Purpose: To demonstrate that arsenic trioxide (As(2)O(3)) 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 As(2)O(3). Buthionine sulfoxide (BSO) and ascorbic acid (AA) rendered these cells more sensitive to As(2)O(3), 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 As(2)O(3)-mediated growth inhibition, disruption of MMP, activation of caspase-3 and apoptosis of cells. Clinically relevant doses of AA enhanced the anticancer effects of As(2)O(3) via the disruption of MMP, activation of caspase-3, and induction of apoptosis. In contrast, increase GSH levels with NAC attenuated all of these As(2)O(3)-mediated actions.
Conclusions: The sensitivity of MOLT-4 and MOLT-4/DNR cells to As(2)O(3) was associated with the intracellular GSH content. As(2)O(3) 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.