Relationship between cytocidal activity and glutathione-S-transferase inhibition using doxorubicin coupled to stereoisomers of glutathione with different substrate specificity.
To determine the cytotoxic mode of action of a glutathione (GSH)--doxorubicin (DXR) conjugate, which exhibited potent cytotoxicity against various multidrug-resistant as well as DXR-sensitive cell lines, the molecular interaction between covalent GSH--DXR conjugates and glutathione-S-transferase (GST), a possible molecular target of the conjugates, was investigated. The following four GSH molecules with stereoisomeric forms were prepared: L-Glu--L-Cys--Gly (LL-GSH), D-Glu--L-Cys--Gly (DL-GSH), L-Glu--D-Cys--Gly (LD-GSH) and D-Glu--D-Cys--Gly (DD-GSH). The enzymic activity of GST against each GSH stereoisomer was 88, 38, 8 and 4 nmol/mg/min, respectively, suggesting that the L-form cysteine residue in the molecule was an important substrate of GST. Addition of DXR conjugated with each isomer (10 microM) to a GSH-containing GST assay mixture inhibited the GST activity to 32% for LL-GSH--XR, 16% for DL-GSH-DXR and 61% for LD-GSH-DXR as compared with the solvent control. Moreover, IC50 values for these conjugates were 30, 20 and 250 nM, respectively. The cytocidal activity of each conjugate corresponded to the substrate specificity of GST activity for the GSH isomer. These conjugates bound to the GST molecule, and the binding ability was 0.746, 0.627 and 0.462 mol/mol of GST for LL-GSH--XR, DL-GSH-DXR and LD-GSH--XR, respectively. These findings suggested that GSH--DXR interacted with the substrate-binding site of the GST molecule and inhibition of GST activity exhibited potent cytotoxicity.
Hashizume
Y
Y
Departments of Biochemistry I, Jikei University School of Medicine, Tokyo 105-8461, Japan.
Asakura
T
T
Oikawa
T
T
Yamauchi
T
T
Soda
K
K
Ohkawa
K
K
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000633:toxicity; D017209:Apoptosis; D001665:Binding Sites; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000494:pharmacology* / Q000633:toxicity*; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology* / Q000633:toxicity*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D051381:Rats; D013237:Stereoisomerism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; C436625:glutathione-doxorubicin conjugate; D004317:Doxorubicin; D005982:Glutathione Transferase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione