Signaling Balances Between Autophagy And Apoptosis In Sodium Selenite-treated Human Leukemia Cells

[Objective] Autophagy is characterized by the sequestration of cytoplasmic materials into autophagosomes for bulk degradation by lysosomes, and its function in tumorigenesis remains highly controversial. Recent research focused on the complex interplay between autophagy and apoptosis, which are closely related with tumorigenesis. Selenium is an essential trace element with chemopreventive potential against various cancers, raising a new idea for its clinical application. Our previous work indicated that20μmol/L of sodium selenite promotes mitochondrial damage and endoplasmic reticulum stress (ER stress) in human acute promyelocytic leukemia derived-NB4cells with decreased autophagic levels and that the direct inhibition of autophagy enhances selenite-induced apoptosis. However, the detail mechanism of such coordination remains understood. Therefore the aim of this study was to determine the mechanisms involved in selenite-modulated apoptosis and autophagy and provide a theoretical basis for the clinical application of sodium selenite in leukemia.[Methods] Human leukemia-drived NB4, Jurkat and HL60cells were used for this study. To assess autophagy occurrence, we performed Transmission eletron microscopy (TEM), indirect immunofluorescent staining and flow cytometry analysis. To test the alteration of autophagy at the molecular level, we performed Western blot and GFP-LC3transfection methods. Annexin V/PI and cell counting kit-8(CCK-8) assays were performed to detect selenite-induced apoptosis and cell proliferation inhibition. Pathway-focused RT-PCR autophagy microarray technology was performed to screen for important regulators from autophagy machinery components and regulators, with Western blot and RT-PCR for further verification. To study the role of certain protein, RNA interference or overexpression methods were applied to change the protein levels; chemical inhibitors or dominant-negative plasmid transfection were used to suppress protein activity. Co-immunoprecipitation, GST pull-down and immunofluorescent colocalization were applied to study the interaction among proteins. Chromosome immunoprecipitation (ChIP) was used to analysis the transcriptional regulation of certain factor on its target gene. Finally, a tumor xenograft model was established to study the antitumor properties of the drug, using immunohistochemical, TUNEL assay and flow cytometry analysis.[Results] Similar treatments administered to NB4, Jurkat and HL60, resulted in different patterns of cell autophagy, which prompted us to explore the precise regulation mechanisms of apoptosis and autophagy by selenite, with discussing the discrepancy effect of autophagy on apoptosis in human leukemia cells. Morever, tumor suppressor gene p53is proved to be a critical factor for such discrepancy.1. Hsp90was identified as a potential key regulator using RT-PCR autophagy microarray, and its importance in autophagy/apoptosis was subsequently confirmed via RNAi and overexpression methods, respectively. Furthermore, we demonstrated the direct interactions of Hsp90with IKK, p38MAPK and DAPK using co-immunoprecipitation, GST pull-down and immunofluorescent colocalization methods.2. Base on findings as described above, client proteins of Hsp90, such as IKK, DAPK and p38MAPK signaling pathways in selenite-treated cells were studied in detail. In NB4cells, downregrlation of Hsp90specifically triggered both IKK protein degradation through ubiquitin-proteasome pathway and autophosphorylation of p38MAPK, led to a decrease in autophagy. In Jurkat cells, co-activation of IKK/NFκB and MKK3/6/p38MAPK cascades by selenite increased autophagosome formation. In HL60cells, selenite involved in stabilization and dephosphorylated activation of DAPK through Hsp90and PP2A, which finally caused apoptosis and autophagic cell death. Hsp90-NFKB-Beclinl axis was established as a potential biological pathway for signaling the switch from autophagy to apoptosis in selenite-treated NB4cells.3. Analysis of the promoters and related regions of autophagy-related genes, we indicated the existence of a potential NFκB binding stie in the first intron of becn1gene through ChIP experiment. Therefore, decreased autophagy through Hsp90-mediated NFκB inactivation was due to the decrease binding of the becnl after selenite treatment. In addition, the expression levels of other components of the Beclinl/Vps34core complex, including Ambra-1, Vps34and UVRAG were also decreased in conjunction with the downregulation of Beclinl. Hsp90/NFκB/Beclin1axis was established as a potential biological pathway for signaling the switch from autophagy to apoptosis in selenite-treated NB4cells.4. Although p38MAPK facilitated selenite-driven apoptosis, the involvement of p38MAPK on autophagy in different cell lines remains elusive. Here we elucidated a potential role of p38MAPK in signaling balance between apoptosis and autophagy through ER stress pathway PERK/eIF2a/ATF4in leukemia treatment by selenite. PERK-mediated Hsp90inhibition released p38MAPK for transducing external signal to eIF2a in NB4, whereas in Jurkat the main upstream and downstream of p38MAPK are MKK3/6and eIF4E. Both eIF2α and eIF4E participated in selectively translation of ATF4, leading to transcriptional initiation of apoptosis-related gene chop or autophagy-related gene mapllc3b with preference themselves.5. In addition, the antitumor efficacy and mechanism of action of selenite were also characterized in vivo using the NB4tumor xenograft model as described, which opens the doors to many new possibilities on human leukemia cancer therapy. In basis of immunohistochemistry and immunoblotting results, we indicated that p38MAPK cascades potentially functioned in autophagy suppression and eventually leading to cell demise in vivo.[Conclusion] Our observations highlight the mechanisms of antitumor effect of sodium selenite on human leukemia treatment both in vitro and in vivo. We show that:(1) reduction of Hsp90in selenite-exposed NB4cells attenuates the activities of the IKK/NFκB signaling pathway and leads to the cell signaling switch from autophagy to apoptosis through becnl transcriptional inhibition;(2) p38MAPK contributes to signaling balance between apoptosis and autophagy in leukemia cell lines, especially in NB4, with establishment of PERK/eIF2a/ATF4axis in directing the cell towards an ultimate fate of either autophagy or apoptosis. These findings may shed light on the exploration and development of new therapeutic strategies for diseases.