Rho-Associated Kinase Inhibitor (Y-27632) Attenuates Doxorubicin-Induced Apoptosis of Human Cardiac Stem Cells

Background

Recent clinical trials using c-kit+ human cardiac stem cells (CSCs) demonstrated promising results in increasing cardiac function and improving quality of life. However, CSC efficiency is low, likely due to limited cell survival and engraftment after transplantation. The Rho-associated protein kinase (ROCK) inhibitor, Y-27632, significantly increased cell survival rate, adhesion, and migration in numerous types of cells, including stem cells, suggesting a common feature of the ROCK-mediated apoptotic pathway that may also exist in human CSCs. In this study, we examine the hypothesis that pretreatment of human CSCs with Y-27632 protects cells from Doxorubicin (Dox) induced apoptosis.

Methods and Results

c-kit+ CSCs were cultured in CSC medium for 3–5 days followed by 48hr treatment with 0 to 10μM Y-27632 alone, 0 to 1.0μM Dox alone, or Y-27632 followed by Dox (48hrs). Cell viability, toxicity, proliferation, morphology, migration, Caspase-3 activity, expression levels of apoptotic-related key proteins and c-kit+ were examined. Results showed that 48hr treatment with Y-27632 alone did not result in great changes in c-kit+ expression, proliferation, Caspase-3 activity, or apoptosis; however cell viability was significantly increased and cell migration was promoted. These effects likely involve the ROCK/Actin pathways. In contrast, 48hr treatment with Dox alone dramatically increased Caspase-3 activity, resulting in cell death. Although Y-27632 alone did not affect the expression levels of apoptotic-related key factors (p-Akt, Akt, Bcl-2, Bcl-xl, Bax, cleaved Caspase-3, and Caspase-3) under basal conditions, it significantly inhibited the Dox-induced increase in cleaved Caspase-3 and reduced cell death under Dox treatment.

Conclusions

We conclude that preconditioning human CSCs with Y-27632 significantly reduces Dox-induced cell death and possibly involves the cleaved Caspase-3 and ROCK/Actin pathways. The beneficial effects of Y-27632 may be applied to stem cell-based therapy to increase cell survival rates after transplantation or to act as a cardiac protective agent for Dox-treated cancer patients.