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

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.