Downregulation of Survivin by Arsenic Trioxide in Acute Myeloid Leukemia Cell Line.

Caspases are proteins that play a central role in apoptosis. Therefore, triggering apoptosis through chemotherapeutical caspases inductor drugs is the major path in cancer treatment. However, hindering apoptosis by inhibitor of apoptosis proteins (IAPs) overexpression, have been described in many cancer types including leukemia and, is frequently related to drug resistance. Survivin, a member of IAPs family, is expressed in most human cancers but undetectable in the majority of normal adult tissues. In acute myeloid leukemia (AML), Survivin expression has been correlated with poor prognosis and chemotherapy resistance. These characteristics make Survivin eligible for a promising target for AML treatment. To explore the relationship between Survivin and drug resistance we investigated the alteration of Survivin expression in two AML cell lines HL60 (AML-M2) and U937 (AML-M5) and one chronic myeloid leukemia cell line in blast crisis for M6 (K562) treated with two chemotherapeutic drugs used in leukemia treatment: arsenic trioxide (As₂O₃) and doxorubicin (Dox). MTT assay was performed to determine the dose of drugs capable to induce cell death in 50% of treated cells (DL₅₀). To verify the percentage of apoptosis induced by As₂O₃ and Dox at DL₅₀ concentrations determined by MTT, the annexin V/propidium iodide-staining assay was performed and analyzed by flow cytometer. Western blot was used to analyze Survivin expression before and after drugs treatment at DL₅₀ concentrations. Among the cell lines studied, HL60 was the most sensitive for both drugs tested. The DL₅₀ concentrations obtained for As₂O₃ were 2, 4 and 5 μM at 24 hours for HL60, U937 and K562, respectively. Dox DL₅₀ concentrations were 10 μM at 24 hours for HL60, 5 μM at 48 hours for K562 and 1 μM at 72 hours for U937. The annexin-V/PI staining showed that these drugs were capable to induce apoptosis in all cell lines tested. The percentages of apoptosis induction for As₂O₃ were 50% for HL60, 21,84% for U937 and 32,7% for K562 in comparison with control cells, while for Dox, the index of apoptosis were 86,8%, 35,7% and 2,2% for HL60, U937 and K562, respectively. Interestingly, at DL₅₀ concentrations As₂O₃ and Dox inhibited Survivin expression in 72,7% and 69,2%, respectively. No significant alteration in Survivin expression was observed in U937 and K562 cell lines. Thus, HL60 cell line was the most sensible cell line studied and it was correlated with downregulation of Survivin expression. It suggests that Survivin could be considered a therapeutic target for AML-M2 and that As₂O₃ and Dox are capable to decrease drug resistance. However, the mechanism of action of As₂O₃ and Dox in Survivin expression seems to be cell type specific.