Research paper Discovery of VEGFR-2 inhibitors exerting significant anticancer activity against CD44þ and CD133þ cancer stem cells (CSCs): Reversal of TGF-b induced epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma Siddharth J. Modi, Vithal M. Kulkarni * Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth (Deemed to be University), Pune, 411038, Maharashtra, India article info Article history: Received 25 May 2020 Received in revised form 2 September 2020 Accepted 13 September 2020 Available online 18 September 2020 Keywords: Hepatocellular carcinoma (HCC) Hep G2 Cancer stem cells (CSCs) VEGFR-2 Angiogenesis Epithelial-mesenchymal transition (EMT) abstract Hepatocellular carcinoma (HCC) is a malignancy characterized by neoangiogenesis, which is an augmented production of proangiogenic factors by the tumor and its adjacent infected cells. These dysregulated angiogenic factors are the therapeutic targets in anti-angiogenic drug development. The signaling pathway of vascular endothelial growth factor (VEGF)/VEGFR-2 is crucial for controlling the angiogenic responses in endothelial cells (ECs). In this study, we carried out a rational drug design approach wherein we have identified the novel orally bioavailable compound VS 8 as a potent VEGFR-2 inhibitor, which remarkably suppresses hVEGF and hVEGFR-2 expression in HUVECs and exhibits sig- nificant anti-angiogenic effects in CAM assay. Besides, VS 8 significantly induces apoptosis in HCC cell line (Hep G2). Later we examined its effectiveness against CD44þ and CD133þ CSCs. Here, VS 8 was found to be active against CSCs, and adequate for the cessation of the cell cycle at ‘G 0 /G 1 ’ and ‘S’ phase in CD44þ and CD133þ CSCs respectively. Factually, transforming growth factor-b (TGF-b) stimulated epithelial-mesenchymal transition (EMT) induces invasion and migration of HCC cells, which results in the metastasis. Therefore, we studied the effect of VS 8 on EMT markers using flow cytometry, which suggested that VS 8 significantly upregulates E-cadherin (epithelial biomarker) and downregulates vimentin (mesenchymal biomarker). Further, VS 8 downregulates the expression of EMT-inducing transcription factors (EMT-TFs), i.e., SNAIL. Altogether, our findings indicate that VS 8 could be a promising drug candidate for cancer therapy. © 2020 Elsevier Masson SAS. All rights reserved. 1. Introduction Regulated angiogenesis has a climacteric role in embryonic development and the female fertilization process through cell growth, tissue regeneration, and wound healing [1]. However, dysregulated angiogenesis is a critical hallmark of cancer and tu- mor growth. Newly formed blood vessels invade tumors, furnish them with O 2 and supplements, and promote the metastasis [2]. Thus, targeting dysregulated angiogenesis can be a functional and persuasive therapy to inhibit neoplasm as well as metastasis, which is better than the existing chemotherapy that causes adverse effects on the healthy cells and leads to the drug resistance in the longer run. Growth factors, viz. VEGF, PDGF, FGF, and cytokines take part in angiogenesis [3]. Amidst different growth factors, VEGFs (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PLGF) and their respective cell sur- face tyrosine kinases (VEGFR-1; Flt-1, VEGFR-2; KDR, and VEGFR-3; Flt-4) exhibit a remarkable impact on the angiogenesis [4]. These VEGFRs reside on the exterior of vascular endothelial cells, lymph vessels, and hematopoietic stem cells [5]. Among three VEGFRs, overexpression of VEGFR-2 is prominent in the various cancers, viz. HCC, RCC, NSCLC, TNBC, breast cancer, bladder cancer, and cervical cancer, thus VEGFR-2 is a recognized target for the discovery of anti-angiogenic agents to prevent tumor progression [6e12]. There are three types of VEGFR-2 inhibitors: ATP competitive inhibitors (type I), allosteric inhibitors (type II), and covalent inhibitors (type III) [13]. Among these, type II inhibitors such as Sorafenib, Regor- afenib, Carbozantinib, Lenvatinib, and Lucitanib demonstrate clin- ical benefits in VEGFR-2 inhibition [14, 15]. Notably, VEGFR-2 * Corresponding author. E-mail address: vmkulkarni60@gmail.com (V.M. Kulkarni). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech https://doi.org/10.1016/j.ejmech.2020.112851 0223-5234/© 2020 Elsevier Masson SAS. All rights reserved. European Journal of Medicinal Chemistry 207 (2020) 112851