Suppression of GST-P by treatment with glutathione-doxorubicin conjugate induces potent apoptosis in rat hepatoma cells
✍ Scribed by Tadashi Asakura; Yukio Hashizume; Ken-ichi Tashiro; Yasuyuki Searashi; Kiyoshi Ohkawa; Jun Nishihira; Masaharu Sakai; Toshiaki Shibasaki
- Publisher
- John Wiley and Sons
- Year
- 2001
- Tongue
- French
- Weight
- 162 KB
- Volume
- 94
- Category
- Article
- ISSN
- 0020-7136
- DOI
- 10.1002/ijc.1465
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✦ Synopsis
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 M 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 M 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 M) 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-(placental type of human GST isozymes) by transfection of GST-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.