Induction of Apoptosis in K562 Human Chronic Myeloid Leukemia Cells by Tetra-Arsenic Tetra-Sulfide.

Realgar has been used as a traditional medicine in China for more than 1500 years. Some studies found that tetra-arsenic tetra-sulfide (As₄S₄), the main ingredient of realgar, used alone was highly effective and safe for all stages of acute promyelocytic leukemia. To explore the effects of As₄S₄ in treatment of human chronic myeloid leukemia K562 cells, we used microculture MTS assay to measure the growth inhibition of K562 cells. The morphologic change was determined by Wright’s staining and Hoechst33342 assay. Cell apoptosis was evaluated by DNA agarose gel electrophoresis. The apoptotic rate and cell cycle were measured by flow cytometry. The changes of transcript and protein levels were determined by real-time reverse transcription-PCR and Western blot analysis, respectively. As₄S₄ had signigicant cytotoxicity on K-562 cells. At the concentration of 2.0μmol/L, the cell viability decreased significantly after 24 hours cultured with the reagent. When the concentration was lower than 0.5μmol/L, As₄S₄ had little effect on K562 cells. The effect of As₄S₄ on K562 was time and concentration dependent. After cultured with As₄S₄ at the concentration of 2.0μmol/L for 24 to 48 hours, K562 cells appeared typical morphological changes of apoptosis. At a concentration greater than or equal to 2.0μmol/L, As₄S₄ could induce apoptosis significantly. After 12 hours of incubation with 2.0μmol/L As₄S₄, the apoptosis rate increased from 2.05% to 12.03%. At the same time, the percentage of cells in G₁ phase decreased from 69.65% to 50.53%, whereas the percentage of G₂/M phase increased from 9.56% to 25.91%. The mRNA levels of BCL-2, BCL-X_L, BAD and BAX, and the protein levels of Akt and pAkt down-regulated after the inhibition of As₄S₄. The transcript and protein levels of BCR-ABL had no change after incubation with As₄S₄. These results indicated that As₄S₄ can inhibit the growth of K562 cells efficiently through inducing apoptosis and cell cycle arrest. It seems that As₄S₄ interferes with Akt pathway and down-regulate BCL-2, BCL-X_L, BAD and BAX, which may be involved in the response of K562 to this agent. As₄S₄ could be beneficial for treatment of CML in combination with conventional drugs.