Cytoprotective role of Phyllanthus urinaria L. and glutathione-S transferase Pi in doxorubicin-induced toxicity in H9c2 cells.

ABSTRACT

OBJECTIVE: To examine cytoprotective effect of Phyllanthus urinaria (PU) ethanolic extract in doxorubicin (DOX)-induced toxicity. The research focus was on the mechanism of action in association with the expression and localization of glutathione-S transferase (GST) in cardiac H9c2 cells. MATERIAL AND METHOD: The presence of GST isoforms was evaluated in H9c2 cells using western blot analysis and confocal immunofluorescence visualization. Cells were then treated with DOX in the presence and absence of PU and several cytoprotective indices were evaluated, including the expression of the rate-limiting enzyme for glutathione synthesis, gamma-glutamylcysteine synthetase (gamma-GCS), manganese superoxide dismutase (MnSOD), copper-zinc SOD (CuZnSOD), and GST activity from cell lysate. The investigations for GST-mediated cytoprotection from DOX-induced oxidative damage were further carried out by SiRNA transfection and apoptosis detection using TUNEL assay. RESULTS: GST Pi (GSTP) was predominantly expressed in H9c2 cells compared with GST Alpha and GST Mu. Treatment with PU protected against the cardiotoxicity of DOX by influencing the nuclear localization of GSTP without significantly affecting the enzymatic activity. Suppression of GSTP expression by RNA interference potentiated the accumulation of DOX in the nucleus and enhanced apoptosis as evaluated by TUNEL assay. Treatment with PU had a cytoprotective effect by reducing cellular levels of DOX with enhanced nuclear localization of GSTP in myocardiac cells. CONCLUSION: The cytoprotective mechanism of PU against DOX cardiotoxicity partially involved the presence of GSTP. Thus, PU extracts may be used as an alternative source of antioxidants with distinctive mechanisms of action that may be suitable for specific types of oxidative insults.