ABSTRACT
Background and Purpose: Metastasis in breast cancer is a leading cause of mortality among women in many countries. This study investigated the anti-cancer role of benzoimidazoquinazoline and benzimidazotriazin; two novel compounds that were designed, synthesized, structurally elucidated, and biologically evaluated as potent anti-angiogenic agents that act through inhibition of vascular endothelial growth factor receptor-2 (VEGFR2). A model of breast cancer was induced by inoculation of Ehrlich Ascites Carcinoma (EAC) cells.
Experimental Approach: Seventy swiss albino mice were randomly divided into 7 groups, 10 animals each: (1) normal, (2) control EAC group, (3) cisplatin treated group, (4&5) benzoimidazoquinazoline treated (5mg/kg and 10mg/kg), (6&7) benzimidazotriazin treated (5mg/kg and 10 mg/kg). The expression of miRNA-122 was assessed in the tumor tissue by quantitative PCR, and the VEGF level was determined in serum by ELISA. VEGFR2 and cluster of differentiation (CD)34 were assessed by immunohistochemistry. Serum levels of ALT, AST, creatinine, and urea were measured.
Key Results: Treatment with benzoimidazoquinazoline and benzimidazotriazin caused a decrease in tumor weight and a significant decrease in the serum levels of VEGF and the expression of VEGFR2 and CD34 in the tumor tissue. MiRNA-122 was significantly upregulated especially in the group treated by benzimidazotriazin (10mg/kg). Interestingly, the new compounds had less renal toxicity compared to cisplatin.
Conclusion and Implication: The designed small molecules are promising anti-cancer candidates that act through inhibition of angiogenesis and can provide a new strategy for the advancement of chemotherapy through modulation of miRNA.
Key words : Breast cancer; Angiogenesis; VEGF; VEGFR-2; miRNA-122; Benzoimidazoquinazoline; Benzimidazotriazin.
INTRODUCTION
Breast cancer is the most widespread cancer among women around the world, affecting more than 2 million new cases and resulting in 600,000 deaths in 2018 (Bray et al., 2018 ). Despite the increased number of patients treated with surgery, radiotherapy or chemotherapeutics, 5-11% of patients exist with metastatic disease (Tao et al., 2017 ), and a large number of early breast cancer patients have a micro-metastatic disease resistant to systemic treatment. These patients may eventually relapse. New treatment strategies of breast cancer are required (Colozza et al., 2007).
Angiogenesis and lymphangiogenesis stimulate the formation of new blood and lymph vessels from pre-existing vasculature. They occur through the proliferation, migration, and maturation of nearby blood or lymph vessel endothelial cells (Lala et al., 2018 ). Angiogenesis is a basic biological process that is essential for development, reproduction, and wound repair. Angiogenesis is a key feature and distinctive marker of cancer (Otrock et al., 2007 ). Since tumors cannot grow to 1–2 mm without an adequate blood supply, angiogenesis is fundamental for uncontrolled growth of tumors to supply of adequate oxygen and nutrients (Lou et al., 2017 ) Similarly, lymphangiogenesis is involved in many cancer types, including breast cancer. The primary sites of metastasis in breast cancer are often the regional lymph nodes, where lymphangiogenesis can facilitate the migration of tumor cells to these sites (Ran et al., 2010 ) In the last two decades, vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have been identified as the key drivers of lymphangiogenesis and angiogenesis in the vascular systems (Shibuya et al., 2011 ) The VEGF signaling pathway is a crucial regulator in many tissues and plays a vital role in the pathogenesis of cancer, cardiovascular and intraocular neovascular diseases.
VEGFRs play a vital role in vasculogenesis and angiogenesis in embryonic development, wound repair, menstruation, and pregnancy. VEGFR-2, a member of the VEGFR family, helps in the generation of new blood vessels from existing tumor mass. Disruption of VEGFR-2 signaling has resulted in inhibition of angiogenesis and prevention of oxygen, nutrients from tumor cells, and decreasing clearance of catabolic products from tumor cells (Ferrara et al., 2010). Inhibition of VEGF/VEGFR signaling cascade has emerged as an attractive therapeutic tactic for inhibition of tumor angiogenesis and tumor proliferation (Ferrara et al., 2010, Olsson et al, 2006 ) Additionally, the cluster of differentiation (CD) 34 is a specific biomarker of cellular vascular endothelium. CD34 is especially sensitive to tumor angiogenesis, as it can clearly identify the condition of neovascularization during tumor growth (Zorgetto, 2013 ). In this manner, CD34 and VEGF are two significant markers of tumor angiogenesis and the relationship between the expression of CD34 and VEGF and the pathological attributes of patients with malignancy have been recently reported (Chen et al., 2015 ) Many molecules have been implicated in angiogenesis regulation. Among them, VEGF is the most pivotal angiogenic factor and their expression is regulated by many factors such as hypoxia-inducible factors, chemokines, and microRNAs (Olsson et al, 2006, Zhong et al., 2006)
MicroRNAs (miRNAs) are small non‑coding RNA molecules ~22 nucleotides in length (Hu et al., 2017 ) MiRNAs play a major role in the regulation of the expression of 30–60% of human genes and are considered as important modulators of cell differentiation, proliferation, cell-cycle progression, epithelial-mesenchymal transition, angiogenesis, stem cell renewal, apoptosis and cell migration, invasion, and metastasis (Hamam et al., 2017 ) Imbalance in miRNA expression is associated with several diseases such as cancers, where they can act as promoters or suppressors of tumorigenesis (Hamam et al., 2017, Song et al., 2013 ). Many miRNAs have been reported to be downregulated in malignant tumors including lung, breast, ovarian, bladder and colon. Dysregulation in the miRNA machinery is a plausible cause for the beginning and evolution of human cancers (Svoronos et al., 2016, Kian et al., 2018 ). MiRNAs can be biological markers for diagnosis, follow-up, and prognosis prediction of cancer patients. miRNA‑122 is downregulated in many types of cancer, including liver, breast and renal cancer (Pan et al., 2016, Ahsani et al., 2017, Maierthaler et al., 2017 ) In breast cancer, miRNA-122 was suggested to act as a tumor suppressor and to inhibit the tumorigenesis through targeting insulin-like growth factor 1 receptor (IGF1R) and regulating PI3K/Akt/mTOR/p70S6K pathway (Wang et al., 2012 )
Since VEGF/VEGFR signaling pathway and miRNAs have been utilized as a valuable target for cancer management, developing novel molecules with dual activity is important. Inhibition of VEGFR occurs through two mechanisms. The first mechanism is to block ligand binding to the extracellular domain with monoclonal antibodies (e.g. bevacizumab). The other mechanism relies on using small-molecule receptor tyrosine kinase (TK) inhibitors that function at intracellular domain. Gefitinib, Sorafenib, pazopanib, and Sunitinib are 4-anilinoquinazoline derivatives which competitively bind to the adenosine triphosphate (ATP) binding pocket of intracellular VEGFR domain (Fig 1) , thereby inhibiting downstream signaling essential for tumor survival and proliferation (Musumeci et al., 2012, Fan-Wei et al., 2017 ) Despite high response rate of patients to these agents, drug resistance, which occurs because of a secondary mutation, limits the therapeutic benefits of these drugs. Therefore, development of second-generation VEGFR tyrosine kinase inhibitors through the discovery of new scaffold can be beneficial for gefitinib-resisting patients.
Modulations to the central core pyridine ring becomes a feature of the medicinal chemistry strategy to look for potency, structure-activity relationship (SAR), and reducing log p of TK inhibitors. A range of heteroatoms have been reported to provide alternatives to the pyridine and/or pyrimidine ring of Sorafenib and Pazopanib. Amongst these rings, quinazoline and naphtamide derivatives, Furo[2,3-d] pyrimidines, pyridinyltriazines, and pyrimidinylindazoles have been reported as VGEFR-2 inhibitor.24 In the current study, diverse structure modifications in hit compounds have been studied to improve its potency against VEGFR. On another hand, unusual regulation of microRNAs has been connected to different human diseases, in particular cancer. Small molecule mediation of microRNA misregulation thus can give new therapeutic approaches to manage such disease (Holmes et al., 2007 ). In this study, the effect of two novel compounds, benzoimidazoquinazoline and benzimidazotriazin in treatment of breast cancer induced in mice was studied. Their effect on the expression of miRNA-122, CD34, VEGFR, and VEGF in the tumor tissues was assessed.
  1. EXPERIMENTAL SECTION
  2. Chemicals and reagents:
The synthesized compounds used in treatment (benzoimidazoquinazoline and benzimidazotriazine) were provided by the medicinal chemistry department and the complete physicochemical data are provided in supporting information. Cisplatin was purchased from ”EIMC united pharmaceuticals, Badr City, Cairo, Egypt”. Disodium EDTA was bought from Alpha Chem (USA). Phosphate buffered saline (PBS) was purchased from BioWhittaker® Lonza (Switzerland). Monoclonal mouse antibodies for CD34 VEGFR-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Synthesis of designed compounds
Benzoimidazoquinazoline (C1) and benzimidazotriazine (C2) molecules were synthesized for initial testing as a promising anticancer lead molecule modulating the activity of VEGFR, CD34, and microRNA-122; full synthesis and characterization details are given in the Supporting Information . o -Phenylenediamine was refluxed with an equimolar concentration of anthranilic acid producing 2-(1H-benzo[d]imidazol-2-yl) aniline in high yield. The 2-(1H-benzo[d]imidazol-2-yl) aniline then was condensed with carbon disulfide in KOH producing a cyclized benzo[4,5]imidazo[1,2-c]quinazoline-6-thiol. The free thiol was reacted with chloroacetylchloride under basic condition to release S-(benzo[4,5]imidazo[1,2-c]quinazolin-6-yl) 2-chloroethanethioate in reasonable yield. Finally, chloroethanethioate derivative was condensed in basic media with dichloroaniline giving the target compound (C1). Similarly, o -phenylenediamine was condensed in acidic media with 2-mercaptoacetic acid releasing (1H-benzo[d]imidazol-2-yl)methanethiol in good yield, which then refluxed for 20 hr. with an excess concentration of hydrazine hydrate in ethanol producing 2-(hydrazinylmethyl)-1H-benzo[d]imidazole. The hydrazine derivative was cyclized producing imidazo[1,2-d][1,2,4]triazine after condensation with trimethoxymethane. Lastly, the cyclized triazine was condensed with salicylaldehyde producing compound C2 in good yield. The complete reaction steps, condition, and yields are found in Scheme-1 .
Tumor cells:
Human breast cancer cell line MCF-7 and Ehrlich Ascites Carcinoma (EAC) cells were purchased from Tumor Biology Department, National Cancer Institute, Cairo University. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 µg/ml gentamycin. All cells were maintained at 37ºC in a fully humidified air atmosphere containing 5% CO2 and were sub-cultured two times a week.
EAC is a murine spontaneous breast cancer that served as the original tumor from which an ascites variant was obtained. On intraperitoneal inoculation, an ascitic fluid rich in tumor cells was produced. The tumor cell line was maintained in our laboratory by serial intraperitoneal passages into Swiss female albino mice at 7-10 days interval.
Experimental Animals:
Seventy swiss albino mice weighing 25-30g were obtained from the Egyptian Organization for Biological Products and Vaccines (Vacsera, Egypt). Animals were housed in plastic cages with mesh floor and hardwood bedding. They were kept under controlled laboratory conditions with normal light/dark cycle at 25-30°C. Food and water were provided ad libitum during the study period. Mice were left to acclimatize for 1 week before the experiments. All animal procedures and experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory animals. The study was approved by the ethical committee of Faculty of Pharmacy, Suez Canal University (201704AM1)
In vitro study:
Anticancer activity of imidazothiazole and pyridazobenzimidazole was tested in the MCF-7 cell line by using HTScan® VEGF Receptor 2 Kinase Assay Kit. The kit provides a means of performing kinase activity assays with recombinant human VEGFR-2 kinase. It includes active VEGFR-2 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine antibody for detection of the phosphorylated form of the substrate peptide. Products Included were Phospho-Tyrosine Mouse mAb (P-Tyr-100), HTScan® Tyrosine Kinase Buffer (4X), DTT (1000x, 1.25 M), ATP (10 mM), Gastrin Precursor (Tyr87) Biotinylated Peptide, VEGF Receptor 2 Kinase (recombinant, human).
Preparation of Ehrlich Ascites Carcinoma (EAC) cells:
Ascetic fluid was withdrawn under aseptic conditions from tumor-bearing mice by needle aspiration from peritoneal cavity. Seven to ten days after EAC cells implantation, EAC cells were tested for viability and contamination using Trypan blue dye exclusion technique (Lazarus et al., 1966 ). Only EAC cells with at least 90% viability were used. EAC cells were suspended in normal saline so that each 0.1 ml contains 2.5x106 cells. Cells were counted under the microscope using hemocytometer.
Induction of solid tumors
Each mouse was inoculated intradermally at 2 sites bilaterally on the lower ventral side (after shaving this area) with 100 μl EAC suspension (2.5×106 cells) on each site.