Sodium Selenite Induction Of Mnsod In The Mechanism Of The Process Of Apoptosis Of Nb4 Cells

Selenium is one of the essential trace elements with anti-tumor properties. Large amount of evidence indicates that supranutritional selenium is able to induce apoptosis in diverse tumor cell lines, including prostatic cancer, hepatoma, colorectal cancer, lung cancer, breast cancer and leukemia. In the previous study of our group, we found 20μM of sodium selenite induced pronounced apoptosis in NB4 cells derived from human acute promyelocytic leukemia (APL), while reactive oxygen species (ROS) played an upstream and critical role. Under selenite exposure, NB4 cells produced considerable ROS, by which mitochondrial apoptosis pathway and endoplasmic reticulum stress (ER stress) pathway were initiated. However, the detailed molecular alterations in selenite-induced apoptosis still require further investigations.Based on our previous work, we further explored the alterations of some ROS-regulated important molecules, as well as their regulation pathways and the influences on selenite-induced apoptosis. Here we discovered by Western blot that the expression of manganese superoxide dismutase (MnSOD) was dramatically elevated in selenite-induced apoptosis of NB4 cells. ROS scavagener MnTMPyP completely abrogated such upregulation, suggesting ROS acted as the upstream regulator. ROS detection revealed that two intracelluler ROS, superoxide radicals and hydrogen peroxide were immediately generated after selenite exposure, in which superoxide radicals might be critical for elevation of MnSOD. As to the detailed mechanisms, we elucidated that ERK and p53 were closely involved. cDNA sequencing validated that NB4 cells used in the present study expressed wild-type p53. Western blot of the nuclear fractions and immunofluorescence staining indicated that after production of ROS induced by selenite, ERK2 was driven by ROS to be translocated from the cytoplasm into the nucleus, and subsequently phosphorylated p53 at the vital site Ser15. Immunoprecipitation results demonstrated ERK2 phosphorylated p53 by a direct bingding, leading to activation of p53 by dissociation from its inhibitory protein mouse double minute 2 (MDM2). Inhibition of p53 transactivation ability by its selective inhibitor Pifithrin-a (PFT) or suppression of its expression by siRNA abolished selenite-induced upregulation of MnSOD, indicating p53 mediated elevated expression of MnSOD as its upstream transcription factor. Additionally, in leukemia cell lines with null or mutant p53 such as HL-60 and U937, selenite could not induce upregulation of MnSOD, further corroborating the critical role of p53 in this modulation. We speculate a ROS-ERK-p53-MnSOD pathway exists in NB4 cells when apoptosis is induced by selenite. This is probably an important mechanism by which NB4 cells counteract oxidative stress induced by selenite.It was revealed by our previous research that selenite repressed autophagy accompanied by the induction of apoptosis in NB4 cells. In the present study, we further explored the mechanisms underlying the switch of NB4 cells from autophagy to apoptosis, and elucidated p53 played a key role. Western blot indicated selenite induced phosphorylation of p53 at the vital site ser15 via ERK and p38MAPK. Immunofluorescence staining and immunoprecipitation showed p53 dissociated with MDM2, while the nucleolar protein B23 transferred from the nucleolus to the nucleoplasm and associated with MDM2, probably stabilizing p53. p53 inhibitor PFT and siRNA interference notably reversed selenite-induced apoptosis, caspases activation, and decrease of autophagic protein Beclin-1 and LC-3, manifesting that active p53 mediated induction of apoptosis and inhibition of autophagy, thus switching NB4 cells from autophagy to apoptosis.We also revealed that protein kinase Calpha (PKCalpha) was dramatically reduced at later stage of selenite-induced apoptosis, which was mediated by ROS. Besides, by means of PKCalpha inhibitor and siRNA interference, we confirmed that PKCalpha played an anti-apoptotic role through its effects on ERK 1/2 and Akt. ROS detection indicated PKCalpha also inhibited generation of intracellular ROS. At later stage, PKCalpha was downregulated at protein level and phosphorylation level by caspase-3 and PP2Ac respectively under the regulation of ROS. In summary, we speculate that PKCalpha functions to counteract apoptosis in NB4 cells, and its downregulation seems an important mechanism maintaining and promoting apoptosis induced be selenite, especially at later stage.COX IV (cytochrome c oxidase subunit IV) is an essential component of mitochondrial respiratory chain. At later stage of selenite-induced apoptosis of NB4 cells, COX IV was remarkably downregulated at protein level without evident alteration in mRNA level, suggesting this downregulation was independent of transcriptional regulation. Further investigation illuminated that ROS mediated COX IV downregulation through caspase-3 activation. COX IV inhibition by shRNA expression vectors markedly enhanced selenite-induced apoptosis of NB4 cells, indicating selenite probably damaged the integrity and function of mitochondrial respiratory chain through the ROS-caspase-3-COX IV pathway, thus accelerating the proceeding of apoptosis.