Indirubin-3-Monoxime Induces Apoptosis and Autophagy in Human Lymphocytic Leukemia Cells and Human Chronic Myelogenous Leukemia Cells.

Indirubin-3-monoxime (IO) is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine. It is a potent inhibitor of cyclin-dependent kinases (CDKs), especially CDK2. In clinical studies demonstrated that indirubin did not cause major side effects in patients with chronic myelogenous leukemia. However, the functional action of indirubin on acute lymphoblastic leukemia (ALL) is still unclear. In the present study, we investigated that cytotoxic effect and mechanism study of IO in human acute lymphocytic leukemia cell line JM1 and human chronic myeloid leukemia cell line K562. We also analyzed the viability influence of IO in normal human granulocytes and lymphocytes.

After the treatment of IO, cell viability of JM1 and K562 cells was determined by WST-1 assay. The influence of IO on cell-cycle distribution was analyzed by FACScan. To identify which cell death types were induced during IO treatment we measured the caspase-3 activity for analysis the induction of apoptosis; evaluated the LDH release in the necrotic analysis and the expression level of LC3-II was determined by Western blotting analysis for autophagic cell death. Furthermore, we also analyzed the toxicity of IO in normal human granulocytes and lymphocytes by the treatment dose of IO with value of IC₅₀.

IO significantly affected the cell viability on JM1 but also on K562 cells in a dose dependent manner. The G2/M phase of cell cycle was arrested and sub-G1 proportion was relative increased. In addition, the finding showed that IO initiated caspase-3-dependent apoptosis in JM1 and K562 cells. The expression of autophagosome-incorporated LC3-II protein was also clearly increased once cells treated with IO. However, the necrotic phenomenon through measuring LDH release from K562 and JM1 cells could not be observed. Excitingly, by the treatment dose of IO with value of IC₅₀, the cell viability of lymphocytes was marginally affected, whereas the cell cytotoxitity of granulocytes was not induced.

IO has the ability to induce potent cytotoxic effect on K562 and JM1 cells. The cell death after IO treatment is mainly caused by apoptosis and autophagy, not by necrosis. This effect is also associated with interrupted cell cycle. Importantly, IO has few or little cytotoxic effect on human lymphocytes and granulocytes. The results suggest that IO might be useful for clinical anti- ALL treatment.

No relevant conflicts of interest to declare.