| Human acute promyelocytic leukemia NB4 cell is a cell line derived from the patient ofHuman Acute Promyelocytic leukemia. It is a good material to study the mechanism of thisdisease. The previous work in our laboratory proved that at certain concentration andtreating time, sodium selenite could induce apoptosis of NB4 cells. The purpose of thisarticle is to invesgate further the detailed mechanism of sodium selenite induced NB4 cellapoptosis and its specific cell signaling transduction pathways, focusing mainly on ROSand ROS induced PKC activation, so as to provide theoretical support for clinical use ofsodium selenite in treating this kind of leukemia.Reactive oxygen species (ROS) include oxygen ions, free radicals and peroxides bothinorganic and organic. They are generally very small molecules and are highly reactive dueto the presence of unpaired valence shell electrons.(Wikipedia-the free incyclopedia).Using ROS specific fluorescence probe, we observed that immediately after sodiumselenite treatment, the ROS level within NB4 cells has been promoted rapidly and the ROSproduction has a linear relationship with the treating time. Besides, long-time treatment ofselenite could inhibit gene-expression of MnSOD. Using an effective and widelyrecognized antioxidant, an artificial MnSOD mimic, to pretreat NB4 cells, couldmiraculously inhibit the apoptosis effect produced by sodium selenite. Hence, it is regardedthat ROS is the conductor of sodium selenite induced apoptosis in NB4 cells.Further investigating of the detailed mechanism of ROS induced apoptosis reflects thatfirstly the phosphorylation and cleavage of several Protein Kinase C (PKC) familymembers could be regulated by selenite treatment to various extents. Among them, selenitecould induce the phosphorylation of Protein Kinase Cαand Protein Kinase Cδ, aswell as their cleavage. And these are the signs of PKC activation. Moreover, there is acausal relationship between ROS production and PKC activation: the phosphorylation andcleavage both could be prevented if pretreat cells with antioxidant before sodium selenite.Secondly, the mitochondrial transmembrane potential loss caused by sodium selenite couldbe prevented by antioxidant pretreatment, too, which suggests the transmembrane potentialloss is also mediated by ROS. Meanwhile, antioxidant pretreatment inhibit the cleavage of Bid, suggesting that the cleavage of Bid might be responsible for the activation ofmitochondrial dependent apoptosis pathway in this model. Thirdly, pretreatment withCaspase3 specific inhibitor could prevent the cleavage of PKC, suggests that themitochondrial-dependent apoptosis pathway could be activated by ROS, which in turnenhance the activation of PKC. Finally, in order to further exam the role played by PKC insodium selenite induced apoptosis, PKC inhibitor GF294003 and PKC activator TPA wereused. Pretreatment with GF294003 could partly inhibit the pro-apoptotic effect of sodiumselenite while pretreatment with TPA intensified apoptosis.To summarize, treatment by sodium selenite could induce ROS production within NB4cells, which cause the mitochondrial transmebrane potential loss and in turn the activationof Caspase3. Activated Caspase3 cleave the PKC, leading to its activated fragmentation. Atthe mean time, ROS could induced over-phosphorylation of PKC which contribute toPKC's activation. As reported by latest literatures, ROS could react with certain amino acidin active site of Phosphatase, lead to the inactivation of Phosphatase, and disturb thebalance between phosphorylation and dephosphorylation. The over-phosphorylation ofPKC could be result from this. It is also reported that the cleaved fragment of PKCcould induce apoptosis in various cancer cell lines. Thus, selenite induce the production ofROS, which in turn activate PKC, leading to a series of downstream signal transductionevents, is supposed to be an critical mechanism in sodium selenite induced apoptosis inNB4 cells. |
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