Mechanism Of The Underlying The Arsenic Trioxide And Its Intermediate Metabolites Induced-Autophagy In Acute Promyelocytic Leukemia NB4Cell

Objective: To investigate the mechanism of arsenic trioxide and itsintermediate metabolites such as MMA~Ⅲand DMA~Ⅲ–induced autophagy incute promyeloid leukemia NB4cells.Methods: Acute Promyeloid Leukaemia (APL) NB4cells and three arseniccompounds (i.e., arsenite (iAs~Ⅲ) and methylated trivalent metabolites MMA~Ⅲand DMA~Ⅲ) were used for present study. MTT assay was used to detect thecell viability. Detection of autophagic bodies and autophagosomes weredetermined by transmission electron microscopy (TEM) andimmunofluorescence. Western-blot was used to detect the expressions ofindividual proteins in NB4cells following exposure to arsenic compounds.Results: MTT assay showed that DMA~Ⅲwas the most toxic form among itsthree arsenicals to NB4cells, followed by MMA~Ⅲand iAs~Ⅲ. In ourpreliminary experiment, we have found that arsenic intermediate metabolite,especially, MMA~Ⅲis able to induced apoptosis via the mitochondria pathway,while induction of apoptosis by DMA~Ⅲpredominantly through theendoplasmic reticulum stress (ER stress). In order to better understand the toxic mechanism, cells were pretreated with caspase inhibitors and then toexposure to different arsenicals to determine the cell viability. Unfortunately,induction of the apoptosis could not completely reversed by its inhibitors,suggesting that a part of the induction of the apoptosis may dependent on theon other pathway. Thus, we hypothesize that the arsenic intermediatemetabolites induced apoptosis (i.e., a part of cellular apoptosis) maydependence on autophagy. As the results, the number of autophagosomessignificantly increased in cells after exposure to arsenic trioxide. In addition,autophagy-related protein LC3and Beclin-1were also remarkably increasedfollowing exposure to As2O3, while it seems that MMA~Ⅲand DMA~Ⅲinducedautophagy is quite different from the As2O3. On the other hand, the confocalmicroscopy experiment has also found that LC3B colocalized with PML byexposure to iAsIII, but there was no signal detected in the cells followingexposure to MMA~Ⅲand DMA~Ⅲ. Although autophagy can be induced by threedifferent arsenic species (e.g., iAsIII, MMAIIIand DMAIII) in NB4cells,MMA~Ⅲand DMA~Ⅲare unable to degrade the PML-RARα fusion protein.In order to understand the different roles of the autophagy in NB4cellsafter exposure to arsenicals, we further determine the expression of autophagyrelated proteins such as p-Akt473and p-mTOR. Our results show that thephosphorylated Akt473(p-Akt473) was transient increased at12h afterexposure to iAsIII, while phosphorylated mTOR (p-mTOR) was significantlydown-regulated. In MMAIIItreated cells, p-Akt473was decreased in a time-dependent manner, and p-mTOR was significantly decreased at12h.interestingly, phosphorylated Akt473was transient increased, and p-mTORwas significantly down-regulated at24h by exposure to DMA~Ⅲ. These resultssuggest that iAsIII, MMAIII, and DMAIIIinduced cellular autophagy areassociated with p-Akt and m-TOR. Especially, induction of autophagy byiAsIIIand DMAIIImay through activated PI3K/Akt—mTOR transient, butMMAIIIinduced autophagy seems to be inhibiting the Akt phosphorylation.Conclusions: Induction of autophagy by iAsIIIand DMAIIImay throughactivated PI3K/Akt—mTOR transient, but MMAIIIinduced autophagy seemsto be inhibiting the Akt phosphorylation.