2D-Gel Electrophoresis Analysis of Proteomic Changes in Three Human Cell Lines; HEK 293, HepG2 and 1321N1 Cells in Response to Cadmium
Akeem O. Lawal and Elizabeth M. EllisAffiliation:
Department of Medical Biochemistry, Faculty of Basic Medical Science, Lagos State University College of Medicine (LASUCOM), Lagos State, Nigeria.
AbstractCadmium (Cd) is a well known environmental and industrial heavy metal with multi-organ toxic effects. In this study, we examined the effects of Cd as cadmium chloride (CdCl2) on the proteomic profiles of exposed HEK 293, HepG2 and 1321N1 cells in an attempt to develop suitable biomarkers for Cd toxicity. 2D-gel electrophoresis (2DE) was performed on the cell extracts after 24 hr exposure to 5, 10 and 50 µM Cd and protein spots were compared using Phoretix TM 2D analysis software. Comparisons were made between Cd treated and untreated cells and spots were identified by mass spectroscopy using peptide-mass fingerprinting and database searching. The results show that the different concentrations (5-50 µ M) of CdCl2 used in this study caused at least 1-5-fold induction in some proteins in the three cell lines. A common feature in the proteomic profile was identified in HepG2 and HEK 293 cells after exposure to 5 µ M CdCl2 and this was the induction of one of the subunits of ATP synthase. 2DE analysis shows a 2.95- and 2.54-fold induction in ATP synthase in HEK 293 and HepG2 cells, respectively after 24 hr exposure to 5 µ M CdCl2. However, western blot validation shows 4.8- and 3.54-fold induction in ATPase in HEK 293 and HepG2 cells respectively. Both 2DE and western blot analysis shows a 2.2-fold induction in calreticulin, a Ca2+-binding protein, in HepG2 cells after 24 hr exposure to 5 µ M CdCl2.Though 2DE analysis shows a 1.7-fold induction in Albumin (ALB) protein in HEK 293 cells exposed to 50 µ M CdCl2, western blot analysis, however, shows a 10-fold increase. Exposure to 5 µM Cd also induced (1.8-fold) C-protein expression in 1321N1 cells. However, western blot analysis shows a 4.5-fold increase. These results suggest that Cd drastically alters the proteomic profiles of exposed cells, which include alterations in the expressions of proteins, involve in metabolism and intracellular Ca2+ homeostasis. These alterations may be important hallmarks in identifying Cd toxicity.
Albumin, ATP synthase, cadmium toxicity, calreticulin, C-protein, 2D-electrophoresis.
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