Comparative anti-proliferative effects of potential HER2 inhibitors on a panel of breast cancer cell lines

Comparative anti-proliferative effects of potential HER2 inhibitors on a panel of breast cancer cell lines
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  Vol.:(0123456789)  1 3 Breast Cancer ORIGINAL ARTICLE Comparative anti-proliferative effects of potential HER2 inhibitors on a panel of breast cancer cell lines Hiba Zalloum 1  · Tuka AbuThiab 1  · Tareq Hameduh 1  · Sara AlBayyari 1  · Waleed Zalloum 2  · Basha’er Abu-Irmaileh 1  · Mohammad S. Mubarak  3  · Malek Zihlif  4   Received: 27 March 2019 / Accepted: 14 September 2019 © The Japanese Breast Cancer Society 2019 Abstract Background  Breast cancer is one of the most lethal types of cancer in women worldwide. The human epidermal growth factor receptor 2 (HER2) is considered as a validated target in breast cancer therapy. Previously, we have used quantitative structure activity relationship QSAR equations and their associated pharmacophore models to screen for new promising HER2 structurally diverse inhibitory leads which were tested against HER2-overexpressing SKOV3 ovarian cancer cell line. Objective  In this study, we sought to explore the effect of most active ligands against different normal and breast cancer cell lines that represent different breast cancer subtypes with distinguished expression levels in HER2 and HER1. Methods  We have tested the promising compounds against SKBR3, MDA-MB-231, MCF7, human fibroblast, and MCF10 cell lines. To understand the inhibitory effects of the active ligands against HER2 over expressed breast cancer cell lines, all inhibitors and the control compound, lapatinib, were docked into the active site of HER2 enzyme performed using Ligand Fit docking engine and PMF scoring function. Results  Five ligands exhibited promising results with relatively low IC 50  values on cells that amplify HER2 and high IC 50  on those that do not express such a receptor. The most potent compound (compound 13) showed an IC 50  of 0.046 µM. To test their toxicity against normal cells, the active compounds were tested against both normal fibroblast and normal breast cancer cell MCF-10 and relatively high IC 50  values were scored. The IC 50  values on HER2 over-expressed breast cancer and normal fibroblast cells provided a promising safety index. Docking results showed the highest similarity in the binding site between the most active ligand and the lapatinib. Conclusion  Our pharmacophore model resulted in a high potent ligand that shows high potency against HER2 positive breast cancer and relatively low toxicity towards the normal human cells.   *  Hiba Zalloum;  *  Malek Zihlif 1  Hamdi Mango Research Center for Scientific Research, The University of Jordan, Amman 11942, Jordan 2  Department of Pharmacy, Faculty of Health Science, American University of Madaba, P.O. Box 2882, Amman 11821, Jordan 3  Department of Chemistry, Faculty of Science, The University of Jordan, Amman 11942, Jordan 4  Department of Pharmacology, Faculty of Medicine, The University of Jordan, Amman 11942, Jordan   Breast Cancer  1 3 Graphic abstractKeywords  Human epidermal growth factor receptor-2 (HER2) · Breast cancer · Docking · Cell line Introduction Breast and many other human cancers are distinguished by an overexpression of proteins that play a vital role in cell proliferation, division, differentiation, and survival [1]. HERs, tyrosine kinase receptor family members which include HER1/ErbB1, HER2/  neu  /ErbB2, HER3/ErbB3, and HER4/ErbB4 [2], are a transmembrane glycoprotein recep-tors that contain an intracellular tyrosine kinase domain. Human epidermal growth factor receptor-2 (HER2) is an “orphan receptor”; a receptor that does not contain a spe-cific ligand. Instead, it forms heterodimers with other mem-bers for signal transduction [1, 3]. The gene of this protein (HER2/neu)  is found to be amplified in 30% of breast can-cers and a variety of cancers such as ovarian [2, 4], stomach, lung (especially lung adenocarcinomas) [2, 5–7] prostate [2, 6, 8], and biologically aggressive forms of uterine cancer [9, 10]. HER tyrosine kinase receptor family members are acti-vated upon binding of their ligands to the extracellular domain, in case of HER1 and HER3, or upon overexpres-sion of HER2 receptors on the surface of cells [11–14]. This binding induces conformational changes on the extracellu-lar domain and subsequent homodimers (HER2/HER2) and heterodimers (HER2/HER1 and HER2/HER3) formation [3, 11, 15]. Dimerization then activates the intrinsic auto- phosphorylation and/or transphosphorylation activity of their intracellular tyrosine kinase domains and leads to the phosphorylation of a specific tyrosine in the COOH-termi-nus [3, 4, 13, 16]. Moreover, these phosphorylated tyrosines provide binding sites for subsequent downstream signaling molecules which expose the antiapoptotic and malignant behavior of breast cancer cells [4, 15, 17]. Breast cancer subtypes have different expression patterns for ErbB receptors [18, 19]. MCF7 breast cancer cell line is a triple negative subtype, which only overexpresses HER1, while SKBR3 cells have an amplification and overexpres-sion of HER2 [20, 21]. Table 1 shows the molecular and morphological features of breast cancer cell line subtypes used in this study.Chemotherapy was found to be an inefficient treatment for HER2-overexpressing tumors. In vitro studies proved the resistance of such tumors to chemotherapy treatment [22, 23]. In this context, therapies targeting HER2 have Table 1 Molecular and morphological features of breast cancer cell line subtypesThe protein markers used for the identification of each breast cancer cell line subtype, as well as their morphological differences are sum-marizedCell line subtypeProteinMorphologyMCF7 (Luminal)ER, GATA3, KRT19 [51]More differentiated; tight cell–cell  junctions [52]SKBR-3 (HER2 positive)HER2Breakdown of cell–cell junctions [53]MDA-MB-231, triple nega-tive A (basal A)EGFRKRT5/6CD10MET [51]Core basal-like [52]  Breast Cancer  1 3 been considered a promising way to control HER2-overex-pressing breast cancers [24, 25]. Such therapies regulate the non-stop cell proliferation and recorded an improvement of chemotherapy efficacy [26, 27]. Trastuzumab [26] is the first approved humanized monoclonal antibody that targets the extracellular domain of HER2 [11, 28]. It is used in HER2- positive breast cancers [29, 30] and has proved its efficacy against in adjuvant setting and in the metastatic disease [26, 31]. Trastuzumab improves the overall survival rates and reduces cancer recurrence [11, 32, 33]. Lapatinib is a small dual intracellular tyrosine kinase inhibitor for epidermal growth factor and HER2 [28, 34, 35]. It is a reversible com- petitive inhibitor that competes with the adenosine triphos-phate (ATP) to the ATP-binding domain of HER2 receptor, preventing the phosphorylation of the intracellular domain of HER2 and subsequent activation of downstream AKT and MAPK pathways [28, 36, 37]. Interestingly, a large fraction of patients showed a resistant behavior to trastuzumab or lapatinib alone, suggesting that combining these two treat-ments adjuvant to chemotherapy would be promising and more beneficial for HER2-positive breast cancer patients [36, 38, 39]. In our initial work, we used quantitative structure activity relationship (QSAR) equations and their associated pharma-cophore models to screen the national cancer institute (NCI) list of compounds and Drug Bank database to search for new promising HER2 structurally diverse inhibitory leads. Inhibitory activities of the resulted compounds were tested against HER2-overexpressing SKOV3 ovarian cancer cell line and promising IC 50  values were scored [40, 41]. Accord- ingly, the present work aims to explore the effect of the most active ligands previously tested against SKOV3 ovarian cells on different normal and breast cancer cell lines that represent different breast cancer subtypes with distinguished expres-sion levels of HER2 and HER1. Materials and methods Cell lines and cell culture SKBR3, MDA-MB-231, MCF7, human fibroblast, and MCF10 were obtained from the American Type cul-ture collections (ATCC) and were cultured in McCoy’s, DMEM, RPMI, DMEM, and MEBM, respectively. Media were obtained from Euroclone (Itlay) except MEBM was obtained from Lonza (Basel, Switzerland). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco Invitrogen), 1% of 2 mM L -glutamine (Lonza), 50 IU/mL penicillin (Lonza), and 50 μg/mL streptomycin (Lonza). Cells were maintained at 37 °C, 5% CO 2  humidi-fied incubator. MCF10 medium was obtained from Lonza Corporation as a kit: MEGM, Kit Catalog no. CC-3150. 100 ng/ml cholera toxin obtained from Lonza was added to the MCF10 medium (Basel, Switzerland). Cell proliferation assay MCF-7, MDA-MB-231, and MCF10 cells were seeded at a density of 1 × 10 7  per well in 96-well plates in appropri-ate medium. Human fibroblast was seeded at a density of 1 × 10 5  per well. For the IC 50  determination, cells were treated with increasing concentrations of the tested com-pound (0.39–100 µM). In all assays, the drugs were dis-solved in DMSO immediately before addition to cell cul-tures and equal amounts of solvent were added to control cells. Concentration of the DMSO never exceeded 0.5%. Cell viability was measured after 3 days (72 h) of treatment with tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT), acquired from Promega (Wisconsin, US). IC 50  concentrations were obtained from the dose–response curves using Graph Pad Prism Software 5 (San Diego California USA, https ://www.graph Structure preparation The 2D chemical structures of compounds 7 , 12 , 13 , 26 , and 28  were sketched in Chemdraw Ultra (12.0.2) and saved in MDL molfile format. Subsequently, a group of energeti-cally accessible conformers was generated using OMEGA2 software [42]. OMEGA rapidly generates conformational ensembles of small molecules using a fragment-based library to build initial models of structures by assembling these fragment templates followed by rule-based torsion search stage. Generated conformers are saved in SD format. Docking experiments The 3D geometry of HER2 enzyme was retrieved from the Protein Data Bank (PDB code: 3pp0, resolution; 2.25 Å) [37]. No energy minimization for the protein structure was done. The docking study was conducted in the presence of explicit water molecules. Inhibitors were docked into the binding pocket of HER2 using hybrid module of Openeye OEDocking software, which needs a target protein structure, a pre-generated multi-conformer database of ligands, a box defining the active site of the protein, and several optional parameters as its input parameters. Ligand conformers and protein structure are treated as rigid entities during the dock-ing process. The ligand in the X-ray structure was a selec-tive HER2 inhibitor. A very close pose of the co-crystalized inhibitor was reproduced by the used docking parameters which ensures the quality of the docking procedure.   Breast Cancer  1 3 Fig. 1 The chemical structure of tested hits (1–30) 1 23 45 67 8 ClOH NHH NClOHOH N N NOFFF FS N N NOHFF ClClHNHNClCl Cl NH N N NHN NCl NClNNHONClONHOHO  N N NH N NH N N NHH N N O OHOHOOO N NOHO OOHOO 9 10 OHOOOHO N NOOHOOH Cl N N NClH 2  N NOH HO N NH OHNO NHO O 11 12  Breast Cancer  1 3 13 1415 16 O N NOOO NClClO ClCl N N NH 2  N NH 2 ClO N NHHO N N  NClS N NHO 17 1819 2021 ClCl NH NH N NH NHO NH N ClCl NH NH N N NH NHN  NH N NHO O NSH N N N  N N NH NH N NO + O + Fig. 1 (continued)
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