Cancer Chemotherapy and Pharmacology

, Volume 52, Issue 1, pp 47–58

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

Original Article

DOI: 10.1007/s00280-003-0629-5

Cite this article as:
Hu, X., Hirano, T. & Oka, K. Cancer Chemother Pharmacol (2003) 52: 47. doi:10.1007/s00280-003-0629-5

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.

Keywords

Arsenic trioxideApoptosisGlutathioneMitochondrial membrane potentialCaspase-3

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Department of Clinical Pharmacology, School of PharmacyTokyo University of Pharmacy and Life ScienceTokyoJapan
  2. 2.National Therapeutic Center of Hematology of Traditional Chinese Medicine, XiYuan HospitalChina Academy of Traditional Chinese MedicineBeijingP.R. China