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.
- EXPERIMENTAL SECTION
- 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.