No abstract

Effects of human selenoprotein SelK on the adhesion and migration ability of human gastric cancer BGC-823 cells using Matrigel adhesion and transwell migration assays, respectively, were investigated in this study. The Matrigel adhesion ability of BGC-823 cells that overexpressed SelK declined extremely significantly (p < 0.01) compared with that of the cells not expressing the protein. The migration ability of BGC-823 cells that overexpressed SelK also declined extremely significantly (p < 0.01). On the other hand, the Matrigel adhesion ability and migration ability of the cells that overexpressed C-terminally truncated SelK did not decline significantly. The Matrigel adhesion ability and migration ability of human embryonic kidney HEK-293 cells that overexpressed SelK did not show significant change (p > 0.05) with the cells that overexpressed the C-terminally truncated protein. In addition to the effect on Matrigel adhesion and migration, the overexpression of SelK also caused a loss in cell viability (as measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) colorimetric assay) and induced apoptosis as shown by confocal microscopy and flow cytometry. The cytosolic free Ca2+ level of these cells was significantly increased as detected by flow cytometry. But the overexpression of SelK in HEK-293 cells caused neither significant loss in cell viability nor apoptosis induction. Only the elevation of cytosolic free Ca2+ level in these cells was significant. Taken together, the results suggest that the overexpression of SelK can inhibit human cancer cell Matrigel adhesion and migration and cause both the loss in cell viability and induction of apoptosis. The release of intracellular Ca2+ from the endoplasmic reticulum might be a mechanism whereby the protein exerted its impact. Furthermore, only the full-length protein, but not C-terminally truncated form, was capable of producing such impact. The embryonic cells were not influenced by the elevation of free Ca2+ level in cytosol, probably due to their much greater tolerance to the variation.

dSelK (G-rich), a homolog of human and mouse SelK, is one of three selenoproteins in Drosophila melanogaster. It is the only trans-membrane selenoprotein in D. melanogaster integrated into both the endoplasmic reticulum (ER) membrane and the Golgi apparatus. The gene expression profile of Drosophila Schneider 2 (S2) cells after the dsRNA interference (dsRNAi) targeting of dSelK was examined with the GeneChip Drosophila Genome 2.0 Array (Affymetrix), a high-density oligonucleotide microarray encompassing nearly the full Drosophila genome. The results showed that the transcriptional expression of eight genes whose proteins are located on (or related to) the ER or the Golgi apparatus was highly induced or repressed by the dsRNAi treatment. The mRNA levels of the inositol 1,4,5-tris-phosphate receptor (IP3 receptor), whose gene product is integrated into the ER membrane and regulates the release of Ca2+ from the ER to the cytosol, were significantly downregulated. In contrast, the expression of inositol 1,4,5-tris-phosphate kinase 1, which is a cytosolic protein with opposing functions to the IP3 receptor, was significantly upregulated. Quantitative real-time PCR verified these results. The concentration of intracellular free Ca2+ of the Drosophila S2 cells was significantly decreased after the knockdown of dSelK, whereas overexpression of dSelK significantly increased the intracellular free Ca2+ concentration. These results indicate that dSelK in D. melanogaster is involved in regulating the release of Ca2+ from the ER to the cytosol and may play important roles in the signal transduction pathways involving Ca2+ mobilization.

Lead (Pb) is a well-known type of neurotoxin and chronic exposure to Pb induces cognition dysfunction. In this work, the potential role of early growth response gene 1 (EGR1) in the linkage of Pb exposure and disrupted in scherophernia-1 (DISC1) activity was investigated. Human neuroblastoma cell line SH-SY5Y was subjected to different concentrations of lead acetate (PbAc) to determine the effect of Pb exposure on the cell viability, apoptosis, and activity of EGR1 and DISC1. Then the expression of EGR1 in SH-SY5Y cells was knocked down with specific siRNA to assess the function of EGR1 in Pb induced activation of DISC1. The interaction between EGR1 and DISC1 was further validated with dual luciferase assay, Supershift electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP)-PCR. Administration of PbAc decreased cell viability and induced apoptosis in SH-SY5Y cells in a dose-dependent manner. Additionally, exposure to PbAc also up-regulated expression of EGR1 and DISC1 at all concentrations. Knockdown of EGR1 blocked the effect of PbAc on SH-SY5Y cells, indicating the central role of EGR1 in the function of Pb on activity of DISC1. Based on the results of dual luciferase assay, Supershift EMSA, and ChIP-PCR, EGR1 mediated the effect of Pb on DISC1 by directly bound to the promoter region of DISC1 gene. The current study elaborated the mechanism involved in the effect of Pb exposure on expression of DISC1 for the first time: EGR1 activated by Pb substitution of zinc triggered the transcription of DISC1 gene by directly binding to its promoter.