Cytoprotective Role of Phyllanthus urinaria L. and Glutathione-S Transferase Pi in Doxorubicin-induced Toxicity in H9c2 Cells
Linda Chularojmontri PhD*, Yoshito Ihara MD, PhD**, Eiji Muroi MD, PhD***, Shinji Goto PhD***, Takahito Kondo MD, PhD***, Suvara K Wattanapitayakul PhD****
Affiliation : * Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Patumthani, Thailand ** Department of Biochemistry, Wakayama Medical University, Kimiidera, Wakayama, Japan *** Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan **** Department of Pharmacology, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand
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, γ-glutamylcysteine synthetase (γ-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.
Keywords : Apoptosis, Doxorubicin, Glutathione S-transferase pi, Myoblasts, cardiac, Oxidative stress, Phyllanthus
