Comparative Proteomics Analysis Of Sodium Selenite Induced Acute Promyelocytic Leukemia Nb4 Cells And The Role Of Rho The Gdi 1 And 2

Selenium (Se) is an essential trace element possessing anticarcinogenic properties. Sodium selenite (Na₂SeO₃) induced apoptosis in human acute promyelocytic leukemia (APL) cell line NB4 with dose and time dependency. In this study, proteomic techniques were used to study the apoptosis of NB4 cells induced by sodium selenite. Twenty-six down-regulated and four up-regulated proteins were identified which exhibited a 1.5 fold change or greater. The identified proteins included key regulators of signal transduction such as Rho GDP dissociation inhibitors (Rho GDIs)1 and 2, members of the MAPK family, and proteins involved in the regulation of e-fos or c-myc expression. Importantly, the identified proteins, hnRNP DOB and Rho GDP dissociation inhibitor (GDI) beta, which were related with the regulation of c-myc, c-fos and c-jun, were determined by reverse transcription-polymerase chain reaction (RT-PCR) to confirm their down-regulation in proteomic study. Western blotting analysis and RT-PCR were then performed on three associated proteins: c-Myc, c-Fos and c-Jun, and their expression were observed to be significantly down-regulated. Results showed that certain regulation involved in c-myc, c-fos and c-jun was present in the apoptosis, and the c-Myc dependent-on and Jun N-terminal kinase (JNK) pathway also play roles.Among all the identified proteins, the Rho GDP dissociation inhibitors (Rho GDIs) are a major class of regulators of Rho GTPases and play essential roles in normal cell growth and malignant transformation. RhoGDIs are aberrantly over-expressed in human acute promyelocytic leukemia (APL) cell line NB4. Although RhoGDIs are known to inhibit Rho activities, recent studies indicate that RhoGDIs can also act as positive regulators. In our study, both Rho GDI1 and Rho GDI2 are cleavaged into 23kDa, and the performance can be ensured by the western blotting results of the nuclear protein and the Confocal test of the apoptoticNB4 cells. The further studies show that the cleavage of Rho GDI 1 is not inhibited by the caspase 3 inhibitor, Ac-DMQD-CHO, but by the caspase 2 inhibitor, Ac-VDVAD-CHO. So Rho GDI 1 isn't the substrate of caspase 3. we tried to downregulate the expression of Rho GDI 1 and 2 by using antisense oligonucleotide and We then treated the NB4 cells with sodium selenite. Downregulation of Rho GD 1 and 2 in NB4 cells resulted in decreased sensitvity to sodium selenite-induced apoptotic cell death. And, Gelsolin, a Ca2+-dependent actin regulatory protein, is verified as an interacting protein of Rho GDI 1, and Gelosin is believed to regulate the intracellular movements which are necessary for cell growth, proliferation, and differentiation. Expression of the gelsolin cleavage product was reported to cause the cells to round up, detach from the plate, undergo nuclear fragmentation and DNA fragmentation, following apoptosis induction. In this study, cleaved gelsolin was upregulated after the treatment of sodium selenite, and it may be one physiological effector of morphologic change during apoptosis.