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The chemopreventive agent N-(4-hydroxyphenyl)retinamide induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family

Abstract

N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a potent chemopreventive agent whose effect has been suggested to involve apoptosis induction. 4-HPR induces a loss of the mitochondrial transmembrane potential and the mitochondrial release of cytochrome c before caspase activation. Inhibition of mitochondrial membrane permeabilization (MMP) by transfection with Bcl-2 or the Cytomegalovirus UL37 gene product vMIA prevented caspase activation and cell death. In contrast to other retinoid derivatives, 4-HPR has no direct MMP-inducing effects when added to isolated mitochondria or when added to proteoliposomes containing the MMP-regulatory permeability transition pore complex (PTPC). Moreover, although reactive oxygen species (ROS) overproduction appears to be instrumental for 4-HPR-induced MMP and apoptosis, inhibition of the NF-κB or p53-mediated signal transduction pathways failed to modulate 4-HPR-induced apoptosis. 4-HPR was found to cause an antioxidant-inhibitable conformational change of both Bax and Bak, leading to the exposure of their N-termini and to the mitochondrial relocalization of Bax. Cells with a Bax−/− Bak−/− genotype were resistant against the 4-HPR-induced MMP, overproduction of ROS and cell death. Altogether, these data indicate that 4-HPR induces MMP through an ROS-mediated pathway that involves the obligatory contribution of the proapopotic Bcl-2 family members Bax and/or Bak.

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Abbreviations

4-HPR:

4-hydroxyphenylretinamide

ANT:

adenine nucleotide translocator

Asp2R110:

rhodamine 110-Asp2

Atr:

atractyloside

atRA:

all-trans-retinoic acid

Casp-3a:

activated caspase-3

CMXRos:

chloromethyl-X-rosamine

Cyt c:

cytochrome c

ΔΨm:

mitochondrial transmembrane potential

DAPI:

4′,6-diamidino-2-phenylindole,dihydrochloride

DiOC6(3):

3,3′dihexyloxacarbocyanine iodide

Eth:

ethidium

HE:

hydroethidine

MMP:

mitochondrial membrane permeabilization

4-MU:

4-methylumbelliferone

4-MUP4-methylumbelliferylphosphatePTPC:

permeability transition pore complex

PI:

propidium iodide

ROS:

reactive oxygen species

vMIA:

viral mitochondrial inhibitor of apoptosis

Z-VAD.fmk:

N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone

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Acknowledgements

We thank Dr Victor Goldmacher (ImmunoGen, Cambridge, MA, USA) and Dr Bern Vogelstein (John Hopkins Cancer Center, Baltimore, MA, USA) for cell lines and Nathanael Larochette and Didier Métivier (CNRS, Villejuif, France) for assistance. This work has been supported by a special grant from the Ligue Nationale contre le Cancer, as well as grants from Ligue départmentale contre le Cancer (Seine St Denis), ANRS, FRM, ARC, and European Commission (QLG1-CT-1999-00739 and QLK3-CT-20002-01956) (to GK). PB received a fellowship from the European Commission (MCFI-2000-00943).

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Correspondence to Guido Kroemer.

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Boya, P., Morales, M., Gonzalez-Polo, RA. et al. The chemopreventive agent N-(4-hydroxyphenyl)retinamide induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family. Oncogene 22, 6220–6230 (2003). https://doi.org/10.1038/sj.onc.1206827

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