Dual role of glutathione in modulating camptothecin activity: Depletion potentiates activity, but conjugation enhances the stability of the topoisomerase I-DNA cleavage complex

Abstract

Depletion of glutathione (GSH) in MCF-7 and MDA-MB-231 cell lines by pretreatment with the GSH synthesis inhibitor buthionine sulfoximine potentiated the activity of 10,11-methylenedioxy-20(S)-camptothecin, SN-38 [7-ethyl-10-hydroxy-20(S)-camptothecin], topotecan, and 7-chloromethyl-10,11-methylenedioxy-20(S)camptothecin (CMMDC). The greatest potentiation was observed with the alkylating camptothecin CMMDC. Buthionine sulfoximine pretreatment also increased the number of camptothecin-induced DNA-protein crosslinks, indicating that GSH affects the mechanism of action of camptothecin. We also report that GSH interacts with CMMDC to form a stable conjugate, 7-(glutathionylmethyl)-10,11-methylenedioxy-20(S)- camptothecin (GSMMDC), which is formed spontaneously in buffered solutions and in MCF-7 cells treated with CMMDC. GSMMDC was synthesized and found to be nearly as active as 10,11-methylenedioxy-20(S)-camptothecin in a topoisomerase (topo) mediated DNA nicking assay. The resulting topo I cleavage complexes were remarkably stable. In cell culture, GSMMDC displayed potent growth-inhibitory activity against U937 and P388 leukemia cell lines. GSMMDC was not active against a topo I-deficient P388 cell line, indicating that topo I is its cellular target. Peptide-truncated analogues of GSMMDC were prepared and evaluated. All three derivatives [7-(gamma-glutamylcysteinylmethyl)-10,11-methylenedioxy-20(S)- camptothecin, 7-(cysteinylglycylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, and 7-(cysteinylmethyl)-10,11-methylenedioxy-20(S)- camptothecin] displayed topo I and, cell growth-inhibitory activity. These results suggest that 7-peptidyl derivatives represent a new class of camptothecin analogues