Reduced cellular accumulation of topotecan: a novel mechanism of resistance in a human ovarian cancer cell line

Abstract

Summary In order to unravel possible mechanisms of clinical resistance to topoisomerase inhibitors, we developed a topotecan-resistant human IGROV-1 ovarian cancer cell line, denoted IGROVTlOOr, by stepwise increased exposure to topotecan (TPT). The IGROVT100r cell line was 29-fold resistant to TPT and strongly cross-resistant to . However, the IGROVTlOOr showed only threefold resistance to camptothecin (CPT). Remarkably, this cell line was 32-fold resistant to mitoxantrone, whereas no significant cross-resistance against other cytostatic drugs was observed. No differences in topoisomerase protein levels and catalytic activity as well as topoisomerase cleavable complex stabilization by CPT in the IGROV-1 and IGROVTIOOr cell lines were observed, indicating that resistance in the IGROVTlOOr cell line was not related to topoisomerase I-related changes. However, resistance in the resistant IGROVTlOOr cell line to accompanied by decreased accumulation of the drugs to approximately 15% and 36% of that obtained in IGROV-1 respectively. No reduced accumulation was observed for CPT. Notably, accumulation of TPT in the IGROV-1 cell line decreased under energy-deprived conditions, whereas the accumulation in the IGROVTlOOr cell line increased under these energy-deprived conditions. The efflux of TPT at 370C was very rapid in the IGROV-1 as well as the IGROVT100r cell line, resulting in 90% efflux within 20 min. Importantly, the efflux rates of TPT in the IGROV-1 and IGROVTlOOr cell lines were not significantly different and were shown to be independent on P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP). These results strongly suggest that the resistance of the IGROVTlOOr cell line to TPT and SN-38 is mainly caused by reduced accumulation. The reduced accumulation appears to be mediated by a novel mechanism, probably related to impaired energy-dependent uptake of these topoisomerase drugs.

Novel mechanisms of drug resistance in leukemia

“…78 The resulting drug-resistant cells are cross-resistant to mitoxantrone, SN-38, and 9-aminocamptothecin, have diminished accumulation and retention of topotecan, do not overexpress Pgp or MRP, and have no alteration in the catalytic activity or expression of DNA topoisomerase I. 78 In contrast to the mitoxantrone-selected cells described above, the topotecan-selected IGROV1 cells are not cross-resistant to doxorubicin or daunorubicin. Recently, marked overexpression of BCRP was reported in the topotecan-selected IGROV1 cells, confirming that selection with topotecan can induce BCRP as a cellular defense mechanism.…”

Mechanisms of resistance to topoisomerase I-targeting drugs

“…Both active and passive transport mechanisms are implicated in intestinal cell uptake of CPT (Gupta et al, 2000). Additionally, ovarian cancer cells contain active transporters that are required for the influx of topotecan and SN-38 (Ma et al, 1998). In addition to uptake, cellular metabolism may be particularly important for the prodrug CPT-11, which is converted to its active form, SN-38, by cellular carboxylesterases (Danks et al, 1998;Ahmed et al, 1999;Humerickhouse et al, 2000;Khanna et al, 2000).…”

Reversal of MDR1-associated resistance to topotecan by PAK-200S, a new dihydropyridine analogue, in human cancer cell lines

“…Of importance is that irinotecan (1-piperidino)-1-piperidino]-carbonyloxy-CPT]) and its active metabolite SN-38 (10-hydroxy-7-ethyl-campto- thecin) appear not to be substrates for P-gp-mediated drug resistance in vitro and in vivo (Jansen et al, 1998). In addition, P-gp-and MRP-independent mechanisms of membrane proteinassociated resistance to topotecan have been observed (Yang et al, 1995;Ma et al, 1998). Yang and co-workers (1995) reported in a mitoxantrone-resistant cell line an energy-dependent efflux of topotecan associated with a high level of drug resistance and reduced steady-state levels of topotecan-mediated TOP-I enzyme/DNA complexes.…”