Arsenic Trioxide Induces Apoptosis in Molt-4 Cell Lines: Caspase 8-Dependent and Caspase 3-Independent.

Abstract

Arsenic Trioxide (As2O3) has been used successfully in the treatment of patients with relapsed or refractory acute promyelocytic leukemia (APL) without severe marrow suppression. Currently, the action of As2O3 on many other hematopoietic malignancies is under investigation. Much evidence has shown that caspase-3 plays essential executing role in apoptosis of many leukemia cell lines. But, the exact mechanism of As2O3-induced apoptosis in Molt-4 cell line which is originated from acute lymphoblastic leukemia is not well understood. Here, we investigate the action of As2O3 on Molt-4 cells and involved mechanism. Significant dose- and time-dependent inhibition of cell growth was observed by MTT assay. Following the treatment of As2O3 for 72 h, As2O3 at 4 μM exhibited 50% inhibition of growth in Molt-4 cells. The effect of As2O3 on the cell cycle was determined in Molt-4 cells by FACS analysis. DNA flow cytometric analysis with three independent experiments indicated that As2O3 induced a G1 and a G2-M phase arrest in Molt-4 cells following 6μM of exposure. Similar results were observed in Molt-4 cells following 2μM and 4μM exposure. These results indicated that As2O3 inhibited the cellular proliferation of Molt-4 cells via a G1 and a G2-M phase arrest of the cell cycle. To confirm and evaluate the induction of apoptosis, we performed the staining of cells with annexin V and PI. As with the percentages of sub-G1 group by FACS analysis, the proportion of apoptotic cells was increased in a dose-and -time dependent manner. Taken together, these results indicate that induction of apoptosis can be another mechanism of the antiproliferative effect of As2O3 besides G1 and G2-M phase arrests of the cell cycle in Molt-4 cells. We subsequently studied the activation of initiator caspase-8 and executioner caspase-3 in Molt-4 cells by Western blotting. Molt-4 cells that had undergone apoptosis on culturing with As2O3 displayed the initial activation of caspase-8 with the appearance of the large cleavage fragment of 43 to 41 kd. Despite the higher basal level of procaspase-3 expression in the Molt-4 cells prior to As2O3 treatment, we were unable to detect cleaved, activated caspase-3 following As2O3 treatment. Next, we checked whether inhibition of caspases-3 could abrogate the proapoptotic effects of As2O3. For this purpose the caspase-3 inhibitor, z-DEVD-fmk, was used. The results shown that addition of z-DEVD-fmk did not rescue Molt-4 cells from apoptosis induced by As2O3. These results clearly differ from other observations made with other leukemia cells and might explain, at least in part, that As2O3 induces apoptosis in Molt-4 cells is caspase 8-Dependent and caspase 3-Independent.