Sodium Selenite Induced Acute Promyelocytic Leukemia Nb4 Cell Apoptosis Mechanism

The trace element selenium is an essential dietary nutrient that has been extensively studied as a potential anticancer agent. Recent reports show that sodium selenite, a common dietary form of selenium, can effectively induce several cancer cell lines to undergo apoptosis, raising a new idea for its clinical application. Our pioneering work has shown that sodium selenite, at concentrations of 5- 20μM, can disrupt△ψm and induce apoptosis in NB4 cells in a caspase 3-dependent manner, and this proapoptotic activity of sodium selenite was mainly attributed to its ability of inducing oxidative stress and downregulating anti-proapoptotic protein Bcl-2. But the detailed mechanism is still unclear.In the present study, we studied the involvement of Bcl-2 family members, MAPK members and Akt/PKB in the apoptosis in NB4 cells induced by sodium selenite and the findings are shown below.Treatment of NB4 cells with 20μM sodium selenite could disrupt△ψm and cause the release of cytochrome c from mitochondrion, caspase 9 specific inhibitor pretratment reduced the apoptosis by sodium selenite. Further study showed that antiapoptotic protein Bcl-xL was downregulated during the apoptosis, meanwhile, proapoptotic Bax and Bak levels elevated after sodium selenite treatment when Bax was cleaved to produce a 18 kD fragment at the later stage of apoptosis. Study also found the translocation of Bax/Bak to mitochondrion and dimerization of Bax was confirmed in a DSS crosslinking essay. Proapoptotic protein Bad was upregulated and its phosphorylation of Set 112 and Ser 136 decreased in the apoptosis. Taken together, these results indicated that mitochondrion apoptosis singaling pathway played an essential role in apoptosis in NB4 cell by sodium selenite and Bcl-2 family members Bax, Bak, Bad, Bcl—xL played important regulatory roles in this signaling pathway.Further study was conducted on the roles that MAPK family members played in the apoptosis, treatment with 20μM sodium selenite could downregulate the activation of Erk and JNK, upregulate the activation of p38 MAPK strongly. The components of Erk pathway, including Raf, MEK and p9ORSK, was downregulated with similar kinetics at the same time. Pretreament with PD98059, a specific chemical inhibitors of the MEK/ERK signaling pathway, were shown to strongly prevent the apoptosis process, while ERK activator TPA enhanced the process. It is also known that p38 MAPK inhibitor SB203580 and JNK inhibitor SP 600125 had slight effects on apoptosis. Further study indicated that ERK exerted its proapoptotic effect only at the early stage of apoptosis and played an antiapoptotic role at the later stages. Taken together, our findings suggest that ERK plays an active role in mediating sodium selenite-induced apoptosis in NB4 cells.Then we found that treatment with 20μM sodium selenite caused rapid decrease of Akt activation. Meanwhile the activation of PDK1 was downregulated and phosphorylation of Ser 259 in Raf and Ser 9 in GSK-3βreduced. Treatment with LY 294002 alone could cause apoptosis in NB4 cells in a dose dependent manner and combination of LY 294002 and sodium selenite could enhance apoptosis induction. Inhibition of Akt pathway with LY 294002 caused inactivation of Erk, dephosphrylation of Bad Ser 112 and upregulation of p38 MAPK and JNK. All those findings mentioned above suggest that Akt activity is vital for the survival of NB4 leukemia tumor cells and intervention with Akt pathway will cause apoptosis in NB4 cells. Inactivation of Akt was a key event in apoptosis caused by sodium selenite, which led to a series of downstream events including inactivation of Erk pathway, activation of p38 MAPK and JNK and dephosphorylation of Ser 112 and Ser 136 in Bad proapoptotic protein, finaly growth arrest and cell death. It was also shown that Akt and MAPK responded in distinct ways to different doses of sodium selenite. Low concentrations of sodium selenite stimulated the activation of Akt and Erk while higher oncentrations of sodium selenite inhibited that. And sodium selenite could upregulated p38 MAPK activation in a dose-dependent way when higher oncentrations of sodium selenite inhibited the activation of JNK. Those differential reactions of MAPK are probably responsible for the distinct effects on NB4 cells of different doses of sodium selenite.