Because cancer cells evade programmed cell death to support malignant growth, understanding the mechanisms involved in programmed cell death is critical to developing treatments for various cancers. The work presented here demonstrates a striking difference in the response to sodium selenite between NB4 cell lines and lines derived from other leukemia types with respect to both autophagic levels and NF-κB signaling and provides evidence that Hsp90 is a key regulator responsible for IKK/NF-κB cascade dysfunction in NB4 cells. Hsp90 mediates the eventual transition from autophagic protection to irreversible apoptotic death. These findings also extend our previous observations regarding the importance of Hsp90-mediated autophagy.
Sodium selenite is a common dietary form of selenium, which has chemotherapeutic potential because of its ability to induce cancer cell apoptosis with minimal side effects on normal cells. Our lab has focused on the therapeutic effect of sodium selenite in human leukemia-derived NB4 cells and colorectal cancer–derived SW480 cells. We have shown that augmented reactive oxidant species triggered by sodium selenite can induce NB4 cell apoptosis mainly through the p53-dependent mitochondrial and ER stress–mediated pathways (Li et al.
; Guan et al.
). Experiments in vivo have demonstrated the ability of selenite to inhibit colorectal tumor growth in mice without obvious adverse effects on body weight and activities (Huang et al.
), providing the possibility of selenite-based therapies for humans in the future. To obtain a more precise mechanistic understanding of the potential role of selenite in cancer therapy, we recently turned our attention to another important cellular process, autophagy, and have detected gradually suppressed autophagy during selenite-induced apoptosis in NB4 cells, indicating that autophagy may antagonize apoptosis by supporting survival in untreated NB4 cells (Ren et al.
). Kim et al.
reported, however, that the generation of superoxide anion triggered by sodium selenite induced mitochondrial damage and subsequent autophagic cell death in malignant glioma cells (Kim et al.
), suggesting the concurrence of autophagy and apoptosis in some tumor cells exposed to cytotoxic drugs. Bursch et al.
also suggested that tamoxifen (TAM) caused dose-dependent autophagy or apoptosis in HL60 cells (Bursch et al.
); this finding emphasized the complex interplay between apoptosis and autophagy. Furthermore, autophagic cell death was reported to avoid apoptosis under certain conditions (Hansen et al.
). Therefore we examined the effect of selenite on autophagy in two other human leukemia cell lines, HL60 and Jurkat, and they unexpectedly exhibited increased autophagic levels during apoptosis identical to those of glioma cells, but unlike those of NB4 cells. These findings suggested that the effect of selenium on autophagy varied in different cell types, and the actual role of autophagy in cell-fate determination depended on particular circumstances, including the cell types, cell contexts, and properties of the agents.
Hsp90 is a molecular chaperone that contributes to prosurvival signaling in tumor cells. Some studies have characterized Hsp90 as the main chaperone required for the stabilization of multiple oncogenic kinases in the development of acute myelogenous leukemia (Reikvam et al.
). Many small molecular chemicals, such as 17-AAG, targeted to Hsp90 inhibition have been used in clinical cancer therapy, suggesting the importance of Hsp90 inhibition in treating the disease (Moser et al.
; Taiyab et al.
; Wu et al.
; Holzbeierlein et al.
). Recently we showed that reduced Hsp90 expression was correlated with decreased autophagic levels and higher apoptotic rates in NB4 cells, but not in HL60 and Jurkat cells, indicating a novel function of Hsp90 in signaling switching during selenite treatment.
Having identified that, after selenite exposure, only NB4 cells exhibited reduced Hsp90 expression and that this inhibition was necessary for selenite-triggered apoptosis and autophagy suppression, we aimed to investigate the responsible signaling molecules in this process. Our study identified IKKα as an interacting protein of Hsp90 in all three cell lines, but we examined the dissociation of these proteins in only NB4 cells during selenite-induced apoptosis. IKK is an upstream regulator responsible for the nuclear translocation and activation of NF-κB (Luo et al.
). Constitutive activation of the NF-κB pathway is involved in some forms of cancer, such as leukemia, lymphoma, colon cancer, and ovarian cancer (Rayet and Gélinas, 1999
). Recently, the direct cross-talk between NF-κB and autophagy was demonstrated. Tumor necrosis factorα–induced NF-κB activation was shown to suppress autophagy, and NF-κB inhibition was found to increase starvation-induced cell death. Autophagy was also described as regulating NF-κB activity (Djavaheri-Mergny et al.
; Djavaheri-Mergny and Codogon, 2007
; Nivon et al.
). In our experimental settings, endogenous NF-κB was inactivated in NB4 cells after selenite exposure. Overexpression of Hsp90, however, restored the nuclear translocation of NF-κB and partially attenuated selenite-induced cell apoptosis, suggesting that the involvement of Hsp90 down-regulation in selenite-induced cell death occurred mainly through NF-κB activity inhibition. Importantly, the Hsp90–NF-κB link seemed to be present in autophagy as well; thus the cell signaling switch was precisely controlled.
Current knowledge suggests that the Beclin1–Vps34 kinase complex is one of the functional groups in the autophagy machinery, which mediates the localization of other autophagy proteins to the preautophagosomes and is involved in the nucleation of autophagosome formation (Kihara et al.
). Beclin1, Vps34, Vps15, and Ambra-1 are considered the common core complex, and UVRAG is usually involved in autophagosome formation and maturation via the regulation of the lipid kinase activity of Vps34 (Itakura et al.
). From ChIP assays, we identified becn1
as the direct target of NF-κB. In addition, the expression levels of other components of the Beclin1/Vps34 core complex were also decreased in conjunction with the down-regulation of Beclin1. Therefore decreased autophagy through Hsp90-mediated NF-κB inactivation was due to the decreased binding of the becn1
promoter after selenite treatment.
In addition, we found that 17-AAG treatment did not cause decreases in the expression of Hsp90 and Beclin1 (), but it impaired the interaction of Hsp90 with IKK (unpublished data). The different effects of selenite and 17-AAG may be determined by different inhibitory mechanisms. 17-AAG, the inhibitor of Hsp90, has been demonstrated to active a heat shock response and possibly acts through the increased expression of molecular chaperones, in particular through Hsp70 (Niikura et al.
; Banerji et al.
). Riedel et al.
reported that 17-AAG induced cytoplasmic alpha-synuclein aggregate clearance by induction of autophagy, suggesting the possible aggregate clearing and autophagy-inducing effects of 17-AAG (Riedel et al.
). Selenite, however, functioned in Hsp90-regulated autophagy mainly through decreasing expression of Hsp90. Hence Hsp90 siRNA and 17-AAG exhibited different effects on autophagy. Moreover, our data from two other leukemia cell lines reflected the opposite but supported our views: Following selenite exposure, unchanged Hsp90 expression levels resulted in Hsp90 always binding to the IKK protein such that constitutively activated NF-κB could translocate into the nucleus and initiate becn1
transcription. Thus these cells exhibited excessive autophagic levels and led to apoptotic and autophagic cell death.
The tumor suppressor p53 plays a vital role in safeguarding the integrity of the genome. Recently, an emerging area of research has indicated additional activities for p53 in the cytoplasm, where it regulates both apoptosis and autophagy (Levine and Abrams, 2008
; Green and Kroemer, 2009
; Scherz-Shouval et al.
). Our results, schematically summarized in , show that Hsp90 was the core regulator in this pathway. The discrepancies in Hsp90 expression among the three selenite-treated leukemia cell lines were possibly due to p53 activity. Recent research revealed that p53 can either inhibit or enhance autophagy and that autophagy can increase or reduce cell survival as a result of certain cellular events and certain signals (Tasdemir et al.
). Induction of autophagy by p53 depends on the transactivation of genes such as DRAM (Crighton et al.
) and also on the inactivation of the mTOR pathway (Feng et al.
). p53 inhibits autophagy through a cytoplasmic (nonnuclear) effect that is correlated with enhanced mTOR activity. Preliminary studies from our laboratory on the p53 status (cloning and sequencing of p53) of our cell lines have suggested that the p53 expressed in the NB4 cell line was wild type (Li et al.
) and that the importance of p53 nuclear translocation and activation in selenite-triggered NB4 apoptosis has been elucidated clearly (Guan et al.
; Li et al.
). Therefore this dual function of p53 indicates a possible mechanism for Hsp90-mediated cell-fate decisions, but the exact mechanism still needs further investigation.
FIGURE 8: The schematic diagram delineating the Hsp90–NF-κB–Beclin1 pathway involved in the selenite-induced apoptosis and autophagy of NB4 cells. Sodium selenite decreased Hsp90 expression, which occurred through some unknown transcription (more ...)
In conclusion, our findings highlight the mechanisms through which Hsp90 regulates the sodium selenite–induced NB4 cell–programmed death process. We show that reduction of Hsp90 in selenite-exposed NB4 cells attenuates the activities of the IKK/NF-κB signaling pathway and leads to the cell signaling switch from autophagy to apoptosis through becn1 transcriptional inhibition. Importantly, we establish a novel link between apoptosis and autophagy and provide a theoretical basis for the clinical application of sodium selenite in leukemia.