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Arsenic trioxide induces apoptosis in cells of MOLT-4 and its daunorubicin-resistant cell line via depletion of intracellular glutathione, disruption of mitochondrial membrane potential and activation of caspase-3

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Abstract

Purpose

To demonstrate that arsenic trioxide (As2O3) induces apoptosis via a mitochondrial pathway in both parent T lymphoblastoid leukemia MOLT-4 cells and cells of its daunorubicin-resistant subline, MOLT-4/DNR, expressing functional P-gp.

Methods

Cell growth was measured using an MTT assay. Cell viability was determined using a dye exclusion test. Intracellular glutathione (GSH) was measured using a glutathione assay kit. Mitochondrial membrane potential (MMP) was assessed by rhodamine 123 (Rh123) staining intensity on flow cytometry. Caspase-3 activity was evaluated using a commercially available assay kit on flow cytometry. The percentage of cells undergoing apoptosis was estimated in terms of caspase+/PI− cells on flow cytometry after assessment for activation of caspase-3 by adding PI.

Results

MOLT-4 cells and MOLT-4/DNR cells were similarly sensitive to the apoptosis-inducing effect of As2O3. Buthionine sulfoxide (BSO) and ascorbic acid (AA) rendered these cells more sensitive to As2O3, whereas N-acetylcysteine (NAC) reduced this sensitivity. BSO and AA decreased, but NAC increased, the intracellular GSH contents of both MOLT-4 and MOLT-4/DNR cells. Decreasing GSH with BSO potentiated As2O3-mediated growth inhibition, disruption of MMP, activation of caspase-3 and apoptosis of cells. Clinically relevant doses of AA enhanced the anticancer effects of As2O3 via the disruption of MMP, activation of caspase-3, and induction of apoptosis. In contrast, increase GSH levels with NAC attenuated all of these As2O3-mediated actions.

Conclusions

The sensitivity of MOLT-4 and MOLT-4/DNR cells to As2O3 was associated with the intracellular GSH content. As2O3 induced apoptosis in parent MOLT-4 cells and MOLT-4/DNR cells expressing functional P-gp via depletion of intracellular GSH, and subsequent disruption of MMP and activation of caspase-3.

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  1. Xiao-Mei Hu

    Present address: National Therapeutic Center of Hematology of Traditional Chinese Medicine, XiYuan Hospital, China Academy of Traditional Chinese Medicine, 100091, Beijing, P.R. China

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  1. Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, 192-0392, Tokyo, Japan

    Xiao-Mei Hu, Toshihiko Hirano & Kitaro Oka

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Correspondence to Toshihiko Hirano.

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Hu, XM., Hirano, T. & Oka, K. Arsenic trioxide induces apoptosis in cells of MOLT-4 and its daunorubicin-resistant cell line via depletion of intracellular glutathione, disruption of mitochondrial membrane potential and activation of caspase-3. Cancer Chemother Pharmacol 52, 47–58 (2003). https://doi.org/10.1007/s00280-003-0629-5

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