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) inhibited glutathione S-transferase (GST) activity of rat hepatoma AH66 cells, and treatment of the cells with GSH-DXR induced caspase-3 activation and DNA fragmentation. After treatment of AH66 cells with 0.1 microM GSH-DXR, GST-P (placental type of rat GST isozymes) mRNA and its protein increased transiently and then decreased thereafter compared with the levels in nontreated cells. Caspase-3 activation and DNA fragmentation were induced following the suppression of GST-P expression by treatment with GSH-DXR. When the cells were treated with 100 microM ethacrynic acid (ECA), an inhibitor of GST, DNA fragmentation and caspase-3 activation were observed. In contrast, treatment of AH66 cells with a low concentration of ECA (1 microM) that showed little inhibition of GST activity induced slight, but significantly enhanced expression and activity of GST-P, and consequent prevention of DXR- and GSH-DXR-induced DNA fragmentation. Overexpression of GST-pi (placental type of human GST isozymes) by transfection of GST-pi sense cDNA into AH66 cells decreased sensitivities to DXR and GSH-DXR, and the suppression of GST-P by transfection of the antisense cDNA into the cells increased drug sensitivity. On the other hand, there was little change in drug sensitivity caused by overexpression of site-directedly mutated GST-P in which the active-site residue 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 apoptosis.
Asakura
T
T
Department of Biochemistry (I), Jikei University School of Medicine, Tokyo, Japan. tad_asakura@jikei.ac.jp
Hashizume
Y
Y
Tashiro
K
K
Searashi
Y
Y
Ohkawa
K
K
Nishihira
J
J
Sakai
M
M
Shibasaki
T
T
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000502:physiology; D053938:DNA Fragmentation; D004317:Doxorubicin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000473:pathology*; D010920:Placenta / Q000201:enzymology*; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D004317:Doxorubicin; D005982:Glutathione Transferase; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione