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Inhibition of Ehrlich ascites cancer, hypoxia-inducible factor-1 alpha, and the kinase insert domain-containing receptor/fms-like tyrosine kinase-binding domains of vascular endothelial growth factor by Thiazole Acetamide Derivatives


1 Department of PG Studies and Research in Biochemistry, St. Aloysius College, Mangalore, Karnataka, India
2 Department of PG Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, Shimoga, Karnataka, India
3 Nitte University Centre for Stem cell Research and Regenerative Medicine, Nitte University, Mangalore, Karnataka, India
4 Department of Chemistry, Mangalore University, Mangalore, Karnataka, India
5 Department of Industrial Chemistry, Mangalore University, Mangalore, Karnataka, India

Correspondence Address:
LN Madhu,
Department of PG Studies and Research in Biochemistry, St. Aloysius College, Mangalore - 575 003, Karnataka
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_577_16

 > Abstract 


Background: Tumor cells that have the ability to express vascular endothelial growth factor (VEGF) are more competent to growth and metastasize by the adequate amount of blood and oxygen supply by the blood vessels to the growing mass of cells. Hypoxic tumors are known for its aggressiveness and resistance to the treatment. Targeting VEGF and hypoxia-inducible factor-1 alpha (HIF-1α) is an attractive strategy to interrupt the multiple pathways crucial for tumor growth. In the present study, two thiazole acetamide derivative's anticancer property, anti VEGF and HIF-1α inhibitory property were investigated.
Methodology: Two thiazole acetamide compounds were synthesized, TA1 and TA2 and its anticancer property was studied in Erlich's ascites cancer cells. To evaluate the anticancer property the assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA diffusion assay for apoptosis, and lactate dehydrogenase leakage assay were carried out. The cell culture media was used to assess the secreted VEGF level. Molecular docking studies were performed to analyze the binding efficiency of the study compounds to the kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (FLT)-binding domains of VEGF protein. HIF-1α inhibitory study was performed by flow cytometry analysis using HUVEC cell line.
Results: The study compounds inhibited HIF-1α and VEGF secretion, these data shown positive prop up for the anticancer property of the derivatives. The docking studies showed moderate binding of study compounds to KDR and FLT-binding domains of VEGF protein.
Conclusion: These results conclude the anticancer and anti-angiogenic property of the synthesized thiazole-acetamide derivatives.

Keywords: Angiogenesis, anticancer, hypoxia-inducible factor-1 alpha, thiazole acetamide, vascular endothelial growth factor



How to cite this URL:
Madhu L N, Telkar S, Kamath D, D. Souza ME, Rao S, Nayak PS, Sarojini B K. Inhibition of Ehrlich ascites cancer, hypoxia-inducible factor-1 alpha, and the kinase insert domain-containing receptor/fms-like tyrosine kinase-binding domains of vascular endothelial growth factor by Thiazole Acetamide Derivatives. J Can Res Ther [Epub ahead of print] [cited 2019 Jul 17]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=261560




 > Introduction Top


Angiogenesis or neovascularization is a process of formation of new blood vessels from preexisting blood vessels by activation, adhesion, proliferation, and transmigration of endothelial cells. It has a vital role in the normal physiological process such healing of wound but also a component in number of pathological processes for the occurrence of diabetic retinopathy, chronic inflammation, certain immune responses and neoplasia, arthritis, and the growth of tumors cells.[1],[2],[3],[4]

There are >20 angiogenic growth factors and 300 antiangiogenic molecules which target different signaling pathways are being tested for their anticancer properties at preclinical and clinical trials. Because of the tumor resistance, the therapy with angiogenesis inhibitors does not prolong survival of cancer patients for more than months.[5]

Vascular endothelial growth factor (VEGF) is a cell-specific growth factor which induces the necessary events for angiogenesis and plays an important role in the growth of the tumor.[6]

Two tyrosine kinase receptors, the kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase-1 (FLT-1), have been reported for having high affinity toward VEGF.[7],[8] Later Keyt et al. conducted a mutation experiment and identified that Arg82, Lys84, and His86 within VEGF were critical for interaction with KDR and the amino acids Asp63, Glu64, and Glu67, were associated with FLT-1 binding.[9] Hypoxia-inducible factor (HIF), HIF-1α, and HIF-2α overexpression upregulates the genes such as VEGF involved in the proliferation and angiogenesis mechanism.[10]

Thiazole is a heterocyclic compound featuring both a nitrogen atom and sulfur atom as part of the aromatic five-membered ring. Thiazoles are reported to possess a wide spectrum of biological activities such as antibacterial, anti-inflammatory, antifungal, antitubercular, antitumor, antioxidant, antiparkinsonism, antiviral, and analgesic activities.[11],[12],[13],[14],[15],[16]

The present study focused to find the VEGF inhibition property and anticancer property of thiazole acetamide derivatives against Erlich's ascites cancer (EAC) cancer cell line model. Furthermore, the inhibitory property of the study compounds on HIF-1α was shown in HUVEC cells.


 > Methodology Top


Chemistry

General procedures for synthesis of amides were followed as Prakash et al.: Substituted phenylacetic acid (1 mmol) and heterocyclic amine (1 mmol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (1.0 g, 0.01 mol) were dissolved in dichloromethane (20 mL). The reaction mixture was stirred in the presence of triethylamine (2.78 ml, 0.02 mmol) at 0°C for about 3 h. The contents were poured into ice cold aqueous hydrochloric acid (100 ml) with stirring, which were extracted thrice with dichloromethane. Organic layer was washed with saturated NaHCO3 solution and brine solution, dried and concentrated under reduced pressure. The resultant crude residue was recrystallized by a slow evaporation method using different combinations of solvents to give pure amides.[17]

Cell culture and maintenance

The EAC cell line were purchased from NCCS Pune, the cells were grown in MEM medium supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified 5% CO2 atmosphere at 37°C.

Experimental design

Cultured EAC cells were exposed to both the thiazole acetamide derivatives at different concentration ranging 0–1000 μg/ml. 24 h postexposure, the following assays were conducted to analyze the effect of compounds on EAC cells.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay

Drug-induced cytotoxicity will be evaluated by conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay as previously reported.[18] Briefly, 1 × 104/cells are seeded in 96-well plates and cultured in MEM media supplemented with 10% FBS for 8 h. They will be exposed to various concentrations of TA derivatives for 24 h in a CO2 incubator. Ten microlitres of 5 g/L MTT solution was added to each well for 4 h at 37°C. Subsequently, the formazan crystals were solubilized with 100 μl of 10% sodium dodecyl sulfate in 0.01 M HCl for 24 h. Absorbance at 570 nm relative to a reference wavelength of 630 nm is determined with a microplate reader. The concentrations resulting in 50% inhibition of cell growth (IC50 values) will be calculated.

DNA diffusion assay

To estimate the percentage of apoptotic cells, DNA diffusion assay was performed as described by Singh, 2000.[19] The percentage of apoptosis measured by counting the apoptotic cells and normal cells. In brief, slide preparation: Base layer 50 μl of agarose smeared and air-dried. A volume of 50 μl of the cell-agarose suspension was layered over the base layer and covered with coverslip. Slides were lysed by treating with alkaline lysing solution further slides were neutralized by treating with DNA precipitating solution for 30 min. This step repeated two times. Finally, slides were stained with 20 μg/ml ethidium bromide and slides were analyzed under fluorescent microscope (Olympus).

Membrane integrity

Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme present in most eukaryotic cells, released into culture medium on cell death due to damage of plasma membrane. The increase of the LDH activity in culture supernatant is proportional to the number of lysed cells. LDH test was used to assess the cell membrane integrity.[20] EAC cells were plated in the 6-well plates (5 × 105 cells per well) and incubated for 24 h. Thiazole acetamide samples were introduced separately to the cells with different concentrations (0, 50, 100, 500, and 1000 μg/mL) and incubated for another 24 h. The positive control was prepared by adding of lysis solution to the control cells at 45 min before the centrifugation. Then, the centrifugation (1200 rpm × 5 min) was performed. One hundred microliters of supernatant was taken out from each well for LDH assay following the instruction of the kit. The absorbance at 340 nm was recorded on a Microplate Reader (Thermo scientific). The LDH leakage is expressed as U/L.

Assessment of vascular endothelial growth factor secretion by Erlich's ascites cancer cells in vitro

The supernatant media used for EAC cell culture with or without thiazole acetamide was used for the VEGF estimation. After 24 h, basal levels of VEGF in cell culture supernatants were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's recommendations. The optical density of the developed ELISA plate was measured on a Thermo Scientific reader using standard correction factors.

Flow cytometry analysis of hypoxia-inducible factor-1 alpha

Cells (HUVEC cell line-PAN-Biotech, Germany) were cultured in a 6-well plate at a density of 3 × 105 cells/2 ml and incubated in a CO2 incubator overnight at 37°C for 24 h. Cells were treated with Dibenzoylmethane (DBM) (Sigma cat no: D33454)– 2 μM to over-express the HIF-1α. For 24 h. Spent medium was aspirated, and cells were treated with 100 μg of experimental compounds and control in 2 ml of culture medium and incubated the cells for 24 h. At the end of the treatment, the media was removed from all the wells and washed with PBS. After removal of the PBS, 200 μl of trypsin-EDTA solution was added and incubated at 37°C for 3–4 min. 2 ml culture medium was added to harvest the cells directly into 12 × 75 mm polystyrene tubes. Tubes were centrifuged for 5 min at 300 ×g at 25°C. The cells were washed again with PBS. Ice cold 70% ethanol was added dropwise by gentle vortexing and incubated at 4°C for overnight. Cells were pelleted by centrifugation at 2500 rpm for 5 min and incubated with 100 μl of blocking and permeabilization solution (1% BSA, 0.2% Triton X-100 in 1× PBS) at room temperature for 20 min. Cells were washed with wash buffer (1× PBS + 0.1% bovine serum albumin) at 2000 rpm for 5 min and treated with the anti-HIF 1α-PE antibody (Biolegend cat no: 359703) (5:100) and incubated at room temperature in dark for 60 min. Cells were washed with wash buffer at 2000 rpm for 5 min and 0.5 ml of PBS was added. Flow cytometry is performed by using the BD FACSCallibur.

In silico studies

An entirely in-house developed drug discovery informatics system OSIRIS was used to perform ADMET based calculations. It is a Java-based library layer that provides reusable cheminformatics functionality and was used to predict the toxicity risks and overall drug score via in silico.[21] The structure of synthesized molecules and the standards were drawn in ChemBioDraw tool (ChemBioOffice Ultra 14.0 suite) assigned with proper two-dimensional (2D) orientation and structure of each was checked for structural drawing error. Energy of each molecule was minimized using ChemBio3D (ChemBioOffice Ultra 14.0 suite). The energy minimized ligand molecules were then used as input for AutoDock Vina, to carry out the docking simulation (Trott and Olson, 2010). The protein databank (PDB) coordinate file with the name “1VPF. pdb” was used as receptor molecule. All the water molecules were removed from the receptor and SPDBV DeepView was used to automatically rebuild the missing side chains in receptor. The Graphical User Interface program “MGL Tools” was used to set the grid box for docking simulations. The grid was set so that it surround the region of interest (active site) in the macromolecule.

In the present study, the active site was selected based on the amino acid residues of VEGF that are involved in interaction with FLT-1 and KDR receptors.[9] Grid box was set so that it surrounded the active site. For all the synthesized molecules, molecular docking simulation was run twice at two different binding domains (considered active site) of KDR and FLT-1 receptors. At the KDR binding domain of VEGF, the grid box volume was set to 18, 14, and 16 Š for x, y, and z dimensions, respectively, and the grid center was set to −3.472, 15.351, and −6.617 for x, y, and z center, respectively, which covered the 3 amino acid residues that involved in interaction with KDR. Similarly, at the FLT-1 binding domain of VEGF, the grid box was set to 14, 12, and 16 Š for x, y, and z dimensions, respectively, and the grid center was set to 37.473, 13.467, and 6.46 for x, y, and z center, respectively, which covered the 3 amino acid residues that involved in interaction with FLT-1 receptor. The molecules 17a and Mp2 were compared in silico with a novel benzophenone-thiazole derivative that is the molecule 10 h of Prashanth et al.[22]

AutoGrid 4.0 Program, supplied with AutoDock tools was used to produce grid maps.[23] The docking algorithm provided with AutoDock Vina was used to search for the best-docked conformation between ligand and protein. During the docking process, a maximum of 10 conformers was considered for each ligand. All the AutoDock docking runs were performed in Corei7 Intel processor CPU with 16 GB DDR3l RAM. AutoDock Vina was compiled and run under Windows 8.0 professional operating system. LigPlot+[24] and educational version of PyMol[25] was used to deduce the pictorial representation of interaction between the ligands and the target protein.


 > Results Top


Thiazole acetamide derivatives were prepared according to the methods proposed in the literature. The synthesized compound was then screened for its solubility and found that both the compounds were soluble in DMSO and partially soluble in water. The purity of the compounds was confirmed by thin layer chromatography using Merck silica gel 60F254-coated aluminum plates using ethyl acetate: n-hexane (3:7, v/v) as the solvent system [Figure 1].
Figure 1: Synthesized Thiazole acetamide derivatives (i) TA1 (ii) TA2

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MTT assay results revealed the IC50 values of TA1 and TA2 against EAC cells. The calculated IC50 value of TA1 by probit analysis was found to be 251.1 μg and 398.1 μg for TA2 [Table 1].
Table 1: Percentage of nonviability of Erlich's ascites cancer treated with different concentration of drugs

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LDH leakage assay results showed that there was statistically significant (P < 0.01) increase in LDH leakage in TA1 treated group when compared to control and there was highly statistically significant (P < 0.001) increase in LDH leakage in TA2 treated group [Table 2].
Table 2: Comparison of lactate dehydrogenase level in thiazole acetamide treated Erlich's ascites cancer cells

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Percentage of apoptotic cells was calculated by counting the number of DNA diffused cells. A total of 500 μg TA1 and TA2 treated cells showed 59.58% and 50.99% of apoptotic cells after 24 h of incubation, at 24 h control group showed only 3.55% apoptotic cells. These results showed the property of TA compounds to induced apoptosis in EAC cells [Figure 2] and [Figure 3].
Figure 2: Comparison of percentage of apoptosis in TA treated Erlich's ascites cancer cells with control

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Figure 3: Photograph of DNA diffusion assay (normal and apoptotic cells)

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Anti-angiogenesis studies

VEGF level was measured in the culture medium of thiazole acetamide treated cultured EAC. EAC cells secreted 292.78 ± 13.33 pg/ml of VEGF, which was reduced 2.91 times in 1000 μg TA1 treated group and reduced up to 2.63 times in TA2-treated cells [Figure 4].
Figure 4: Comparison of secreted vascular endothelial growth factor levels in the Erlich's ascites cancer cell treated with TA

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DBM-induced HIF-1α overexpression was found to be down-regulated by TA1 and TA2 treatment. The Geo-mean values were reduced up to 26.75 and 24.72 in TA1 and TA2 treated HUVEC cells when compared with DBM alone (48.03) treated cells [Figure 5] and [Figure 6].
Figure 5: Hypoxia-inducible factor-1 alpha counts in the flow cytometry for dibenzoylmethane treated HUVEC with and without thiazole acetamide derivatives. (Gray - Control, Red - dibenzoylmethane control, Green - TA1, Blue - TA2)

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Figure 6: Comparison of FSC values in gated graph for the hypoxia inducible factor-1 alpha (a). Graph showing comparison of geo-mean value among treatment groups obtained in flow cytometry analysis of hypoxia inducible factor-1 alpha (b)

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In silico studies

Considering the results obtained earlier, it was thought worthy to perform molecular docking studies by substantiating the in vitro results with in silico studies. The comparative docking of VEGF with both synthesized molecules exhibited good affinity. They established bonds with one or more amino acids in the receptor active pocket as represented in [Table 3]. [Table 3] explains the protein-ligand interaction of both the considered active pockets.
Table3:Bindingaffinity(kcal/mol),H-bonds,H-bondlengthandH-bondformationofthestandardsandthesynthesizedmolecules after in silico docking

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Ligand-protein interactions can be further extrapolated from [Figure 7] and [Figure 8]. In both figures, a, b, and c depicts the 2D representation of the best conformation of TA1, TA2 and 10 h docked against KDR domain and FLT domain of VEGF, respectively. Here, the ligand is highlighted in purple color. The set of conserved residues that are commonly involved in interaction with all the molecules are encircled with red color.
Figure 7: (a-c) represents the two-dimensional illustration of the interaction of molecules TA1, TA2 and 10 h with the kinase insert domain-containing receptor-binding domain of vascular endothelial growth factor. (d-f) Represents the three-dimensional illustration of the interaction of the same molecules respectively

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Figure 8: (a-c) represents the two-dimensional illustration of the interaction of molecules TA1, TA2 and 10 h with the fms-like tyrosine kinase-binding domain of vascular endothelial growth factor. (d-f) Represents the three-dimensional illustration of the interaction of the same molecules respectively

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Further, for the same, the 3D interaction was visualized and represented in [Figure 7] and [Figure 8]d,[Figure 8]e,[Figure 8]f. The ligands are represented in green color, H-bonds with their respective distances are represented with pale blue color, and the interacting residues are represented in ball and stick view.


 > Discussion Top


Cancer cell multiplication and its metastasis were well studied by Folkman and Shing[26] The study investigators show the relationship between tumor growth and angiogenesis. Nuclear events which regulate the cellular expression of the genes of different signaling pathways and growth factors involved in the angiogenesis process.[27] VEGF is a potent angiogenic agent that acts as a specific mitogen for vascular endothelial cells through specific cell surface receptors. VEGF will be released by a variety of tumor cells for its growth and metastasis.[28] The recent investigations shown, the carboxy terminus of VEGF-A was considered to be potential target for anti-angiogenic therapy.[29] The pathways and mechanism of action of anti-angiogenic tyrosine kinases inhibitors was reviewed and summarized by Gotink and Verheul 2010.[30]

The three signaling tyrosine kinase receptors VEGFR-1 (flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (flt-4) consist of immunoglobulin-like structures in the extracellular domain. These domains are responsible to carry the signal from the VEGF.[31] The investigations of the present study showed the binding efficiency of the Thiazole acetamide derivatives toward flt-1 and KDR-binding domains of VEGF. The study also showed the inhibition of VEGF secretion from the EAC cells treated with both TA1 and TA2. VEGF level was declained up to 2.9 times in 1000 μg TA2-treated group and 2.6 times in TA1 treated cells.

The study compound docking reports were compared with a thiazole acetamide derivative, i.e., 10 h-2-[4-(4-Fluoro-benzoyl)-2-methyl-phenoxy]-N-[4-(4-methoxyphenyl)-thiazol-2-yl]-acetamide. 10 h was studied on in vitro and in vivo models for antoangiogenic property. It was found to be reducing the VEGF secretion from cancer cell in a dose-dependent manner.[22] The compound TA1 showed hydrophobic interaction with Cys68, Gly59, Cys60, Cys61, Asp63, Glu64, Leu66 of FLT-binding domain. TA2 was found to be interacting with Glu64, Leu66, Glu67, Gly59, Cys68, and Cys60. It also showed the interaction with KDR binding domain at Gln89, Ile83, Ile46, Pro85, and His86. The previously reported study explains that the positively charged surface of VEGF, i.e., Arg82, Cys84 and His86 located in a hairpin loop are responsible for the binding of KDR/Flk-1, while negatively charged amino acid residues, Asp63, Glu64, and Glu67 were associated with the FLT-1 binding.[9]

Overexpression of HIF, HIF-1α and HIF-2α leads to the up-regulation of genes involved in proliferation and angiogenesis.[10] Inhibition of HIF-1α gene expression by siRNA significantly decreases the VEGF production and angiogenesis in ovarian cancer cells.[32] Nontoxic doses of antimycin A1 was reported for its antiangiogenic property by inhibiting the HIF-1α and decreasing the production of VEGF in mouse sarcoma-180 cells.[33] DBM is a natural dietary compound which can increase the HIF-1α protein level and expression of VEGF.[34] In the current study, DBM was used to induce the overexpression of HIF-1α in HUVEC cells. Treatment of study compounds on these cells showed a reduction in the HIF-1α expression. HIF-1α inhibitory action of thiazole acetamide derivatives may help in lowering the VEGF secretion.

The obtained results conclude the anticancer property of the studied thiazole acetamide derivatives. It also explains the mode of VEGF inhibitory action of study compounds.

Financial support and sponsorship

This study was supported by the UGC Minor Research Grant.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

 
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