Previously, we demonstrated that the Rho/ROCK pathway is involved in ouabain-induced apoptosis in HUVEC. In the current work, we investigated whether the Rho/ROCK pathway is functional during cardiac glycosides-induced cytotoxic effects in cancer cell lines, as well as in non-tumor cells. For that purpose, we evaluated the role of ROCK activation in bleb formation and cell migration over upstream and downstream effectors in addition to ROCK cleavage after cardiac glycosides treatment. All three cardiac glycosides (ouabain, digoxin and bufalin) induced cell death in HeLa and HepG2 cells and increased the formation of blebbing in HeLa cells. In contrast to our previous study, ROCK inhibitor Y27632 did not prevent bleb formation. Observation of ROCK II cleavage after ouabain, digoxin and oxaliplatin treatments in HeLa and/or HepG2 cells suggested that cleavage is independent of cell type and cell death induction. While inhibiting cleavage of ROCK II by the caspase inhibitors z-VAD-fmk, z-VDVAD-fmk and z-DEVD-fmk, evaluation of caspase 2 siRNA ineffectiveness on this truncation indicated that caspase-dependent ROCK II cleavage is differentially regulated in cancer cell lines. In HeLa cells, ouabain induced the activation of ROCK, although it did not induce phosphorylation of ERM, an upstream effector. While Y27632 inhibited the migration of HeLa cells, 10 nM ouabain had no effect on cell migration. In conclusion, these findings indicate that the Rho/ROCK pathway is regulated differently in cancer cell lines compared to normal cells during cardiac glycosides-induced cell death. (C) 2016 Elsevier Inc. All rights reserved.