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Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation

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Abstract

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7β-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7β-hydroxycholesterol, cholesterol-5β, 6β-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7β-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.

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Abbreviations

7KC:

7-Ketocholesterol

7β-OH:

7β-Hydroxycholesterol

25-OH:

25-Hydroxycholesterol

CLSM:

Confocal laser scanning microscopy

DNR:

Daunorubicin

FAMIS:

Factor analysis of medical image sequences

NR:

Nile Red

PI:

Propidium iodide

RAPA:

Rapamycin

TUNICA:

Tunicamycin

VP-16:

Etoposide

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Acknowledgments

We thank Pr Eric Solary (INSERM UMR 866/IFR 100, Dijon, France) for his helpful comments. The authors are indebted to Mrs. Mary Bouley for reviewing the English version of the manuscript.

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Authors and Affiliations

  1. INSERM UMR 866/IFR100, CHU-Hôpital du Bocage, BP77908, 21079, Dijon Cedex, France

    Anne Vejux, Thomas Montange & Gérard Lizard

  2. INSERM U678/UMR-S UPMC, IFR 14, CHU Pitié-Salpêtrière, 75634, Paris Cedex 13, France

    Edmond Kahn

  3. Centre de Microscopie Appliqué à la Biologie et à la Médecine, INSERM IFR 100, 7 Bd Jeanne d’Arc, 21033, Dijon Cedex, France

    Franck Ménétrier

  4. Service Commun de Microscopie Electronique, INRA, BP 86510, 21065, Dijon Cedex, France

    Jeannine Lherminier

  5. Centre Anti-Cancer GF Leclerc, Laboratoire de Biologie Médicale, 1 bis rue du Pr Marion, 21000, Dijon, France

    Jean-Marc Riedinger

  6. LBMC, Faculté des Sciences Gabriel, Université de Bourgogne, 6 Bd Gabriel, 21000, Dijon, France

    Anne Vejux, Thomas Montange & Gérard Lizard

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  1. Anne Vejux
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Correspondence to Gérard Lizard.

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This work was supported by grants from the INSERM, the Ligue Contre Le Cancer (Comité de Côte d’Or) and the Conseil Régional de Bourgogne.

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Vejux, A., Kahn, E., Ménétrier, F. et al. Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation. Histochem Cell Biol 127, 609–624 (2007). https://doi.org/10.1007/s00418-006-0268-0

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