CELLULAR DETERMINANTS OF RESISTANCE TO INDOLOCARBAZOLE ANALOG LO[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 determ inants of resistance to the indolocarbazole compound NB-506 [2,3-alpha ]pyrrolo[3,4-c]carbazole-5,7(6H)-dione], a structurally novel TOP-I-in teractive drug, in parental and multidrug-resistant tumor cells expres sing either the P-170 glycoprotein (Pgp(170)) or multidrug resistance protein (MRP). MRP-expressing 250-fold doxorubicin-resistant human fib rosarcoma HT1080/DR4 tumor cells were drug sensitive to NB-506 and cam ptothecin (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 ov arian A2780/Dx5 tumor cells [Pgp(170) phenotype] were 6.2-fold resista nt to NB-506, whereas resistance to CPT was 2.6-fold. HPLC analysis of cellular NB-506 accumulation showed no significant differences betwee n A2780 and A2780/Dx5 cells (peak intracellular concentrations after 1 20-min exposure to 10 mu M NB-506: 400 +/- 85.0 and 352 +/- 95.1 nmol NB-506/mg protein, respectively). However, resistant A2780/Dx5 cells e xpressed a lower amount of TOP-I mRNA and 29% protein levels of TOP-I compared to parental A2780 cells, resulting in decreased TOP-I catalyt ic activity (3.17 +/- 0.02 vs. 1.16 +/- 0.15 rel.U/mu g nuclear protei n) and reduced induction of NB-506-mediated cleavable complex formatio n in A2780/Dx5 cells. Furthermore, the lower induction of NB-506-induc ed protein-linked DNA breaks (PLDB) in A2780/Dx5 cells correlated with significantly decreased DNA 12.2-440 kb size fragmentation in these c ells. The present study demonstrates that expression of MRP and Pgp(17 0) does not confer resistance to NB-506. Resistance to indolocarbazole substance NB-506 in A2780/Dx5 cells was only related to downregulatio n of TOP-I associated with lower induction of cleavable complex format ion 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.