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Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine

Oncogene volume 22, pages 3927–3936 (2003)Cite this article

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 A1), MMP (Bcl-XL, 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.

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Abbreviations

AO:

acridine orange

Baf A1:

bafilomycin A1

Casp-3a:

activated caspase-3

CB:

cathepsin B

ΔΨm:

mitochondrial transmembrane potential

DAPI:

4′,6-diamidino-2-phenylindoledihydrochloride

DiOC6(3):

3,3′-dihexyloxacarbocyanine iodide, DN,dominant negative

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

JC-1,5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; HCQ:

hydroxychloroquine

LMP:

lysosomal membrane permeabilization

MMP:

mitochondrial membrane permeabilization

PMP:

plasma membrane permeabilization

PS:

phosphatidylserine

z-FA-fmk:

N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone

Z-VAD.fmk:

N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone

vMIA:

viral mitochondrial inhibitor of apoptosis

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Acknowledgements

We thank Drs Victor Goldmacher (ImmunoGen, Cambridge, MA, USA) for cell lines, David Andrews (Hamilton University, Ontario, Canada), Nicole Israel (Pasteur Institute, Paris, France), Nathanael Larochette, and Didier Métivier (CNRS, Villejuif, France) for assistance, Dominique Coulaud (CNRS, UMR5826, Villejuif, France), and the NIH AIDS reagents program (Bethesda, MD) for cell lines.This work has been supported by a special grant from LNC, as well as grants from ANRS, FRM, and European Commission (QLG1-CT-1999-00739) (to GK). PB receives a fellowship from the European Commission (MCFI-2000-00943).

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  1. Centre National de la Recherche Scientifique, UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, Villejuif, F-94805, France

    Patricia Boya, Rosa-Ana Gonzalez-Polo, Delphine Poncet, Karine Andreau, Helena LA Vieira, Thomas Roumier, Jean-Luc Perfettini & Guido Kroemer

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Correspondence to Guido Kroemer.

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Boya, P., Gonzalez-Polo, RA., Poncet, D. et al. Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene 22, 3927–3936 (2003). https://doi.org/10.1038/sj.onc.1206622

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