfection; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000900:Anti-Bacterial Agents; C494772:BAK1 protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; C494773:Bak1 protein, mouse; C494774:Bak1 protein, rat; C494827:Bax protein, mouse; C494828:Bax protein, rat; C494812:Bcl2l1 protein, mouse; C494813:Bcl2l1 protein, rat; D015853:Cysteine Proteinase Inhibitors; D018942:Macrolides; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D006886:Hydroxychloroquine; C040929:bafilomycin A1; D020169:Caspases