Selenium-induced Autophagy And Apoptosis Molecular Mechanism Of Leukemia

ObjectivePrevious study had discovered super-nutritional selenite induced leukemia NB4cells death through switching protective autophagy to apoptosis and this mechanism was unclear, while in other leukemia cell lines the anti-tumor effect of selenite was still unknown. In this investigation, selenite was used as an anti-tumor agent and the mechanisms by which selenite inhibited autophagy in NB4cells were explored. Additionally, we also determined the mechanisms by which selenite induced apoptosis in other leukemia cell lines. Finally, we established two leukemia cells bearing nude mice models and confirmed selenite therapeutic effects and some proteins alterations in vitro. All of the results helped us explore the regulatory status of some molecules in autophagy network and also supported the theoretical and experimental basis of selenite clinical application.MethodsWestern blots, indirectly labeling LC3and calculating the ratio of dots positive cells and transmission electron microscopy were used to detect the alteration of autophagy. Flow cytometry was used to analyze the change of apoptosis ratio and cell cycle distribution. To measure the cell viability, cell counting kit-8was purchased. Protein interaction was determined with immunoprecipitation, GST pull-down assay and immunofluorescence. To investigation the interaction between transcription factor and DNA sequence, chromatin immunoprecipitation was performed. Revers transcription-polymerase chain reaction was used to test the change of mRNA level. Special inhibitors uses and transfection of siRNA and plasmids were used to investigate the protein function. Leukemia cells bearing nude mice model was established to detect selenite therapeutic effects through TUNEL technology and hematoxylin-eosin staining assay. Immunohistochemistry and western blots were carried out to detect alterations of some proteins in vivo.ContentsIn this study, we investigated the mechanisms of selenite-induced leukemia cells death. On the one hand, we detected the alterations of survivin-2B, IKK alpha, P73and UVRAG, the relationship between these proteins and the role of them in autophagy process in selenite-treated NB4cells. And then, we concluded their function in cell fate determination through exploring the relationship between autophagy and apoptosis. Finally, we confirmed selenite anti-tumor activity and alterations of these proteins in a nude mice model which had been demonstrated previously.Additionally, because effects of selenite on other leukemia cells were unknown, we investigated the apoptotic mechanisms in HL60cells. In this study, we also found selenite could affect the dynamics of microtubule and further inhibited HL60cycle distribution. Further experiments indicated the alteration of the CDK activity should be responsible for the change of apoptosis-related proteins. Finally, we also established an HL60cells bearing nude mice model to confirm selenite anti-tumor activity, its microtubule inhibitory effects and alterations of cyclin Bl and Mcl-1.ResultsSunivin2B/IKK alpha/P73/UVRAG axis involved in the regulation of protective autophagy in NB4cells.First of all, we confirmed autophagy was down-regulated in selenite-treated NB4cells and inhibition of autophagy could promote selenite-induced apoptosis. Further experiments found survivin-2B interacted with IKK aloha and further accumulated it in the nucleus. The nuclear IKK alpha stabilized P73which promoted the expression of UVRAG, known as the initiator of autophagy. Finally, selenite was proved to have anti-tumor activity and alter these proteins just as it was indicated in vitro. Collectively, our results discovered the regulatory status of survivin-2B in the network of autophagy and provided a experimental basis for the selenite clinical application.Selenite induced apoptosis in HL60cells through altering microtubule dynamics At first, we determined the growth inhibitory effects and apoptosis induction energy of selenite in HL60cells and made sure that20μM selenite induced apparent apoptosis in HL60cells which was related with mitochondrial apoptotic pathway. Then, we also discovered selenite prevented microtubule assembly and arrested HL60cells at G2/M phase. In this process, increases of cyclin B1interacted with CDK1. And either transfection of siRNA targeting cyclin B1or inactivation of CDK1could reverse the down-regulation of anti-apoptotic Mcl-1.Finally, in HL60cells bearing nude mice model, selenite anti-tumor activity, its microtubule inhibitory effects and the alterations of cyclin B1and Mcl-1were confirmed. Collectively, both of in vitro and in vivo experiments confirmed selenite depolymerized microtubule by which selenite induced HL60cells death.Conclusions1、Survivin-2B accumulated IKK alpha in the nucleus which further regulated the stability of P73and UVRAG expression by which selenite regulated autophagy in selenite-treated NB4cells.2、Selenite regulated the microtubule dynamics and inhibited cell cycle distribution which finally resulted in the cyclin B1-dependent degradation of Mcl-1and apoptosis in HL60cells.