The dopamine D3 receptor antagonists PG01037 ...

Cancer Letters 396 (2017) 167e180

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Original Article

The dopamine D3 receptor antagonists PG01037, NGB2904, SB277011A, and U99194 reverse ABCG2 transporter-mediated drug resistance in cancer cell lines Noor Hussein a, 1, Haneen Amawi a, 1, Chandrabose Karthikeyan b, F. Scott Hall a, Roopali Mittal c, Piyush Trivedi b, Charles R. Ashby Jr. d, **, Amit K. Tiwari a, * a

Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, OH 43614, USA School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, MP 462036, India Pediatric Gastroenterology, OU Medical Center, Children's Ave, Oklahoma City, OK 73104, USA d Pharmaceutical Sciences, College of Pharmacy, St. John's University, Queens, NY 11432, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 February 2017 Received in revised form 6 March 2017 Accepted 8 March 2017

The ATP e binding cassette (ABC) family G2 (ABCG2) transporters are known to produce multidrug resistance (MDR) in cancer, thereby limiting the clinical response to chemotherapy. Molecular modeling data indicated that certain dopamine (DA) D3 receptor antagonists had a significant binding affinity for ABCG2 transporter. Therefore, in this in vitro study, we determined the effect of the D3 receptor antagonists PG01037, NGB2904, SB277011A, and U99194 on MDR resulting from the overexpression of ABCG2 transporters. The D3 receptor antagonists, at concentrations >100 mM, did not significantly affect the viability of H460-MX20, S1M1-80, A549-MX10 or wild type ABCG2 overexpressing (HEK293-R2) cells. However, at concentrations ranging from 0.01 to 10 mM, the D3 receptor antagonists PG01037, NGB2904, SB-277011A, and U99194 significantly increased the efficacy of the anticancer drugs mitoxantrone and doxorubicin in ABCG2-overexpressing MDR cells. Efflux studies indicated that both PG01037 and NGB2904, at a concentration of 5 mM, significantly decreased the efflux of rhodamine 123 from H460MX20 cells. Interestingly, 5 mM of PG01037 or NGB2904 significantly decreased the expression levels of the ABCG2 protein, suggesting that these compounds inhibit both the function and expression of ABCG2 transporters at non-toxic concentrations. © 2017 Elsevier B.V. All rights reserved.

Keywords: Multidrug resistance D3 receptor antagonists ABCG2-transporters PG01037 NGB2904 SB277011A

Introduction Multidrug resistance (MDR) is defined as a significant decrease in the efficacy of structurally and mechanistically unrelated compounds in cancer cells and microorganisms [1e3]. Notably, the development of drug resistance poses a continual clinical problem in cancer chemotherapy [4]. It is well established that MDR can be mediated by the overexpression of specific ATP-binding cassette (ABC) proteins in the cell membrane of cancer cells [5]. There are 49 * Corresponding author. Department of Pharmacology and Experimental Therapeutics, College of Pharmacy & Pharmaceutical Sciences, University of Toledo, OH 43614, USA. Fax: þ1 419 383 1909. ** Corresponding author. Department of Pharmaceutical Sciences, St. John's University, Jamaica, Queens, NY 11439, USA. E-mail addresses: [email protected] (C.R. Ashby), amit.tiwari@utoledo. edu (A.K. Tiwari). 1 The first two authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2017.03.015 0304-3835/© 2017 Elsevier B.V. All rights reserved.

known human ABC transporter genes [3] and the ABC transporter superfamily encompasses 7 subfamilies from ABC- A to G based on amino-acid sequence [6]. The structure of ABC transporters contains two highly conserved nucleotide-binding domains (NBDs) [3]. Furthermore, ABC transporter proteins also share the same characteristic motifs, known as Walker A and B motifs, that are separated by ~90e120 amino acids [7]. Additionally, there is a unique linker called the C region, which is located upstream of the Walker B site, distinguishing them from other ATP-binding proteins [7e9]. ABC transporters are expressed in a variety of organs and tissues throughout the human body, including the liver, intestine, kidney, lung, gonadal organs, and endothelial cells of the bloodebrain barrier [10]. Their primary function is to promote the excretion or efflux a wide variety of endogenous compounds and xenobiotics in order to minimize their toxicity [10]. ABC transporters act as active efflux pumps, utilizing the energy derived from the hydrolysis of ATP to extrude substrates against a concentration gradient [11].

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The expression of key ABC transporters, particularly the multidrug resistance 1 transporter (MDR1; p-glycoprotein or ABCB1), various members of the multi-drug resistance protein (MRP or ABCC) family, and breast cancer resistance protein (BCRP; mitoxantrone resistance protein (MXR) or ABCG2) play a major role in mediating MDR in cancer by extruding a variety of chemotherapeutic drugs, reducing intracellular drug concentrations and contributing to the failure of chemotherapy [1,11]. The G2 subfamily of the ATP-binding cassette transporters, ABCG2, also known as the breast cancer resistance protein (BRCP), is a 72 kDa half transporter that is encoded by the ABCG2 gene localized on chromosome 4q22 [12]. In contrast to the ABCB1 transporter, ABCG2 needs to form a homodimer to become functionally active [13]. ABCG2 transporters have broad and sometimes overlapping substrate specificity with ABCB1 and MRP1 transporters [14,15]. The xenobiotic substrate profile for ABCG2 is quite broad as it transports a wide variety of structurally and functionally diverse substrates [16]. Indeed, anthracenes (e.g. mitoxantrone (MX)), camptothecins (e.g. topotecan, SN-38), polyglutamates (e.g. methotrexate), nucleoside analogs, and various tyrosine kinase inhibitors, are substrates for ABCG2 [16]. ABCG2 transporters are expressed in numerous organs and tissues, such as breast, placenta, colon, small and large intestine, alveoli, islets and acinar cells of the pancreas, canalicular membrane of the liver and bile, venous endothelium and in capillaries, adrenal glands, cortical tubules of the kidney and prostate epithelium, and on the luminal surface of the endothelial cells of human brain micro-vessels [17,18]. ABCG2 transporters are also 1) expressed on the plasma membrane of erythrocytes; 2) present in stem cells from various human tissues and 3) highly expressed in the placenta [16]. The overexpression of ABCG2 transporters mediates the development of MDR to many anti-cancer drugs in both solid tumors, such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), colon carcinoma, breast cancer, and hepatic cancer and hematological malignancies, like acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myelogenous leukemia (CML) [4,19,20] ABCG2 transporters confer resistance to various anticancer drugs that are ABCG2 substrates, such as nucleoside analogs, tyrosine kinase inhibitors, anthracyclines (e.g. doxorubicin (DOX) and MX), topoisomerase inhibitors (e.g. topotecan, SN-38), methotrexate and flavopiridol [21e23]. Thus, developing compounds that inhibit the efflux function and/or decrease ABCG2 protein expression, thereby increasing the concentrations of chemotherapeutic compounds in resistant cancer cells, represents a therapeutic strategy [24]. Numerous compounds have been reported to reverse MDR mediated by ABCG2 transporters in various types of cancer cells and in xenograft tumor mouse models [25]. Currently, however, no ABCG2 modulators have been clinically approved for treating MDR cancers [26,27]. Numerous studies have reported that tyrosine kinase inhibitors (TKIs) are efficacious against various cancers via inhibition of proliferation, invasion, metastasis, angiogenesis and induction of apoptosis in cancer cells [28,29]. Interestingly, in vitro studies have shown that TKIs modulate the function of ABC transporters like ABCB1, ABCG2, and ABCC1, at plasma concentrations obtained after therapeutic dosing [30,31]. For example, the Bcr-Abl kinase inhibitor, imatinib (STI 571 or Gleevec), and the epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib (Iressa/ZD1839), reverse ABCG2-mediated MDR by inhibiting ABCG2 efflux functions [32,33]. Mouse xenograft data indicate that the systemic administration of TKIs (i.e. imatinib, gefitinib, lapatinib and nilotinib) have been shown to significantly augment the efficacy of antineoplastic drugs that are substrates for the ABCG2 transporter, without producing significant toxicity [34e37]. According to pharmacophore modeling studies, pharmacophore models for the ABCG2 inhibitors encompass the following major

features: one hydrogen bond acceptor, one hydrogen bond donor, one hydrophobic group, and three aromatic rings [38]. For example, certain tyrosine kinase inhibitors (TKI), such as nilotinib, align with this six feature binding model, enabling optimum binding with the ABCG2 transporter [39]. This feature is coupled with the hydrophobic nature of nilotinib, including the hydrogen bond donor feature mapped onto the amide eNH group [39]. We postulated that certain highly selective dopamine D3 receptor antagonists may interact with the ABCG2 transporter as they have certain structural features (C]OeNH (carboxamide), at least 3 aromatic rings and a long molecular axis (hydrophobicity)) like some TKI compounds features such as imatinib, dasatinib, and nilotinib, that have high affinity for ABCG2 transporters. In support of our hypothesis, our preliminary molecular docking studies with highly selective D3/D2 receptor antagonists PG01037 [40], NGB2904 [41] and SB277011A [42] indicated computational binding affinity for ABCG2 (Fig.1). Furthermore, NGB2904, PG01037, and SB-277011A share common structural features with each other [43]. However, U99194, which has a significantly lower D3/D2 receptor affinity ratio in vitro [44] than NGB2904, PG01037, and SB-277011A, also had a lower computational docking score for ABCG2 compared to the other D3 receptor antagonists. Thus, given this finding, we conducted experiments to determine the efficacy of PG01037, NGB2904 and SB277011A to surmount resistance to MX and DOX, and to determine whether these effects are mediated by the overexpression of the ABCG2 transporter in certain cancer cell lines. The compound U99194, which in vitro has a 10e22-fold affinity for D3 over D2 receptors, was used as it has structural features that are distinct from PG01037, NGB2904 and SB277011A and a lower D3/D2 receptor selectivity, which might indicate reduced effects on ABCG2. In addition, we conducted experiments to determine the mechanism by which the D3 receptor antagonists might surmount ABCG2-mediated resistance. To our knowledge, our study is the first to investigate the efficacy of PG01037, NGB2904, SB277011A and U99194 to surmount ABCG2-mediated resistance. Materials and methods Materials NGB2904, PG01037, SB277011A dihydrochloride, and U99194 maleate were purchased from Tocris Bioscience (Bristol, UK). Mouse monoclonal antibody (BXP-21) for ABCG2 was purchased from Novus Biologicals (Littleton, CO, USA). Nilotinib was purchased from SigmaeAldrich (St. Louis, MO, USA). Mitoxantrone was purchased from Selleck Chemicals (Houston, TX, USA). Doxorubicin was purchased from Enzo Life Science, Inc. (Farmingdale, NY, USA). Rhodamine 123 fluorescent dye was purchased

Fig. 1. Chemical structures of the D3 Receptor antagonists used in this study. A. PG01037 B. NGB-2904 C.SB-277011A D. U99194 maleate.

N. Hussein et al. / Cancer Letters 396 (2017) 167e180 from Marker Gene Technologies, Inc. (Eugene, OR, USA). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was purchased from Calbiochem EMD Millipore (Billerica, MA, USA). Dulbecco's modification of Eagle's medium (DMEM) and 0.25% trypsin þ 2.2 Mm ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS without calcium or magnesium), and DMEM (phenol red - free) were purchased from Mediatech, Inc. (Corning subsidiaries, Manassas, VA, USA). Fetal Bovine Serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). Penicillin/streptomycin was purchased from Lonza, Inc. (Allendale NJ, USA). Propidium Iodide (PI) and 40 ,6-diamidino-2-phenylindole, dihydrochloride (DAPI) was purchased from Molecular Probes (Eugene, OR, USA). Dimethyl sulfoxide (DMSO) was purchased from VWR Analytical (Radnor, PA, USA). Monoclonal antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), monoclonal antibody against beta-actin (b-actin), anti-mouse secondary antibody, and anti-mouse Alexa Fluor® 488 were purchased from Cell Signaling Technology (Danvers, MA, USA). Bovine serum albumin (BSA) standard protein, bicinchoninic acid (BCA) solution, and copper solution were purchased from G-Biosciences (St. Louis, MO, USA). Paraformaldehyde (powder form) was purchased from Fisher Scientific (Hampton, NH, USA). Mini-Protean® TGX™ precast Gels, Clarity™, and Clarity Max™ Western ECL Blotting Substrates were purchased from BIO-RAD Laboratories (Hercules, CA, USA). Polyvinylidene difluoride (PVDF) membrane was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Non-fat dry milk was purchased from Cell Signaling (Danvers, MA, USA). Tween-20 was purchased from Fisher Scientific (Springfield Township, NJ, USA). Cell lines and cell culture The S1, S1M1-80, A549, A549-MX10, H460 and H460-MX20 cell lines were a gift from the late Dr. Gary Kruh (University of Chicago, IL). All of the cell lines were grown as adherent monolayers in flasks with DMEM cultured media supplemented with 10% FBS and 1% streptomycin/penicillin in a humidified incubator with 5% CO2 at 37  C. The human embryonic kidney 293 (HEK293)/pcDNA3.1 and wild type HEK293-R2 (ABCG2-482-R2) cell lines were originally generated by transfecting HEK293 cells with either the empty pcDNA3.1 vector or the pcDNA3.1 vector containing the full length ABCG2 gene, coding for arginine (R) at amino acid 482. The resistant colon cancer cell line, S1M1-80 was originally established by maintaining colon cancer S1 cells in increasing concentrations of MX up to 80 mM [45]. The ABCG2 overexpressing, non-small cell lung cancer (NSCLC) cell line, A549-MX10, was established by selecting and maintaining A549 cells with MX up to 10 mM [37,45,46]. All of the aforementioned cell lines were cultured in media with 2 mg/ml of G418 [47] until 1 week before experimentation.

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buffer A [1M HEPES-KOH (pH 7.9), 45 mM MgCl2, 300 mM KCL, 50 mM dithiothreitol (DTT), 100 mM phenylmethylsulfonyl fluoride (PMSF), and 3 ml of a protease inhibitor cocktail (ThermoScientific)] to each sample. The sample pellets were resuspended through vortexing and transferred to Eppendorf tubes. The cell homogenates were kept on ice for 15 min and 23 mL of NP40 was added to each sample for 2 min. The samples were centrifuged at 13,000 rpm for 1 min and the supernatant layer, which contains membranous proteins, was taken by aspiration with a pipette and kept at 20  C until analysis. The concentrations of the proteins in the samples were determined using the BCA assay. A standard curve, using 8 different concentrations of bovine serum albumin, was prepared. The working solution used was composed of BCA solution and copper solution in a ratio of 50 parts BCA: 1 part copper solution. Two hundred microliters of the working solution was added to the samples and standard wells. After incubating the plate at 37  C, the absorbance was measured at 562 nM using the Synergy H1 Multi-Mode Hybrid plate reader from BioTek (Winooski, VT, USA) with Gen 5 software. The calibration curve was used to determine the concentration of protein samples. Western blot analysis Protein samples were analyzed by loading equivalent amounts (20 mg) onto 7.5% SDS-polyacrylamide gels using Mini-PROTEAN® Tetra Cell from Bio-Rad (Hercules, CA, USA). The proteins were separated based on their molecular weight and then transferred to a PVDF membrane, which was previously activated using methanol. In order to prevent non-specific binding, the membrane was incubated 1 h with 5% non-fat milk in TBS-T (10 Tris buffered saline (TBS) [24.2 g Tris base, 80 g NaCl, 1N HCl, 38 ml] containing 0.1% Tween-20) before adding the primary BXP-21 antiABCG2 antibody (1:1000). The membrane was incubated with the primary antibody overnight at 4  C with gentle shaking. After overnight incubation, the membrane was washed three times with TBS-T washing buffer. Before adding the horseradish peroxide (HRP)-linked anti-mouse secondary antibody (1:3000; cell signaling), the membrane was incubated with blocking buffer. Subsequently, the membrane was incubated with the secondary antibody for 90 min at room temperature and washed 3 times with TBS-T buffer. The Western ECL blotting substrates (Western peroxide reagent, and clarity Western luminol enhancer) were added in a 1:1 ratio before reading the blots with a Molecular imager® ChemiDoc™ XRSþ from BIO RAD (Hercules, CA, USA). Either b-actin or GAPDH was used as a house-keeping control for protein samples. Image quantification analysis of Western blots was done using either Image Lab or Image J software (NIH, Bethesda, USA). Immunocytochemistry

Determination of cell cytotoxicity by the MTT assay The MTT colorimetric assay was used to analyze the sensitivity of cells to anticancer drugs, as previously described [48]. Briefly, after harvesting the cells with 0.25% trypsin, both resistant and parental cancer cell lines were re-suspended in DMEM. Cells were seeded evenly (160 mL/well) into 96 well-plates in triplicate at 5000 cells/well and the plates were returned to the incubator, allowing cells to attach to the wells for up to 24 h. For the reversal experiments, various concentrations of NGB2904, PG01037, SB-277011A dihydrochloride, U99194 maleate, or 5 mM of nilotinib (20 mL/well) were added during the second day of the experiment, followed by different concentrations of DOX and MX (20 mL/well) ranging from 0.1 to 100 mM into the same wells. After 72 h of incubation, 20 mL of the MTT solution (4 mg/ml) was added to each well. Subsequently, the plates were further incubated for 4 h to allow the viable cells to biotransform the yellow-colored MTT into darkblue formazan crystals. The media was aspirated and 100 mL of DMSO was used to dissolve the formazan crystals. The absorbance was measured using the Synergy H1 Multi-Mode Hybrid Reader from BioTek (Winooski, VT, USA) at 570 nm. The IC50 was determined using the Bliss method [49], which is based on the change in the percentage of viable cells after the addition of chemotherapeutic drugs, with or without the reversal compounds. Resistance was determined by dividing the IC50 obtained in the resistant cancer cells (with or without the reversal compounds) by the IC50 of the non-resistant, parental cancer cells. Cell morphological analysis Cell images were taken using an inverted microscope (Olympus, BX53F) (Center valley, PA) with a fluorescent lamp and digital camera and morphological changes were observed 72 h after incubation with the chemotherapeutic drugs MX and DOX alone, or the combination of the chemotherapeutic drugs with the D3 receptor antagonists at different concentrations. Protein estimation: cell lysate preparation and bicinchoninic acid (BCA) analysis H460-MX20 cells, in T25 flasks, were incubated with 5 mM of either NG2904 or PG01037 for either 24 or 48 h. The media was aspirated and the cells were rinsed with 5 ml of PBS drop by drop, followed by a gentle shaking and aspiration of PBS. Two ml of PBS was added three times to the cells, which were collected for lysis with the use a cell scraper to detach the cells. During the transfer of the cells from flasks to 15 ml tubes, they were kept on ice. The tubes were centrifuged at 1500 rpm for 3 min and the supernatant was discarded and the white pellets were kept for the cell lysis step. The cytosolic fraction was extracted by the addition of 400 mL of ice-cold lysis

The H460-MX20 cells were seeded at a density of 2  105 per ml on coverslips inside six well plates and left overnight. Coverslips had been sterilized in 1N HCl and washed with distilled water and soaked with 100% ethanol 24 h previously. On the second day, 5 mM of NGB2904 or PG01037 were added for 24 or 48 h, respectively. The cells were fixed with 4% paraformaldehyde for 20 min and then rinsed three times with PBS. A 7.5% bovine serum albumin (BSA) blocking buffer (SigmaeAldrich, St. Louis) (MO, USA) was added to the cells for 1 h at room temperature and the cells were incubated with a monoclonal antibody BXP-21 against ABCG2 (1:200) overnight at 4  C. The cells were washed three times with PBS and blocked with BSA before incubation with anti-mouse Alexa Fluor fluorescent secondary antibody (1:500) for 1 h at room temperature. Coverslips were placed over labeled glass slides and mounted with 1e2 drops of DAPI for staining the nucleus, and were sealed. The slides were allowed to dry for about 1e3 h before taking images. The images were taken using a Nikon TE-2000s fluorescence microscope (Melville, NY, USA). Fluorescein isothiocyanate (FITC) was excited at 488 nm and light emission measured at 558 nm, whereas DAPI was excited at 350 nm and light emission measured at 470 nm. The images were analyzed using the Nikon NIS Elements microscope imaging software and image J software. Rhodamine 123 accumulation and efflux assay The ABCG2 overexpressing NSCLC H460-MX20 cells were seeded at a density of 5  105 cells per ml in six-well plates and allowed to attach overnight. Each plate was incubated for 1 h with 5 mM of NGB2904, PG01037, or nilotinib. Thereafter, 5 mM rhodamine 123 was added and the cells further incubated for 1 h. The cells were rinsed twice with ice-cold complete phenol red-free media. The cells were further incubated at 37  C in a rhodamine-free media in the presence or absence of 5 mM of NGB2904 or PG01037 for 1 or 2 h, respectively, to allow the efflux of rhodamine. Subsequently, the cells were harvested at the indicated efflux times (0, 1 or 2 h) and centrifuged at 400 g for 5 min, washed twice with ice-cold PBS, and dispersed in 1 ml of PBS. Cellular rhodamine 123 accumulation was analyzed in 10,000 events per 1 ml of the sample using a flow cytometer from Accuri C6 Cytometers, Inc. (Ann Arbor, MI, U.S) and the data was captured using C Flow® Plus software. The data were further analyzed using either FCS Express 5 Plus (Glendale, CA, U.S) and/or Flow Jo V10 software. The effect of the D3 receptor antagonists on the efficacy of MX and DOX In order to determine if DA D3 receptor antagonists can increase the efficacy of anticancer drugs, such as MX and DOX, the combination index (CI) was determined based on the Chou and Talalay method [50]. The CI represents a quantitative

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measure of the magnitude of the additive, synergistic or antagonistic drug effects [50]. The data were analyzed using CompuSyn software (CompuSyn, NJ). The CI values are interpreted as follows: antagonism, CI > 1; addition, CI ¼ 1; and synergism, CI < 1. The fraction affected value (Fa) corresponds to the percent of cells that survive: Fa ¼ 0 when the percentage of cells surviving ¼ 100%, Fa ¼ 1 when the percentage of cells surviving is equal to 0%. Molecular docking studies Ligand structure preparation The structures of the four D3 receptor antagonists were constructed using the builder module of Maestro v 9.3.5 and the energy minimized by Macromodel pro€ dinger, Inc., New York, NY, 2012), using the OPLSAA force field with gram v9.9 (Schro the steepest descent, followed by a truncated Newton conjugate gradient protocol. The low-energy 3D structures of the four D3 receptor antagonists were generated by LigPrep v2.5 and the parameters were defined based on different protonation states at physiological pH ± 2, and all possible conformations were filtered with a maximum relative energy difference of 5 kcal/mol to exclude redundant conformers. The ligand structures obtained from the LigPrep v2.5 run were further used for generating 100 ligand conformations for each structure using the default parameters of mixed torsional/low-mode sampling. The output conformational search (Csearch) file containing 100 unique conformers of each D3 receptor antagonist was used as input for docking simulations at the binding site of human ABCG2. Protein structure preparation A homology model of ABCG2 was constructed as previously reported [51], using mouse apoprotein (PDB ID: 3G5U) [52] as a template for the molecular docking studies. The homology model of ABCG2 was optimized using the 'Protein Prepara€dinger molecular modeling suite tion Wizard' workflow implemented in the Schro €dinger, Inc., New York, NY, 2012). This optimization includes adding hydrogen (Schro atoms, assigning correct bond orders and building disulfide bonds. The protonation states of all of the ionizable residues were predicted by PROPKA provided in the protein preparation wizard. An optimized structure model was energy minimized (only hydrogen atoms) using the OPLS2005 force field. The refined protein model was used to generate various grids based on the following residues as centroids: Arg482 (grid 1), Asn629 (grid 2), Arg383 (grid 3), and Leu241 along with Gly83 (grid 4). The selection of these residues was based on their significant involvement in ABCG2 function as demonstrated in mutational experiments [47,53e55]. Grid 2 was generated using Asn629 as the centroid was found to have the best docking score; hence, docking analyses were based on binding mode of four D3 receptor antagonists at this site. Docking protocol The diverse conformational library of NGB2904, SB-277011A, PG01037, and U99194 were docked at grid 2, using the ‘‘Extra Precision’’ (XP) [56] mode of Glide €dinger, Inc., New York, NY, 2012), with the default functions. program v5.8 [57] (Schro The best-docked conformations of the D3 receptor antagonists were established by high XP GScore and used for further analysis. Statistical analysis At least 2 to 3 independent experiments were done for each of the abovementioned assays. Data were expressed as the mean ± standard deviation (SD) in cell survival assays and as the mean ± standard error of the mean (SEM) in all other assays. Statistical analyses were performed using Graph Pad prism software 5.04 from Graph Pad Software, Inc. (La Jolla, CA, U.S). The data were analyzed using a oneway analysis of variance (ANOVA). Post hoc analysis was performed using Dunnett's test, a test for linear trends to show dose- or time-dependent effects or Tukey's multiple comparison tests. The a priori significance level was set at P < 0.05.

Results The effect of D3 receptor antagonists on the efficacy of MX and DOX in cell lines overexpressing ABCG2 transporters There are two critical aspects to interpreting MDR reversal: the choice of comparison cell lines, and determining if the drugs being studied affect cell viability. First, using the appropriate pairs of MDR-ABC transporter overexpressing (i.e. resistant cell lines) and non-resistant cell lines, is crucial. Therefore, we chose both drug selected cell line pairs, H460, H460-MX20, A549, A549-MX10, S1, and S1M1-80, as well as transfected ABCG2 cell lines, including HEK293 and HEK293-R2, as shown in Figs. 2 and 3, 2S, 3S, 4S, and 5S. Second, we determined the effect of the D3 receptor antagonists on cell viability. The IC50 values of U99194, NGB2904, PG01037, and SB-277011A alone in H460 cells were 87, 70, 63, 73 mM,

respectively. In contrast, the IC50 values of U99194, NGB2904, PG01037, and SB-277011A alone in ABCG2-overexpressing cells H460-MX20 were 133, 159, 122, and 130 mM, respectively as shown in Fig. 1S. The aforementioned IC50 values suggest that relatively high concentrations of the D3 receptor antagonists alone are required to significantly decrease cell viability. Based on the above data, we used concentrations from 0.1 to 10 mM for each D3 receptor antagonist to determine their efficacy for sensitizing ABCG2mediated MDR to MX and DOX, which are ABCG2 transporter substrates [58]. PG01037, at concentrations of 0.1, 0.5, 1, 2.5, 5, and 10 mM, significantly reduced the IC50 of MX in both the H460-MX20 and A549-MX10 cell lines (Table 1, Table 2, and Fig. 2). Additionally, PG01037 significantly reduced the IC50 of MX in S1-M180 cells at concentrations of 1, 2.5, 5, and 10 mM (Table 3, Fig. 2). Similarly, 5 mM nilotinib (an inhibitor of ABCB1 and ABCG2 [59]) significantly reduced the IC50 of MX in H460-MX20, A549-MX10 and S1M1-80 cell lines (Tables 1e3 and Fig. 2). However, neither NGB2904 nor PG01037 significantly altered the cytotoxicity of MX in the parental H460, A549, or S1 cell lines (Tables 1e3). NGB2904, at concentrations of 0.01, 0.1, 0.5, 1, 2.5, 5, or 10 mM, significantly reduced the IC50 of MX in H460-MX20 cells (Table 1 and Fig. 3). Furthermore, NGB2904 significantly reduced the IC50 of MX in A549-MX10 (0.1, 0.5, 1, 2.5, 5, or 10 mM) and S1-M180 (1, 0.5, 1, 2.5, 5, or 10 mM) cell lines (Tables 2 and 3, and Fig. 3). SB277011A, at concentrations of 1, 2.5, 5, and 10 mM, significantly reduced the IC50 of MX in both H460-MX20 and A549-MX10 cells (Tables 2 and 3, and Fig. 2S). SB-277011A also significantly reduced MX's IC50 in H460-MX20, A549-MX10, and S1-M180 cell lines, in a concentrationedependent manner, at concentrations of 2.5, 5, and 10 mM (Tables 1e3, and Fig. 2S). U99194, at concentrations of 1, 2.5, 5, and 10 mM, significantly reduced the IC50 of MX in both H460MX20 and A549-MX10 cells (Tables 2 and 3, and Fig. 3S). U99194A significantly reduced the IC50 of MX in S1-M180 cells at concentrations of 2.5, 5, and 10 mM (Table 3 and Fig. 3S). Also, U99194, in a concentration-dependent manner, significantly reduced the IC50 of MX in H460-MX20, A549-MX10, and S1M180 at 2.5, 5, and 10 mM (Tables 1e3 and Fig. 3S). At concentrations up to 5 mM, NGB2904, PGO1037, and SB-277011A produced a significant reduction in the IC50 of MX in ABCG2 overexpressing transfected wild-type (HEK293-R2) cells (Table IS and Fig. 4S). However, U99194 did not produce a significant change in the IC50 of MX. Furthermore, U99194, at a concentration of 5 mM, significantly reduced the IC50 of DOX (Table IIS and Fig. 5S). The combination of 5 mM of PG01037 or NGB2904 with MX produces cell morphological changes exemplified by a significant reduction in the number of living cells and the shifting of the IC50 for MX in H460-MX20 ABCG2 overexpressing cells, corroborating the synergistic effects of D3 receptor antagonists (Fig. 2G and 3G). The rank order of potency for the reversal of MX resistance in the H460-MX20 cell was: NGB2904 > PG01037 > U99194 > SB277011A. In contrast, the rank order of potency for the reversal of MX resistance in A549-MX10 cells was: NGB2904 z PG01037 > SB277011A > U99194. In S1M1-80 MX resistance cells, the rank order of reversal was: NGB2904 > PG01037 > SB277011A > U99194A. D3 receptor antagonists synergistically increase the efficacy of MX and DOX Achieving a synergistic therapeutic effect and reducing the onset of drug resistance are the main goals of drug combinations in cancer treatment [60]. Thus, we determined if 5 or 10 mM of NGB2904, PG01037, SB-277011A, or U99194 produced synergistic effects in combination with MX or DOX, in H460-MX20 cells. The CI values were determined using the CI ranges values table (Table IIIS)

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Fig. 2. The effect of PG01037 on resistance to MX mediated in the ABCG2 overexpressing cell lines H460-MX20, A549-MX10, and S1M1-80. MTT cytotoxicity expressed as IC50 values for MX alone (control) or in combination with different concentrations of PG01037 or with 5 mM of nilotinib (a known inhibitor of the ABCG2 transporter) in H460-MX20 cells (A), A549-MX10 cells (C), and S1M1-80 cells (E). The results also show the changes in cell survival percentages in H460 and H460-MX20 cells (B), A549 and A549-MX10 cells (D), and S1 and S1M1-80 cells (F). G: Microscopic images illustrating the synergistic effects of PG01037 on the potentiation of MX cytotoxicity at different concentrations. Data points represent the means ± SD of at least three independent experiments, each with triplicate determinations. The data were analyzed using a one-way ANOVA with Dunnett's post hoc to compare the IC50 of MX in combination with different concentrations of PG01037 or with 5 mM of nilotinib (a known inhibitor of the ABCG2 transporter) in H460-MX20 cells (***P < 0.001) vs. control cells treated with MX alone.

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Fig. 3. The effect of NGB2904 on ABCG2-mediated resistance to MX in H460-MX20, A549-MX10, and S1M1-80 cell lines. MTT cytotoxicity expressed as IC50 values for MX alone (control) or in combination with different concentrations of NGB2904 or with 5 mM nilotinib (a known inhibitor of the ABCG2 transporter) in H460-MX20 cells (A), A549-MX10 cells (C), and S1M1-80 cells (E). The results also show the changes in the percentage of surviving cells in H460 and H460-MX20 cells (B), A549 and A549-MX10 cells (D), and S1 and S1M1-80 cells (F). G: Microscopic images illustrating the synergistic effects of NGB2904 on the potentiation of MX cytotoxicity at different concentrations. Data points represent the means ± SD of at least three independent experiments, each with triplicate determinations. The data were analyzed using a one-way ANOVA with Dunnett's post hoc comparisons of IC50 values of MX in combination with different concentrations of NGB2904 or with 5 mM nilotinib (a known inhibitor of the ABCG2 transporter) in H460-MX20 cells (***P < 0.001) vs. control cells treated with MX alone.

N. Hussein et al. / Cancer Letters 396 (2017) 167e180 Table 1 The effect of NGB2904, PG01037, SB-277011A, U99194A, and nilotinib on the cytotoxicity of MX in ABCG2-overexpressing H460-MX20 cells. Treatment

IC50 ± SD (mM)a H460

Mitoxantrone þ NGB2904 e 0.01 mM þ NGB2904 e 0.1 mM þ NGB2904 e 0.5 mM þ NGB2904 e 1 mM þ NGB2904 e 2.5 mM þ NGB2904 e 5 mM þ NGB2904 e 10 mM þ Nilotinib e 5 mM

Table 2 The effect of NGB2904, PG01037, SB-277011A, U99194A, and nilotinib on the cytotoxicity of MX in ABCG2-overexpressing A549-MX10 cells. Treatment

FRb

H460-MX20

FRb

0.25 0.27 0.14 0.15 0.13 0.11 0.18 0.16 0.16

± ± ± ± ± ± ± ± ±

0.01 0.02 0 0 0.02 0 0 0.05 0.04

1 1 0.6 0.6 0.5 0.4 0.7 0.6 0.6

6.26 0.6 0.44 0.5 0.47 0.45 0.44 0.45 0.4

± ± ± ± ± ± ± ± ±

0.03 0.05*** 0.02*** 0.02*** 0.01*** 0*** 0.09*** 0*** 0***

25 2.4 1.8 1.8 1.8 1.7 1.7 1.7 1.5

þ þ þ þ þ þ þ

PG01037 e 0.1 mM PG01037 e 0.5 mM PG01037 e 1 mM PG01037 e 2.5 mM PG01037 e 5 mM PG01037 e 10 mM Nilotinib e 5 mM

0.2 0.14 0.13 0.17 0.14 0.18 0.16

± ± ± ± ± ± ±

0.01 0.01 0.01 0 0 0.02 0.04

0.8 0.6 0.5 0.6 0.6 0.7 0.6

2.2 2 0.46 0.46 0.4 0.4 0.4

± ± ± ± ± ± ±

0.08*** 0.05*** 0.05*** 0*** 0.04*** 0.05*** 0***

8.5 8 1.8 1.8 1.6 1.6 1.5

þ þ þ þ þ

SB-277011A e 1 mM SB-277011A e 2.5 mM SB-277011A e 5 mM SB-277011A e 10 mM Nilotinib e 5 mM

0.14 0.18 0.21 0.17 0.16

± ± ± ± ±

0.1 0 0.04 0 0.04

0.5 0.7 0.8 0.66 0.6

3.75 3.6 1.8 0.84 0.4

± ± ± ± ±

0.05*** 0*** 0.1*** 0.04*** 0***

15 14 7 3.32 1.5

þ þ þ þ þ

U99194 e 1 mM U99194 e 2.5 mM U99194 e 5 mM U99194 e 10 mM Nilotinib e 5 mM

0.23 0.16 0.26 0.13 0.16

± ± ± ± ±

0 0.06 0.05 0.02 0.04

0.9 0.6 0.9 0.5 0.6

5 4.2 3.5 0.8 0.4

± ± ± ± ±

0.05*** 0.2*** 0.08*** 0.02*** 0***

20 16.6 14 3 1.5

Statistical significance was determined in comparison to the control condition (MX alone) *P < 0.05; **P < 0.01; ***P < 0.001. a IC50 data is presented in the two columns H460 and H460-MX20 for the two cell lines as mean ± SD (standard deviation) of at least three experiments conducted in triplicate. b Fold resistance (FR) is presented in the adjacent columns, calculated as described in the materials and methods. The percentage of surviving cells was determined using the MTT assay.

[61,62] with an Fa range from 0 to 1. The combination of 10 mM PG 01037 and 0.1e100 mM of MX produced a strong synergistic effect (CI ¼ 0.1e0.3; Fig. 6A) and the combination of 1 mM of MX and 10 mM of PG 01037 produced a very strong synergistic effect (CI < 0.1; Fig. 6A). Similarly, the combination of 10 mM of NGB2904 and 1e30 mM of MX produced a very strong synergistic effect (CI < 0.1; Fig. 6B). The combination of 10 mM of NGB2904 with 0.1 mM MX, as well as 100 mM MX combined with 10 mM of NGB2904, produced synergism, albeit to a lesser extent (CI ¼ 0.3e0.7; Fig. 6B). However, the combination of 0.3 mM of MX and 10 mM of NGB2904 produced a strong synergism (CI value ¼ 0.1e0.3; Fig. 6B). The combination of 10 mM of SB-277011A with 0.3e100 mM of MX produced a strong synergistic effect (CI value ¼ 0.1e0.3; Fig. 6C). However, the combination of 10 mM of SB277011A and 0.1 mM of MX showed synergism (CI value ¼ 0.3e0.7; Fig. 6C). Finally, the combination of 10 mM of U99194 and 0.3e100 mM of MX produced a strong synergistic effect (CI 0.1e0.3; Fig. 6D), although 10 mM of U99194 and 0.1 mM of MX elicited only moderate synergism (CI ¼ 0.7e0.85; Fig. 6D). The combination of 5 mM of PG01037 and 0.03e1 mM of DOX produced a strong synergism (CI value ¼ 0.1e0.3; Fig. 6SA), whereas 3 mM of DOX and 5 mM of PG01037 produced synergism (CI value ¼ 0.3e0.75; Fig. 6SA). In contrast, 5 mM of PG01037 and 10 mM of DOX produced an antagonistic effect (CI ¼ 1.45e3.3; Fig. 6SA). The combination of 5 mM of NGB2904 and 0.1e3 mM of DOX produced synergism (CI value ¼ 0.3e0.75; Fig. 6SB). However, 5 mM of NGB2904 and 0.03 mM of DOX and 5 mM of NGB2904 and 10 mM

173

IC50 ± SD (mM)a A549

FRb

A549-MX10

FRb

Mitoxantrone þ NGB2904 e 0.1 mM þ NGB2904 e 0.5 mM þ NGB2904 e 1 mM þ NGB2904 e 2.5 mM þ NGB2904 e 5 mM þ NGB2904 e 10 mM þ Nilotinib e 5 mM

0.26 0.27 0.22 0.18 0.21 0.2 0.22 0.23

± ± ± ± ± ± ± ±

0.02 0 0.02 0.07 0.05 0 0.01 0.01

1 1 0.9 0.7 0.8 0.7 0.9 0.9

1.51 0.5 0.27 0.24 0.24 0.25 0.22 0.27

± ± ± ± ± ± ± ±

0.05 0*** 0.01*** 0.05*** 0*** 0.02*** 0.02*** 0.01***

6 1.7 0.9 0.9 0.9 0.9 0.9 1

þ þ þ þ þ þ þ

PG01037 e 0.1 mM PG01037 e 0.5 mM PG01037 e 1 mM PG01037 e 2.5 mM PG01037 e 5 mM PG01037 e 10 mM Nilotinib e 5 mM

0.25 0.21 0.18 0.2 0.23 0.12 0.23

± ± ± ± ± ± ±

0.03 0.02 0.03 0 0.03 0.01 0.01

1 0.8 0.7 0.8 0.9 0.44 0.9

0.5 0.24 0.24 0.25 0.27 0.24 0.27

± ± ± ± ± ± ±

0.06*** 0.04*** 0.06*** 0.03*** 0.01*** 0.02*** 0.01***

1.8 0.9 0.9 0.9 0.9 0.9 1

þ þ þ þ þ

SB-277011A e 1 mM SB-277011A e 2.5 mM SB-277011A e 5 mM SB-277011A e 10 mM Nilotinib e 5 mM

0.24 0.24 0.25 0.23 0.23

± ± ± ± ±

0.03 0.04 0.04 0.03 0.01

0.9 0.9 0.9 0.9 0.9

0.7 0.5 0.43 0.21 0.27

± ± ± ± ±

0.1*** 0.03*** 0.07*** 0.03*** 0.01***

2.6 1.9 1.6 0.9 1

þ þ þ þ þ

U99194 e 1 mM U99194 e 2.5 mM U99194 e 5 mM U99194 e 10 mM Nilotinib e 5 mM

0.17 0.2 0.25 0.23 0.23

± ± ± ± ±

0.01 0.07 0.02 0.01 0.01

0.7 0.8 1 0.9 0.9

0.4 0.4 0.26 0.23 0.27

± ± ± ± ±

0.06*** 0.05*** 0.02*** 0.01*** 0.01***

1.6 1.5 1 0.9 1

Statistical significance was determined in comparison to the control condition (MX alone) *P < 0.05; **P < 0.01; ***P < 0.001. a IC50 data is presented in the two columns A549 and A549-MX10 for the two cell lines as mean ± SD (standard deviation) of at least three experiments conducted in triplicate. b Fold resistance (FR) is presented in the adjacent columns, calculated as described in the materials and methods. The percentage of surviving cells was determined using the MTT assay.

of DOX produced antagonism (CI ¼ 1.45e3.3; Fig. 6SB). The combination of 5 mM of SB-277011A and 0.03e10 mM of DOX produced synergism (CI ¼ 0.3e0.75, Fig. 6SC). However, 10 mM of DOX and 5 mM of SB-277011A produced a moderate antagonism (CI value ¼ 1.2e1.5; Fig. 6SC). The combination of 5 mM of U99194 and 0.3e3 mM of DOX produced synergism (CI ¼ 0.3e0.75; Fig. 6SD). Similarly, 0.03 mM of DOX and 5 mM of U99194, as well as 0.1 mM of DOX and 5 mM of U99194 produced moderate synergism (CI ¼ 0.7e0.85, Fig. 6SD). However, 10 mM of DOX and 5 mM of U99194 produced moderate antagonism (CI ¼ 1.2e1.45; Fig. 6SD) (Tables IVS, and VS summarize the CI values for the combinations of D3 receptor antagonists and MX or DOX). PG01037 and NGB2904 significantly decrease the protein expression levels of the ABCG2 transporter protein NGB2904 and PG01037-induced increase in the efficacy of MX and DOX in cells overexpressing ABCG2 transporters could result from 1) inhibiting the efflux function of ABCG2 transporter; 2) a reduction in the ABCG2 protein level; 3) a decrease in the number of ABCG2 transporters present in the cell membrane. Based on cell viability results, PG01037 and NGB2904 were the most efficacious compounds for reversing ABCG2-mediated MDR. Therefore, we tested the in vitro effects of NGB2904 and PG01037 on the expression of ABCG2 protein levels. The incubation of H460-MX20 cells (which overexpress ABCG2 transporters) with 5 mM of PG01037 for 24 or 48 h significantly reduced the protein expression

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Table 3 The effect of NGB2904, PG01037, SB-277011A, U99194A, and nilotinib on the cytotoxicity of MX in ABCG2-overexpressing S1M1-80 cells. Treatment

IC50 ± SD (mM)a S1

FRb

S1M1-80

FRb

Mitoxantrone þ NGB2904 e 1 mM þ NGB2904 e 2.5 mM þ NGB2904 e 5 mM þ NGB2904 e 10 mM þ Nilotinib e 5 mM

0.9 0.8 0.42 0.6 0.8 0.14

± ± ± ± ± ±

0.08 0.06 0.05 0.03 0.1 0.03

1 0.9 0.5 0.7 0.83 0.15

80 23 10 6 6 0.81

± ± ± ± ± ±

0.08*** 0.01*** 0.1*** 0.08*** 0.02*** 0.02***

88 25 11 6 6 0.9

þ þ þ þ þ

PG01037 e 1 mM PG01037 e 2.5 mM PG01037 e 5 mM PG01037 e 10 mM Nilotinib e 5 mM

0.74 0.8 0.74 0.74 0.14

± ± ± ± ±

0.03 0.06 0.01 0.07 0.03

0.8 0.9 0.8 0.8 0.15

27 9 6 6 0.81

± ± ± ± ±

0*** 0.01*** 0.01*** 0.09*** 0.02***

29 10 6.6 6.6 0.9

þ þ þ þ

SB-277011A e 2.5 mM SB-277011A e 5 mM SB-277011A e 10 mM Nilotinib e 5 mM

0.8 0.83 0.83 0.14

± ± ± ±

0.1 0 0.07 0.03

0.9 0.9 0.9 0.15

27 20 8 0.81

± ± ± ±

0*** 0.1*** 0.09*** 0.02***

30 21.4 8 0.9

þ þ þ þ

U99194 e 2.5 mM U99194 e 5 mM U99194 e 10 mM Nilotinib e 5 mM

0.9 0.9 0.7 0.14

± ± ± ±

0 0.03 0 0.03

1 1 0.8 0.15

24 23 16 0.81

± ± ± ±

0.09*** 0.07*** 0.1*** 0.02***

26 25 17 0.9

Statistical significance was determined in comparison to the control condition (MX alone) *P < 0.05; **P < 0.01; ***P < 0.001. a IC50 data is presented in the two columns S1 and S1M180 for the two cell lines as mean ± SD (standard deviation) of at least three experiments conducted in triplicate. b Fold resistance (FR) is presented in the adjacent columns, calculated as described in the materials and methods. The percentage of surviving cells was determined using the MTT assay.

levels of the ABCG2 transporter (Fig. 4B). Additionally, the incubation of cells with 5 mM of NGB2904 for 48 h significantly reduced the protein expression levels of ABCG2 transporter (Fig. 4B). The above results were further corroborated by immunocytochemistry assays, which showed a significant down-regulation of the ABCG2 transporter after 48 h of incubation of cells with 5 mM of PG01037 or 5 mM of NGB2904 (Fig. 4C). The fluorescence-integrated density of cells incubated with either PG01037 or NGB2904 was significantly reduced compared with the control cells (Fig. 4C). Thus, NGB2904 and PG01037 significantly reverse ABCG2-mediated MDR through the down-regulation of the ABCG2 protein. Future experiments should be done to determine the effect of D3 receptor antagonists on ABCG2 mRNA (transcriptional level) and the role of proteasomal degradation in the down regulation of ABCG2. However, these compounds may also attenuate ABCG2-mediated MDR by inhibiting its function as discussed below. PG01037 and NGB2904 significantly inhibit the efflux function of the ABCG2 transporter In order to further elucidate the mechanisms by which PG01037 and NGB2904 attenuate ABCG2-mediated MDR, we determined their effect on the cellular accumulation of the fluorescent dye rhodamine 123, a substrate for ABC transporters [63,64]. Based on cell viability results, PG01037 and NGB2904 were the most potent compounds for reversing ABCG2-mediated MDR. Therefore, we tested the in vitro effect of NGB2904 and PG01037 on ABCG2 efflux. The intracellular levels of rhodamine 123, which correspond to the fluorescence intensity, were measured in the presence or absence of NGB2904, PG01037, or nilotinib (used as a positive control) in H460-MX20 cells. The intracellular accumulation of rhodamine in H460-MX20 cells incubated with 5 mM of PG01037, NGB2904 or

nilotinib was significantly greater than in control (i.e. no treatment) H460-MX20 cells (Fig. 5). This occurred because the rhodamine fluorescence intensity in control H460-MX20 cells was substantially and significantly reduced over time at the 1 and 2 h timepoints, reflecting the continuous efflux, as compared with the 0 h time-point. However, there was no significant change in cells incubated with either PG01037 or NGB2904. NGB2904 (a representative D3 receptor antagonist) produced a similar inhibition of efflux function when tested in a second ABCG2 overexpressing cells S1-M1-80 (Fig. 7S). Molecular docking of SB277011A, NGB2904, PG01037, and U99194A as determined using a homology model of the ABCG2 transporter Molecular docking studies were performed for the four D3 receptor antagonists using a homology model of ABCG2 following a previously reported protocol [65]. The docking models predicted by Glide indicated that all of the D3 receptor antagonists bind at the substrate-binding site of the ABCG2 transporter, predominantly through hydrophobic interactions. The binding conformation of PG01037 is similar to NGB2904, whereas SB-277011A and U99194 exhibit a different binding pattern compared to PG01037 and NGB2904. As shown in Fig. 7A, the piperazinyl ring, with an attached 2,3 dichlorophenyl moiety in PG01037, is positioned in a hydrophobic pocket of the ABCG2 transporter formed by Tyr464, Phe489, Phe511, Ile573, Tyr576, and Leu581, thereby enabling hydrophobic interactions. This placement is further stabilized by an electrostatic interaction between the 3-Cl atom in the phenyl ring and the eOH group of Tyr576. The 4-(pyridin-2-yl) benzamide of PG01037 also binds favorably in another hydrophobic pocket formed by the side chains of Leu626, Trp627, and His630, along with Val631. The orientation of the 2-pyridinyl ring in the hydrophobic pocket also facilitates a polar interaction with Arg465. The benzamido eNH group forms a hydrogen bond with the imidazole ring nitrogen of His630 (NH/N-His630, 2.1 Å). This interaction is significant as it had been demonstrated previously that His630 from each monomer plays a critical role in function of ABCG2 [66]. The compound with the next highest docking score, NGB2904, adopts a similar binding conformation to that of PG01037, differing only in the orientation of 9H-fluorenyl ring in the hydrophobic pocket formed by the side chains of Leu626, Trp627, and His630, along with Val631. This is mainly due to steric hindrance caused by the increased planarity of 9H-fluorenyl ring compared to the 2phenylpyridine ring in PG01037 (Fig. 7B). The Glide predicted docking models for SB-277011A and U99194 (Fig. 7C, and D) revealed differences in the binding mode compared to PG01037 and NGB2904 at the Asn629 centroid-based grid of ABCG2. This is not surprising given the polyspecificity associated with substrates for ABCG2. For SB-277011A, the quinoline ring is stabilized by hydrophobic contacts with amino acid residues Ile573, Tyr464, Phe489, Tyr570, Ile573, Pro574, and Leu581. Furthermore, the quinoline ring nitrogen atom also forms a hydrogen bond with the hydroxyl group of Tyr464 (N/HO-Tyr464, 2.13 Å). The cyclohexyl ethyl linker between the quinolinyl-4-carboxamide and 5,6,7,8-tetrahydronaphthalene-2-carbonitrile moieties are involved in extensive hydrophobic interactions with Ala580, Leu581, Phe511, Trp627, and His630. The orientation of the 5,6,7,8tetrahydronaphthalene-2-carbonitrile moieties is stabilized through hydrophobic contacts with side chains of Phe507, Val508, and Leu633. In addition, the nitrile group present in the 5,6,7,8tetrahydronaphthalene ring is also involved in electrostatic interactions with the basic amino group of Lys628. U99194, which had the lowest docking score, adopts a binding conformation that positions the 5,6-dimethoxy-2,3-dihydro-1H-

N. Hussein et al. / Cancer Letters 396 (2017) 167e180

175

Fig. 4. The effects of PG01037 and NGB2904 on protein expression of ABCG2 transporter. A: The baseline expression of the ABCG2 protein in H460 and H460-MX20 cell lines. B: H460-MX20 cells incubated with both 5 mM NGB2904 and 5 mM PG01037 for 24 and 48 h, respectively. Columns in the lower lanes represent the mean of the Western blot quantification values. The error bars represent the SEM. At least two other trials showed a similar result to the representative figure results. The data were analyzed using a one-way ANOVA and Dunnett's post hoc comparisons (*P < 0.05 vs. control group). C: Immunocytochemical analysis of H460-MX20 cells after incubation with 5 mM of both NGB2904 and PG01037 for 48 h. In the immunofluorescence image, the ABCG2-specific antibody is shown in green and the nuclear staining with DAPI is shown in blue. In addition to the representative figure, at least three independent experiments were performed using different batches of cells at different times. The fluorescence integrated density was quantified and is represented by the lower lane bar graph. The data were analyzed using a one-way ANOVA, followed by Dunnett's post hoc comparisons (***P < 0.001 vs. control group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

indene ring in the hydrophobic pocket lined by side chains of amino acid residues Phe511, Ala580, His630, Leu633, and Trp627. The oxygen atom in the methoxy group attached to the indene ring forms a hydrogen bond with the caboxamido eNH2 group of Asn629 (O/HN-Asn629, 2.1 Å). The n-propyl side chains of the 2-

dipropylamino group attached to C2 of the 2,3-dihydro-1Hindene ring were stabilized through hydrophobic contacts with the side chains of Ile412, Phe489, Gly577, Leu581, Pro574, and Trp627. The XP GScores for PG01037, NGB2904, SB-277011A, and U99194 were 9.704, 9.299, 9.068, and 6.525, respectively.

Fig. 5. The effect of PG01037, NGB2904, and nilotinib on the accumulation and efflux of intracellular rhodamine 123 in ABCG2 overexpressing H460-MX20 cells. A: Rhodamine efflux at 0, 1, and 2 h is shown. The treatment groups were: black: control untreated H460-MX20 cells; red: 5 mM NGB2904; blue, 5 mM PG01037; green: 5 mM nilotinib. Rhodamine efflux is determined by comparing treated and untreated groups in H460-MX20 cells, using a one-way ANOVA, followed by a post hoc test for linear trends. B: The intracellular rhodamine accumulation was expressed as the units of mean fluorescent intensity. The data represent the mean ± SEM of triplicate determinations. The data for the efflux experiments at 0, 1, and 2 h was analyzed using a one-way ANOVA, followed by Tukey's multiple comparison post hoc test (#, P < 0.05 and ##, P < 0.01 vs. control 1 h; ***, P < 0.001 vs control 2 h). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. The combination index values resulting from combining PG01037, NGB2904, SB-277011A, and U99194A with MX. CI values for 10 mM PG01037 (A), 10 mM NGB2904 (B), 10 mM SB-277011A (C), and 10 mM U99194 (D) in combination with 0.1, 0.3, 1, 3, 10, 30, 100 mM of MX for Fa range from 0 to 1. CI < 1, synergism; CI ¼ 1, additive effect; CI > 1, antagonism. DATA represent the mean ± SD of three independent experiments, each in triplicate.

Fig. 7. Model for the binding of PG01037, NGB2904 and SB-277011A, U99194 with the ABCG2 transporter. XP-Glide predicted binding mode of PG01037 (A), NGB2904 (B), SB-227011A (C), and U99194 (D) with homology modeled ABCG2. Important amino acids are depicted as sticks with the atoms colored as: carbon ¼ green; hydrogen ¼ white; nitrogen ¼ blue; oxygen ¼ red; and sulfur ¼ yellow. The ligand PG01037 is shown with the same color scheme except for the carbon atoms, which are represented by yellow and chlorine atoms are represented by dark green. The red dotted lines represent hydrogen bonding. A schematic diagram of the proteineligand interaction is shown for PG01037 (E), NGB2904 (F), SB-227011A (G) and U99194 (H). Blue circles represent polar amino acids, green circles represent hydrophobic amino acids and purple arrows represent side chain donor/acceptor interactions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

N. Hussein et al. / Cancer Letters 396 (2017) 167e180

Discussion This study is the first to report that D3 receptor antagonists increase the efficacy of ABCG2 substrate cancer therapeutics. Specifically, the D3 receptor antagonists PG01037, NGB2904, SB-277011A, and U99194 significantly increase the efficacy of MX and DOX (ABCG2 substrates) in the ABCG2 over-expressing cancer cell lines H460-MX20, A549-MX10, and S1M1-80. Furthermore, at 5 mM, PG01037, NGB2904, SB-277011A, and U99194 significantly enhanced the efficacy of MX in a transfected wild-type ABCG2 overexpressing cell line, HEK293-R2. By contrast, the above listed D3 receptor antagonists did not significantly sensitize the parental cell lines H460, A549, S1, or HEK293, which do not overexpress ABCG2 transporters, to MX or DOX. This finding suggests that the D3 receptor antagonists selectively reverse ABCG2-mediated MDR. It is unlikely that the D3 receptor antagonists used in this study were producing toxicity alone as the concentrations used (0.1e10 mM) were all below concentrations that decreased cell viability. PG01037 and NGB2904, at a concentration of 0.1 mM, significantly reduced fold-resistance (e.g. the sensitized cell line compared to the non-sensitized cell line) in A549-MX10 cells from 6 to 1.8, and from 6 to 1.7, respectively. Both SB-277011A and U99194, in a concentration-dependent manner, significantly reduced fold-resistance and restored sensitivity in H460-MX20, S1M1-80, and A549-MX10 cell lines to MX. However, SB-277011A and U99194 were not as efficacious as PG01037 and NGB2904 in decreasing the resistance fold of MX in the same cell lines. Although it is unknown why NGB2904 and PG01037 are more efficacious than SB277011A and U99194A, these findings do appear to follow a similar pattern to aspects of the molecular docking analyses. Although U99194 has a significantly lower docking score than NGB2904 and PG01037, the docking score for SB277011A was not significantly different from NGB2904 and PG01037. Thus, factors other than affinity for the ABCG2 transporter may explain the differences in efficacy. The mode of binding was most similar between NGB2904 and PG01037, compared to SB277011A and U99194A. As previously reported, nilotinib significantly increased the efficacy of MX and DOX [59]. Nilotinib, at concentration of 1, and 2.5 mM, significantly reduces the fold-resistance of HEK293-R2 cells to MX and dox as compared with the FTC [59]. Previous studies have identified selective and efficacious ABCG2 modulators, but these compounds produced significant toxicity and/or drugedrug interactions, thereby preventing their use in the clinic [67]. For example, in vitro studies show that 5 mM FTC augments the efficacy of MX, DOX and topotecan by 93-, 26- and 24fold, respectively [68]. However, in vivo studies indicate that FTC is extremely toxic, thus precluding its clinical use [67]. Other studies have shown that nilotinib is an ABCG2 substrate and inhibitor [59,69]. Nilotinib binds and interacts with the ABCG2 transporter substrate binding sites and inhibits the efflux function of the ABCG2 transporter [69]. We postulated that D3 receptor antagonists could inhibit the ABCG2 transporter and/or be a substrate, based on structural homology to TKIs. However, MTT results alone cannot determine if a compound is an ABCG2 substrate. Mechanistic assays, such as the ATPase assay, photo-affinity labeling, and membrane vesicle uptake assays are necessary to ascertain if D3 receptor antagonists bind to the ABCG2 substrate sites. Numerous studies have reported that using drug combinations in cancer treatment has many advantages compared to use of a single drug [62,70,71]. The combination treatment data from the present studies presented here corroborate previous MTT results, where the most potent compounds in term of reversing ABCG2mediated MDR were NGB2904 and PG01037, which produced strong and very strong synergism, respectively. The combination of 10 mM of PG01037 and 0.1e100 mM of MX produces strong to very

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strong synergism. Similarly, the combination of 10 mM of NGB2904 with 1e30 mM MX produces strong to very strong synergism. The combination effects of both SB-277011A and U99194 ranged from synergism to strong synergism as well. However, the results obtained with combinations of 0.03e30 mM of DOX and 5 mM of the D3 receptor antagonists were more variable, producing effects ranging from synergism to antagonism. It is possible that using lower concentrations of the D3 receptor antagonists, which significantly augmented MX efficacy, may produce less variable results (i.e. synergism only). Another major finding of this study was that PG01037 and NGB2904 maintained concentrations of rhodamine 123, an ABCG2 substrate, in H460-MX20 cells by preventing drug efflux. In addition, as previously reported [72], 5 mM nilotinib also maintained intracellular levels of rhodamine 123. Moreover, the magnitude of the effects of NGB2904 and PG01037 on the maintenance of intracellular rhodamine levels was similar to what was observed with nilotinib treatment. These results, in combination with the docking analyses for NGB2904 and PG01037, suggest that these compounds surmount ABCG2-mediated resistance to DOX and MX by inhibiting the efflux function of the ABCG2 transporter. However, additional studies must be conducted to determine if the D3 receptor antagonists are binding directly to the efflux site of ABCG2 transporters or act through another mechanism. Our results with the D3 receptor antagonists are consistent with those reported for other compounds, such as Ko143 [73], novobiocin [74], elacridar [75], tariquidar analogue [76], imatinib [32], lapatinib [77], and nilotinib [59], which have been shown to inhibit ABCG2 efflux function in cell lines overexpressing ABCG2 transporters. The D3 receptor antagonists used in this study surmounted ABCG2-mediated resistance by downregulating the levels of the ABCG2 protein. PG01037 and NGB2904 were the most efficacious drugs at reversing ABCG2-mediated resistance to MX and DOX. Our results indicated that the incubation of H460-MX20 cells for 24 or 48 h with 5 mM of PG01037 or 5 mM of NGB2904 significantly decreased levels of the ABCG2 protein. Previous studies showed that one of the mechanisms responsible for reversing ABCG2 mediated MDR is the down regulation of the ABCG2 transporter [78e80]. For example, YHO-13177 and artesunate have shown to reverse ABCG2 mediated MDR through downregulation of ABCG2 [80,81]. There are data suggesting that c-jun N-terminal kinase, mitogen-activated protein kinases, phosphate and tensin homolog, epidermal growth factor receptor and human epidermal growth factor (EGFR), among others, mediate the expression of ABCG2 in various cancer cell lines [82]. PD153035 (an EGFR antagonists) could significantly decrease the expression level of ABCG2 at the transcription [83], as well as post-translational level, but further study is needed to determine how PD153035 downregulates ABCG2 expression. Perhaps PG01037 and NGB2904 decrease ABCG2 protein levels by inducing rapid internalization and degradation via lysosomes and/or proteasomes [84]. Finally, PG01037 and NGB2904 may decrease protein expression levels by decreasing ABCG2 gene transcription or decreasing mRNA stability. Clearly, future studies must be conducted in order to delineate the mechanism(s) by which PG01037 and NGB2904 downregulate ABCG2 protein expression. Our in vitro immunocytochemistry data also indicated that 5 mM of NGB2904 or PG01037 significantly decreased ABCG2 fluorescence in H460-MX20 cells after 48 h of incubation compared to cells incubated with vehicle. These results were congruent with the Western blot data indicating that 5 mM of NGB2904 or PG01037 significantly decreased the expression of the ABCG2 protein in H460-MX20 cells. Thus, the reversal of resistance to MX in cancer cells lines overexpressing ABCG2 transporters by the D3 antagonists used in this study may results

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from inhibition of the efflux activity of ABCG2, as well as decreased expression of the ABCG2 protein. The XP scores obtained from our docking analysis for NGB2904, PG01037, SB277011A and U99194A were 9.704, 9.299, 9068 and 6,525, respectively, It should be noted that the rank order of the docking scores for the D3 receptor antagonists used in this study was similar to the rank order of potency for the magnitude of reversal of resistance to MX in the MX-resistant cell lines. In addition, based on in vitro binding data for human receptors [85], the rank order of D3/D2 receptor selectivity for NGB2904, PG01037, SB-277011A and U99194 is similar to their rank order of potency for reversal of ABCG2-mediated resistance and the docking scores. U99194's low docking score for the ABCG2 transporter results from the absence of a number of the structural features in NGB2904, PG01037 and SB-277011A that contribute to their high affinity and selectivity for D3 receptors [86], and high affinity for ABCG2. Data indicate that depending on the context, autophagy can increase tumor growth [87]. Interestingly, a recent study has shown that autophagy elicited by certain stressors is augmented in cancer cell sublines that overexpress ABCG2 transporters [88]. In addition, in HeLa cells, ammonia increases autophagy via activation of dopamine D3 receptors and inhibition of mTOR [89]. Therefore, it is possible that the efficacy of the D3 receptor antagonists, in part, may result from a decrease in autophagy, which can contribute to a decrease in cancer cell survival. However, experiments must be conducted to verify the aforementioned hypothesis. In conclusion, the results presented here indicate that the D3 receptor antagonists PG01037, NGB2904, SB-277011A, and U99194 significantly reversed ABCG2-mediated MDR in both lung and colon cancer cell lines overexpressing ABCG2 transporters. This reversal effect produced by the compounds used in this study occurred at in vitro concentrations significantly below than those decreased cell viability. PG01037 and NGB2904 were the most potent in reversing ABCG2-mediated MDR. In addition, NGB2904 and PG01037 significantly inhibited the intracellular ABCG2 efflux function. Furthermore, both compounds significantly decreased the expression of ABCG2 protein levels. A synergistic response was obtained with the combination of the D3 receptor antagonists and the ABCG2 transporter substrates, MX and DOX. Tentatively, our results, if replicated in vivo, suggest that D3 receptor antagonists could be evaluated as adjuvant therapeutic compounds with traditional chemotherapeutics to improve the clinical response in the clinic. However, due to physiochemical and/or pharmacokinetic liabilities [90], D3 receptor antagonists in this study face challenges with their use in the humans. Finally, further studies, such as photo affinity labeling and ATPase assay, are needed to elucidate the interaction of D3 receptor antagonists with the ABCG2 transporter. Acknowledgments This research was funded by the University of Toledo start-up (F110760) to A.K.T. N.H. was financially supported by the higher committee for Education Development in Iraq (HCED) (http:// www.hcediraq.org/). We would like to thank Charisse Montgomery, University of Toledo for editorial assistance. Conflicts of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2017.03.015.

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