Dual effects of ouabain on the regulation of proliferation and apoptosis in human prostatic smooth muscle cells

Purpose: To elucidate the role of ouabain in the pathophysiology of benign prostatic hyperplasia we examined the effects of ouabain on the proliferation and apoptosis of human prostatic smooth muscle cells. Materials and Methods: Primary cultures of human prostatic smooth muscle cells were obtained from 7 patients with bladder outlet obstruction caused by benign prostatic enlargement. A cell proliferation study was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method to examine the effects of different concentrations of ouabain and various inhibitors. Western blot analysis was done to determine mitogen activated protein kinase (MAPK) activation. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method and caspase-3 activity assay were also performed to examine the apoptotic mechanism. Results: Ouabain exhibited a modest but significant proliferative effect in nanomolar concentrations; whereas it induced cell apoptosis at higher concentrations. Ouabain caused rapid activation of p42/44 MAPKs. The proliferative effect of ouabain was completely flattened by W-7 and MAPK kinase (MEK) inhibitor, suggesting the requirement of Ca²⁺ mobilization and the involvement of the MEK-p42/44 MAPK cascade. The cytotoxic effect by ouabain was defined as apoptosis and necrosis using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction technique and lactate dehydrogenase release assay, respectively. In addition, ouabain induced profound caspase-3 activity in the cytotoxic concentrations and DEVD-CHO reversed the cytotoxic action to ouabain, demonstrating the involvement of caspase-3 activation in the cytotoxic action. Conclusions: Ouabain at different concentrations caused dual effects on proliferation and apoptosis in human prostatic smooth muscle cells. At low concentrations ouabain promoted cell proliferation via a Ca²⁺ dependent mechanism and activation of the MEK-p42/44 MAPK pathway; whereas it induced cell apoptosis via the activation of caspase-3 activity at higher concentrations.