Cellular determinants of resistance to indolocarbazole analogue 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (NB-506), a novel potent topoisomerase I inhibitor, in multidrug-resistant human tumor cells.
Membrane protein-associated alterations in cellular drug accumulation have been recently implicated in resistance to topoisomerase I (TOP-I)-interactive drugs. The present study investigated the cellular determinants of resistance to the indolocarbazole compound NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7(6H)-dione], a structurally novel TOP-I-interactive drug, in parental and multidrug-resistant tumor cells expressing either the P-170 glycoprotein (Pgp170) or multidrug resistance protein (MRP). MRP-expressing 250-fold doxorubicin-resistant human fibrosarcoma HT1080/DR4 tumor cells were drug sensitive to NB-506 and camptothecin (CPT) (resistance factor: 0.7 and 0.8, respectively) with no alterations of TOP-I parameters including DNA relaxation, expression of TOP-I protein and mRNA. In contrast, doxorubicin-resistant human ovarian A2780/Dx5 tumor cells [pgp170 phenotype] were 6.2-fold resistant to NB-506, whereas resistance to CPT was 2.6-fold. HPLC analysis of cellular NB-506 accumulation showed no significant differences between A2780 and A2780/Dx5 cells (peak intracellular concentrations after 120-min exposure to 10 microM NB-506: 400+/-85.0 and 352+/-95.1 nmol NB-506/mg protein, respectively). However, resistant A2780/Dx5 cells expressed a lower amount of TOP-I mRNA and 29% protein levels of TOP-I compared to parental A2780 cells, resulting in decreased TOP-I catalytic activity (3.17+/-0.02 vs. 1.16+/-0.15 rel.U/microg nuclear protein) and reduced induction of NB-506-mediated cleavable complex formation in A2780/Dx5 cells. Furthermore, the lower induction of NB-506-induced protein-linked DNA breaks (PLDB) in A2780/Dx5 cells correlated with significantly decreased DNA 12.2-440 kb size fragmentation in these cells. The present study demonstrates that expression of MRP and Pgp170 does not confer resistance to NB-506. Resistance to indolocarbazole substance NB-506 in A2780/Dx5 cells was only related to downregulation of TOP-I associated with lower induction of cleavable complex formation and DNA fragmentation. The data reported herein may indicate that the new indolocarbazole compound NB-506 has potent antitumor efficacy in membrane-associated multidrug resistance.
Vanhoefer
U
U
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Voigt
W
W
Hilger
R A
RA
Yin
M B
MB
Harstrick
A
A
Seeber
S
S
Rustum
Y M
YM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D015152:Blotting, Northern; D002166:Camptothecin / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology*; D002452:Cell Count / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005354:Fibrosarcoma / Q000188:drug therapy* / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology*; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D002166:Camptothecin; D002227:Carbazoles; D004264:DNA Topoisomerases, Type I; D004317:Doxorubicin; D004791:Enzyme Inhibitors; D005960:Glucosides; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; D059004:Topoisomerase I Inhibitors; C092476:NB 506