Inhibition of the action of the topoisomerase II poison amsacrine by simple aniline derivatives: Evidence for drug-protein interactions

The action of the anticancer drug amsacrine appears to involve molecular in teractions with both DNA and topoisomerase II. It has been shown previously that DNA intercalators can inhibit the action of amsacrine and several oth er topoisomerase II poisons, presumably as a result of interference with th e DNA binding sites for the enzyme. We show here that drug molecules such a s N-phenylmethanesulfonamide, which mimic the anilino side chain of amsacri ne, inhibit the cytotoxicity against cultured Lewis lung murine carcinoma o f amsacrine, amsacrine analogues including asulacrine and DACA (N-[2-(dimet hylamino)-ethyl]acridine-4-carboxamide dihydrochloride), and etoposide, In contrast, the cytotoxicity of doxorubicin was slightly increased by co-incu bation with N-phenylmethanesulfonamide. The cytotoxicity of amsacrine was a lso modulated in human Jurkat leukemia, HCT-8 colon, and HT-29 colon cell f ines. Because o-AMSA, an amsacrine analogue containing a methoxy group in t he ortho rather than in the meta position, is known to be inactive as an an titumor drug, the abilities of the ortho and meta methoxy-substituted deriv atives of methyl-N-phenylcarbamate to reverse the cytotoxicity of amsacrine , asulacrine, and DACA were compared. The ortho substitution decreased acti vity while meta substitution slightly increased it, suggesting that the sid e chains were binding to a similar site to that occupied by amsacrine. To d etermine whether the side chain variants actively inhibited the formation o f DNA-topoisomerase II covalent complexes, cultured cells were treated with amsacrine or asulacrine, harvested, and lysed directly on acrylamide gels before electrophoresis and Western blotting to identify non-DNA-bound topoi somerase II. Extractable topoisomerase II was depleted in cells incubated w ith amsacrine but partially restored by coculture with methyl-N-phenylcarba mate. The findings are consistent with the hypothesis that low molecular we ight molecules can modulate the effects of topoisomerase II poisons by dire ctly interacting with the enzyme.