Yoji Ikegami

A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

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.

Re-evaluation and functional classification of non- synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2

Impacts of genetic polymorphisms of the ATP‐binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re‐evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site‐directed mutagenesis and stably expressed each of them in Flp‐In‐293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp‐In‐293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp‐In‐293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN‐38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN‐38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above‐mentioned non‐synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2‐mediated drug resistance without any influence from major non‐synonymous polymorphisms. (Cancer Sci 2007; 98: 231–239)

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.

Acidic phospholipids directly inhibit DNA binding of mammalian DNA topoisomerase I

Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein‐DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreoever, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme.

Transport Mechanism-Based Drug Molecular Design: Novel Camptothecin Analogues to Circumvent ABCG2-associated Drug Resistance of Human Tumor Cells

Acquired and intrinsic drug resistance in cancer is the major obstacle to long-term, sustained patient response to chemotherapy. Irinotecan (CPT-11) is a widely-used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, we have synthesized and characterized a total of fourteen new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition activity. In this context, we have synthesized new CPT analogues with different substitutions at positions 10 or 11 of the A ring. All of the tested CPT analogues strongly inhibited the Topo I activity in a cell-free system. Accordingly, we have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from ABCG2-overexpressing cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are good substrates for ABCG2 and therefore effectively extruded from cancer cells. Thus, hydrogen bond formation is considered to be involved in substrate recognition and/or transport processes of ABCG2. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.

Critical role of ABCG2 in ALA-photodynamic diagnosis and therapy of human brain tumor.

Primary brain tumors occur in around 250,000 people per year globally. Survival rates in primary brain tumors depend on the type of tumor, patients age, the extent of surgical tumor removal, and other factors. Photodynamic diagnosis (PDD) is a practical tool currently used in surgical operation of aggressive brain tumors, such as glioblastoma and meningiomas, whereas clinical application of photodynamic therapy (PDT) to brain tumor therapy has just recently started. Both PDD and PDT are achieved by a photon-induced physicochemical reaction, which is induced by the excitation of porphyrins exposed to light. In fluorescence-guided gross-total resection, PDD can be achieved by the administration of 5-aminolevulinic acid (5-ALA) as the precursor of protoporphyrin IX (PpIX). Exogenously administered ALA induces biosynthesis and accumulation of PpIX, a natural photosensitizer, in cancer cells. However, ATP-binding cassette transporter ABCG2 plays a critical role in regulating the cellular accumulation of porphyrins in cancer cells and thereby its expression and function can affect the efficacy of PDD and PDT. In response to the photoreaction of porphyrins leading to oxidative stress, the nuclear factor erythroid-derived 2-related transcription factor can transcriptionally upregulate ABCG2, which may reduce the efficacy of PDD and PDT. On the other hand, certain protein kinase inhibitors potentially enhance the efficacy of PDD and PDT by blocking ABCG2-mediated porphyrin efflux from cancer cells. In this context, it is of great interest to develop ABCG2 inhibitors that can be applied to PDD or PDT for the therapy of brain tumor and other tumors.

Metabolism of sulphobromophthalein. II. Species differences between rats, guinea-pigs and rabbits

Interesting species differences in the metabolism of sulphobromophthalein sodium have been observed between rats, guinea‐pigs and rabbits. The species difference was measured in terms of sulphobromophthalein monoglutathione conjugate (mGSH) positional isomer formation. After an intravenous injection of sulphobromophthalein to rats, 92% of sulphobromophthalein‐mGSH excreted into bile was the α ‐isomer. In contrast, in guinea‐pigs the three isomers α, β and δ were excreted in equivalent amounts. In rabbits, the majority of sulphobromophthalein‐mGSH was excreted as the β‐isomer. The formation ratio of glutathione (GSH) conjugates in‐vitro using cytosolic glutathione S‐transferases (GSTs) prepared from livers generally accounted for the biliary excretion ratio of α‐, β ‐ and δ‐monomercaptide isomers in‐vivo. GSTs from the livers of rat, guinea‐pig, and rabbit were purified and characterized. Although their main GSTs produced different isomers, their 20 amino acid residues showed that they belonged to the same class mu of GSTs. The results suggested differences of the three‐dimensional structure of GSTs that formed sulphobromophthalein‐mGSH isomers between the three animal species.

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.

High-performance liquid chromatographic determination of sulphobromophthalein and its conjugates.

A simple, sensitive and selective high-performance liquid chromatographic method for the determination of sulphobromophthalein and its mercaptide conjugates in rat bile was developed. These pigments, which have an absorption maximum at 580 nm in alkaline solution, were separated isocratically on an alkali-resistant ODS column by paired-ion chromatography. Analysis of bile samples obtained after intravenous administration of sulphobromophthalein to rats showed the presence of at least twenty peaks of metabolites, of which thirteen were identified and seven quantified.