No 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.

The parental IGROV-1 human ovarian adenocarcinoma cell line was intermittently exposed to increasing concentrations of cisplatin to obtain resistant sublines. A stable resistant subline with a resistance factor of 8.4 had been developed after 9 months and 28 passages, which was denoted IGROV(CDDP). A high correlation coefficient of 0.97 was found between the log cell survival and the DNA-adduct peak level during the process of resistance development. IGROV(CDDP) was strongly cross-resistant to carboplatin and doxorubicin and moderately cross-resistant to etoposide, docetaxel, and topotecan. Only minor resistance against 5-fluorouracil was observed, whereas IGROV(CDDP) was not cross-resistant to methotrexate. Intracellular accumulation of cisplatin was 65% lower in IGROV(CDDP) as compared with parental IGROV-1 at 37 degrees C under normal conditions. Coincubation of cisplatin with the Na+/K+-ATPase inhibitor ouabain resulted in a more pronounced decrease in platinum accumulation in IGROV-1 (44% decrease) than in IGROV(CDDP) (26% decrease). Under energy-depleting conditions the accumulation of cisplatin in the parental cell line was approximately 60% lower than that observed under normal (energy [i.e., ATP] rich) culture conditions. In contrast, the accumulation in IGROV(CDDP) was not affected by ATP-depletion. There appeared to be no significant difference between the intracellular accumulation of platinum in the resistant and sensitive cells under conditions of energy deprivation or when the uptake was studied at 0 degrees C. In conclusion, abrogation of energy-dependent accumulation in IGROV(CDDP) seems to be a major mechanism of resistance to cisplatin in this cell line.

SummaryThe profiles of an i.v. bolus and 3 h and 20 h infusion of cisplatin (CDDP) were simulated in vitro by using a culture of the IGROVI human ovarian cancer cell line. Disappearance of pharmacologically active unbound CDDP was accomplished by adding human albumin to the medium. Total and unbound CDDP and CDDP-DNA adduct levels were quantitated by atomic absorption spectroscopy (AAS), and tumour cell survival was measured by the clonogenic assay. The design of the experiment resulted in non-significant differences in the magnitude of the area under the concentration-time curve (AUC) of unbound CDDP between the three dose-input functions (AUC i.v. bolus, 6.34 ± 0.36; 3 h infusion, 6.35 ± 0.59; and 20 h infusion, 6.76 ± 0.40 pg h ml-'). Also, the differences between the area under the CDDP-DNA adduct -time curves (AUA) of the three dose-input functions were not significant. The initial rate of decline of the CDDP-DNA adduct-time curve was significantly higher for the i.v. bolus and 3 h infusion than for the 20 h infusion. There was a log-linear relationship between the AUC of unbound CDDP and cell survival. These relationships were not significantly different between the three dose-input functions. Variation in the rate of input of CDDP leads to differences in the shape of the AUC and AUA without significant effects on cell survival.

The purpose of this study was to determine the mechanism of the pharmacodynamic interaction between docetaxel/paclitaxel and cisplatin. Cisplatin-induced DNA-adducts and cisplatin accumulation were quantitated in peripheral blood leukocytes (WBC). The WBC were obtained from patients treated with docetaxel or paclitaxel in phase I/II studies and were incubated in vitro with cisplatin. In addition, blank whole-blood samples were obtained from patients and healthy subjects and incubated in intro with cisplatin or docetaxel/paclitaxel and cisplatin. The cisplatin-induced DNA-adduct levels measured in WBC after treatment with docetaxel or paclitaxel were significantly lower than those determined in non-pretreated WBC. Docetaxel and paclitaxel reduced the intracellular accumulation of cisplatin in WBC by 46-47%. If the pharmacodynamic interaction between docetaxel/paclitaxel and cisplatin also occurs in other normal tissues such as bone marrow, it may well contribute to the sequence dependent toxicity that has been observed in clinical studies.