Indirubin-3'-Monoxime Induces Apoptosis and Autophagy In Acute Lymphoblastic Leukemia Cells and Chronic Myelogenous Leukemia Cells, Exhibits Limited Cytotoxicity In CD34⁺ Hematopoietic Stem Cells, Lymphocytes and Granulocytes

Abstract 3984

The rates of treatment-related mortality (TRM) and relapse are unacceptably high in adults undergoing antileukemia treatments for acute lymphoblastic leukemia (ALL). So far has no better therapy with low side effects to improve long-term survival in these patients. Indirubin, a Chinese translational anti-chronic myelogenous leukemia (CML) agent, is able to induce cellular apoptosis. However, until now the functional action of IO on ALL remains still unknown. Therefore, here we investigated and compared the cytotoxic efficacy and action of indirubin-3'-monoxime (IO) in JM1 (ALL cell) and K562 (CML cell). ALL and CML cells were treated with a series of IO dose for 24 and 48h, and cell survival was determined by WST-1 assay. ALL and CML cells were shown to be similar susceptible to IO cytotoxicity. In order to clear in which way of cell death induced by IO, we performed analyses for apoptosis, necrosis and autophagy, respectively. After IO treatment, both ALL and CML cells were arrested in the G2/M cell cycle phase. In addition, an increase of sub-G1 proportion was caused. We found also increasing of caspase-3 activation and formation of cleaved PARP in a dose dependent manner. These were associated with the form of apoptosis. However, the caspase inhibitor Z-VAD-FMK only could partially prevent cell death in ALL and CML cells. When further analyzing the necrotic phenomenon through measuring LDH release, the result clearly showed that LDH release was not remarkable after cell treatment with high dose of IO. Besides, we observed surprisingly in Western blot the increasing expression of microtubule-associated protein light chain 3-II (LC3-II), which generally correlates to formation of autophagosomes. Because better antileukemic drug should not induce toxicity in normal blood cells as much as possible, so the cytotoxic effect of IO in CD34⁺ hematopoietic stem cells, lymphocytes and granulocytes was analyzed. Excitingly, results showed that IO could not affect cell viability of granulocytes, and IO cytotoxicity in lymphocytes was only marginal. If CD34⁺ hematopoietic stem cells were treated with IO, the rate of cell survival and their ability of differentiation were almost identical in contrast with non-treated control. Apparently, these data indicated that IO possesses the capability to induce apoptosis and autophagy in both CML and ALL cells. The most important is that the IO hardly influences the cell survival and the differentiation of CD34⁺ hematopoietic stem cells, the cytotoxic effect in granulocytes and lymphocytes is only limited. In conclusion, IO can be considered as a potential agent for clinical anti- ALL treatment.