Antiangiogenic activity of 5-bromoindole carbothioamide derivative in ex vivo , in vivo , and in vitro experimental study

activity of 5-bromoindole carboth-ioamide derivative in


Introduction
New blood vessels are formed by two basic processes, namely vasculogenesis and angiogenesis (D' Alessio et al. 2015).The initial stage is believed to be triggered by the activation of endothelial cells in existing blood arteries in response to angiogenic stimuli.This process is usually triggered in hypoxic tissues, where there is a need for new blood vessels to ensure adequate oxygenation and feeding (Egginton and Gaffney 2010).Stimulation of angiogenesis can be therapeutic in ischemic heart disease, peripheral arterial disease, and wound healing (Deveza et al. 2012;Johnson et al. 2019;Veith et al. 2019).Decreasing or inhibiting angiogenesis can be therapeutic in cancer and other diseases, such as colitis (Kadhim 2016;Manna et al. 2019;Kamal and Khadhim 2021).
When the tissue experiences low oxygen levels (hypoxia), cellular processes that detect oxygen levels are triggered, leading to the activation of genes that produce different proteins that promote the growth of blood vessels (pro-angiogenic proteins) (Ali et al. 2016;Raghif 2016;Khamees et al. 2018); The HIFs (hypoxia-inducible factors) are activated in hypoxia, which increases the expression of various pro-angiogenic genes either directly or indirectly (Fong 2008;Mourad et al. 2023).VEGF-A (vascular endothelial growth factor-A) is the primary upregulated gene that plays a crucial role in promoting cell proliferation and migration throughout this phase (Al-Zubaidy et al. 2016;Hussein et al. 2018).Cellular hypoxia stimulates the cells to produce HIF, which stimulates the release of VEGF-A, among other functions (including modulation of erythropoiesis) (Holmes et al. 2007).Circulating VEGF-A binds to VEGF receptors on endothelial cells, triggering a tyrosine kinase pathway leading to angiogenesis (Shibuya 2011).
Indole is a naturally heterocyclic compound with a benzene ring attached to a pyrrole ring with electrophilic substitution exposure (Chadha and Silakari 2018).Indole derivatives, such as carbothioamide, oxadiazole, and triazole, showed anticancer activities because they can disrupt the mitotic spindle and prevent the proliferation, expansion, and invasion of human cancer cells (Dhuguru and Skouta 2020).Several therapeutic compounds containing an indole moiety are now being studied for their potential in managing various disease states, including bacterial, malaria, fungal, viral, tubercular, and HIV infections (Kumar et al. 2022).The study aimed to investigate the antiangiogenic, antioxidant, and cytotoxicity activity of a carbothioamide indole derivative (2-NPHC) using ex vivo and in vivo experimental models and an MTT assay on HUVEC and then examine its effect on VEGF gene expression in colon cancer cell line (HCT116).

Cell lines and culture
The Human umbilical vein endothelial (HUVECs) cells and colon cancer cell line (HCT116) were obtained from the American Type Culture Collection.Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) and a mixture of antibiotics (penicillin, streptomycin, amphotericin B) under normal conditions (5% CO 2 , 37 °C, 95% humidity).The cells were distributed into six-well plates at roughly 6 × 10 4 cells/mL density.They were then incubated for two to three days to facilitate cell growth and achieve a confluency level of approximately 70%-80% (Unterleuthner et al. 2020).

Study design
The antiangiogenic activity of the test indole derivative was evaluated using the ex vivo rat aorta ring (RAR) assay and the in vivo chick chorioallantois membrane (CAM) assay.To elucidate the most probable mechanism of action of its antiangiogenic activity, the DPPH assay was performed to explore the ability of the test agent at free radical scavenging, MTT assay on HUVEC, as well as its ability to reduce the expression of the VEGF gene in HCT116.

Ethical considerations and study settings
The investigation was carried out in the tissue culture laboratory of the Pharmacology Department at the College of Medicine, Al-Nahrain University.The project commenced in August 2023 and was completed in March 2024.The studies were carried out after the approval of the Ethics Committee of the College of Medicine -Al-Nahrain University (approval number: UNCOMIRB202405015, date: 22 August 2023).
The rats were obtained from the animal house in the College of Medicine, Al-Nahrain University.They were kept in a hygienic environment at 23 ± 2 °C and a humidity level of approximately 45%.They were exposed to a twelve-hour cycle of light and darkness and given one week to adapt to the new habitat.During this time, they had unrestricted access to water and food.

Anti-angiogenic ex vivo RAR assay
Eight female Sprague Dawley rats, aged twelve to fourteen weeks, were given intraperitoneal anesthesia with a dosage of 80 mg/kg of ketamine and 10 mg/kg of xylazine.After being fully anesthetized, the rats were euthanized by puncturing their hearts (Pierozan et al. 2017;Underwood and Anthony 2020).The thoracic aorta is dissected using a microscope, washed with serum-free medium M199, cleared of surrounding fibro adipose material and remaining blood clots, and cut into thin rings measuring 1 mm thick.The experiment utilized a 48-well tissue culture plate.Each well was supplemented with 300 µl of fibrinogen (3 mg/ml) and aprotinin (5 mg/ml) in serum-free media (M199).Subsequently, ring tissues were positioned in the center of each well.Each well was supplemented with 10 μl of thrombin, produced at a concentration of 50 NIH U/mL in a solution of 0.15 M NaCl.The tissue culture was then kept in a humidified 5% CO 2 incubator (Lab Tech, Korea) at 37 °C for 30 minutes to allow for solidification and the creation of a fibrin gel.Next, 300 µl of medium M199, which was well-received, was supplemented with 0.1% E-aminocaproic acid, 1% L-glutamine, and 0.6% gentamycin.2-NPHC was dissolved in 1% DMSO to create a stock solution with a concentration of 0.1% (10 mg of the drug in 1 ml of DMSO).This stock solution was then diluted in M199 medium to create a series of dilutions with the following concentrations: 100, 50, 25, 12.5, and 6.25 μg/ml.Every concentration was conducted in three replicates (Brown et al. 1996).
The culture plates were incubated in a humidified incubator at 37 °C with 5% CO 2 for five days.The uppermost layer of the medium was substituted with a new medium on the fourth day.Tissue rings exposed to only 1% DMSO are classified as negative control.The vessel growth inhibition was evaluated by calculating the average percentage of inhibition compared to the negative control (Nicosia 2009).On the sixth day of the operation, the quantification was performed manually using a 40× magnification inverted microscope (Optika, Italy), a camera, and specialized software.

In vivo CAM assay
Fourteen fertilized chicken eggs were acquired from a hatchery in AL-Taji/Baghdad, Iraq.The specimens were decontaminated using a solution of 70% ethanol alcohol and Povidone iodine 10%.Subsequently, they were placed in an incubator at 37 °C for approximately 72 hours, with a relative humidity of 60%.Following 72 hours of incubation, a small hole was created in the shell.Using a 2-ml syringe with a 21G needle, 2 ml of albumin was extracted.This process facilitates the separation of the CAM from the shell, allowing for a clearer observation of the formed CAM.Subsequently, the hole was closed.The eggs were horizontally incubated for an additional 24 hours.
Subsequently, a precision drill bit was employed to create a square-shaped opening (with a diameter of approximately 3-4 cm) in the shell.The square section of the shell was then extracted using pointed forceps.The resulting square opening was covered with sterile surgical adhesive tape.The drug under investigation, 2-NPHC, was produced at a concentration of 10 mg/ml (the final dose in each disc was 100 μg).It was then applied onto filter paper discs and allowed to dry before being transferred to the CAM.The embryos were incubated for 72 hours at 37 °C.Finally, the area of inhibition was captured and quantified.The entire treatment was carried out in a sterile environment (Marchesan et al. 1998).

Free radical scavenging activity with DPPH assay
The DPPH technique was employed to test the free radical scavenging activity of 2-NPHC.100 µl of the tested substance at concentrations of 500, 250, 125, 62.5, 31.25, and 15.625 µg were mixed with 200 µl of 0.1 mM DPPH dissolved in methanol.The mixture was then incubated for 30 minutes.The experiment used 96 well plates, with each concentration tested in triplicate.Subsequently, the absorbance was measured at 517 nm using an ELISA reader (Human ® , USA).The negative control consisted of 100 µl of methanol and 200 µl of DPPH (Blois 1958).

Cell viability in vitro assay
The MTT assay, based on the Mosmann method (Mosmann 1983), was employed to determine the proliferation ability of the cell line.All those cells were located within passages four to seven.The cells were subsequently exposed to a series of concentrations (400,200,100,50,25) µg/ml of the substances for forty-eight hours.The MTT was made by adding a 5 mg/ml concentration in PBS (phosphate buffer saline).A volume of 20 µl of MTT was added to each well, and the plates were placed in an incubator at a temperature of 37 °C, with a CO 2 concentration of 5%, for four hours.The plates were subsequently withdrawn from the incubator, and the supernatant layer was extracted.DMSO, in a volume of 200 µl, was added to each well.Next, the plates were forcefully shaken for one minute at room temperature to dissolve the blue crystals completely.An ELISA reader was utilized to measure the absorbance at a wavelength of 570 nm.The absorbance of the cell culture in the control media was considered to indicate 100% vitality.The vitality of treated cells was assessed by comparing it to the untreated control, expressed as a percentage.Each concentration was tested four times, and the experiment was repeated twice.The cell density in each well is 1 × 10 4 cells.

Gene expression analysis
The HCT116 cell line was individually subjected to two distinct doses of the test substance, namely 400 μg/ml (considered high) and 100 µg/ml (considered low), for 12 hours (based on the cytotoxicity analysis from the MTT assay, the serial concentration started at 400 µg/ml and ended at 25 µg/ml.We concluded that 400 µg/ml is preferably selected as the highest concentration for gene expression analysis, and 100 µg/ml was selected as the low concentration based on the RAR and CAM assays as the dose that gave visible angiogenic activity).Every group was replicated three times.After the incubation, the plates were removed and examined for any changes in shape or structure using an inverted microscope.The media was eliminated, and the cells were collected and kept in TriZol® reagent for gene expression evaluation.The gene that was specifically focused on in this experiment was the VEGF gene (Adams 2020).VEGF gene (GAGATGAGCTTCCTA-CAGCAC, and TCACCGCCTCGGCTTGTCACAT; forward and reverse primer), and for GAPDH gene (TG-CCACCCAGAAGACTGTGG, and TTCAGCTCAGG-GATGACCTT forward and reverse primer).
The RNA was extracted from the cell samples following the TRIzol® Reagent technique (Thermo Scientific, USA).The concentration of extracted RNA was determined using a Quantus Fluorometer (Promega, USA).The SYBR Green kit and the one-step real-time qPCR analysis equipment (BioMolecular equipment, Australia) were used to quantify the total mRNA expression.This was done to evaluate the relative mRNA expression of VEGF in the HCT116 cell line.The results were standardized using the housekeeping gene (GAPDH) with the delta-delta Cycle Threshold (DDCT) approach.Analysis of data was done based on the relative quantitation approach, which is the ratio between the RG (reference gene GAPDH) and GOI (gene of interest) and is represented by the (2 -ΔΔCT method) (Chomczynski and Sacchi 1987).

Statistical analysis
The results were reported as the mean ± standard deviation (SD).The CAM assay and gene expression findings were analyzed using one-way ANOVA and declared statistically significant at a significance level of P < 0.05.The IC50 value was determined using logarithmic regression equations (Hussein et al. 2024).The statistical analysis, figures, and graphs were conducted using GraphPad Prism 8.0.

Ex vivo RAR assay of 2-NPHC
The assay demonstrated that the test drug effectively suppressed the growth of micro-blood vessels in a concentration-dependent manner.This inhibition was observed on day 6 of the experiment compared to the negative control treated with merely 1% DMSO.The IC50 value for 2-NPHC was determined to be 13.42 µg/ml, as illustrated by Figs 1, 2.

In vivo CAM assay of 2-NPHC
The blood vessels in the CAM exhibited regression in response to 2-NPHC, resulting in disorganization and a pale yellowish appearance.The avascular zone encircling the disc containing the test drug indicated the inhibition, and the size of the inhibition zone was quantified.The assay result demonstrated a statistically significant regression in the blood vessels of the CAM when exposed to the test agent, as compared to the negative control CAM; this is displayed in Fig. 3, with an inhibition score of 6.5 (++) for 2-NPHC, as indicated in Table 1.

Free radical scavenging activity of 2-NPHC using the DPPH assay
The findings demonstrated that the test agent (2-NPHC) effectively decreased the DPPH free radical in a manner that was dependent on its concentration, as illustrated in Fig. 4. The nonlinear regression analysis calculated the IC50 value for the test agent, which was determined to be 175.3µg/ml for 2-NPHC.

Effect of 2-NPHC on HUVEC viability
The results indicated that 2-NPHC had low to non-toxic effects on the HUVEC cell line, with an IC50 value of 711.7 μg/ml, as illustrated in Fig. 5.

Effect of 2-NPHC on the VEGF gene expression in HCT116 cell line
The HCT116 gene expression analysis results indicated that the control cells, exposed to the media, consistently expressed the VEGF gene at a stable level.Comparatively, the cells treated with 100 µg/ml and 400 µg/ml of 2-NPHC exhibited a decrease in the expression of the VEGF gene compared to the control cells.There was no significant   distinction between the two concentrations, as depicted in Fig. 6.Ultimately, the gene expression findings were congruent and cooperate the ex vivo RAR assay results and the in vivo CAM experiment.

Discussion
The idea of using angiogenesis as a target for treating diseases was introduced over 50 years ago.The first medicine to specifically target VEGF, bevacizumab, was developed for the treatment of cancer and neovascular ocular conditions (Cao et al. 2023).Exploring and identifying new substances that potentially exhibit a beneficial impact as antiangiogen-    ic agents is a significant area of study.This study investigated the properties of a newly discovered chemical, 2-NPHC, and assessed its effectiveness in inhibiting angiogenesis.The findings of this investigation demonstrate that the heterocyclic indole derivatives effectively and proportionally in a dose-dependent manner limit the sprouting of micro-vessels.The 2-NPHC's antiangiogenic effect followed a dose-dependent pattern.Its angiogenic activity started at 12.5 μg/ml concentration (IC50 13.42 μg/ml) and showed maximum inhibition of sprouting blood vessels at 100 μg/ml concentration.
Ostoot et al. have found that Indolephenoxyacetamide (IPA) analogs can inhibit the formation of new blood vessels and have therapeutic properties.The antiangiogenic efficacy of IPA was confirmed using CAM, rat corneal, tube formation, and migration assays; in these assays, IPA, an indole derivative, is a highly effective antiproliferative compound with powerful antiangiogenic activity (Al-Ostoot et al. 2021).
A recent study investigated the anti-angiogenesis effect of new indole derivatives chemical in vitro using a tube formation assay.The HUVEC were seeded on matrigel, a standard method for confirming anti-angiogenesis properties.The new indole derivatives exhibited inhibitory potency, specifically on HUVEC invasion; this indicates that the compound's inhibitory action on invasion relies on its concentration (Yao et al. 2022); these studies corroborate the results of this investigation, indicating the suppression of microvessel formation caused by 2-NPHC may be attributed to its indole properties.
In this investigation, 2-NPHC strongly impacted the CAM assay.The 2-NPHC inhibited angiogenesis in the CAM assay (at a dose of 0.1 mg) beneath the disc by preventing the growth of many blood vessels.In addition, the blood vessels treated with the 2-NPHC became less dense, disorganized, and pale yellow.
The current findings were in line with another study that conducted the CAM assay on the 11 compounds of indolyl chalcones.The compounds exhibited excellent antiangiogenic properties and effectively suppressed the formation of capillaries (Wanegaonkar et al. 2021).A recent study utilized zebrafish embryos with fluorescent blood vessels as a model to screen for in vivo antiangiogenic chemicals, specifically focusing on indoloquinazoline alkaloids.It exhibited notable inhibitory activity and demonstrated the highest effectiveness in reducing the formation of blood vessels in zebrafish embryos (Guo et al. 2023).These studies provide evidence of the fundamental concept for advancing Indols as a substance that inhibits the formation of new blood vessels.
The ROS scavenging activity of 2-NPHC was assessed in this work using the DPPH radical.The 2-NPHC showed dose-dependent scavenging activity.
A previous study found that ethenyl indoles showed ROS scavenging activity in DPPH assay; this antioxidative properties due to the transfer of hydrogen atoms from the indolic NH group and/or electron transfer followed by proton transfer (Kumar et al. 2020).Previous studies have demonstrated that the antioxidant activity of compounds containing C-3 substituted indole derivatives is attributable to multiple pathways.It has been confirmed that the presence of indole structures influences the antioxidants' efficacy.The heterocyclic nitrogen atom in indole acts as an active redox center due to the presence of a free electron pair (Estevão et al. 2010;Jasiewicz et al. 2021).It is widely recognized that compounds with strong antioxidant activity often have antiangiogenic effects (Iacopetta et al. 2020).
The equilibrium between producing and removing ROS is disrupted in disorders.Consequently, producing free radicals might lead to many medical conditions (Pan et al. 2007).External ROS can promote the growth of new blood vessels by enhancing the production of VEGF in many types of cells, including endothelial cells, smooth muscle cells, and macrophages; this, in turn, contributes to the development of numerous illnesses associated with the formation of new blood vessels (Wang et al. 2011).
A study investigated the in vitro effects of recently developed indole compounds (indole-2-carbohydrazide derivatives).The findings revealed that these compounds exhibited strong cytotoxic effects on HCT116 and SW480 cell lines while showing no activity against MRC-5 cell lines originating from lung cells.In addition, its anti-angiogenesis characteristics were assessed by CAM, HUVEC migration, endothelial microtubule formation assays, and VEGF gene expression.The study demonstrated that the indole compound hinders the activity of VEGFR-2 and its associated proteins downstream, which suggest that indole compounds are promising angiogenesis inhibitors (Zhang et al. 2018).Another study that examined thiadiazole-carboxamide bridged β-carboline-indole hybrids showed cytotoxic potential against the A549 cell line, with safety profile against normal human lung epithelial cells (BEAS-2B cell line) (Tokala et al. 2020).
In the present study, the HCT116 gene expression analysis results revealed that cells treated with 2-NPHC exhibited a notable decrease in VEGF gene expression compared to the control cells.No notable variation was observed between the two concentrations regarding their impact on the expression of the VEGF gene.
The VEGF gene's expression change for 2-NPHC can be attributed to their strong antioxidant properties, as observed in the DPPH results, or their ability to inhibit cell proliferation, as mentioned previously (Hassan et al. 2023).The study showed that 2-NPHC could block the tyrosine kinase signaling of EGFR, similar to the standard TK inhibitor (erlotinib); based on data obtained from molecular docking analysis, 2-NPHC demonstrated good inhibition in this regard (Hassan et al. 2023).In addition, the recently produced indole derivatives exhibited lethal effects on three human cancer cell lines, namely HepG2, A549, and MCF-7.The indole derivative had the highest potency, resulting in the lowest IC50 value against HepG2 cancer cells.Therefore, it was chosen for further in-depth investigation as an anticancer agent.Furthermore, the IC50 was like that of erlotinib (Hassan et al. 2023).
Another study examined a series of salicylic acid-modified indole tri-methoxy-flavone derivatives designed and synthesized by introducing benzotrimethoxy-structure commonly used in blood vessel blockers, and their antitumor activities were evaluated.One of the compounds exhibited significant antiproliferative activity against two hepatoma cells, HepG-2 and SMMC-7721, the expressions of tumor angiogenesis-related vascular endothelial growth factor of the glycolytic pathway were down-regulated (Zou et al. 2023).

Study limitations
There are a lot of limitations involved in the screening of antiangiogenic activity for new substances, especially when it comes to the choice of the screening assay.The in vitro and ex vivo (exclusively RAR) assays are the most commonly used for screening analysis because of the ease of access and the resources required for these assays.On the other hand, the most limited models to further explore angiogenesis are in vivo, in which the CAM assay is the only available model to study in Iraq.The vast majority of these are unavailable, including the zebrafish, the corneal, the sponge implantation, and the dorsal air sac, but most importantly, there is a lack of animal strains specific for tumor and angiogenesis induction.

Conclusion
The 2-NPHC exhibited significant inhibition of the angiogenesis process, either directly by inhibiting the release or activity of VEGF or indirectly through its antioxidant properties.It also showed a minimal cytotoxic profile against the HUVEC cell line.These activities indicate the potential molecular mechanism for its antiangiogenic and antiproliferative activity.

Figure 2 .
Figure 2. Cultured ex vivo RAR assay concentration-dependent inhibition of micro blood vessel growth for the test agent (2-NPHC).

Figure 3 .
Figure 3.The in vivo CAM assay showing a marked regression in the blood vessel growth surrounding the implanted disk (indicated by the black arrows).A. Negative control; B. 2-NPHC (100 μg/disk).

Figure 5 .
Figure 5. MTT analysis of the cell viability of the HUVEC cell line after treatment with serial concentrations of 2-NPHC.

Figure 6 .
Figure 6.Violin plot of the relative gene expression of VEGF in the HCT116 cell line treated with 2-NPHC.* Indicate p-value ≤0.05.

Table 1 .
Scoring of the inhibition zone for the in vivo CAM assay for 2-NPHC.