Gefitinib (''Iressa'', ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRPoverexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K i value was 1.01 F 0.09 Mmol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP. (Cancer Res 2005; 65(4): 1541-6)

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Breast cancer resistance protein (BCRP/ABCG2) of an ATP-binding cassette half-transporter confers resistance against mitoxantrone and camptothecin derivatives of topotecan and irinotecan. Novobiocin, a coumermycin antibiotic, is known to enhance anticancer drug sensitivity of cancer cells in vitro and in vivo, the mechanism of which remains undetermined. Here we focused on drug efflux pump and examined whether novobiocin reversed drug resistance in multidrug-resistant cells highly expressing BCRP. To explore the reversal mechanisms, intracellular drug accumulation was measured by flow cytometry, and a topotecan transport study using plasma membrane vesicles was performed. We used PC-6/SN2-5H2 small cell lung cancer and MCF-7/MX breast cancer cells selected with SN-38 of the active irinotecan metabolite and mitoxantrone, respectively, and the BCRP cDNA transfectant MCF-7/clone 8 cells. These cells expressed high levels of BCRP mRNA but not other known transporters. Compared to the parental PC-6 cells, PC-6/ SN2-5H2 cells were 141-, 173-and 57.2-fold resistant to topotecan, SN-38 and mitoxantrone, respectively. Novobiocin at 60 M decreased the degree of the above resistance by approximately 26-fold in PC-6/SN2-5H2 cells, and similarly reversed resistance in MCF-7/MX, MCF-7/clone 8 and unselected NCI-H460 cells highly expressing BCRP. Furthermore, novobiocin increased the intracellular topotecan accumulation in these cells and inhibited the topotecan transport into the membrane vesicles of PC-6/SN2-5H2 cells. No effects of novobiocin in these assay were observed in the parental PC-6 and MCF-7 cells. The kinetic parameters in the transport study indicated that novobiocin was a inhibitor for BCRP, resulting in competitive inhibition of BCRP-mediated topotecan transport. These findings suggest that novobiocin effectively overcomes BCRP-mediated drug resistance at acceptable concentrations.

Key words: SN-38; CPT analogue; ABCG2; SAR; DNA topoisomerase I inhibitorCamptothecin (CPT) is an antitumor alkaloid that was originally isolated from Camptotheca acuminata, a tree native to southern China. 1 CPT has been demonstrated to inhibit mammalian DNA topoisomerase I (Topo I), the nuclear enzyme that changes the topologic state of duplex DNA by single-strand breakage and resealing. Stabilization of the covalent Topo I-DNA complex (so-called cleavable complex) by CPT is a critical step in its antitumor action where Topo I-mediated DNA breaks are induced via prevention of DNA relegation. 2 Clinical and preclinical studies, however, revealed reversible bone marrow depression and hemorrhagic cystitis as the major dose-limiting toxicities of CPT. Thus, efforts have been directed at finding new CPT analogues with higher antitumor activity and less toxicity.Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin; CPT-11) has been synthesized and developed as a new water-soluble analogue of CPT with wide-spectrum antitumor activity against many types of human tumor cells. 3,4 CPT-11 is a prodrug, the antitumor activity of which is exerted by 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of CPT-11. 5 Despite enormous expense and efforts spent on the development of cancer chemotherapies, acquired and intrinsic drug resistance in tumors is the major obstacle to long-term sustained patient response to chemotherapy. Hitherto several mechanisms for the resistance to SN-38 and its analogues have been proposed, e.g., mutations or decreased expression of Topo I, increased expression of the UGT1A protein or single nucleotide polymorphisms (SNPs) of the UGT1A gene, increased activity of O 6 -methylguanine-DNA-methyltransferase, a DNA repair protein, decreased activity of carboxylesterase that catalyzes the biosynthesis of SN-38 from CPT-11 in the plasma and liver and overexpression of drug export pumps. 6 -11 It has been documented that several ATP-binding cassette (ABC) transporters, such as P-glycoprotein (ABCB1/MDR1/P-gp) and multidrug resistance-associated protein 1 (ABCC1/MRP1), can cause drug resistance in tumor cells by actively extruding antitumor drugs. 12,13 Recently, a novel ABC transporter, ABCG2, also known as breast cancer-resistant protein (BCRP/MXR1/ ABCP), has been discovered in drug-resistant cell lines selected for by mitoxantrone or Topo I inhibitors. 14,15 Overexpression of ABCG2 has been shown to confer resistance to doxorubicin, mitoxantrone and various CPT analogues. 14,16,17 In a recent study with plasma membrane vesicles prepared from ABCG2-overexpressing SN-38-selected human small cell lung carcinoma cells, we found that ABCG2 transported SN-38 and its glucuronide metabolite in an ATP-dependent manner. 18 Thus, it is highly likely that ABCG2 actively extrudes SN-38 from tumor cells and thereby confers drug resistance.To circumvent ABCG2-associated drug resistance, in the present study we have synthesized a total of 14 different analogues of CPT and examine...

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