Cellular pharmacology of azatoxins (topoisomerase-II and tubulin inhibitors) in P-glycoprotein-positive and -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 (methylazatoxin) 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 methyl-azatoxin-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 nitroanilinoazatoxin 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.
Eymin
B
B
Laboratory of Onco-Hematology and Pharmacology, Faculty of Medicine, Dijon, France.
Solary
E
E
Chevillard
S
S
Dubrez
L
L
Goldwasser
F
F
Duchamp
O
O
Genne
P
P
Leteurtre
F
F
Pommier
Y
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D001483:Base Sequence; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003603:Cytotoxins / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D017931:DNA Primers / Q000737:chemistry; D018432:Drug Resistance, Multiple; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003603:Cytotoxins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D007211:Indoles; D009153:Mutagens; D012333:RNA, Messenger; D059005:Topoisomerase II Inhibitors; C075996:azatoxin; D014700:Verapamil