Molecular Mechanism Of Autophagy Induced By Arsenic Trioxide In Human Myeloid Leukemia Cell HL-60

Objective:To investigate the molecular mechanism of induction of autophagy in human myeloid leukemia HL-60 cells following exposure to arsenic trioxideMethod:Human myeloid leukemia HL-60 cells, HEK 293 T cells, acute promyelocytic leukemia (APL) NB4 cells were exposed to different concentrations of arsenic trioxide (i.e., iAsⅢ) at different times. Western-blot was used to detect the expressions of individual protein expression in different cell lines following exposure to iAsⅢ. In addition, we predominantly used HL-60 cells for subsequent experiments for determination of ATP contents by ATP kit or mitochondrial membrane potential (MMP) by using JC-1 fluorescence after exposure to iAsⅢ. Likewise, pyruvate dehydrogenase Enzyme Activity Microplate Assay Kit, L-Lactate Assay Kit and Pyruvate Assay Kit were used to detect activity of Pyruvate dehydrogenase complex and the content of lactate and pyruvate in HL-60 cells treated with arsenic trioxide. Finally, we attempt to determine the effects of pyruvate and lactate on induction of autophagy in HL-60 cells as compare with iAsⅢ-treated HL-60 cells. In the experiment, changes in LC3 protein were used to evaluate the induction of autophagy in HL-60 cells after exposure to pyruvate and lactate.Results: We found that autophagy was significantly induced in HL-60, HEK293T and NB4 cells after exposure to iAsⅢ. Moreover, cellular energy metabolism (i.e., ATP) was not affected in HL-60 cells by treatment of arsenic at 6h. Likewise, there was no appreciable changes observed in mitochondrial membrane potential (MMP) in HL-60 cells at 1 or 2μM of iAsⅢ, while a little change was found in MMP at 5μM of iAsⅢ. On the other hand, we also interested in determination of pyruvate dehydrogenase complex (PDHc-LA) expression after exposure to different concentrations of iAsⅢ(e.g.,1,2 and 5μM) for 6h. Interestingly, we observed the pyruvate dehydrogenase complex (PDHc-LA) expression was decreased significantly with increasing of exposure concentrations or time dependent manner. Surprisingly, the concentrations of intracellular lactate and pyruvate were remarkably increased after treatment of iAsⅢ. At the same time, we further examined effects of iAsⅢ on inhibition of the pyruvate dehydrogenase complex activity in HL-60 cells, and the activity of PDHc-LA was completely inhibited by iAsⅢ. Thus, we opt to evaluate whether the exogenous lactate or pyruvate is able to induce the autophagy in HL-60 cells, we found that only lactate was able to induce autophagy in cells, implying that the iAsⅢ induced autophagy in HL-60 cells predominantly mediate by increasing of lactate, but not by itself.Conclusion:It was first time to show that iAsⅢ is able to induce autophagy in various cell lines, and the inhibition of activity of pyruvate dehydrogenase complex by iAsⅢ may result in induction of autophagy. In addition, inhibition of activity of PDHc-LA further resulted in increasing lactate concentration in HL-60 cells, then its lactate induces the autophagy in HL-60 cells (i.e., arsenic induced autophagy probably be mediated by lactate).