Abstract
Vascular endothelial growth factor receptor-2 is a dynamic target for therapeutic intervention in various types of cancers. This study was aimed to explore the anti-angiogenic activity of a novel vascular endothelial growth factor receptor–specific inhibitor named F16 in both in vitro and in vivo experimental models. This compound effectively reduced cell proliferation, tube formation, and migration of human umbilical vein endothelial cells in a concentration-dependent manner by directly inhibiting vascular endothelial growth factor binding and subsequent vascular endothelial growth factor receptor-2 phosphorylation. The F16 was also able to inhibit the phosphoinositide 3-kinase/protein kinase B–mediated survival and migration pathways in cancer in addition to inhibiting the focal adhesion kinase and mitogen-activated protein kinases–mediated signaling in GI-101A cancer cells. The chorioallantoic membrane assay followed by tumor growth inhibition measurements with GI-101A breast cancer xenograft implanted athymic nude mice confirmed the in vivo tumor reductive effects of F16. It was interesting to observe a decrease in tumor burden after F16 treatment which correlated very well with the decrease in the plasma levels of mucin-1 (MUC-1). Our studies so far have confirmed that F16 is a specific inhibitor of angiogenesis in both in vitro and in vivo models. The F16 also works very efficiently with Taxol in combination by limiting the tumor growth that is better than the monotherapy with any one of the drugs that were tested individually. Thus, F16 offers a promising anti-proliferative and anti-angiogenic effects with better specificity than some of the existing multi-kinase inhibitors.
Keywords
Introduction
Angiogenesis, the process of new blood vessel formation, plays a central role in both local tumor growth and distant metastasis of various cancers including the breast cancers. 1 Tumor angiogenesis is a complex multistep process involving extracellular matrix remodeling, endothelial cell migration, proliferation, capillary differentiation, and vascular anastomosis that are regulated by pro-angiogenic signaling. 2 The vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a strong endothelial cell mitogen that increases the permeability of microvessels. 3 Increased expression of VEGF has been reported in several types of human tumors and has been shown to correlate with poor prognosis. 4 The effects of VEGF are mediated through two distinct high-affinity endothelial cell surface receptors that are known as Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), both of which are type III tyrosine kinase receptors. 5 The interaction of VEGF with VEGFR-2 causes endothelial cell proliferation, survival, migration and capillary-tube formation, which occur rapidly to support the growth and metastasis of the tumors. 6 Following VEGF binding, the downstream signaling pathway is initiated by the dimerization of VEGFR-2 and subsequent phosphorylations of several tyrosine residues including Tyr951, Tyr1175, and Tyr1214. Among these residues, VEGFR-2-Tyr1175 is the major autophosphorylation site that controls the pro-angiogenic responses in relation to the status of tumor microenvironment (TME) and the pro-angiogenic molecules that are released in response to the TME. 7 The Akt activation is an almost indispensable signal transduction process for regulating tumor angiogenesis, and endothelial nitric oxide synthase (eNOS) pathways are also known to contribute in this process.7,8 Therefore, antagonizing VEGF/VEGFR-2 axis remains as one of the most common modes of cancer therapy for its role in inhibiting tumor growth. 9
The current therapeutic approaches that are utilized to inhibit angiogenesis include neutralizing antibodies against VEGF, small molecules that can target the receptor tyrosine kinase–mediated signal transduction mechanisms and gene silencing approaches. 10 A variety of anti-angiogenesis therapies directed against the VEGFR kinases have been validated till date and are currently in therapeutic use. Several other anti-angiogenic agents are under active evaluation for their efficacy and safety in multiple clinical trials.11,12 Most of the currently approved small-molecule inhibitors of VEGFR kinase are adenosine triphosphate (ATP)-analogues that can bind to the ATP-pocket of the kinase domain. Among the first-generation VEGFR kinase inhibitors, the indolinones, SU5416, and SU6668 produced disappointing outcomes in clinical settings due to their non-specific, multi-kinase effects and the consequent adverse reactions. 13 However, some of the promising effects of compounds such as PTK787 on the growth of colon tumors14,15 led to the development of second-generation VEGFR kinase inhibitors. Intrigued by the importance of VEGFR targeting to treat solid tumors, we embarked on the search for novel compounds that could act as potential inhibitors of angiogenesis through direct binding with VEGFR-2. 16 Our lead compound (1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)-amide code named as F16 (Figure 1) is emerging as a promising third-generation anti-angiogenic drug with a novel ability for inhibiting VEGFR-2 by competing for the VEGF binding site at the N-Terminal portion of the receptor.17,18
F16 chemical structure. F16 has a molecular formula C13H7O6N3 with a molecular weight of 301.21 g/mol.
To obtain further evidence for the anti-cancer effects of F16, in vivo studies were conducted with a patient-derived xenograft (PDX) experimental model that was developed using the cell line derived from a reoccurring ductal adenocarcinoma breast cancer sample of a 57-year-old cancer patient. This PDX is not only tumorigenic but also shows frequent metastasis to the lungs and lymph node in 90%–100% of animals. 19 Based on the higher tumor take rate, pro-angiogenic gene expressions, and metastatic potential of this PDX model, 20 we evaluated the effects of F16 by measuring the tumor volume (TV) at different time points after the start of the treatments along with the measurement of the mucin-1 (MUC-1) biomarker level in the serum. In addition to being effective and safe during monotherapy, F16 was working very well in combination with cytotoxic agents such as Taxol that is commonly used for effective control of the tumors when anti-angiogenic therapeutics such as Avastin® and Sutent® are used for increasing the overall survival (OS) rate or progression-free survival (PFS) of the patients.21,22 The pre-clinical data presented here bring a valuable confirmation that F16 can become a very useful anti-angiogenic agent with novel mechanism.
Material and methods
Cell lines and reagents
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Walkersville, MD, USA) and maintained in the endothelial growth medium-2 (EGM-2) with BulletKit containing VEGF and all other required growth factors. Cells were incubated at 37°C with 95% air and 5% CO2 in a humidified incubator. HUVECs were used in assays when the cell passages were between 3 and 8. The PDX was established from the GI-101A cell line that was derived from a 57-year-old female patient with recurrent ductal adenocarcinoma (stage IIIa, T3N2MX) who had not previously received any chemotherapy or radiation therapy other than surgery.
23
The GI-101A and PC12 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM
Matrigel tube formation assay
In vitro anti-angiogenesis assay of F16 was performed on Matrigel (ECM625; Millipore, Billerica, CA, USA) according to the manufacturer’s instructions. The EC Matrix™ kit consists of laminin, collagen type IV, heparan sulfate, proteoglycans, entactin, and nidogen. The incubation condition was optimized for maximal tube formation as follows: 50 µL of EC Matrix was suitably diluted in the ratio of 9:1 with 10× diluent buffer and used for coating the 96-well plate. The coated plates were incubated at 37°C for 1 h to allow the Matrix solution to solidify. After 1 h pre-incubation of the plate with Matrix solution, the HUVECs were plated at 104 cells/well in the absence or in the presence of F16 (0.05–10 µM). After 8 h of incubation at 37°C, tube formation was assessed by counting the capillary tube branch points in five randomly selected fields for each well using Leica microscope (DMI 3000 B; IL, USA) and assigned the score for semi-quantitative comparison. 16
Cell proliferation assay
The proliferation of HUVECs was determined by BrdU (bromodeoxyuridine) labeling assay. Briefly, HUVECs were plated at 5 × 103 cells/well in 96-well plates and allowed to attach for 24 h in EGM-2 BulletKit (100 μL/well). Cells were exposed to different concentrations of F16 and incubated in the presence of 1× BrdU for 24, 48, and 72 h and then assayed with BrdU Cell Proliferation Kit (Danvers, MA, USA) following the manufacturer’s instructions.
Cytotoxicity assay using PC12 cells
To further confirm the VEGF-specific binding of F16, cytotoxicity on VEGF-independent PC12 cells was carried out by following the trypan blue dye exclusion method. Briefly, the cells were treated with different concentrations of F16 (0.1, 1.0, 5.0, 10.0, and 20.0 µM) for 24 h. Then, the cell death was determined using trypan blue dye exclusion method to calculate the relative percentage of live cells.
Cell migration assay
The effect of F16 on cell migration was assessed using both transwell and scratch assays. For the transwell migration assay, 6.5-mm transwell plates with 8 µm polycarbonate membrane inserts (Corning, NY, USA) were used. The upper chamber of transwell contained HUVECs suspended in basal medium and exposed to different concentrations of F16. The lower chamber was filled with EGM-2 supplemented with 50 ng/mL VEGF-A as a chemoattractant. Then, transwell plates were incubated for 24 h to allow for migration of HUVECs across the porous membrane. Transwell filters were fixed in 70% ethanol and stained with crystal violet and examined under Leica microscope. Migratory cells were detached from the lower chamber and counted using Bio-Rad TC10™ Automated Cell Counter. For the scratch assay, monolayer of HUVECs was grown on 24-well plates close to 80% confluency. Using a sterile 200 µL tip, single straight line scratches were made in each well. The wells were washed with phosphate-buffered saline (PBS) and refilled with growth medium containing various concentrations of F16. The images were captured using Leica microscope at 12 h post-scratch.
Displacement binding assay with Fluorokine-conjugated VEGF
F16 mediated displacement of VEGF binding to its specific receptor was determined by employing the Fluorokine-labeled biotinylated-VEGF (R&D Systems, Minneapolis, MA, USA). The binding of F16 was confirmed by displacement of the fluorokine-labeled VEGF by F16. Briefly, the cells were incubated with 500 nM concentration of fluorokine-labeled VEGF for saturation of binding. After 30 min of incubation, the F16 was added at different concentrations (0.001–10.0 µM), and then, the HUVEC cells were further incubated for 1 h at 4oC. After completing the incubation, the cells were washed twice with the wash buffer, resuspended in the PBS, and the fluorescence was measured at 485/535 (excitation/emission wavelengths) using a Victor3 spectrofluorometer (Perkin Elmer, Waltham, MA, USA).
Inhibition of VEGF-stimulated VEGFR-2 phosphorylation
The ability of F16 to inhibit VEGFR-2 phosphorylation was determined using western blotting assay. In this experiment, the HUVECs were treated with F16 (1 and 10 µM) in the presence and absence of VEGF (50 ng/mL). After 24 h of treatment, the cells were lysed on ice as described below; proteins were extracted and analyzed by western blotting method using the p-VEGFR-2-specific antibody.
Cytotoxicity assay using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Cell viability was measured by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) in which MTT was reduced by mitochondrial succinate dehydrogenase and generating a purple formazan for colorimetric measurement. During the assay, the GI-101A cells were plated at a density of 5 × 103 cells/well in 96-well plates and incubated at 37°C under 5% CO2 for 24 h, and then, the cells were treated with different concentrations (0.1–50 µM) of F16 for an additional 24 h. After the drug treatment, the old medium was aspirated, the cells were washed twice with fresh medium to remove phenol red and then 10 µL of MTT (0.5 mg/mL) was added to each well with the final incubation at 37°C for 3 h. At the end of last incubation, the MTT solution was removed, and purple formazan crystals were solubilized in 100 µL of dimethyl sulfoxide (DMSO) and the absorbance was measured at 540 nm using a VersaMax microplate reader.
Preparation of whole cell extracts and western blotting
To prepare the whole cell extract, after treatment with F16 (2 µM) for a period of 24 h, cells were washed with PBS and suspended in radioimmunoprecipitation assay (RIPA) cell lysis buffer (20 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1% NP 40, 0.5% deoxycholic acid, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/mL leupeptin, 20 mg/mL aprotinin). After 15 min of shaking at 4°–8°C, the cells were sonicated for 90 s and then were centrifuged (10,000 g) for 20 min, and the supernatant was collected as the whole cell extract. Total protein was quantified using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Equal amounts (25 µg) of protein were resolved on 7.5%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% (w/v) non-fat dry milk and then incubated with the primary antibodies (FAK, p-FAK, Akt, p-Akt, PI3K, and MAPK) diluted (1:1000) in antibody diluent buffer (1× Tris-buffered saline and 0.3% Tween-20 with 5% bovine serum albumin (BSA)) with gentle shaking at 4°C–8°C overnight. The membranes were then incubated with peroxidase-conjugated secondary antibodies. Signals were detected by enhanced chemiluminescence detection method and captured using UVP image analyzer (EC3 Chemi HR 410 imaging system). In all cases, the blots were stripped using a stripping buffer (Tris (62.5 mM), SDS (2%), and 2-mercaptoethanol (90 mM) with the pH 6.7) and reprobed with β-actin antibody as a loading control.
Chorioallantoic membrane angiogenesis assay
The chorioallantoic membrane (CAM) assay was performed according to the method described by Tamilarasan et al. 24 The chicken eggs were obtained and incubated in an automated digital egg incubator at 37°C with 67% relative humidity. On the fourth day, the incubated eggs were broken and gently plated on petri dishes under sterile conditions. F16 at 5 and 10 µM concentrations was added to a paper disk containing 50 ng/mL VEGF and incubated for 12 h. Images were captured using a digital camera (Canon PowerShot A810). Angiogenesis was quantified by counting the number of branching blood vessels.
Treatment with F16 and Taxol as mono and combination therapy to GI-101A tumor xenograft implanted athymic nude mice
The female athymic NCr–nu/nu nude mice (8–9 weeks old and weight 22–25 g) were housed under environmentally controlled conditions (22°C; 12:12-h light–dark cycle) and were provided with pathogen-free food and water. All animal care and experiments were performed in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) of Nova Southeastern University, Ft. Lauderdale, FL. GI-101A human breast cancer cells (5 × 106) in 100 µL of PBS mixed with Matrigel (BD Biosciences) were injected into the mid-right flank of the mice, which developed tumors in approximately 2 weeks. The tumor-bearing mice were divided randomly into four groups: group I was the untreated control, group II was treated with F16 (100 mg/kg), group III was treated with Taxol (10 mg/kg) and group IV was treated with F16 (100 mg/kg) and 3 h later Taxol (10 mg/kg). The experimental mice were treated once in every 2 days for the period of 22 days. The TV was calculated according to the formula: TV = ½ × (L × W2) where L is the tumor length, whereas W is the tumor width. All the animals in the control and experimental groups were euthanized at the end of treatment periods. Blood samples were collected and plasma was separated for MUC-1 analysis.
Analysis of plasma MUC-1 levels
Enzyme-linked immunosorbent assay (ELISA) was used for measuring the levels of MUC-1 following the manufacturer protocol (Sigma-Aldrich, St. Louis, MO, USA). Briefly, the plasma collected from control and experimental animals was added to microwells coated with anti-MUC-1 antibody and allowed to incubate for 2 h at 37°C. Then, the microwells were incubated with rabbit anti-MUC-1 detection antibody followed by horseradish peroxidase (HRP)-conjugated secondary antibody. The color for the final measurement was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The absorbance was read at 450 nm after adding the stop solution and the results were recorded using a microplate reader.
Statistical analysis
The data presented here represent mean ± SD values from at least three independent experiments. Statistical analyses were performed using a one-way analysis of variance and the differences between means were tested by Tukey’s multiple comparison test. The value of
Results
Matrigel tube formation assay
The efficiency of F16 for inhibiting the VEGFR function was assessed initially by its ability to slow down the progression of angiogenesis under in vitro conditions. There are many assays in existence that are known to be representative of in vivo angiogenesis. 25 The formation of the three-dimensional web-like structures of interconnected cells (cords) by the endothelial cells is one of the most popular and widely used qualitative assays. 26 Our compound F16 was also tested using the above-mentioned assay to determine its ability to inhibit the activity of endothelial cells. The results are depicted in Figure 2. The score is based on the extent of cellular networks: (+++++): well-separated individual cells, (++++): cells begin to migrate and align, (+++): cells lineup but do not sprout, (++): visible sprouting, and (+): closed polygons begin to form. As can be seen, F16 was able to significantly inhibit the HUVEC cell alignment and network formation at 1.0 µM concentration confirming its anti-angiogenic ability.
Inhibition of HUVECs tube formation. HUVECs were seeded on Matrigel layer and treated with different concentrations of F16 (0.05–10 μM). HUVECs tube formation was assessed 8 h later. The score is based on the extent of cellular networks: (+++++): well-separated individual cells; (++++): cells begin to migrate and align; (+++): cells lineup but do not sprout; (++): visible sprouting; and (+): closed polygons begin to form.
Effects of F16 on the HUVECs proliferation and migration
We initially sought to confirm the inhibitory effects of F16 on endothelial cell proliferation. As shown in Figure 3(a), the proliferation of HUVEC stimulated by VEGF was markedly decreased after F16 treatment in a dose- and time-dependent manner. However, no significant cell death was observed when VEGF-independent PC12 cells were treated with F16 for 24 h (Figure 3(b)). To further confirm the anti-angiogenic property of F16, we determined the effects of F16 on the migration of HUVECs using the Transwell migration assay. Our results show that F16 was able to significantly inhibit the migration ability of HUVECs in a concentration-dependent manner (Figure 3(c) and (d)). After 24 h, about 90% of HUVECs were trapped in the upper compartment when treated with 20 µM F16 as compared to untreated cells, indicating a potential anti-migration effect of F16. Similarly, F16 exhibits consistent inhibitory effects on cell migration as shown by the results of the Scratch assay.
Inhibition of cell proliferation and migration. HUVECs were treated with F16 (5.0–20.0 μM) for 24, 48, and 72 h. (a) Proliferation was then assessed by BrdU assay. (b) The effect of F16 (0.1–20.0 μM) on PC12 cells. (c) Representative pictures of the inhibitory effects of F16 on cell migration using scratch assay after 12 h of incubation. (d) The effect of F16 on the migratory potential of HUVECs was analyzed using in vitro Transwell assay. (d1) Representative images depicting F16 inhibitory effects on HUVECs migration. (d2) Quantification of migratory cells in treatment groups relative to control (*p < 0.05, ***p < 0.001 compare to control.
Competitive binding of F16 on VEGFR and blocking the phosphorylation of VEGFR-2
To determine the competitive binding of F16 on the VEGF receptor, we employed the displacement binding assay with Fluorokine-Biotinylated-VEGF. Our results clearly depicted that HUVECs treated with F16 showed a dose-dependent decrease in the fluorescence-labeled VEGF binding to the HUVECs compared to the binding in the absence of F16 (Figure 4(a)). Previous studies have clearly indicated that blockage of VEGFR-2 activity could significantly limit the tumoral neo-angiogenesis process. 27 Therefore, to further confirm whether F16 binding could suppress the cellular growth by binding to VEGFR-2, the HUVEC cells were treated with F16 and the expression levels of proteins related to angiogenesis and metastasis, such as p-VEGFR-2 (Tyr 1175), which is the active form of VEGFR-2, was analyzed. Several studies have shown that phosphorylation of VEGFR-2 triggered by VEGF binding could subsequently trigger multiple downstream signals that induced proliferation and differentiation activities of endothelial cells. 28 As shown in Figure 4(b), there was a significant reduction of p-VEGFR-2 levels following incubation of cells with F16, while the total levels of VEGFR-2 had little changes. Furthermore, to verify whether F16 could inhibit the function of VEGFR-2 through blocking trans-phosphorylation of the kinase domain we, determined the effect of F16 on the total tyrosine phosphorylation activity of VEGFR-2 using PathScan Sandwich ELISA Kit. Results presented in Figure 4(c) shows that the trans-phosphorylation activity of VEGFR-2 was increased by the exogenously added VEGF; however, the VEGF-induced phosphorylation activity of VEGFR-2 was significantly attenuated in cells treated with F16.
F16 competitive binding on VEGFR-2. (a) F16 showed a concentration-dependent decrease in the fluorescence-labeled VEGF binding in HUVECs. (b) F16 inhibited VEGF-induced phosphorylation of VEGFR-2 in HUVECs. The phosphorylation activity of VEGFR-2 on Tyr1175 phosphorylation site was measured after treatment with F16 for 12 and 24 h (Figure 4(c)).
Inhibition of cancer cell proliferation and attenuation of the downstream signaling pathway of VEGFR-2
The direct anti-proliferative effects of F16 were assessed using GI-101A breast cancer cells. Though VEGFR-2 is expressed at high levels in endothelial cells, some of the cancer cells express the VEGFR in high levels. 29 Consistent with these previous reports, as shown in Figure 5(a), the proliferation of GI-101A was notably decreased after F16 treatment ranging from 0.1 to 50 µM. To better understand the direct inhibitory effects of F16 on signaling proteins related to cell growth and metastasis, we analyzed several molecules involved in breast cancer cell proliferation using western blot analysis. The phosphorylation of Akt at Ser473 site, a key molecular target downstream of VEGFR-2, was blocked by F16 in the breast cancer cells (Figure 5(b)). These results indicated that F16 had an ability to abolish Akt-dependent cell survival. Furthermore, we identified that FAK activation on Tyr397, which is one of the targets regulating cellular motility and adhesion, was also inhibited by F16 at a concentration of 2 µM. In addition to inhibiting Akt and FAK levels, the F16 treatment reduced the p38 MAPK expression levels also in GI-101A breast cancer cells (Figure 5(b)).
Inhibition of GI-101A proliferation and protein expression levels. (a) The concentration-dependent effect of F16 treatment on GI-101A cell proliferation. The cells were treated with F16 (0–50 μM) for 24 h. The cell proliferation was determined using MTT assay. The percent of viable cells was relative to the untreated cells (100%). (b) Expression of PI3K, Akt, p-Akt, FAK, p-FAK, and MAPK proteins in control and F16-treated GI-101A cells was analyzed by western blotting. F16 altered the PI3K/Akt, FAK, and MAPK proteins in GI-101A cells. β-actin was used as a loading control.
Inhibition of angiogenesis using CAM assay
To confirm the anti-angiogenic effects of F16, CAM assay was performed with 5 and 10 µM concentrations of F16. The number of blood vessel formation in the CAM was drastically suppressed in both 5 and 10 µM F16-treated groups as compared with the controls (Figure 6(a) and (b)). The number of newly formed vessels was suppressed by about 70% (p < 0.001) after 12 h treatment with 5 µM of F16. However, the inhibition of blood vessel formation by F16 was significantly higher at 10 µM after 12 h (94%; p < 0.001) treatment compared to lower concentrations.
Inhibition of angiogenesis using CAM assay. Eggs incubated for 4 days were broken and the contents were transferred into a sterile petri dish. The images of the blood vessels were captured at 0 h on the fourth day. After imaging, the vascular bed was exposed to 0.1, 5, and 10 μM of F16 that was loaded on a sterile filter disk for 12 h. (a) Representative images confirm the inhibition of blood vessel formation in 5 and 10 μM F16-treated eggs after 12 h. (b) Quantification of number of branching blood vessels in the experimental group compared to the controls (***p < 0.001 compared to control).
Inhibition of xenograft breast tumor growth by F16
To further investigate the in vivo tumor growth inhibitory effects of F16, we established a subcutaneous breast cancer xenograft model using GI-101A breast cancer cells. Previous studies have indicated that the GI-101A cell line is highly aggressive with consistent metastatic spread to the lungs and the lymph nodes in xenograft animals. 19 In fact, the GI-101A xenograft model was considered as one of the best breast cancer models available for pre-clinical testing. Therefore, the immune-deficient mice bearing GI-101A xenografts were treated with or without F16, Taxol, and F16 + Taxol combination by intraperitoneal administration for 22 days. Representative pictures of mice with GI-101A xenografts are shown in Figure 7(a). Our results clearly showed that mice implanted with GI-101A tumors showed 40% and 50% suppression of tumor growth after treatment with 100 mg/kg F16 and 10 mg/kg Taxol, respectively, for 22 days (Figure 7(b)). Interestingly, the inhibitory effect of F16 monotherapy was comparable to Taxol at the specified dose with no signs of toxicity in F16 group. However, F16 combination with Taxol treatment caused 85% suppression of tumor growth confirming the additive effects of F16 with no observed toxicity symptoms. Thus, F16 monotherapy, as well as the combination with Taxol, was well tolerated, and there were no significant changes in the behavior, food intake, or body weight during the experimental period (Figure 7(d)).
Effects of F16 and Taxol combination on tumor growth of GI-101A xenografts using a mouse model. (a) control (untreated), F16 (100 mg/kg), Taxol (10 mg/kg), and F16 (100 mg/kg) + Taxol (10 mg/kg)-treated groups. (b) The changes in tumor volume in control and treatment groups. (c) Total body weight of mice post tumor implantation. (d) The concentrations of circulating MUC-1 in the plasma of control and experimental groups. The plasma MUC-1 level was measured at the end of the treatment using ELISA kit (***p < 0.001 in comparison to tumor group).
Plasma MUC-1 levels
Since the tumor-associated antigen MUC-1 was found to be overexpressed in various carcinomas of epithelial origin, including breast, pancreatic, and ovarian cancers, we measured the levels of this biomarker in tumor-bearing animals. The MUC-1 levels in the blood can be used not only for confirming the extent of growth of above-listed cancers but also for assessing the therapeutic responsiveness. 30 Since MUC-1 is also used as a prognostic indicator for survival, the effect of F16 on the plasma levels of MUC-1 in control and treated xenograft mice were analyzed using ELISA. As shown in Figure 7(d), the level of MUC-1 in F16-treated animals showed a significant decrease after 22 days of treatment. Interestingly, reduction of the TV and consequent lowering of MUC-1 levels was greater when F16 was treated in combination with Taxol. Thus, the effect of the combination treatment was found to be lot more effective than the individual drug treatments.
Discussion
Angiogenesis can be detected throughout the onset, growth, and metastasis of breast and several other cancers. 31 Therefore, anti-angiogenic therapy has added value to traditional chemotherapy for solid tumors due to the suppression of tumor invasion and metastasis. Several processes such as proliferation of endothelial cells, proteolytic degradation of the extracellular matrix, migration of endothelial cells, and organization of endothelial cells into capillary-like structures are integral part of tumor angiogenesis and thus become targets for anti-angiogenic therapies. 31 Results of our study have shown that F16 exerts inhibitory effects on the critical steps of angiogenesis, including endothelial organization (Figure 2), and proliferation (Figure 3(a)) in a dose-dependent manner. We also revealed that F16, at micromole concentration range, significantly inhibits VEGF-driven migration of endothelial cells (Figure 3(b) and (c)). Among the various types of pro-angiogenic molecules, VEGF165 isoform is one of the most potent factors that send signals through VEGFR-2 on endothelial cells. Therefore, interfering with the binding of VEGF to VEGFR-2 is a major strategy for stopping new blood vessel formation in the tumors. Thus, inhibition of VEGFR-2 has been serving as an effective strategy for the last two decades and several anti-angiogenic drugs are currently in clinical use. 32 Our experimental results confirm that F16 blocks the VEGF-induced phosphorylation of VEGFR-2 (Tyr1175) in a concentration-dependent manner indicating that the direct effects of F16 is through inhibiting the VEGF-VEGFR-2 binding interaction. In addition, inhibition of VEGFR-2 phosphorylation by F16 leads to reduced levels of p-Akt, MAPK, and p-FAK, which are known to mediate pro-angiogenic signals. As mentioned earlier, there are several anti-angiogenic compounds with the ability to inhibit the VEGFR-2 functions by directly blocking the intracellular kinase activity which are in clinical use. These anti-angiogenic compounds are multi-target receptor tyrosine kinase inhibitors and function as ATP analogs that compete with intracellular ATP binding within the catalytic domain of VEGFRs. Such interaction directly halts tyrosine phosphorylation and subsequently downstream VEGFRs’ signaling. 33 However, the toxicities that are typically observed with these tyrosine kinase inhibitors, as a result of promiscuity of their kinase inhibition, limit their use. The toxicities associated with multi-kinase inhibitors include hypertension, hypothyroidism, hematotoxicity, hemorrhage, and potentially life-threatening cardiac toxicities.34,35 However, our F16 distinguishes itself as a novel VEGFR-2 inhibitor targeting the ligand binding domain and not the kinase domain 16 and thereby inhibits angiogenesis and tumor growth. Our experimental findings clearly reveal that F16 suppresses Tyr1175 phosphorylation of VEGFR-2 stimulated by VEGF in HUVECs. In addition, no observation of toxicities associated with F16 treatment in mice suggests F16 as a well-tolerated compound to halt angiogenesis and subsequently tumor growth. The inhibitory effects of F16 observed in this study is consistent with the interruption of the essential steps of the angiogenesis pathway, which ultimately results in the inhibition of vascular tube formation and consequent tumor growth inhibition in the xenograft animals.
VEGFR-2 was initially believed to be expressed exclusively in endothelial cells with weak expression in normal cells. Interestingly, it is reported that 64.5% breast adenocarcinoma expresses VEGFR-2. 36 Moreover, several laboratories have confirmed that VEGF can directly act on breast cancer cells such as MCF-7, MDA-MB-2321, and T47-D, which are known to express Flt-1 and Flk-1 receptors and influence their growth. 29 In addition, VEGFR-2 overexpression was reported in various other cancers including lung, colon, uterus, and ovarian. 37 These studies clearly suggest an incriminating role for both autocrine and paracrine VEGF–VEGFR signaling mechanism during tumor growth.38–40 Some of the earlier studies suggested that not only the overexpression of VEGFR-2 occurs in cancers but also the expression of VEGFR-2 is correlated to the disease stage, recurrence, and poor prognosis. 37 However, when VEGF binds to the VEGFR-2 receptors found on vascular endothelial cells, the association results in the phosphorylation at Tyr1175 sites, within its intracellular kinase domain, with subsequent initiation of downstream signal cascade in the endothelial cells leading to angiogenesis. 41 However, our experimental findings clearly reveal that F16 can effectively suppress the Tyr1175 phosphorylation of VEGFR-2 stimulated by VEGF in HUVECs.
When VEGF binds to the VEGFR-2 receptor, the signaling pathway is initiated by the dimerization of VEGFR-2 and subsequent phosphorylations of several tyrosine residues, including Tyr1175, which is the major autophosphorylation site within intracellular domain of VEGFR-2. 42 As discussed above, the F16 treatment significantly blocks VEGF binding to VEGFR-2 and causes the inhibition of the linked tyrosine kinase and disruption of the downstream signaling cascade. Interestingly, the toxicities associated with F16 is very minimal as compared to Taxol and other anti-angiogenic drugs such as Sutent® that can be attributed to the specificity of F16. In addition, the consequences of VEGFR-2 inhibition, affecting multiple MAPK signaling mediators, including p-ERK, p-Akt, and p-FAK, were clearly evident from our experiments. Since these pathway components can contribute to the survival and proliferation of endothelial cells as well as cancer cells, lowering of their levels is suspected to be an important mechanism for the overall effects of F16. According to several reports found in the literature, the elevation of p42/44 ERK contributes to an increased cell proliferation ability similar to the increase in p-Akt that is responsible for the survival of cancer cells. The FAK was also known to impart growth persistence and metastatic ability to cancer cells. In a previous study focusing on the interaction between VEGFR-2 and FAK in HUVECs and human gliomas, 43 the VEGF was shown to rapidly phosphorylate FAK at Tyr397 and increase F-actin assembly that coincided with the stimulation of PI3K activities 44 and phosphorylation of Akt.45,46 On the contrary, a decline in p-FAK level was found to be associated with the rapid dissolution of cell (cytoskeletal) integrity, disintegration of cell adhesion molecules and initiation of cell migration and related events such as metastasis in cancer cells. Therefore, F16 treatment leading to lowered levels of p-Akt, ERK, and p-FAK in breast cancer cells confirms the abilities of F16 to stop cell cycle progression in addition to the anti-angiogenic effects mediated through the HUVEC cell inhibition.
In addition to the strong in vitro results, evidence concerning the in vivo effects of F16 came from CAM assay and from the experiments with GI-101A tumor xenograft model. F16 inhibited the tumor growth significantly in GI-101A breast cancer xenograft model that was evidenced by the comparison of the TV in F16 and F16 + Taxol combination–treated animals. The reduction of the tumor burden was further evidenced by the decrease in MUC-1 levels in the plasma of the experimental animals. MUC-1 is a transmembrane glycoprotein of the
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by the Royal Dames of Cancer Research, Inc., Ft. Lauderdale, FL, USA.