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

Abstract

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.

Inhibition of the Multidrug Resistance Protein 1 (MRP1) by Peptidomimetic Glutathione-Conjugate Analogs

“…The EA group can dissociate from the GSH-sulfhydryl, thereby regaining its ␣,␤-unsaturated ketone moiety, which may modify cysteine residues in proteins. In addition, the VII-dimethyl ester might induce apoptosis through inhibition of GST, as seen with other GST inhibitors in various systems (Morgan et al, 1996;Asakura et al, 2001;Ruscoe et al, 2001). Because compound VII is also an effective GST inhibitor (D. Burg, R. Hermanns, I. M. C. M. Rietjens, P. van Bladeren, G. van der Marel, G. J. Mulder, submitted for publication), the observed cell death may be caused by such GST inhibition.…”

Prognostic Significance of Glutathione S-Transferase-Pi in Invasive Breast Cancer

“…It is reported to be variably expressed in breast cancer (3) and is associated with estrogen receptor level expressed by the tumor (4,5). Down-regulation of GST-pi activity in a T-cell line study appears to favor apoptosis (6) and inhibition of GST-pi function induces apoptosis in rat hepatoma cells (7).…”

Target chemotherapy of anti-CD147 antibody-labeled liposome encapsulated GSH-DXR conjugate on CD147 highly expressed carcinoma cells

“…We demonstrated in the previous study that GSH-DXR exhibited potent cytotoxicity compared to DXR (17)(18)(19). Here, the effect of drug-delivery of GSH-DXR on target chemotherapy was examined using the aCD147ab-liposome.…”

“…GSH-DXR was prepared as described previously (16)(17)(18)(19). In brief, the combination of 1 mg of each GSH and 0.5 mg of DXR in 0.5 ml of 0.15 M NaCl containing 0.1% glutaraldehyde was incubated at room temperature for 30 min.…”

“…On the other hand, a recent study revealed that a conjugate of doxorubicin (DXR) with glutathione (GSH) (GSH-DXR) potently induced apoptosis in rat hepatoma AH66 cells relative to DXR (15)(16)(17). The same study also showed that GSH-DXR inhibited GST activity and suppressed GST P1 mRNA, but DXR did not, indicating that inhibition of the enzyme makes an important contribution to manifestation of potent GSH-DXR cytotoxicity against AH66 cells (18,19). Therefore, it was predicted that the disappearance of GST P1-1 enzymatic activity did not suppress JNK activity in spite of binding to the JNK molecule and potently inducing apoptosis.…”