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Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10)-mediated multidrug resistance

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Lapatinib and erlotinib are potent reversal agents for MRP7 (ABCC10)-mediated multidrug resistance
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  Lapatinib and erlotinib are potent reversal agents for MRP7(ABCC10)-mediated multidrug resistance Ye-Hong Kuang a,b,‡, Tong Shen a,‡, Xiang Chen b,*, Kamlesh Sodani a, Elizabeth Hopper-Borge c,  Amit Kumar Tiwari a, Jeferson W K K Lee a,d, Li-Wu Fu e, and Zhe-Sheng Chen a,* a  Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St.John’s University, Queens, NY 11439, USA b  Department of Dermatology, Xiang Ya Hospital, Central South University, Changsha, 410008,China c  Molecular Medicine Program, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia,Pennsylvania, USA d  Department of Pharmacy and Pharmacology, University of Bath, Bath, BA27AY, UK e  State Key Laboratory for Oncology In South China, Cancer Center, Sun Yat-Sen University,Guangzhou 510060, China  Abstract In recent years, a number of TKIs (tyrosine kinase inhibitors) targeting epidermal growth factor receptor (EGFR) family have been synthesized and some have been approved for clinical treatmentof cancer by the FDA. We recently reported a new pharmacological action of the 4-anilinoquinazolinederived EGFR TKIs, such as lapatinib (Tykerb®) and erlotinib (Tarceva®), which significantly affectthe drug resistance patterns in cells expressing the multidrug resistance (MDR) phenotype.Previously, we showed that lapatinib and erlotinib could inhibit the drug efflux function of P-glycoprotein (P-gp, ABCB1) and ABCG2 transporters. In this study, we determined if these TKIshave the potential to reverse MDR due to the presence of the multidrug resistance protein 7 (MRP7,ABCC10). Our results showed that lapatinib and erlotinib dose-dependently enhanced the sensitivityof MRP7-transfected HEK293 cells to several established MRP7 substrates, specifically docetaxel, paclitaxel, vinblastine and vinorelbine, whereas there was no or a lesser effect on the control vector transfected HEK293 cells. [ 3 H]-paclitaxel accumulation and efflux studies demonstrated thatlapatinib and erlotinib increased the intracellular accumulation of [ 3 H]-paclitaxel and inhibited theefflux of [ 3 H]-paclitaxel from MRP7 transfected cells but not in the control cell line. Lapatinib is amore potent inhibitor of MRP7 than erlotinib. In addition, the Western blot analysis revealed that both lapatinib and erlotinib did not significantly affect MRP7 expression. We conclude that the EGFR TKIs, lapatinib and erlotinib reverse MRP7-mediated MDR through inhibition of the drug effluxfunction, suggesting that an EGFR TKI based combinational therapy may be applicable for chemotherapeutic practice clinically. * Corresponding author Requests for reprints: 1. Zhe-Sheng Chen: Department of Pharmaceutical Sciences, St. John’s University, Jamaica, New York, 11439, USA. Chenz@stjohns.edu, Phone: 1-718-990-1432, Fax: 1-718-990-1877; 2. Xiang Chen: Department of Dermatology, Xiang Ya Hospital, Central South University, 87 Xiang Ya Road, Changsha, 410008, China. chenxck@yahoo.com, Phone:+86 731 432 7377, Fax: +86 731 432 8478.‡Contributed equally to this work   NIH Public Access Author Manuscript  Biochem Pharmacol . Author manuscript; available in PMC 2010 October 12. Published in final edited form as:  Biochem Pharmacol . 2010 January 15; 79(2): 154–161. doi:10.1016/j.bcp.2009.08.021. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    Keywords tyrosine kinase inhibitor; EGFR; lapatinib; erlotinib; ABC transporters; MRP7/ABCC10; multidrugresistance 1. Introduction The signaling pathways of tyrosine kinases (TKs) are involved in cancer cell proliferation,apoptosis, angiogenesis and metastasis [1,2]. Tyrosine kinase inhibitors (TKIs) are generallyreversible competitors against ATP for binding to the intracellular catalytic domain of the TKs.Consequently, they inhibit autophosphorylation as well as downstream signaling processes,thereby representing a promising class of anticancer agents in the clinic [3,4]. The FDA hasapproved TKIs including imatinib, gefitinib, erlotinib and lapatinib for the treatment of variouscancers in recent years [5,6]. Two predominant classes of TKIs have been developed and used in the clinic or clinical trials. These include BCR-ABL TKIs such as imatinib, nilotinib,dasatinib and bosutinib, and epidermal growth factor receptor (EGFR, HER1) TKIs such asgefitinib, erlotinib, lapatinib, caneritinib and AG1478. Recent studies have identified TKIs asmodulators of ATP-binding cassette (ABC) tr ansporter-mediated multidrug resistance (MDR)in cancer cells [7–9].MDR is the development of  resistance to a variety of anticancer drugs that are structurally and  mechanistically unrelated, presenting a major obstacle to successful chemotherapy treatment[10]. Recently, a significant effort to elucidate the mechanism of MDR has been focused onthe ABC transporters, and their abilities to extrude drugs from the cells [11–16]. Thesetransporter proteins srcinate from one of the largest protein families which is divided intoseven subfamilies (A–G) based on sequence similarities, including ABCB1, also called P-glycoprotein (P-gp) [11], and multidrug resistance proteins (MRPs, ABCCs) [14] and ABCG2[15,16]. These ABC transporters are highly varied transpor ters which function to extrude awide range of str ucturally and mechanistically different drugs from the cells. For example,drugs transported by P-gp include vinca alkaloids, anthracyclines, epipodophyllotoxins and taxanes [17]; drugs transported by MRP1 such as vinca alkaloids, anthracyclines,epipodo phyllotoxins and some heavy metal anions [18]; drugs transported by ABCG2 include anthracyclines, mitoxantrone, antifolates, and flavopiridol [19]. Mechanistically, these ABCtransporters are coupled to an ATP hydrolysis process, thereby utilizing energy to transportdrugs outside of cells. Inhibition of ABC transporter-mediated drug efflux may re-sensitizeMDR cancer cells to an effective MDR tumor treatment with chemotherapeutic agents. Curr ently, three generations of P-gp inhibitors and a number of MRP1 and ABCG2 inhibitorshave been developed to enhance the effect of chemotherapeutic drugs on MDR cancer cells in vitro  and in vivo [20–23]. Recently, we and others have reported that several TKIs are dualmodulators of P-gp and ABCG2. For example, resistance to imatinib was related to P-gpoverexpression and imatinib could reverse P-gp-mediated drug resistance. In addition, imatinibreverses ABCG2-mediated MDR [24,25]. Our recent data suggests that AG1478, an EGFR TKI, interacts with the substrate binding sites of P-gp and ABCG2 [26], we have also shownfor the first time that lapatinib and erlotinib enhance the cytotoxic effects of multiple anti-cancer drugs by increasing the accumulation of P-gp and ABCG2 substrates due to their directinteraction at the substrate  binding site[27,28]. Based on the amino acid sequence similarity,multidrug resistance protein 7 (MRP7/ABCC10) was recently characterized [29]. On the basisof amino acid sequence comparisons, the topology of MRP7 is similar to those of  MRP1, 2, 3 and 6, with two nucleotide-binding domains and three membrane-spanning domains [29].Phylogenetic analysis indicated that MRP7 is related to lipophilic anion pumps and is alsoinvolved in the regulation of ion channels. Previous in vitro  studies on MRP7 transfected celllines suggested that 17- β -estradiol-(17-beta-D-glucuronide), some taxanes and vinca alkaloids Kuang et al.Page 2  Biochem Pharmacol . Author manuscript; available in PMC 2010 October 12. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    are substrates of MRP7 [30,31]. Bessho Y et al recently reported that MRP7 confers resistanceto vinorelbine in non-small cell lung cancer (NSCLC) cells [32]. The discovery of potent and specific inhibitors of MRP7 is of great interest, and may represent a strategy to overcomeclinical drug resistance. It was hypothesized that since MRP7 shares some common substratesand functions with other members in the ABC family, modulators that overcome P-gp or ABCG2-linked MDR may also alleviate MRP7-mediated drug resistance. Indeed, we found that a P-gp inhibitor cepharanthine could also reverse MRP7-mediated resistance to paclitaxel[33]. In the present study, by using our previously established MRP7 transfected HEK293 cells,we conducted experiments to determine whether TKIs such as lapatinib and erlotinib could reverse MRP7-mediated MDR to elucidate their reversal mechanisms. 2. Material and Methods 2.1 Materials Lapatinib and erlotinib were purchased from ChemieTeck Inc. (Indianapolis, IN). [ 3 H]- paclitaxel (3.0 Ci/mmol) was purchased from Moravek Biochemicals. (Brea, CA). Themonoclonal mouse antibody against P-gp (P7965), the polyclonal goat antibody against MRP7(C-19), the secondary horseradish peroxidase-labeled anti-goat or anti-mouse IgG, docetaxel, paclitaxel, vinblastine, vinorelbine and cisplatin were purchased from Sigma-AldrichChemical Co. (St. Louis, MO). A polyclonal antibody against human ABCC1 (MRP1) [34]was kindly provided by Dr. Shin-ichi Akiyama (Kagoshima Univ., Japan). A monoclonalantibody BXP-34 (against ABCG2) was acquired from Signet Laboratories Inc (Dedham, MA).Cepharanthine was generously provided by Kakenshoyaku Co. (Tokyo, Japan). 2.2 Cell lines We used MRP7 expression vector, parental plasmid and MRP7 transfected cell lines previouslydescribed by Chen et al. [30]. The parental drug-sensitive human epidermoid carcinoma cellline KB-3-1 and its corresponding resistant KB-C2 cell line were kindly provided by Drs.Michael M. Gottesman (NCI, NIH, Bethesda) and Shin-ichi Akiyama (Kagoshima Univ.,Japan), respectively. The P-gp-overexpressing KB-C2 cells were established from KB-3-1cells by exposing them to increasing concentrations of colchicine up to 2 μ g/ml, in a gradualmanner [35]. All the cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% bovine serum, 100 units/ml penicillin, and 100 μ g/ml streptomycin ina humidified incubator containing 5% CO 2  at 37°C. 2.3 Cell cytotoxicity by MTT assay Drug sensitivity was analyzed using an MTT colorimetric assay [27]. HEK293-pcDNA3.1 and HEK293-MRP7-2 cells were seeded into 96-well plate in triplicate at 5,000 cells/well. After incubation in DMEM supplemented with 10% bovine serum at 37°C for 24 h, three differentconcentrations of lapatinib and erlotinib (0.625, 1.25, 2.5 μ M) were added 1 h prior to theaddition of the anticancer drugs. After 72 h of incubation, 20 μ l of MTT solution (4 mg/ml)was added to each well. The plate was further incubated for 4 h, the medium discarded, and 100 μ l of dimethylsulfoxide (DMSO) was added into each well to dissolve the formazancrystals. The absorbance was determined at 570 nm by an OPSYS microplate Reader fromDYNEX Technologies, Inc. (Chantilly, VA). The concentrations required to inhibit growth by50% (IC 50 ) were calculated from survival curves. The degree of resistance was calculated bydividing the IC 50  of the MDR cells by that of the parental sensitive cells. 2.4 [ 3 H]-paclitaxel accumulation and efflux The parental HEK293-pcDNA3.1 and HEK-MRP7-2 transfected cells were seeded in two T75flasks and incubated with DMEM supplemented with 10% bovine serum at 37°C. After the Kuang et al.Page 3  Biochem Pharmacol . Author manuscript; available in PMC 2010 October 12. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    cells reached 90% confluency, the cells were trypsinized and two aliquots (48 × 10 6  cells) fromeach cell line were suspended in the medium, pre-incubated with or without lapatinib/erlotinib(2.5 μ M) at 37°C for 1 h. Subsequently, cells were suspended in the medium containing 0.1 μ M [ 3 H]-paclitaxel with or without lapatinib/erlotinib at 37°C for 1 h. The cells were washed with PBS for three times, and then suspended in fresh medium with or without lapatinib/erlotinib at 37°C. Aliquots (1 × 10 6  cells) were collected at various time points (0, 30, 60, 120min), followed by placed in scintillation fluid to measure the radioactivity by a Packard TRI-CARB 1900CA liquid scintillation counter (Packard Instrument Inc., Downers Grove, IL). 2.5 Preparation of cell lysates Cells in T-25 flask treated with lapatinib or erlotinib for different time periods (0, 36, 72 h),then were harvested and rinsed twice with cold PBS. The cell extracts were prepared byincubating the cells with the Radioimmunoprecipitation assay (RIPA) buffer [1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μ M p-aminophenylmethylsulfonylfluoride, 10 μ M leupeptin, and 10 μ M aprotinin] for 30 min on ice with occasional rocking,followed by centrifugation at 12,000 rpm at 4°C for 15 min. The supernatant containing totalcell lysates were collected and stored at − 80°C until future experiments. The proteinconcentration was determined by bicinchoninic Acid (BCA ™ )-based protein assay (ThermoScientific, Rockford, IL). 2.6 Immunoblotting Equal amounts of total cell lysates (40 μ g) were resolved by 4–12% sodium dodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred ontoPVDF membrane, then immersed in blocking solution (5% skim milk in TBST) to block nonspecific binding for 1 h at room temperature. The membrane was then immunoblotted overnight with primary antibodies (polyclonal anti-MRP7, monoclonal anti-P-gp or monoclonal anti-ABCG2 at 1:500 dilution, or polyclonal anti-MRP1 at 1: 3,000 dilution) at 4°C. The following day, the membrane was washed with TBST buffer (0.3% Tris, 0.8% NaCl,0.02% KCl, 0.05% Tween 20) for three times and followed by 3 h incubation with horseradish peroxide (HRP)-conjugated secondary anti-goat IgG for MRP7, anti-mouse IgG for P-gp and ABCG2 (1:1,000) or anti-rabbit IgG for MRP1 (1:1,000) for 3 h, respectively. Proteins weredetected by enhanced chemoluminescence detection system (Amersham, NJ). β -Actin wasused to confirm equal loading in each lane in the samples prepared from cell lysates. We alsoused 10 and 20 μ g protein to detect MRP7 in the treatment of lapatinib/erlotinib experimentsin order to avoid that overloading of the protein may mask the differences in the expression. 2.7 Statistical analysis Unless otherwise indicated, all experiments were repeated at least three times and thedifferences were determined by the two-tailed Student’s t-test. When statistical differences between more than 2 groups were analyzed, one-way ANOVA followed by Tukey’s multiplecomparison test was performed, as indicated. Results are presented as means ± standard deviations (SD). The statistical significance was determined to be P  < 0.05. 3. Results 3.1 Expression of ABC transporters in MRP7-transfected HEK293 cells Since MRP7 and P-gp share some common substrates and some EGFR TKIs were proved to be effective in reversing P-gp-mediated drug resistance, we decided to determine if MRP7 and P-gp were present in HEK293-pcDNA3.1 and HEK-MRP7-2 cells using immunoblottinganalysis. We found that the MRP7 protein, with a molecular weight of 171 kD, was expressed in HEK-MRP7-2 cells but not in HEK293-pcDNA3.1 cells (Fig. 1A). P-gp, with a molecular  Kuang et al.Page 4  Biochem Pharmacol . Author manuscript; available in PMC 2010 October 12. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    weight of 170 kD, was highly expressed in the positive control KB-C2 cell line, but wasundetectable in the negative control KB-3-1 cells as well as in both HEK293-pcDNA3.1 and HEK-MRP7-2 cell lines (Fig. 1B). To determine whether other ABC transporters are expressed and/or contribute to MDR in the cell lines used for this study, we also did Western blot analysesfor MRP1 and ABCG2 expression in HEK293-pcDNA3.1 and HEK-MRP7-2 cells. Weconfirmed that in the MRP7 transfected HEK293 cells used in this study, only MRP7 proteinis overexpressed and the levels of other ABC transporters such as MRP1 and ABCG2 are notup-regulated compared with the empty vector transfected HEK293/pcDNA3.1 cells (data notshown). 3.2 The effect of lapatinib and erlotinib on drug sensitivity of MRP7-transfected HEK293 cells Consistent with our previous findings [33], the colorimetric sensitivity assay revealed thatHEK-MRP7-2 cells, compared to HEK293-pcDNA3.1 cells, exhibited a significant resistanceto various MRP7 substrates such as docetaxel (12-fold), paclitaxel (8.6-fold), vinblastine (5.4-fold), and vinorelbine (3.6-fold), but showed no significant sensitivity difference to cisplatin(0.9-fold), (Table 1, Fig. 2).We tested lapatinib and erlotinib in the combination with the above mentioned MRP7 substratesto determine if they would significantly reverse MRP7-mediated MDR. To avoid toxicity, thehighest concentration of lapatinib and erlotinib used in the reversal experiments was 2.5 μ M,a concentration that caused <10% growth inhibition in all the cell lines (data not shown).Lapatinib and erlotinib at 0.625, 1.25 and 2.5 μ M, dose-dependently decreased the IC 50  valuesof docetaxel, paclitaxel, vinblastine and vinorelbine of HEK-MRP7-2 cells (Table 1). Inaddition, lapatinib, at 2.5 μ M, significantly sensitized the parental HEK293-pcDNA3.1 cells;however, this effect was significantly lower than measured in HEK-MRP7-2 cells. In contrast,lapatinib and erlotinib did not significantly reverse the resistance of cells to cisplatin, a non-MRP7 substrate (Table 1, Fig. 2). Previously, we showed that cepharanthine could reverseMRP7-mediated resistance to paclitaxel in a competitive manner, hence to compare lapatiniband erlotinib we used cepharanthine as a positive control in the present experiment (Table 1).The effect of cepharanthine was comparable to the effect of lapatinib and erlotinib (Table 1). 3.3 The effects of lapatinib and erlotinib on the intracellular accumulation of [ 3 H]-paclitaxel The effects of lapatinib and erlotinib on the accumulation of [ 3 H]-paclitaxel in HEK293- pcDNA3.1 and HEK-MRP7-2 cells were examined. Our data showed that the intracellular concentration of [ 3 H]-paclitaxel in HEK-MRP7-2 cells was significantly lower (41.91%) thanthat in HEK293-pcDNA3.1 cells (Fig. 3, P  < 0.05). After the cells were incubated with either lapatinib or erlotinib at 2.5 μ M for 1 h, intracellular [ 3 H]-paclitaxel accumulation wassignificantly enhanced in HEK-MRP7-2 cells by 2.82-fold and 1.96-fold, respectively(P<0.05). However, in the control HEK293-pcDNA3.1 cells, neither lapatinib nor erlotinibsignificantly altered the intracellular [ 3 H]-paclitaxel accumulation. In consistent with our  previous findings, cepharanthine produced a 2.80-fold increase in the intracellular accumulation of [ 3 H]-paclitaxel in HEK-MRP7-2 cells, which was consistent with our previousfindings [33]. 3.4 The effects of lapatinib and erlotinib on the efflux of [ 3 H]-paclitaxel To ascertain whether the increase in the intracellular [ 3 H]-paclitaxel accumulation caused bylapatinib and erlotinib was due to an inhibition of [ 3 H]-paclitaxel efflux, we conducted a timecourse study to determine [ 3 H]-paclitaxel efflux in the presence of lapatinib or erlotinib. Our results indicated that HEK-MRP7-2 cells extruded a significantly higher percentage of intracellular accumulated [ 3 H]-paclitaxel than HEK293-pcDNA3.1 cells (Fig. 4, P  < 0.05).When we incubated cells with lapatinib or erlotinib at 2.5 μ M, they significantly blocked theintracellular [ 3 H]-paclitaxel efflux at different time periods (0, 30, 60, 120 min) from HEK- Kuang et al.Page 5  Biochem Pharmacol . Author manuscript; available in PMC 2010 October 12. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  

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