Investigation of possible link between RHO kinase and ouabain-induced senescence

Recently drug-induced senescence has gained an importance as a new approach in cancer therapy. It has been demonstrated that cardiac glycosides induce senescence in several cancer cell lines. Moreover, activation of small G protein Rho is required for the formation of morphological features of the senescent cells. Also, it has been shown that inhibition of Rock, one of the downstream effectors of Rho, prevents the Notch-induced senescence in endothelial cells. In the current work, we evaluated the possible role of Rock in ouabain-induced senescence in HeLa cells. The xCELLigence real time cell analyzer system, senescence associated ß-galactosidase staining and cell size were used to determine senescence. Concentration dependent effect of ouabain on cell viability and morphology was evalulated. In contrast to the treatment of ouabain in high concentrations which resulted in cell death, low nanomolar concentrations of ouabain induced senescence in HeLa cells. Although preincubation of specific Rock inhibitor Y-27632 (10?M, 30min) did not inhibit the occurrence of ouabain-induced (30nM, 48h) senescence in cells, it reduced the cell size. Furthermore preincubation of Y-27632 enhanced the cell death in senescence induced by ouabain. Although incubation of ouabain-induced G2/M phase cell cycle arrest, preincubation of Y-27632 had no effect on that induction. Our results indicate that inhibition of Rock with Y-27632 didn`t inhibit the senesence induced by ouabain. However it induced the cell death in senescent cells. These results suggest that Rock may be an important theurapeutic target in ouabain-induced senescence in cancer therapy. Film 1 and film 2 are enclosed herewith.