CELLULAR PHARMACOLOGY OF AZATOXINS (TOPOISOMERASE-II AND TUBULIN INHIBITORS) IN P-GLYCOPROTEIN-POSITIVE AND P-GLYCOPROTEIN-NEGATIVE CELL-LINES

Azatoxin (NSC 640737), a synthetic molecule, was rationally designed as a topoisomerase-II inhibitor and was shown to be a potent cytotoxic: agent that inhibits both tubulin and topoisomerase II. A structure-activity relationship study allowed to select 3 derivatives that inhibit either tubulin (methylalatoxin) only or topoisomerase II (fluoroanilinoazatoxin and nitroanilino-azatoxin) in MTT assays performed on K562 and K562/ADM cells; the latter, expressing P-glycoprotein, indicated cross-resistance of K562/ADM cells to all 4 compounds. DNA double-strand breaks induced by the 3 azatoxins that inhibit topoisomerase II in vitro were decreased in K562/ADM as compared with K562 cells. Nitroanilino-azatoxin was the only compound for which resistance and reduced DNA damage observed in K562/ADM cells was partially reversed by verapamil, suggesting that nitroanilinoazatoxin was a substrate for P-glycoprotein, These results were confirmed by testing the cytotoxic activity of azatoxins on P-glycoprotein-expressing rat colon-carcinoma DHDK12/TRb cells in the absence and the presence of verapamil. Cell-cycle and mitotic-index studies indicated that azatoxin- and methylazatoxin-induced M-phase arrest was less in K562/ADM than in K562 cells. The G2 block induced by fluoro- and nitroanilinoazatoxins was delayed in K562/ADM cells, Verapamil increased cell-cycle inhibition induced by nitroanilinoalatoxin in K562/ADM cells without modifying cell-cycle effects of fluoroanilinoazatoxin, These results (i) are consistent with the specific inhibition of topoisomerase II or tubulin by azatoxin derivatives in cells; (ii) indicate that the nitro group of nitroanilinoazatoxin allows recognition and efflux by the P-glycoprotein; and (iii) suggest that cross-resistance of K562/ADM cells to other azatoxin derivatives is not mediated by P-glycoprotein. (C) 1995 Wiley-Liss, Inc.