| Objective:To investigate the effects of inorganic arsenite and its intermediate metabolites such as MMAⅢ and DMAⅢ on acute promyeloid leukemia (APL) NB4cells.Methods:Acute Promyeloid Leukaemia (APL) NB4cells and three arsenic compounds (i.e., arsenite (iAsⅢ), intermediate metabolites MMAⅢ and DMAⅢ) were used to present study throughout. MTT assay was used to detect the cell viability. Wright-Giemsa stain and flow cytometry were used to detect NB4cells differentiation. Confocal laser scanning microscopy was used to determine formation of neuclear bodies (i.e., determination of PML, SUMO-1and DAPI). Induction of the apoptosis was determined by flow cytometry after staining with FITC-conjugated annexin V and propidium iodide (PI). Mitochondria AΨm was determined by using JC-1staining. Western blotting was used to detect the expressions of individual proteins in NB4cells following exposure to arsenic compounds.Results:MTT assay indicated that DMAⅢ was the most toxic form among its arsenic species, followed by MMAⅢ and iAsⅢ. Regarding the effects of three arsenic compounds on the induction of cells differentiation, iAsⅢ significantly induced the NB4cell differntiaiton and PML-RARa fusion protein degradation in a similar manner to positive control ATRA. By contr as, MMAⅢ and DMAⅢ had no effect on NB4cellular differentiations, and no degradation of PML-RARa was observed. In order to understand the mechanism underlying the arsenicals induced cell differentiation, the formation of nuclear bodies were determined by co focal microscopy after exposure to arsenicals. PML and SUMO-1co localized in nuclear (i.e., restored PML-NBs) after exposure to iAsⅢ, while MMAm and DMAⅢ has no effect on the formation of nuclear bodies (PML in microspeckled form) when compared with iAsⅢ and RATA-treated NB4cells, suggesting the methylated intermediate metabolites are unable to induces NB4cell differentiation.On the other hand, we hypothesize that the methylated metabolites may have potent effects on the induction of apoptosis. In order to address this question, induction of apoptosis by arsenicals was measured by Annexin V/PI assays. After the treatment of arsenicals,1μM MMAⅢ and DMAⅢ induced apoptotic cells up to approximately40%and50%(respectively) in NB4cells afer24h treatment. Howere, no appreciable apoptotic effect was observed in NB4cells after exposure to1μM of iAsⅢ. In addditon, effects of arsenic compound on mitochondrial membrane potential (AΨm) were alsosudied. As the results, arsenic intermiediate metabolites MMAⅢ and DMAⅢ strongly induced loss of mitochondrial membrane potential in NB4cells, but inorganic iAsⅢ did not. Results of immunoblot have clearly found that MMAⅢ and DMAⅢ not only induced Cytochrome C release, but also activatited the caspase3as well as down regulated Bcl-2/Bax, resulted in induction of apoptosis. However, no appreciable effects were observed in NB4cells after iAsⅢ exposure. The above findings were also strongly correlated with the results of flow cytometry.On the other hand, it has found that the ER stress-associated molecules such as phosphorylated protein kinase-like ER kinase (PERK), eukaryotic translation initiation factor-2a (eif2a), glucose-regulated protein78/immunoglobulin heavy chain-binding protein (GRP78), C/EBP-homologous protein (CHOP) were significantly induced by exposure to DMAⅢ (but not of iAsⅢ and MMAⅢ), the induction of ER-stress triggered release of Cyt c from mitochondria and induced the apoptosis in cells. These results indicated that the induction of Cytc release was predominantly caused by ER-stress in DMAⅢ-exposed cells.Conclusions:Inorganic arsenic,its intermetabolites monomethylarsonous acid (MMAⅢ) and dimethylarsinous acid (DMAⅢ) have different effects on the NB4cells. Especially, iAsⅢ can induce NB4cell differentiation specially, while its intermediate metabolites MMAⅢ and DMAⅢ has no effects on the induction of cells differentiation, but they have potent apoptosis-inducing activity... |
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