MCLR Caused PP2A Inhibiition And Cellular Effects In Human Larynageal Epithlial Cells(Hep-2)

Background:Eutrophication and cyanobacterial blooms are serious problems nowadays. The toxic microcystins (Mcs) produced by a large number of blue-green algae have adverse effects on water bodies and threaten on varies organisms including human beings. So the toxic mechanism of microcystins has become the research hotspot for several decades. Microcystin-LR (MCLR) is one of the most toxic members of microcystins. The major toxic mechanism of Microcystin-LR is to inhibite the activity of protein phosphatase 2A (PP2A), resulting in a series of cytotoxicity. PP2A has important implications in the process of tumor cells’proliferation, growth, adhesion and migration. Our previous study demonstrated that MCLR induced toxic effects on normal cells by inhibiting PP2A and also caused some similar but different effects on tumor cell. Therefore, in order to further explore the MCLR toxicity mechanism on tumor cells, the human laryngeal epithelial cell (Hep-2) was selected in this study.Methods:The human laryngeal epithelial cell Hep-2 cells were exposed to the different concentrations of MCLR and effects on molecular and cellular level were determined. PP2A activity kit, flow cytometry, western blot, immunofluorescence assay, immunoprecipitation assay, cell proliferation assay, cell scratch assay and transwell migration assay were used in the study.Results:MCLR could enter into Hep-2 cells and inhibit PP2A activity by directly binding to PP2A/C. The protein level of PP2A/A, PP2A/C, B55 a and the post-translational modification of PP2A/C, including the phosphorylation at Tyr307 site and the methylation at Leu309 site, and the binding of the AC core enzyme as well as the binding of PP2A/C and a4, were all altered, which may be the reasons of PP2A inhibition. MCLR could also induce cytoskeletal reorganization, which was attributed to the increasing phosphorylation of MAPK pathway members, p38, ERK1/2 and the cytoskeleton-associated proteins, VASP, Tau and Ezrin. Furthermore, MCLR promoted cell migration, but did not change the cell cycle, apoptosis and proliferation.Conclusion:Taken together, covalent binding to PP2A/C and altering the protein level and post-translational modification as well as the binding of subunits are the main mechanisms for MCLR affecting PP2A activity in Hep-2. A dose-dependent change of p-Tau and p-Ezrin due to PP2A inhibition may contribute to the change of cytoskeleton and be related to the cell migration in Hep-2. The promotion in the cell migration implies that MCLR maybe have important effect on the tumor invasion. Our data provide a comprehensive exposition to the MCLR mechanism on tumor cells.