Suppression of GST-P by treatment with glutathione-doxorubicin conjugate induces potent apoptosis in rat hepatoma cells

A conjugate of doxorubicin and glutathione via glutaraldehyde (GSH-DXR) inh ibited glutathione S-transferase (GST) activity of rat hepatoma AH66 cells, and treatment of the cells with GSH-DXR induced caspase-3 activation and D NA fragmentation. After treatment of AH66 cells with 0.1 muM GSH-DXR, GST-P (placental type of rat GST isozymes) mRNA and its protein increased transi ently and then decreased thereafter compared with the levels in nontreated cells. Caspase-3 activation and DNA fragmentation were induced following th e suppression of GST-P expression by treatment with GSH-DXR. When the cells were treated with 100 muM ethacrynic acid (ECA), an inhibitor of GST, DNA fragmentation and caspase-3 activation were observed. In contrast, treatmen t of AH66 cells with a low concentration of ECA (1 muM) that showed little inhibition of GST activity induced slight, but significantly enhanced expre ssion and activity of GST-P, and consequent prevention of DXR- and GSH-DXR- induced DNA fragmentation. Overexpression of GST-pi (placental type of huma n GST isozymes) by transfection of GST-pi sense cDNA into AH66 cells decrea sed sensitivities to DXR and GSH-DXR, and the suppression of GST-P by trans fection of the antisense cDNA into the cells increased drug sensitivity. On the other hand, there was little change in drug sensitivity caused by over expression of site-directedly mutated GST-P in which the active-site residu e Tyr39 was replaced with His (W39H) or the substrate-binding site residue Cys48 was replaced with Ser (C48S) by transfection of those cDNAs into AH66 cells. These results suggested that the suppression of GST-P in AH66 cells treated with GSH-DXR must play an important role in the induction of apopt osis. (C) 2001 Wiley-Liss, Inc.