Reactive Oxygen Species Activation Of Endoplasmic Reticulum Stress And Mitochondrial Apoptosis Pathway Mediated By Sodium Selenite Induced Apoptosis Of Nb4 Cells

Selenium,an essential trace element possessing anti-carcinogenic properties,can induce apoptosis in cancer cells,raising a new idea for its clinical application.Previous studies discovered that 20μM selenite markedly inhibited the proliferation and induced apoptosis of acute promyelocytic leukemia NB4 cells in a time-dependent manner. Selenium at low concentrations has a chemopreventive role against cancer,while at high concentrations,it exerts a direct antitumor effect.cDNA microarray analysis showed that different gene expression induced by 2μM and 20μM sodium selenite respectively.However,the mechanisms behind these effects remain elusive.In this article,we delineated the apoptotic signaling pathways activated by sodium selenite in NB4 cells.We found that sodium selenite induces NB4 cell apoptosis through induction of ER stress and p53-dependent mitochondrial dysfunction,both of which act as the downstream events of ROS.In addition,sodium selenite at low concentrations activated pro-survival pathways,whereas high concentrations of selenite induced activation of pro-apoptotic molecules.The findings are shown blow:Selenite specifically activated caspase-9 and -4 rather than caspase-8 in NB4 cells, suggested the possible involvement of both mitochondria and ER-dependent pathways in selenite-induced NB4 cell apoptosis.ER stress triggers the UPR to protect cells against ER stress.The present work showed that three UPR transducer pathways, PERK-eIF2a,IRE1-XBP1S and ATF6,were activated very rapidly following selenite exposure,although their activation receded at later stages.These UPR signaling pathways initiate cell protective mechanisms to protect cells against ER stress-induced damage.However,if the stress level is too severe to be repaired by cells,the apoptotic signals will be triggered.CHOP,also known as growth-arrest and DNA-damage inducible gene 153(GADD153),is a key pro-apoptosis transcription factor that is closely related with ER stress.Selenite induced GADD153 expression,and GADD153-specific siRNA reduced cell apoptosis induced by selenite,suggesting a causal role of GADD153 in selenite-induced NB4 cells apoptosis.The down-stream effectors of GADD153 are poorly understood.Further study found that selenite suppressed the activation of anti-apoptotic kinase AKT,while silencing of GADD153 gene expression by siRNA prevented selenited-induced the inactivation of AKT.These data suggest that GADD153 acts as a critical negative regulator of AKT,therefore pushing the cell in the direction of apoptosis.Since activated AKT can induce the phosphorylation of Bad and caused a decrease of cytochrome c release.Thus GADD153 induction may transmit the signal from ER to mitochondria and execution of death by dephosphorylation of AKT and Bad.Selenite-induced mitochondrial apoptotic pathway has been documented in other studies.In this study,we demonstrated that selenite-induced mitochondrial apoptotic pathway is dependent on p53.P53 can induce cell apoptosis in transcription-dependent and transcription-independent manner.NB4 cells express a mutant form of p53 that is incapable of binding DNA.Inhibition of p53 by PFT prevented selenite-cauced mitochondrial membrane permeabilization and cytotoxicity,but had no effect on Bax protein expression,indicated that this transactivation-deficient mutant of p53 in NB4 cells participates in the apoptotic process in a transcription-independent manner. Immunofluorescence result and western-blot analysis provided evidence of p53 mitochondrial translocation after selenite treatment.PFT suppressed p53 mitochondrial translocation along with Bax mitochondrial translocation that resulted in mitochondrial membrane permeabilization,cytochrome c release and subsequent caspase-9 activation. These suites of results suggest that p53 mediates mtiochondrial apoptotic pathway by direct activation of Bax mtiochondrial translocation.Since selenite induced ER stress and mitochondrial dysfunction,ER and mitochondria are two major sites that are directly involved in the storage and regulation of intracellular Ca²⁺.Time-course analysis in NB4 cells showed a significantly increased[Ca²⁺]c within initial hours of selenite treatment and decreased with time. Further studies showed that Ca²⁺ accumulated in mitochondria,which may promote the loss of mitochondrial permeability.Selenite has been reported to increase the generation of ROS,which is an important signal molecule during apoptosis.A cell-permeable superoxide scavenger, MnSOD mimic,MnTMPyp,abrogated the increase in ROS and simultaneity completely inhibited cell apoptosis induced by selenite,suggesting that ROS plays an important role in selenite-induced cell apoptosis.Antioxidant MnTMPyP attenuated the activation of UPR as well as abolished the expression of ATF4 and GADD153 induced by selenite. Further study found that removal of ROS by MnTMPyP completely prevented p53 translocation to mitochondria,in return blocked Bax mitochondrial translocation, mitochondrial membrane permeabilization,cytochrome c release from mitochondria and caspases activation.ROS also regulates intracellular Ca²⁺ homeostasis.In conclusion,these results show that selenite induces apoptosis by producing ROS to initiate ER stress and p53-dependent mitochondrial dysfunction in NB4 cells.Antioxidant enzyme plays a vital role in controlling the redox status of the cell. Manganese superoxide dismutase(MnSOD) is an essential primary antioxidant enzyme, which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix.MnSOD mimic,MnTMPyp and MnSOD-specific siRNA inhibited and increased selenite-induced cell apoptosis respectively,suggest that MnSOD plays a prominent role in protection against selenite-induced cell apoptosis.In this study,we first found that selenite induced MnSOD translocation from mitochondria to cytosol, which leads to a reduction of superoxide scavenging by MnSOD and subsequent accumulation of ROS.Selenium at low concentrations has a chemopreventive role against cancer,while at high concentrations,selenite exerts a direct antitumor effect.We speculated that ER stress-induced pro-survival and pro-apoptotic pathways probably mediate the chemopreventive and antitumor effects of selenite.MTT assay and flow cytometry analysis show that selenite above 5μM reduces NB4 cell viability and induces apoptosis,while 2μM selenite increases cell viability and has no effect on apoptosis.In this study,we found that low concentrations of selenite elicited mild ER stress and mediated cell survival by activating UPR signaling whereas high concentrations of selenite induced severe ER stress and caused cell death by activation of the pro-apoptotic transcription factors GADD153,p53 and caspase.The net balance between these signaling cascades probably governs cell survival or apoptosis.These findings uncover the molecular mechanisms of the chemopreventive and antitumor effects of different concentrations of selenite.