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

Abstract

In order to unravel possible mechanisms of clinical resistance to topoisomerase I inhibitors, we developed a topotecan-resistant human IGROV-1 ovarian cancer cell line, denoted IGROV(T100r), by stepwise increased exposure to topotecan (TPT). The IGROV(T100r) cell line was 29-fold resistant to TPT and strongly cross-resistant to SN-38 (51-fold). However, the IGROV(T100r) 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 I protein levels and catalytic activity as well as topoisomerase I cleavable complex stabilization by CPT in the IGROV-1 and IGROV(T100r) cell lines were observed, indicating that resistance in the IGROV(T100r) cell line was not related to topoisomerase I-related changes. However, resistance in the resistant IGROV(T100r) cell line to TPT and SN-38 was 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 IGROV(T100r) cell line increased under these energy-deprived conditions. The efflux of TPT at 37 degrees C was very rapid in the IGROV-1 as well as the IGROV(T100r) cell line, resulting in 90% efflux within 20 min. Importantly, the efflux rates of TPT in the IGROV-1 and IGROV(T100r) 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 IGROV(T100r) 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 I drugs.