ABSTRACT Statins, the cholesterol-lowering drugs, are known to exert pleiotropic actions including anticancer and antiviral effects. The interaction between simvastatin and oncolytic vesicular stomatitis virus (VSV), a potential candidate for anticancer therapy, was investigated. HeLa cells in culture were exposed to simvastatin and/or VSV-green fluorescence protein (GFP) at different times and concentrations. VSV protein contents, virus infectivity, apoptosis (PE Annexin V), necrosis (7-aminoactinomycin D) and cell viability were analyzed by immunoblot, plaque-forming units/size, and flow-cytometry. VSV infection was characterized by the presence of infected cells (GFP-stained cells), glycoprotein (G), nucleocapsid/polymerase (N/P) and matrix (M) viral proteins in cell lysates and culture media, production of viable progeny, and induction of apoptosis, necrosis and cell death. The effects were directly related to the exposure time and multiplicity of infection (MOI) employed. Notably, large numbers of apoptotic and/or necrotic cells were GFP-negative (non-GFP stained). Simvastatin induced cell loss, apoptosis and necrosis; however, concentrations > 2 µM were required to observe significant cell damage. Simvastatin (0.25-2 µM; > 4 hours exposure) exerted a potent antiviral action, characterized by decreases in viral proteins, ability of viral progeny to induce plaque formation, number of infected, apoptotic and necrotic cells, and increased cell survival. Greater antiviral action was observed with higher concentrations. VSV-induced cell damage (cell loss + apoptosis + necrosis) was reduced by 79 ± 8% with 4 µM simvastatin (P < 0.01). Simvastatin inhibited VSV replication and infectivity, and markedly decreased VSV-induced cell damage, independently of its pro-apoptotic action. Exposure time and inoculum size are strong determinants of the mechanisms by which VSV induces cell death.
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