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Sanguinarine-induced apoptosis is associated with an early and severe cellular glutathione depletion

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Abstract

Purpose

The quaternary benzophenanthridine alkaloid sanguinarine exhibits a broad range of activity, including cytotoxicity against various human tumour and normal cell lines. Here, we examined its potency as an anticancer drug.

Methods

The differential cytotoxicity against cancer versus normal cells was assessed in vitro by two fluorimetric assays (RRT and Hoechst 33342 dye DNA assays, respectively) in a panel of human solid cancer cell lines and a human fibroblast primary culture. The ability to induce apoptosis was demonstrated in PC3 human prostatic adenocarcinoma cells by analysis of morphological changes, internucleosomal DNA fragmentation, cellular poly(ADP-ribose) polymerase cleavage and caspase 3/7 activation. Production of reactive oxygen species was evaluated by the 2′,7′-dichlorofluorescin diacetate assay. Depletion of cellular glutathione content was assessed with the monochlorobimane assay.

Results

Sanguinarine markedly inhibited the growth of all tested cells (IC50 0.9–3.3 μM) without differential cytotoxicity against normal versus cancer cells. In PC3 cells, continuous treatment with 5 μM sanguinarine induced an early (within 10 min) cellular reduced glutathione depletion insensitive to dithiothreitol or N-acetylcysteine treatment, followed by a caspase 3/7-dependent apoptotic response within 2 h. Complementary assays suggested that the glutathione depletion was initiated by direct reactivity of sanguinarine with reduced glutathione.

Conclusions

Taken together, these results show that (1) sanguinarine exhibits no specificity for cancer cells, and (2) its strong cytotoxicity is probably due to a rapid apoptotic response induced by an early and severe glutathione-depleting effect. They also suggest that the clinical usefulness of this alkaloid as an anticancer drug is limited.

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Abbreviations

DCFH-DA:

2′,7′-dichlorofluorescin diacetate

DEM:

Diethylmaleate

DTT:

Dithiothreitol

GSH:

Reduced glutathione

HBSS/Hep:

Hank's balanced salt solution/Hepes 15 mM, pH 7.4

NAC:

N-acetyl cysteine

PARP:

Poly(ADP-ribose) polymerase

PBS:

Phosphate-buffered saline

PBSMT:

1×PBS, pH 7.4, 5% m/v non-fat powdered milk, 0.1% v/v Tween 20

ROS:

Reactive oxygen species

RP-HPLC:

Reversed-phase high pressure liquid chromatography

RRT:

Resazurin reduction test

SDS:

Sodium dodecyl sulphate

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Authors and Affiliations

  1. UMR-INSERM U-484, Rue Montalembert, BP 184, 63005, Clermont-Ferrand, France

    Eric Debiton, Jean-Claude Madelmont, Jean Legault & Chantal Barthomeuf

  2. Laboratoire de Médecine Moléculaire, Université du Québec à Montréal, Montréal, Québec, Canada

    Chantal Barthomeuf

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  1. Eric Debiton
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  2. Jean-Claude Madelmont
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  3. Jean Legault
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  4. Chantal Barthomeuf
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Correspondence to Chantal Barthomeuf.

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Debiton, E., Madelmont, JC., Legault, J. et al. Sanguinarine-induced apoptosis is associated with an early and severe cellular glutathione depletion. Cancer Chemother Pharmacol 51, 474–482 (2003). https://doi.org/10.1007/s00280-003-0609-9

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  • DOI: https://doi.org/10.1007/s00280-003-0609-9

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