A novel epidermal growth factor receptor inhibitor for treating lung cancer

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1 698358TUB1.1177/ Tumor BiologyXia et al. research-article217 Original Article A novel epidermal growth factor receptor inhibitor for treating lung cancer Tumor Biology April 217: 1 5 The Author(s) 217 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: journals.sagepub.com/home/tub Jiansheng Gao, Yuli Liang, Dongying Zhang, Yi Wang, Jiamin Yang and Hua Liu Abstract To investigate the effects of a novel synthetic epidermal growth factor receptor inhibitor, COMPOUND789, on the inhibition of lung cancer growth in vitro and the underlying mechanisms, we treated three lung tumor cell lines (,, and ) with COMPOUND789 and a Food and Drug Administration approved epidermal growth factor receptor inhibitor. Then, we examined cell growth in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell survival in a Cell Counting Kit-8 assay, and cell apoptosis by Annexin V flow cytometry in the presence of fluorouracil. We found that compared to, COMPOUND789 inhibited cell growth more potentially and induced more cell death in the presence of fluorouracil. Thus, our study demonstrates that COMPOUND789 may be a promising epidermal growth factor receptor inhibitor for human lung cancer therapy. Keywords Epidermal growth factor receptor, lung cancer, inhibitors Date received: 28 July 216; accepted: 24 December 216 Introduction Lung cancer is a commonly occurred malignant cancer in humans. Most types of lung cancer are not sensitive to either chemotherapy or radiotherapy. 1 3 Fluorouracil (5-FU) treatment has been used as a supplementary treatment to surgical removal of the primary cancer, which improves the survival of the patients. 4 8 However, some lung cancers have been shown to be resistant to 5-FU treatment, and the molecular mechanisms underlying this phenomenon are not completely understood. Epidermal growth factor receptor (EGFR) signaling pathway plays a critical role in the growth and metastasis of lung cancer Epidermal growth factor (EGF) is one of the major ligands that binds to EGFR leading to receptor dimerization, resulting in receptor phosphorylation at tyrosine residues, leading to the activation of their downstream signaling, of which the mitogen-activated protein kinase (MAPK) pathways, the Jun N-terminal kinase (JNK) pathway, or the phosphatidylinositol 3-kinase (PI- 3K) pathway mediate signal transduction in different cell types. Since the discovery of protein kinase activity in 1954, many kinase inhibitors have been comprised and used in basic research and clinical practice. At present, first-line treatment with EGFR TKIs (, erlotinib, and afatinib) has been approved for patients who harbor exon 19 deletions or exon 21 (Leu858Arg) substitution EGFR mutations. These agents have been shown to substantially improve response rates, time to progression, and overall survival. Unfortunately, patients develop resistance, limiting patient benefit and posing a challenge to oncologists. Thus, novel and more potent EGFR inhibitors are in great need. 13 High-throughput docking is used for drug discovery since 2, which forecasts an optimized conformation for the protein and ligand molecule Hence, development of novel therapeutic treatments, for example, specific pathway inhibitors for lung cancer, is highly urgent, which may substantially improve the patients 5-year survival ratio. Department of Geriatrics, Fist Affiliated Hospital of Guangdong University of Pharmacy, Guangzhou 518, China Corresponding author: Hua Liu, Department of Geriatrics, Fist Affiliated Hospital of Guangdong University of Pharmacy, 19 Nonglinxia Road, Guangzhou 518, China. swj732@yeah.net Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4. License ( which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (

2 2 Tumor Biology In this study, we investigated the effects of a novel synthetic EGFR inhibitor, COMPOUND789, on the inhibition of lung cancer growth in vitro and the underlying mechanisms, using three lung tumor cell lines (,, and ). We examined cell growth in an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell survival in a Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis by Annexin V flow cytometry in the presence of 5-FU. Our study demonstrates that COMPOUND789 may be a promising EGFR inhibitor for human lung cancer therapy. Materials and methods Study approval All the experimental methods have been approved by the research committee at Union Hospital of Guangdong University of Pharmacy. All the experiments have been carried out in accordance with the guidelines from the research committee at Guangdong University of Pharmacy. Cell line and reagents Three human lung cancer cell lines (origin from carcinoma), (origin from adenocarcinoma), and (origin from non-small-cell lung cancer) were all purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). These three cell lines were used in this study, since they represent different types of lung cancer. Three cell lines were all cultured in Dulbecco s Modified Eagle s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) in a humidified chamber with 5% CO 2 at 37 C. 5-FU (Sigma-Aldrich) was prepared in a stock of 1 mmol/l and applied to the cultured cells at 1 µmol/l. Cell viability assay The CCK-8 detection kit (Sigma-Aldrich) was used to measure cell viability according to the manufacturer s instructions. Briefly, cells were seeded in a 96-well microplate at a density of ml 1. After 24 h, cells were treated with resveratrol. Subsequently, CCK-8 solution (2 ml/well) was added and the plate was incubated at 37 C for 2 h. The viable cells were counted by absorbance measurements with a monochromator microplate reader at a wavelength of 45 nm. The optical density value was reported as the percentage of cell viability in relation to the control group (set as 1%). Cell growth assay A MTT assay was performed to determine cell growth. An amount of 5 cells per well was seeded in a 96-well plate to allow the cells to grow. Then, the media were removed and washed with phosphate-buffered saline (PBS), after which 5 g/l of thiazolyl tetrazolium (Amresco, Indianapolis, IN, USA) was added to each well. After 4 h, MTT was removed and 15 µl of dimethyl sulfoxide (Sigma-Aldrich) was added. The viability of the cells was calculated from the absorption at 57/63 nm with an enzyme-linked immunosorbent assay reader. Apoptosis assay by flow cytometry For analysis of cell apoptosis, the dissociated cultured cells were re-suspended at a density of 1 6 cells/ml in PBS. After double staining with fluorescein isothiocyanate (FITC)- Annexin V and propidium iodide (PI) from an FITC-Annexin V Apoptosis Detection Kit I (Becton-Dickinson Biosciences, San Jose, CA, USA), cells were analyzed using FACScan flow cytometer (Becton-Dickinson Biosciences) equipped with CellQuest software (Becton-Dickinson Biosciences) for determination of Annexin V+ PI apoptotic cells. Statistical analysis All data were statistically analyzed using one-way analysis of variance (ANOVA) with a Bonferroni correction, followed by Fisher s exact test for comparison of two groups (GraphPad Prism, GraphPad Software, Inc., La Jolla, CA, USA). All values are depicted as mean ± standard deviation and are considered significant if p <.5. Results Self-docking study Self-docking was carried out on the X-ray structure using the Surflex-Dock in geom mode. After virtual screening, the top compound named COMPOUND789 was chosen (Figure 1). Next, we compared the biological effects of COMPOUND789 with a Food and Drug Administration (FDA)-approved EGFR inhibitor on the growth and survival of lung cancer cells with and without the presence of 5-FU. Figure 1. Self-docking study. Self-docking was carried out on the X-ray structure using the Surflex-Dock in geom mode. After virtual screening, the top compound named COMPOUND789 was chosen.

3 Xia et al. 3 (a) COMPOUND Time (hours) (b) COMPOUND Time (hours) (c) COMPOUND Time (hours) Figure 2. Anti-proliferative potential of COMPOUND789 in lung cancer cells. (a c) To investigate the effects of COMPOUND789 on the inhibition of lung cancer growth in vitro and the underlying mechanisms, we treated three lung tumor cell lines (,, and ) with COMPOUND789 and an FDA-approved EGFR inhibitor. Then, we examined cell growth in an MTT assay using (a), (b), and (c) cells (p <.5, N = 5).

4 4 Tumor Biology Anti-proliferative potential of COMPOUND789 in lung cancer cells To investigate the effects of COMPOUND789 on the inhibition of lung cancer growth in vitro and the underlying mechanisms, we treated three lung tumor cell lines (,, and ) with COMPOUND789 and an FDA-approved EGFR inhibitor. Then, we examined cell growth in an MTT assay. We found that compared to, COMPOUND789 inhibited cell growth more potentially in (Figure 2(a)), (Figure 2(b)), and cells (Figure 2(c)). These data demonstrate the cellular anti-proliferative activities of COMPOUND789 in lung cancer cells. COMPOUND789 decreases lung cancer cell survival upon 5-FU treatment Next, we examined lung cancer cell survival in a CCK-8 assay in the presence of 5-FU. We found that compared to, COMPOUND789 reduced cell survival more potentially in,, and cells (Figure 3). These data suggest that COMPOUND789 may decrease lung cancer cell survival upon 5-FU treatment. COMPOUND789 decreases lung cancer cell survival upon 5-FU treatment by increasing apoptosis To understand the mechanisms underlying the effects of COMPOUND789 on cell survival in the presence of % cell viability at 1µmol/l 5-FU 1 5 COMPOUND789 Figure 3. COMPOUND789 decreases lung cancer cell survival upon 5-FU treatment. We examined lung cancer cell survival in a CCK-8 assay in the presence of 5-FU (p <.5, N = 5). (a) 1 % Apoptosis of cells at 1µmol/l 5-FU 5 COMPOUND789 (b) COMPOUND789 Cell counts apoptotic cells apoptotic cells apoptotic cells Figure 4. COMPOUND789 decreases lung cancer cell survival upon 5-FU treatment by increasing apoptosis. To understand the mechanisms underlying the effects of COMPOUND789 on cell survival in the presence of 5-FU, we performed an Annexin V flow cytometry assay. (a and b) We found that compared to, COMPOUND789 induced more apoptotic cell death in the presence of 5-FU, shown by (a) quantification and (b) representative flowcharts (p <.5, N = 5).

5 Xia et al. 5 5-FU, we performed an Annexin V flow cytometry assay. We found that compared to, COMPOUND789 induced more apoptotic cell death in the presence of 5-FU, shown by quantification (Figure 4(a)) and by representative flowcharts (Figure 4(b)). Thus, our study demonstrates that COMPOUND789 may be a promising EGFR inhibitor for human lung cancer therapy. Discussion EGFR signaling pathway has been shown to be highly activated in lung cancers and plays a critical role in tumor proliferation and metastasis. Hence, selective Inhibition of EGFR signaling pathway appears to be a promising strategy for future tumor therapy. Thus, in our study, we identified COMPOUND789 as a critical EGFR inhibitor that has a potent effect on lung cancer growth and susceptibility to chemotherapy, as treatment with kinase inhibitors has demonstrative anti-cancer effects including anti-proliferation, cell cycle arrest, and apoptosis. 17 Compared to an FDA-approved EGFR inhibitor, COMPOUND789 showed greater effects on cell growth arrest in an MTT assay, greater decreases in cell survival in an CCK-8 assay by 5-FU, possibly by augmentation of the cell apoptosis, and decrease in cell proliferation. The combination of these effects resulted in a significant enhancement of the anti-cancer effect. In future, the effects of this compound in vivo may be tested in a proper lung cancer animal model to evaluate whether it is similarly effective inside the body as in vitro. Thus, we present a novel compound, which is a potential EGFR inhibitor, which appears to be a better choice than in treating lung cancer. COMPOUND789 may be a promising EGFR inhibitor for human lung cancer therapy. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding This work was supported by Guangdong Province Natural Scientific Fund (no. S211121). References 1. Mitsudomi T, Suda K and Yatabe Y. Surgery for NSCLC in the era of personalized medicine. Nat Rev Clin Oncol 213; 1: Pallis AG and Syrigos KN. Epidermal growth factor receptor tyrosine kinase inhibitors in the treatment of NSCLC. Lung Cancer 213; 8: Zarogoulidis K, Zarogoulidis P, Darwiche K, et al. Treatment of non-small cell lung cancer (NSCLC). J Thorac Dis 213; 5: S389 S Zhao Z, Han F, Yang S, et al. Oxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells: involvement of the Akt-mTOR signaling pathway. Cancer Lett 215; 358: Ge J, Chen Z, Huang J, et al. Upregulation of autophagyrelated gene-5 (ATG-5) is associated with chemoresistance in human gastric cancer. PLoS ONE 214; 9: e Hosogi S, Kusuzaki K, Inui T, et al. Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell. J Cell Mol Med 214; 18: Liu M, Li CM, Chen ZF, et al. Celecoxib regulates apoptosis and autophagy via the PI3K/Akt signaling pathway in SGC-791 gastric cancer cells. Int J Mol Med 214; 33: Tang C, Yang L, Jiang X, et al. Antibiotic drug tigecycline inhibited cell proliferation and induced autophagy in gastric cancer cells. Biochem Biophys Res Commun 214; 446: Jassem J and Dziadziuszko R. EGFR inhibitors for wildtype EGFR NSCLC: to use or not to use? Lancet Oncol 213; 14: Stella GM, Scabini R, Inghilleri S, et al. EGFR and KRAS mutational profiling in fresh non-small cell lung cancer (NSCLC) cells. J Cancer Res Clin Oncol 213; 139: Kim S, Choi JH, Lim HI, et al. EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway, but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line. Cell Signal 29; 21: Pei J, Lou Y, Zhong R, et al. MMP9 activation triggered by epidermal growth factor induced FoxO1 nuclear exclusion in non-small cell lung cancer. Tumour Biol 214; 35: Tan CS, Gilligan D and Pacey S. Treatment approaches for EGFR-inhibitor-resistant patients with non-small-cell lung cancer. Lancet Oncol 215; 16: e447 e Hospital A, Goni JR, Orozco M, et al. Molecular dynamics simulations: advances and applications. Adv Appl Bioinform Chem 215; 8: Ferreira LG, Dos Santos RN, Oliva G, et al. Molecular docking and structure-based drug design strategies. Molecules 215; 2: Rao CM, Yejella RP, Rehman RS, et al. Molecular docking based screening of novel designed chalcone series of compounds for their anti-cancer activity targeting EGFR kinase domain. Bioinformation 215; 11: Lokadasan R, James FV, Narayanan G, et al. Targeted agents in epithelial ovarian cancer: review on emerging therapies and future developments. Ecancermedicalscience 216; 1: 626.