Megumi Yoshikawa

Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin, a coumermycin antibiotic

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 μM 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 un‐selected 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.

Novel camptothecin analogues that circumvent ABCG2-associated drug resistance in human tumor cells

Irinotecan (7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]‐carbonyloxycamptothecin; CPT‐11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I); however, overexpression of ABCG2 (BCRP/MXR/ABCP) can confer cancer cell resistance to SN‐38, the active form of CPT‐11. We have recently demonstrated that plasma membrane vesicles prepared from ABCG2‐overexpressing PC‐6/SN2‐5H cells transported SN‐38 and its glucuronide conjugate in an ATP‐dependent manner (Nakatomi et al., Biochem Biophys Res Commun 2001;288:827–32). In the present study, we have characterized a total of 14 new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. All of the tested CPT analogues, which have different substitutions at positions 10 and 11, strongly inhibited the Topo I activity in a cell‐free system, as did SN‐38. Their antitumor activities in the SN‐38‐resistant PC‐6/SN2‐5H2 cell line greatly varied, however, being correlated with intracellular accumulation levels. We have examined ATP‐dependent transport of those CPT analogues by using plasma membrane vesicles prepared from both PC‐6/SN2‐5H2 cells and ABCG2‐transfected HEK‐293 cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with high polarity are good substrates for ABCG2 and are therefore effectively extruded from cancer cells. In this context, to circumvent ABCG2‐associated drug resistance, low‐polarity CPT analogues are considered to be potent lead compounds. The present study provides a practical approach to discover new CPT‐based drugs for the chemotherapy of drug‐resistant human cancer.

Molecular and cytogenetic characterization of the mouse ATP-binding cassette transporter Abcg4.

We have cloned a new mouse ATP-binding cassette (ABC) transporter, Abcg4, from a complementary DNA (cDNA) library of mouse brain. The cloned Abcg4 cDNA encodes a protein consisting of 646 amino acids and including one ATP-binding cassette and six transmembrane domains. The Abcg4 protein exhibits high identity (96%) with human ABCG4 in terms of the amino acid sequence. Fluorescence in situ hybridization with mouse and rat chromosomes has revealed that the Abcg4 gene is located on chromosomes 9A5.3 and 8q22 distal in mouse and rat, respectively. In these loci on mouse and rat chromosomes, conserved linkage homologies were hitherto identified with human chromosome 11q23, which involves the human ABCG4 gene. The mouse Abcg4 gene as well as the human ABCG4 gene each has a total of 14 exons to encode its respective protein. High transcript levels of mouse Abcg4 were detected in mouse brain, spleen, eye, and bone marrow. Taken together, our data on the chromosomal location, gene homology, protein structure, and phylogenetic relationships strongly support the idea that mouse Abcg4 is orthologue to the human ABCG4. By functionally analyzing the mouse Abcg4 protein, we may better understand the biological role of the human ABCG4 transporter.

Gefitinib Increases Serum Concentrations of Oral Irinotecan and SN-38 without Increasing the Biliary Concentration of SN-38 in Rats

Background: Gefitinib competes with topoisomerase I inhibitors at drug efflux pumps in vitro. We evaluated the effects of oral gefitinib on pharmacokinetic parameters of orally coadministered irinotecan. Methods: We measured the serum pharmacokinetic parameters and biliary excretion of irinotecan, SN-38 and its glucuronide after irinotecan (50 or 100 mg/kg) was orally administered with or without gefitinib 100 mg/kg to rats. We measured the concentrations of irinotecan and SN-38 in the small intestine, liver, lungs and kidneys in each rat. Results: The plasma area under the curve (0–24 h) of irinotecan and SN-38 was increased significantly, while the apparent elimination constant of irinotecan was decreased significantly. Gefitinib significantly increased the biliary cumulative amounts of irinotecan, but not of SN-38, and also influenced the enterohepatic circulation of irinotecan, but not of SN-38. Conclusions: Gefitinib increased the serum concentrations of irinotecan and SN-38 following oral administration of irinotecan without increasing the biliary excretion of SN-38 in vivo.

ABC transporters: a new approach to toxicogenomics

Membrane transporters play a pivotal role in drug absorption in the intestinal tract and in drug distribution to brain and other tissues, as well as in the elimination of toxic metabolites from cells. However, at present, little is known about the toxicogenomic and pharmacogenomic significance of drug transporters. In 1992, Ishikawa first proposed a new concept of the “phase III” detoxification system by emphasizing the biological importance of ATP-dependent transporters, GS-X pumps, for elimination of glutathione S-conjugates in our body (Trends Biochem Sci 7: 463–468, 1992). Since that time, more than 40 different human ATP-binding cassette (ABC) transporter genes have been discovered, and some of them have been documented to be critically involved in transport of drugs and metabolites. This review summarizes the toxicogenomic aspects of ABC transporters and the potential mechanisms underlying their gene expression.