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Gefitinib ("Iressa", ZD1839), an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Reverses Breast Cancer Resistance Protein/ABCG2–Mediated Drug Resistance

¹ Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan; ² Division of Respiratory Medicine, Department of Internal Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan; and ³ Department of Drug Metabolism and Disposition, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan

Requests for reprints: Mikio Oka, Division of Respiratory Medicine, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. Phone: 81-86-462-1111; Fax: 81-86-464-1041; E-mail: moom{at}med.kawasaki-m.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 BCRP-overexpressing 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 ± 0.09 µmol/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.

Key Words: EGFR inhibitor • Drug resistance • ABC transporter • Topoisomerase I inhibitor • Lung cancer

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with specific tyrosine kinase activity, and it serves to regulate proliferation and differentiation of epidermal cells (1, 2). In human solid tumors, overactivation and/or dysregulation of EGFR promotes tumor progression, including invasion, angiogenesis, metastasis, and treatment resistance with blocking apoptosis (3–6). Because EGFR is important in epithelial tumor biology, EGFR-targeted cancer therapy has been developed (7). Gefitinib ("Iressa", ZD1839, AstraZeneca. Co., Macclesfield, United Kingdom) is an orally active, selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways implicated in the proliferation and survival of cancer cells (8). Although gefitinib combined with various cytotoxic agents, such as taxanes, platinums, and topotecan, has been reported to enhance cytotoxicity in vitro and in vivo, the exact enhancement mechanisms still remain undetermined (9–15).

Transporter proteins belonging to the ATP-binding cassette superfamily pump out drugs using the energy from ATP hydrolysis, leading to resistance of cancer cells against multiple anticancer drugs (16). Among them, P-glycoprotein and the multidrug resistance protein family have been extensively investigated (16). The breast cancer resistance protein (BCRP/ABCG2) of an ATP-binding cassette half-transporter was isolated from atypical multidrug-resistant MCF-7 human breast cancer cells selected with doxorubicin and verapamil (17). BCRP-overexpressing cells show cross-resistance to mitoxantrone and DNA topoisomerase I inhibitors of topotecan and SN-38, suggesting strongly that BCRP plays an important role in acquiring resistance against these drugs (16, 18, 19). We have already shown that BCRP efficiently transports SN-38 with a high affinity in vitro (20). Interestingly, CI1033 of the HER family tyrosine kinase inhibitors was reported to enhance the cytotoxicity of topotecan and SN-38 through inhibition of BCRP-mediated drug efflux in cancer cells (21). The HER family of receptors includes EGFR, HER-2, HER-3, and HER-4. Therefore, we hypothesized that gefitinib would reverse drug resistance in cancer cells overexpressing BCRP. Here, using plasma membrane vesicles, we report that gefitinib reverses BCRP-mediated resistance against topotecan, SN-38, and mitoxantrone through direct inhibition of BCRP in multidrug-resistant human small cell lung cancer and breast cancer cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Lines and Reagents. PC-6 and PC-6/SN2-5 human small cell lung cancer cell lines were kindly provided by Dr. Akiko Tohgo (Daiichi Pharmaceutical, Co., Tokyo, Japan). SN-38-resistant PC-6/SN2-5 cells were selected from the parental PC-6 cells by continuous exposure to 4.8 nmol/L SN-38 (22). Highly resistant PC-6/SN2-5H cells were further selected from PC-6/SN2-5 cells by continuous exposure to 25 nmol/L SN-38 in our laboratory. These cells have no mutation at codon 482 of BCRP (23), which determines the specificity of BCRP substrates (16, 18). MCF-7 human breast cancer cells and mitoxantrone-resistant MCF-7/MX cells were provided by Dr. Masayuki Nakagawa (Kagoshima University, Kagoshima, Japan; ref. 24). MCF-7/MX cells have no mutation at codon 482 of BCRP (25). MCF-7/BCRP clone 8 cells, transfectants of the vector pcDNA3-BCRP construct, were kindly provided by Dr. Douglas D. Ross (University of Maryland, Baltimore, MD; ref. 17). The transfectants have a mutant form of BCRP (R482T; ref. 25). These cells were cultured in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Inc., Grand Island, NY) and 50 mg/L kanamycin sulfate (Meiji Seika, Co., Tokyo) in a humidified incubator containing 5% CO₂ at 37°C.

Gefitinib was from AstraZeneca, topotecan from SmithKlineBeecham, Co. (Tokyo), SN-38 from Yakult Honsha, Co. (Tokyo), mitoxantrone hydrochloride from Takeda Yakuhin Kogyo, Co. (Osaka, Japan), and doxorubicin from Kyowa Hakko Kogyo, Co. (Tokyo). Vincristine, etoposide, and novobiocin sodium salt were purchased from Sigma Chemical, Co. (St. Louis, MO).

RNA Extraction and Reverse Transcription-PCR Analysis. Total RNA extraction from culture cells and reverse transcription-PCR were done as previously described (19). The first-strand cDNA was 2-fold diluted from 2.5- to 320-fold in reverse transcriptase buffer. Target sequences for the EGFR and glyceraldehyde-3-phosphate dehydrogenase genes were separately amplified in 5-fold dilutions for 28 and 26 cycles, respectively. The amplifying conditions were 94°C for 90 seconds, 63°C for 60 seconds, and 72°C for 60 seconds in a 9600 Thermocycler (Perkin-Elmer Cetus, Norwalk, CT). Preliminary runs were done to determine the maximum number of cycles that could be carried out with cDNA derived from A431 human lung adenocarcinoma cells of positive control cells for EGFR. The set of primers for the EGFR gene was as follows: forward 5'-CTCACGCAGTTGGGCACTTT-3' (accession no. AF125253, nucleotide: nt 357-376) and reverse 5'-TCATGGGCAGCTCCTTCAGT-3' (nt 638-657), for a 261-bp product. The set of primers for the glyceraldehyde-3-phosphate dehydrogenase gene was described previously (19). Ten microliters of PCR products were separated by 2% agarose gel electrophoresis and were then visualized by ethidium bromide staining.

Anticancer Drug Sensitivity Assay. The sensitivity of the cells to each anticancer drug was determined using the tetrazolium dye assay as reported previously (19). Briefly, cells (7,500 cells per well) were seeded in 96-well plates in medium containing 10% FCS. Cells were incubated for 96 hours, and then WST-1 reagent (Boehringer Mannheim GmbH, Mannheim, Germany) was added, and they were subsequently incubated for 4 hours. Absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA). The drug concentration producing 50% cell growth inhibition (IC₅₀) was calculated for each drug from linear regression analysis of the linear portion of the growth curves. Each experiment was done at least in triplicate.

Flow Cytometric Detection of Intracellular Topotecan. Cells (2 x 10⁶) were exposed to 30 µmol/L topotecan for 15 minutes at 37°C with or without 0.05 to 10 µmol/L gefitinib or 300 µmol/L novobiocin, and then washed twice in ice-cold saline, as described previously (26). Novobiocin of the relatively specific BCRP inhibitor was used as a positive control (26). Fluorescence of topotecan was analyzed with a fluorescence-activated cell sorting scan flow cytometer (Becton Dickinson, Mountain View, CA) equipped with a 15-mW argon laser. The topotecan-derived fluorescence of 30,000 events was measured through a 488 nm band-pass filter at an excitation wavelength of 585 nm. Topotecan accumulation after incubation for 15 minutes was expressed in fluorescence units. In all fluorescence assays, parallel samples were stored on ice to control for nonspecific binding of the drugs to plasma membranes, and cells without topotecan were included as a control for autofluorescence.

Topotecan Transport Into Plasma Membrane Vesicles. The transport buffer [sucrose, Hepes/Tris (pH 7.4), CaCl₂, and MgCl₂ at 250, 10, 0.2, and 10 mmol, respectively] contained various concentrations of topotecan, 5 mmol ATP and the ATP-regenerating system (10 mmol creatine phosphate and 100 µg/mL creatine kinase). The transport reaction was initiated by adding 10 µL of the vesicle suspension (50 µg protein) to 70 µL of the transport buffer, preincubated for 2 minutes at 37°C. The substrate and ATP reactions were given in addition to 1 mL of ice-cold stop solution [sucrose, NaCl, Hepes/Tris (pH 7.4), and CaCl₂ at 250, 100, 10, and 0.2 mmol, respectively]. The stopped reaction mixtures were centrifuged at 16,000 x g for 30 minutes at 4°C, and then washed thrice with 1.5 mL of stop solution. The mixtures were then dissolved in 110 L of 0.05 mol/L NaOH, sonicated for 3 minutes and centrifuged at 16,000 x g for 2 minutes at 4°C. Next, the supernatants were subjected to high-performance liquid chromatography analysis to determine the uptake of topotecan into the plasma membrane vesicles. In the control experiments, uptake reaction was done at 0°C. The rates of net ATP-dependent transport were calculated by subtracting values obtained at 0°C, as a blank, from those at 37°C. For inhibition studies, topotecan transport was measured at 30 mol/L and 14 escalating concentrations of gefitinib (0-30 mol/L). For determination of kinetic constants, transport rates were measured at four independent concentrations of topotecan (5-30 mol/L). K_m and V_max values were determined as concentrations of topotecan at half-maximal velocity of transport under these conditions. For determination of the inhibition constant (K_i) for competitive inhibition kinetic constants in the Dixon method (27), transport rates were measured at each independent concentration of topotecan.

High-performance Liquid Chromatography Analysis of Gefitinib. The intracellular accumulation of gefitinib was measured by the modified high-performance liquid chromatography method reported by Warner et al. (28). The high-performance liquid chromatography system consisted of a Jusco PU-1580 pump, a Jusco 870-UV UV/VIS detector (Jusco Inc., Tokyo) and a Shimazu C-R4A integrator (Shimazu, Kyoto, Japan). Isocratic elutions were done using a Mightysil RP-18 (L) GP column (5 µmol/L, 4.6 x 150 mm; Kanto Chemical, Co., Tokyo) with a guard column (5 µmol/L, 4.6 x 5 mm). The excitation setting was 253 nm. The mobile phase, consisting of 0.1 mol/L triethylamine (pH 8.0)/acetonitrile/tetrahydrofuran (40:60:2, v/v/v), was used for the separation of gefitinib. The flow rate was 1.0 mL/min, and all separations were carried out at room temperature.

Statistical Analysis. Data in the drug sensitivity assay were presented as mean ± SD. Differences between groups were tested by the Student's t test. All P values were two-sided, and P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
EGFR and BCRP Expression in Cell Lines. EGFR mRNA expression was not detected in PC-6 cells and PC-6/SN2-5H cells (data not shown). On the other hand, BCRP mRNA and protein were overexpressed in PC-6/SN2-5H cells but were not detectable in PC-6 cells, as reported previously (19, 20). Other transporter genes of MDR1, MRP2, and MRP3 mRNA were not detectable in PC-6 and PC-6/SN2-5H cells in reverse transcription-PCR analyses (19).

Reversal of Drug Resistance by Gefitinib. The IC₅₀ values of each drug in the PC-6 and MCF-7 cell panels are shown in Table 1. Compared with the respective parental cells, PC-6/SN2-5H, MCF-7/MX, and MCF-7/BCRP clone 8 cells were resistant to topotecan, SN-38, and mitoxantrone. MCF-7/MX and MCF-7/BCRP clone 8 cells showed weak and moderate cross-resistance to doxorubicin, respectively, as reported previously (17, 24). In the preliminary experiments, the IC₁₀ (the drug concentration producing 10% growth inhibition) of gefitinib was 11.8 ± 0.41, 10.4 ± 0.84, 2.45 ± 0.24, 2.45 ± 0.27, and 2.98 ± 0.94 µmol/L in PC-6, PC-6/SN2-5H, MCF-7, MCF-7/MX, and MCF-7/BCRP clone 8 cells, respectively. Based on these data, 10 and 2 µmol/L gefitinib, of a noncytotoxic maximum dose, were used for PC-6 and MCF-7 cell panels, respectively, in the present experiments (Table 1). In PC-6/SN2-5H cells, gefitinib completely reversed topotecan resistance from 50.8- to 1.44-fold (P < 0.0001). Furthermore, the degree of SN-38 and mitoxantrone resistance in the subline were decreased from 30.8- to 1.23-fold and from 33.4- to 1.20-fold, respectively (P < 0.0001). In contrast, no effects were observed on sensitivity to doxorubicin, vincristine, and etoposide. On the other hand, in MCF-7/MX cells, gefitinib reversed topotecan, SN-38, mitoxantrone, and doxorubicin resistance from 34.5- to 8.82-fold (P < 0.0001), from 12.7- to 1.65-fold (P < 0.0001), from 115- to 17.6-fold (P = 0.0035), and from 3.15- to 0.94-fold (P < 0.0001), respectively. In MCF-7/BCRP clone 8 cells, gefitinib reversed these resistances from 48.4- to 3.81-fold (P = 0.0005), from 9.45- to 0.85-fold (P < 0.0001), from 940- to 14.9-fold (P < 0.0001), and from 30.5- to 1.30-fold (P < 0.0001), respectively.

View larger version (19K): [in this window] [in a new window]   Figure 1. Intracellular topotecan accumulation in PC-6/SN2-5H and PC-6 cells in the presence (solid line) or absence (shadow) of gefitinib. Dotted lines, fluorescence without exposure to topotecan.

View larger version (30K): [in this window] [in a new window]   Figure 2. A, intracellular topotecan accumulation in PC-6 cells in the presence (bold line) or absence (shadow) of gefitinib. Dotted lines, fluorescence without exposure to topotecan; thin lines, topotecan fluorescence in the presence of novobiocin. B, intracellular topotecan accumulation in MCF-7 cells in the presence (bold line) or absence (shadow) of gefitinib. Dotted lines, fluorescence without exposure to topotecan; thin lines, topotecan fluorescence in the presence of novobiocin.

View larger version (12K): [in this window] [in a new window]   Figure 3. A, effects of gefitinib on topotecan transport. The amounts of ATP-dependent topotecan transport were determined in the membrane vesicles of PC-6/SN2-5H cells at 14 different gefitinib concentrations. The plasma membrane vesicles were incubated with 30 µmol/L topotecan in the presence () or absence () of 5 mmol ATP, and the topotecan concentration was determined as described in Materials and Methods. The amounts of topotecan transport were calculated by subtracting the values at 0°C, as a blank, from those obtained at 37°C. Data represent mean ± SD in at least three different experiments. B, the effects of gefitinib on the kinetics of topotecan transport in the membrane vesicles of PC-6/SN2-5H cells. The amounts of topotecan transport were analyzed in the presence of topotecan at 5 (), 10 (), 20 (), and 30 () µmol/L. Points, mean (SD) from at least three different experiments. C, the inclinations of the topotecan transport graphs are plotted in B. Each inclination was obtained in the presence of topotecan at 5 (), 10 (), 20 (), and 30 () µmol/L, and the inhibition constant (Ki) was calculated by the Dixon analysis method.

View larger version (8K): [in this window] [in a new window]   Figure 4. Uptake of 30 µmol/L gefitinib into the vesicles of PC-6/SN2-5H cells in the presence of 30 µmol/L topotecan. The plasma membrane vesicles were incubated with 30 µmol/L topotecan in the presence () or absence () of 5 mmol ATP; (x) control level of 30 µmol/L gefitinib in the absence of ATP at 0°C. Gefitinib concentration was determined as described in Materials and Methods. The amounts of gefitinib transport were calculated by subtracting the values at 0°C, as a blank, from those obtained at 37°C. Points, mean from at least three different experiments; bars, SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Gefitinib at 0.5 to 10 µmol/L dose-dependently increased the topotecan accumulation within PC-6/SN2-5H cells (Fig. 1), whereas 2 and 10 µmol/L gefitinib reversed drug resistance in three BCRP-expressing cell lines (Table 1). Because the 0.5 to 10 µmol/L gefitinib used here was in clinically achievable plasma concentrations (29), the reversal effects as shown in the present study would be observed in vivo. Ciardiello et al. (13) reported that gefitinib enhanced the cytotoxicity of paclitaxel and docetaxel in multidrug-resistant breast cancer cells overexpressing P-glycoprotein and EGFR in vitro. In a tumor-bearing mouse model, gefitinib has shown a synergistic effect in combination with various cytotoxic agents, resulting in prolonged survival, and the phenomenon was not associated with the EGFR expression status in the cancer cells (9–14). However, the enhancement mechanisms have not been explored, and interactions between gefitinib and the expression or function of ATP-binding cassette transporters were not analyzed in these prior reports. On the other hand, as shown in our study, the enhancement effect of gefitinib with topoisomerase I inhibitors is probably due to the direct inhibition of BCRP-mediated drug efflux.

To date, several BCRP inhibitors with different structures have been reported, but the exact inhibition mechanisms have not been sufficiently investigated and explored (18). Very recently, we also showed that novobiocin of a coumermycin antibiotic reversed BCRP-mediated resistance using membrane vesicles (26). Fumitremorgin C of a specific BCRP inhibitor, a mycotoxin from Aspergillus fumigatus, and its structural analogues, are likely to reverse BCRP-mediated resistance through inhibition of BCRP-ATPase activity (30–32). The acridone carboxamide derivative GF120918 of an antagonistic P-glycoprotein inhibitor also inhibits BCRP-mediated drug efflux and resistance (33, 34). Thus, probable direct interactions between BCRP and gefitinib, as shown here, are of great interest, and experiments are extensively ongoing for the exploration of the reversal mechanisms of gefitinib.

When combined with gefitinib and BCRP substrate drugs in a clinical setting, gefitinib may affect the plasma concentrations of these drugs, leading to adverse effects because gefitinib may inhibit BCRP-mediated drug transport in normal cells. In fact, coadministration of the BCRP inhibitor GF120918 has significantly increased the plasma levels of topotecan in mice and humans (34, 35), especially when topotecan was given p.o. Thus, BCRP is probably involved in the regulation of drug uptake and excretion from the gastrointestinal tract because BCRP is expressed in the epithelium of the small intestine and colon (36). In addition, the effects of gefitinib on other ATP-binding cassette transporters would be of great interest. In our preliminary experiments, no modulating effects on MRP1/ABCC1 were observed, but there were moderate effects on P-glycoprotein (detailed data not shown).

In conclusion, the EGFR tyrosine kinase inhibitor gefitinib effectively reversed drug resistance through inhibition of drug efflux in three multidrug-resistant cancer cell lines overexpressing BCRP. In addition, gefitinib inhibited BCRP-mediated topotecan transport in the plasma membrane vesicles of these cells by mechanisms other than competitive inhibition. The cellular pharmacologic effect of combining gefitinib with topoisomerase I inhibitors in BCRP-overexpressing cells requires further clarification.

	Acknowledgments

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/ 5/03. Revised 6/ 7/04. Accepted 12/ 1/04.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Goustin AS, Leof EB, Shipley GD, Moses HL. Growth factors and cancer. Cancer Res 1986;46:1015–29.[Abstract/Free Full Text]
2. Aaronson SA. Growth factors and cancer. Science 1991;254:1146–53.[Abstract/Free Full Text]
3. Di Fiore PP, Pierce JH, Kraus MH, Segatto O, King CR, Aaronson SA. erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science 1987;237:178–82.[Abstract/Free Full Text]
4. Sibilia M, Wagner EF. Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 1995;269:234–8.[Abstract/Free Full Text]
5. Threadgill DW, Dlugosz AA, Hansen LA, et al. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 1995;269:230–4.[Abstract/Free Full Text]
6. Miettinen PJ, Berger JE, Meneses J, et al. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 1995;376:337–41.[CrossRef][Medline]
7. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 2002;7:2958–70.
8. Baselga J, Averbuch SD. ZD1839 (‘Iressa’) as an anticancer agent. Drugs 2000;60:33–40.
9. Sirotnak FM, Zakowsky MF, Miller VA, Scher HI, Kris MG. Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 2000;6:4885–92.[Abstract/Free Full Text]
10. Ohmori T, Ao Y, Nishio K, Saijo N, Arteaga CL, Kuroki T. Low dose cisplatin can modulate the sensitivity of human non-small cell lung carcinoma cells to EGFR tyrosine kinase inhibitor (ZD1839; ‘Iressa’) in vivo. Proc Am Assoc Cancer Res 2000;41:482.
11. Ciardiello F, Caputo R, Bianco AR, et al. Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin Cancer Res 2000;6:2053–63.[Abstract/Free Full Text]
12. Ciardiello F, Caputo R, Bianco AR, et al. Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clin Cancer Res 2001;7:1459–65.[Abstract/Free Full Text]
13. Ciardiello F, Caputo R, Borriello G, et al. ZD1839 (Iressa), an EGFR-selective tyrosine kinase inhibitor enhances activity of taxanes in bcl-2 overexpressing, multidrug resistant MCF-7 ADR human breast cancer cells. Int J Cancer 2002;98:463–9.[CrossRef][Medline]
14. Naruse I, Ohmori T, Ao Y, et al. Antitumor activity of the selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) Iressa (ZD1839) in an EGFR-expressing multidrug-resistant cell line in vitro and in vivo. Int J Cancer 2002;98:3010–5.
15. Mendelsohn J, Fan Z. Epidermal growth factor receptor family and chemosensitization. J Natl Cancer Inst 1997;89:341–3.[Free Full Text]
16. Borst P, Oude Elferink R. Mammalian ABC transporters in health and disease. Annu Rev Biochem 2002;71:537–92.[CrossRef][Medline]
17. Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 1998;95:15665–70.[Abstract/Free Full Text]
18. Allen JD, Schinkel AH. Multidrug resistance and pharmacological protection mediated by the breast cancer resistance protein (BCRP/ABCG2). Mol Cancer Ther 2002;1:427–34.[Free Full Text]
19. Kawabata S, Oka M, Shiozawa K, et al. Breast cancer resistance protein directly confers SN-38 resistance of lung cancer cells. Biochem Biophys Res Commun 2001;280:1216–23.[CrossRef][Medline]
20. Nakatomi K, Yoshikawa M, Oka M, et al. Transport of 7-ethyl-10-hydroxycamptothecin (SN-38) by breast cancer resistance protein ABCG2 in human lung cancer cells. Biochem Biophys Res Commun 2001;288:827–32.[CrossRef][Medline]
21. Erlichman C, Boerner SA, Hallgren CG, et al. The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux. Cancer Res 2001;61:739–48.[Abstract/Free Full Text]
22. Joto N, Ishii M, Minami M, Kuga H, Mitsui I, Tohgo A. DX-8951f, a water-soluble camptothecin analog, exhibits potent antitumor activity against a human lung cancer cell line and its SN-38-resistant variant. Int J Cancer 1997;72:680–6.[CrossRef][Medline]
23. Nakatomi K, Oka M, Shiozawa K, et al. Mutation and quantitative analysis of breast cancer resistance protein (BCRP/MXR/ABCG2) gene in lung cancer cells. Proc Am Assoc Cancer Res 2002;43:778.
24. Nakagawa M, Schneider E, Dixon KH, et al. Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells. Cancer Res 1992;52:6175–81.[Abstract/Free Full Text]
25. Honjo Y, Hrycyna CA, Yan QW, et al. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells. Cancer Res 2001;61:6635–9.[Abstract/Free Full Text]
26. Shiozawa K, Oka M, Soda H, et al. Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin, a coumermycin antibiotic. Int J Cancer 2004;108:146–51.[CrossRef][Medline]
27. Dixon M. The determination of enzyme inhibitor contents. Biochem J 1953;55:170–1.[Medline]
28. Warner DL, Burke TG. Simple and versatile high-performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of camptothecin anticancer drugs. J Chromatogr B Biomed Appl 1997;691:161–71.
29. Ranson M, Hammond LA, Ferry D, et al. ZD1839, a selective oral epidermal growth factor receptor-tyrosine kinase inhibitor, is well tolerated and active in patients with solid, malignant tumors: results of a phase I trial. J Clin Oncol 2002;20:2240–50.[Abstract/Free Full Text]
30. Robey RW, Honjo Y, van de Laar A, et al. A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2). Biochim Biophys Acta 2001;1512:171–82.[Medline]
31. Özvegy C, Litman T, Szakacs G, et al. Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. Biochem Biophys Res Commun 2001;285:111–7.[CrossRef][Medline]
32. Rabindran SK, Ross DD, Doyle LA, Yang W, Greenberger LM. Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein. Cancer Res 2000;60:47–50.[Abstract/Free Full Text]
33. de Bruin M, Miyake K, Litman T, Robey R, Bates SE. Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR. Cancer Lett 1999;146:117–26.[CrossRef][Medline]
34. Jonker JW, Smit JW, Brinkhuis RF, et al. Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan. J Natl Cancer Inst 2002;92:1651–6.
35. Kruijtzer CMF, Beijnen JH, Rosing H, et al. Increased oral bioavailability of topotecan in combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin Oncol 2002;20:2943–50.[Abstract/Free Full Text]
36. Maliepaard M, Scheffer GL, Faneyte IF, et al. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 2001;61:3458–64.[Abstract/Free Full Text]

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				Z. Shi, X.-X. Peng, I.-W. Kim, S. Shukla, Q.-S. Si, R. W. Robey, S. E. Bates, T. Shen, C. R. Ashby Jr., L.-W. Fu, et al. Erlotinib (Tarceva, OSI-774) Antagonizes ATP-Binding Cassette Subfamily B Member 1 and ATP-Binding Cassette Subfamily G Member 2 Mediated Drug Resistance Cancer Res., November 15, 2007; 67(22): 11012 - 11020. [Abstract] [Full Text] [PDF]

				C. Kersting, C. Gebert, K. Agelopoulos, H. Schmidt, P. J. van Diest, H. Juergens, W. Winkelmann, M. Kevric, G. Gosheger, B. Brandt, et al. Epidermal Growth Factor Receptor Expression in High-Grade Osteosarcomas Is Associated with a Good Clinical Outcome Clin. Cancer Res., May 15, 2007; 13(10): 2998 - 3005. [Abstract] [Full Text] [PDF]

				W. Liu, M. R. Baer, M. J. Bowman, P. Pera, X. Zheng, J. Morgan, R. A. Pandey, and A. R. Oseroff The Tyrosine Kinase Inhibitor Imatinib Mesylate Enhances the Efficacy of Photodynamic Therapy by Inhibiting ABCG2 Clin. Cancer Res., April 15, 2007; 13(8): 2463 - 2470. [Abstract] [Full Text] [PDF]

				I Chau, D Cunningham, T Hickish, A Massey, L Higgins, R Osborne, N Botwood, and A Swaisland Gefitinib and irinotecan in patients with fluoropyrimidine-refractory, irinotecan-naive advanced colorectal cancer: a phase I-II study Ann. Onc., April 1, 2007; 18(4): 730 - 737. [Abstract] [Full Text] [PDF]

				G. Cusatis, V. Gregorc, J. Li, A. Spreafico, R. G. Ingersoll, J. Verweij, V. Ludovini, E. Villa, M. Hidalgo, A. Sparreboom, et al. Pharmacogenetics of ABCG2 and Adverse Reactions to Gefitinib J Natl Cancer Inst, December 6, 2006; 98(23): 1739 - 1742. [Abstract] [Full Text] [PDF]

				Y. Zhuang, C. H. Fraga, K. E. Hubbard, N. Hagedorn, J. C. Panetta, C. M. Waters, and C. F. Stewart Topotecan Central Nervous System Penetration Is Altered by a Tyrosine Kinase Inhibitor Cancer Res., December 1, 2006; 66(23): 11305 - 11313. [Abstract] [Full Text] [PDF]

				E. E.W. Cohen Role of Epidermal Growth Factor Receptor Pathway-Targeted Therapy in Patients With Recurrent and/or Metastatic Squamous Cell Carcinoma of the Head and Neck J. Clin. Oncol., June 10, 2006; 24(17): 2659 - 2665. [Abstract] [Full Text] [PDF]

				H. E. M. zu Schwabedissen, M. Grube, A. Dreisbach, G. Jedlitschky, K. Meissner, K. Linnemann, C. Fusch, C. A. Ritter, U. Volker, and H. K. Kroemer EPIDERMAL GROWTH FACTOR-MEDIATED ACTIVATION OF THE MAP KINASE CASCADE RESULTS IN ALTERED EXPRESSION AND FUNCTION OF ABCG2 (BCRP) Drug Metab. Dispos., April 1, 2006; 34(4): 524 - 533. [Abstract] [Full Text] [PDF]

				C. Xue, J. Wyckoff, F. Liang, M. Sidani, S. Violini, K.-L. Tsai, Z.-Y. Zhang, E. Sahai, J. Condeelis, and J. E. Segall Epidermal Growth Factor Receptor Overexpression Results in Increased Tumor Cell Motility In vivo Coordinately with Enhanced Intravasation and Metastasis Cancer Res., January 1, 2006; 66(1): 192 - 197. [Abstract] [Full Text] [PDF]

				G. Gasparini, R. Sarmiento, S. Amici, R. Longo, D. Gattuso, M. Zancan, and M. Gion Gefitinib (ZD1839) combined with weekly epirubicin in patients with metastatic breast cancer: a phase I study with biological correlate Ann. Onc., December 1, 2005; 16(12): 1867 - 1873. [Abstract] [Full Text] [PDF]

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