Acacetin Inhibits In Vitro and In Vivo Angiogenesis and Downregulates Stat Signaling and VEGF Expression

Angiogenesis is the formation of new blood vessels and capillaries either de novo or from the preexisting ones, and is an important target in the treatment and management of solid tumors (1). Besides playing an important role in the normal embryonic development, angiogenesis is involved in many adult physiologic processes such as tissue remodeling during wound healing, female endometrial development as well as few pathologies such as atherosclerosis, macular degeneration, psoriasis, tumor growth, and metastasis. Onset of angiogenesis breaks down tumor dormancy and tumors enter into an active state of growth (1–3). Solid tumors cannot grow beyond 2 to 3 mm diameter due to limits of diffusion for nutrients, metabolic wastes, and gases, and resume active growth only when new vasculature is recruited, facilitating nutrients and gaseous exchange to support tumor growth (2, 3). Tumor vasculature is a prognostic marker and a predictor of stages and malignant potential of a tumor (4, 5). Tumor vasculature is leaky and supported by less number of pericytes, hence, making tumors prone to physiologic concentrations of inhibitory agents. So, targeting angiogenesis could be one of the promising antiangiogenic or angiopreventive strategies in cancer management (6–8). Angiogenesis involves critical steps including vasodilation, endothelial permeability, periendothelial support, endothelial cell proliferation, migration, survival, and subsequent organization and remodeling into three-dimensional network of tubular structures (9, 10). Hence, efficacy of antiangiogenic agents could be directly correlated with the extent and number of these steps being targeted.

Many anticancer agents target tumor angiogenesis in their overall efficacy, and dedicated efforts to this field have led to the identification of novel antiangiogenic agents; a few of these have been approved by U.S. Food and Drug Administration (FDA) and some are at different stages of clinical trials (11–13). Radio- and chemotherapies for cancer are often associated with the burden of high cost, serious side effects, toxicity, and tumor relapse; therefore, approaches that are safe, nontoxic, cost-effective, mechanism-based, and easily available are desired to control tumor growth and progression. Asian populations show lesser risk of acquiring various types of cancers including prostate cancer than their western counterparts and this has been linked to the larger consumption of vegetarian diets including fruits and vegetables (14–16). Attenuating effects of this fruitarian and vegetarian lifestyle on the risks of acquiring specific cancers has been attributed to the presence of certain novel phytochemicals that modulate signaling pathways deregulated in cancer (11, 17). For a decade now, phytochemicals have been the prime focus of scientific investigations for anticancer and antiangiogenic activities (11, 17, 18).

Present study evaluates the role of acacetin, an O-methylated flavone present in plants such as Robinia pseudoacacia (black locust) and Turnera diffusa (damiana), as an antiangiogenic agent. We and others have shown that acacetin possesses anticancer activity against many types of cancer cells including T-cell leukemia, lung, prostate, and breast cancer (19–25); however, its antiangiogenic activity is poorly known. One study reported that acacetin inhibits ovarian cancer cell–induced angiogenesis in chorioallantoic membrane (CAM) assay (26). Herein, we evaluated its antiangiogenic efficacy and associated mechanisms using well-established models in vitro, ex –vivo, and in vivo. Our results suggest that acacetin possesses strong and promising antiangiogenic activity and inhibits various aspects and stages of angiogenesis process.

Human umbilical vein endothelial cells (HUVEC) and EGM2-MV basal or complete growth media were from Lonza Walkersville Inc., human lung carcinoma cell line A549 and human prostate cancer (22Rv1, DU145, and PC3) cell lines were from American Type Culture Collection. HUVEC was used within 1 year of its procurement and all four cancer cell lines were obtained in 2008, and were tested and authenticated by DNA profiling for polymorphic short-tandem repeat (STR) markers at University of Colorado Cancer Center DNA Sequencing & Analysis Core (Aurora, CO) most recently in August 2010. RPMI-1640 media, antibiotic–antimycotic cocktail, and other culture reagents for tumor cell growth were from HiMedia Laboratories. FBS was procured from HyClone-Thermo Fisher Scientific. Cells were grown in a humidified atmosphere in 5% CO₂ at 37°C temperature. Anti-VEGF antibody was from Abcam; VEGF and complete cocktails of protease and phosphatase inhibitors were from Roche Molecular Biochemicals. Other specific primary and horseradish peroxidase (HRP)–linked secondary antibodies were from Cell Signaling Technology. Enhanced chemiluminescence (ECL) was from Millipore. Acacetin [Fig. 1A; dissolved in dimethyl sulfoxide (DMSO) as 100 mmol/L stock solution] and AG490 were purchased from Sigma-Aldrich. Matrigel was from BD Biosciences. Fertilized chicken eggs for CAM assay were procured from Government Poultry Farm, Chandigarh, Punjab, India.

For cell growth and death assays, HUVEC were seeded at the density of 2 × 10⁴ or 1 × 10⁵ per 35- or 60-mm plate, respectively, in EGM-2MV complete medium, and were treated with either DMSO or indicated concentrations of acacetin for the desired time periods. In ligand-stimulated experiments, HUVEC were seeded in 12-well plates in regular medium and after 24 hours, growth factors and serum-starved overnight followed by treatment with VEGF and/or acacetin. Cells were collected and processed for scoring total and dead cells by Trypan blue dye exclusion assay. Equal volume of DMSO (0.1% v/v) was present in each treatment in all the experiments. Data are represented as mean ± SE of triplicate samples, and repeated with similar findings.

To examine the effect of acacetin on in vitro angiogenesis, two methods were used. In first method, HUVEC (4 × 10⁴ cells/well) were simultaneously seeded with acacetin in 24-well Matrigel-coated plates in EGM-2MV complete media, and tube formation was observed and quantified periodically over specific time periods under phase contrast microscope. In second protocol, acacetin treatment was carried out 6 hours after initial cell seeding. In ligand-induced tube formation, 20,000 cells were seeded in 12-well plates and after overnight starvation, treated with acacetin and/or VEGF (50 ng/mL) for 16 hours. Tubular network was photographed at ×100 magnification and scored by either counting the number of closed structures made by three or more independent cells as one tube, or counting the trisections where at least three cells meet together using an inverted microscope equipped with Zuiko digital camera (Olympus Imaging Corp.). In tumor cells conditioned media (TCM)–stimulated assays, freshly collected TCM at 50% strength (50:50 with basal EGM2-MV) was used to stimulate capillary growth in presence or absence of different concentrations of acacetin. For this, serum-free conditioned media were collected after 24 hours from 70% confluent A549 cells. Data are represented as mean ± SE of triplicate samples, and repeated two to three times with similar findings.

For cell motility assay, HUVEC were plated in 6-well dishes in complete EGM-2MV medium and treated with different concentrations of acacetin for 24 hours followed by wounding and change of treatment media with fresh serum-free media containing only mitomycin C (5 μg/mL). Wound closure was recorded by photography at 0 and 16 hours after injury using an inverted microscope equipped with Zuiko digital camera at ×100 magnification. Five independent areas in each wound (in duplicates) were measured and data were represented as their mean ± SE, and repeated with similar findings.

HUVEC (3 × 10⁴) were seeded in upper chamber of Matrigel-coated Boyden inserts (with 8-μm pore size) and allowed to adhere for 3 hours. Cells were treated with acacetin in serum-free conditions and allowed to invade and migrate for 16 hours toward the lower chamber filled with complete medium. The inserts were processed for counting the invaded/migrated cells at the bottom of the membrane as published elsewhere (12). Photographs (×200 magnification) were taken by inverted microscope equipped with Zuiko digital camera. Experiment was repeated with similar findings.

HUVEC were grown to 70% confluency and treated with the desired concentrations of acacetin for 24 hours. At the end of the treatments, cell lysates were prepared in nondenaturing lysis buffer and protein concentrations were determined as published elsewhere (27). Cancer cells were grown in regular conditions and treated with acacetin as indicated in the figures, and cell lysates were prepared as described previously (27). Protein lysate (50–80 μg) were resolved in 10%, 12%, or 15% SDS-PAGE and were blotted onto nitrocellulose membranes and probed with specific primary antibodies, followed by detection with HRP-conjugated appropriate secondary antibodies using ECL detection system. Experiments were carried out two to three times with similar results.

To assess the effect of acacetin on ex vivo angiogenesis in an organotypic blood vessel culture, 6-week-old male Wistar rats, approved from the Institutional Animal Ethics Committee, were sacrificed and aortae were retrieved after surgery. Aortae were rinsed profusely with antibiotic cocktail solution (1% antibiotic–antimycotic solution in 1× PBS, pH 7.4) and surrounding fibro-adipose tissue was gently removed with the scalpel under sterile condition. Aortae were cut into 1-mm thick sections with sharp scalpel, and implanted on Matrigel-coated tissue culture plates. Additional thin layer of Matrigel was layered onto individual rings to embed them, and plates were kept for 15 minutes in incubator in humidified atmosphere followed by feeding with the complete EGM-2MV media for 2 days and then treatment with specific concentrations of acacetin every 48 hours for 2 weeks. After treatment time was over, media was removed and plates were washed with 1× PBS and photographed at ×200 magnification under the phase contrast microscope.

CAM assay was conducted to assess the effect of acacetin on blood vessel formation and networking in developing fertilized chicken eggs under in vivo–like conditions. Eggs were incubated in 5% CO₂ atmosphere at 37°C in humid conditions for 2 days. On day 3, a small hole was introduced at the pointed end of the egg to remove 1 mL albumin and later sealed with cello-tape and kept in incubator. After 48 hours of incubation, 1 cm × 1 cm window was made to expose the CAM and closed by transparent tape thereafter. Next day (on day 6), acacetin (50 μmol/L final conc.) was mixed with 15 μL of Matrigel and implanted on each CAM showing major blood vessels, followed by incubation till 14th day. Thereafter, CAMs were photographed using a digital camera (Nikon) with ×10 optical zoom. Quantitative data for nascent vessel formation between the major blood vessels are represented as mean ± SE of total number of vessels in five independent areas on CAMs for each treatment. Experiment was repeated with similar results.

HUVEC were seeded (1 × 10⁵ cells) onto coverslips and grown for 24 hours under regular conditions followed by treatment with DMSO or indicated acacetin concentrations for desired time periods. Cells were fixed with paraformaldehyde followed by permeabilization with Triton X-100.

Next, cells were blocked with PBS containing 0.5% bovine serum albumin and 0.15% glycine for 1 hour at room temperature followed by incubation with specific primary antibody overnight at 4°C and then with Alexa Fluor 488–conjugated secondary antibody for 1 hour. Coverslips were mounted on glass slides and examined under LSM780 Laser Confocal Microscope (Carl Zeiss Inc.) at Central University of Gujarat (Gandhinagar, India) for image acquisition and processing. Five fields per sample were analyzed and imaged, and experiments were carried out in duplicates. Experiment was carried out twice with similar observations.

This assay was conducted as described earlier (26). Male Swiss albino mice (∼8 weeks old) were housed in the Central Animal House under standard conditions as approved by Institutional Animal Ethics Committee, Jawaharlal Nehru University (New Delhi, India). Mice were divided into three groups (n = 5/group) and subcutaneously injected with 500 μL of Matrigel alone or with VEGF (50 ng/mL) and/or acacetin (25 or 50 mg/kg body weight of mouse). Fourteen days later, mice were sacrificed and the Matrigel plugs were removed, weighed, and photographed using Nikon digital camera. Amount of hemoglobin (Hb) was measured using HEMOCOR-D kit (Crest Biosystems, Tulip Group,) following the manufacturer's protocol step-by-step. Body weight, diet, and water consumption were monitored every 3 days.

The data were statistically analyzed using the Jandel Scientific SigmaStat 3.5 software. Student t test was used to assess the statistical significance of difference between control and treatment groups. A statistically significant difference was considered to be present at P < 0.05.

Inhibiting HUVEC growth and proliferation is one of the prime aspects of antiangiogensis. Acacetin treatment to HUVEC significantly inhibited cell proliferation and survival in a dose- and time-dependant manner. Treatment with acacetin for 24 and 48 hours in regular growth conditions inhibited cell proliferation by 18% to 51% and 58% to 80%, respectively, at 10 to 50 μmol/L concentrations as compared with DMSO control (P < 0.05–0.001; Fig. 1B). Under similar conditions, acacetin induced cell death by 14% to 19% and 17% to 18% compared with 9% and 12% cell death in DMSO controls after 24 and 48 hours, respectively (Fig. 1C). This also indicates that the decrease in cell number could predominantly be mediated by acacetin-caused inhibition of cell proliferation.

Because VEGF is an essential and potent mitogen for HUVEC growth, proliferation, and survival, we assessed the effect of acacetin on VEGF-induced HUVEC growth and survival. VEGF treatment increased the growth as well as survival of starved cells. Acacetin dose-dependently (P < 0.001) inhibited VEGF-stimulated HUVEC growth and proliferation by 63% to 92% at 10 to 50 μmol/L concentrations (Fig. 1D), and induced cell death by 31% to 53% (P < 0.01) compared with 20% in VEGF treatment (Fig. 1E). Serum and growth factors starvation of HUVEC for 24 hours with an additional 24 hours of starved condition during treatments increased the cell death to 30% as compared with 9% cell death in regular growth condition in control (Fig. 1C and E). HUVEC cell viability was inhibited by 14% to 41% compared with VEGF-stimulated control after 24 hours of treatments under similar conditions (data not shown). These results suggest that acacetin strongly inhibits HUVEC growth, proliferation, and survival in regular as well as VEGF-induced conditions.

Blood capillary formation is a prerequisite step for angiogenesis process and its inhibition has been suggested as a promising approach in angioprevention and cancer control (11). Because acacetin strongly inhibited HUVEC growth and proliferation, therefore, we next assessed its effect on HUVEC capillary tube formation. For capillary network formation, invasion, and migration assays, 24 hours or less treatment times were used to minimize or exclude the cell death effect of acacetin on these parameters. Acacetin suppressed HUVEC capillary tube formation in a de novo mode as well as inhibited the growth of preformed rudimentary capillaries (Fig. 2A). Under regular growth conditions, 10, 25, and 50 μmol/L acacetin treatment at the time of HUVEC seeding inhibited tube formation by 71%, 84%, and 88% (P < 0.001) after 6 hours, and by 85%, 93%, and 98% (P < 0.001) after 16 hours, respectively (Fig. 2B). Acacetin also suppressed the growth of preformed rudimentary capillary tubes, in which HUVEC were seeded for 6 hours under regular growth conditions resulting in rudimentary tube formation, after the treatment with 10, 25, and 50 μmol/L acacetin. This significantly (P < 0.001) disintegrated and suppressed their further growth by 28%, 41%, and 44% after 6 hours and by 74%, 89%, and 91% after 16 hours, respectively (Fig. 2B). Because VEGF is an important proangiogenic factor stimulating HUVEC capillary tube formation, we assessed the effect of acacetin on VEGF-stimulated HUVEC capillary tube formation on Matrigel. After seeding and overnight starvation followed by VEGF/acacetin cotreatment for 16 hours, capillary tube formation was suppressed by 51% and 83% (P < 0.001) at 10 and 25 μmol/L acacetin as compared with VEGF control, respectively (Fig. 2C and D). These findings suggest that acacetin could inhibit angiogenesis by suppressing de novo capillary tube formation as well as disintegrating the preformed/rudimentary capillary networks in a dose- and time-dependent manner.

Tumor cells secrete specific proangiogenic factors in the tumor microenvironment that attract the growth of new capillaries toward the tumor mass pushing tumors to high growth and metastatic progression (11). Therefore, suppressing tumor-induced angiogenesis would be a desired strategy for cancer prevention and control. TCM is enriched with many secreted proangiogenic factors including VEGF, and has the potential to stimulate HUVEC capillary tube formation on Matrigel (Supplementary Fig. S1A). Therefore, TCM-stimulated angiogenesis is an important model to check the efficacy of any angiopreventive agent. We observed that acacetin potently suppressed TCM-stimulated HUVEC capillary tube formation in a dose-dependent manner. Acacetin at 10, 25, and 50 μmol/L concentrations significantly suppressed TCM-stimulated capillary network formation by 34%, 61%, and 80% after 12 hours and 38%, 59%, and 83% after 18 hours, respectively, compared with TCM control (P < 0.001; Supplementary Fig. S1A and S1B). These results suggest that acacetin has the potential to suppress tumor-induced angiogenesis and thus could be an angiopreventive agent in cancer prevention and control.

Endothelial cell migration and invasion events are very critical during angiogenesis process. During tumor angiogenesis, endothelial cells proliferate, degrade, and invade the surrounding basement membrane and migrate into the stroma. Finally, they differentiate and organize themselves into new blood capillaries that are crucial for tumor growth (28). Because acacetin suppressed HUVEC capillary tube formation, we next examined its effects on HUVEC migration and invasion processes (Fig. 3A–C). Acacetin (10, 25, and 50 μmol/L) treatment for 24 hours in regular growth condition followed by16 hours of migration in serum-free condition suppressed HUVEC migration by 67%, 97%, and approximately 100% (P < 0.001), respectively, compared with control (Fig. 3A and B). Similarly, acacetin (10, 25, and 50 μmol/L) treatment suppressed HUVEC Matrigel invasion by 43%, 55%, and 68% (P < 0.05–0.001), respectively (Fig. 3C and D). These observations suggest that acacetin has the potential to suppress angiogenesis via inhibiting endothelial cell invasion and migration.

Stats are important regulators of angiogenesis and can induce the expression of VEGF, inducible nitric oxide synthase (iNOS), and survivin (29–31). In the present study, we observed that acacetin strongly inhibits activation of Stat-1 and -3, leading to concomitant downregulation of downstream targets including VEGF, iNOS, endothelial nitric oxide synthase (eNOS), basic fibroblast growth factor (bFGF), and survivin. Acacetin (10–50 μmol/L) treatment for 24 hours under regular growth condition inhibited phosphorylation of both Stat-1(Tyr701) and Stat-3(Tyr705), and dose-dependently decreased the expression level of their targets including VEGF, iNOS, eNOS, bFGF, and survivin (Fig. 4A). Acacetin treatment for 24 hours under regular growth conditions in the presence or absence of Janus kinase inhibitor, AG490, which inhibits the phosphorylation and activation of Stat-3, suppressed HUVEC proliferation (P < 0.001) and also induced cell death (P < 0.05–0.001) with greater efficacy in combination treatment (Fig. 4B and C). The combination treatment showed significantly (P < 0.001) higher effects from that of their individual treatment for both cell number and cell death; however, the decrease in total cell number is contributed mostly via enhancing cell death. Concomitantly, AG490, acacetin, or their combination also inhibited Stat-3 (Tyr705) phosphorylation levels compared with the DMSO control (Fig. 4D). AG490 also decreased the protein levels of VEGF, matrix metalloproteinase-2 (MMP-2), and survivin, and this effect was further enhanced in combination with acacetin treatment (Fig. 4D). Overall, these observations suggest that one of the mechanisms by which acacetin could inhibit various angiogenic attributes in endothelial cells could be due to the inhibition of Stat signaling and associated with a decrease in VEGF, iNOS, eNOS, and bFGF expression. The decrease in survival could be associated with the decrease in survivin level.

Stat-3, a transcription factor regulating the expression of VEGF and other proangiogenic genes, is activated by phosphorylation at critical residues such as tyrosine 705. After this phosphorylation, it gets translocated into the nucleus to carry out its transcriptional activities. Because acacetin inhibited phosphorylation of Stat-3 (Tyr705), we examined whether it also inhibited its nuclear localization in HUVEC. Acacetin treatment under regular growth conditions at 10, 25, and 50 μmol/L showed the reduced immunofluorescence for pStat-3 (Tyr705) in nucleus after 12 and 24 hours compared with DMSO control (Fig. 4E and F). pStat-3 (Tyr705) levels were decreased in a dose-dependent manner compared with control, and it was virtually undetectable in the nucleus at 50 μmol/L acacetin treatment for 24 hours (Fig. 4F). These observations suggest that acacetin inhibits Stat-3 activation and its nuclear localization and hence could strongly suppress its transcriptional activity. Thus, Stat-3 signaling could be a potential target for acacetin in its angiopreventive efficacy.

Ex vivo models of angiogenesis allow examining the effects of various agents on angiogenesis in more relevant conditions where more cell types are involved in the process. Because we observed that acacetin strongly inhibits various attributes of angiogenesis in vitro, we next assessed its effects on ex vivo angiogenesis in rat aortic ring and CAM angiogenesis models. Under regular growth conditions, acacetin strongly suppressed capillary sprouting as well as networking from rat aortic rings as well as developing egg CAMs as depicted in the representative pictures (Fig. 5A–C). Compared with DMSO control, 25 and 50 μmol/L doses of acacetin inhibited angiogenesis from rat aortic rings by 81% and 100% (P < 0.001), respectively, after 2 weeks of treatment (Fig. 5B). Similarly, acacetin inhibited both capillary sprouting and networking from larger vessels in developing CAMs by 71% at 50 μmol/L dose (P < 0.001) after 8 days of treatment under regular conditions (Fig. 5D). These results indicate that by strongly inhibiting angiogenesis in both rat aortic ring and CAM models, acacetin has the potential to suppress angiogenesis in more complex systems involving complex angiogenic events including endothelial cell proliferation, invasion, migration, differentiation, and capillary organization, and also where involvement of other cell types occurs.

Because acacetin showed strong antiangiogenic activities by inhibiting angiogenesis both in vitro as well as ex vivo, we also examined its in vivo antiangiogenic efficacy using Matrigel plug assay in Swiss albino mice. Briefly, Matrigel was thawed and kept on ice and subcutaneously implanted in the right flanks of mice with or without VEGF (50 ng/mL) and acacetin (25 or 50 mg/kg body weight) for 2 weeks. There was no considerable change in diet or water consumption by mice during the experiment (data not shown). Because VEGF attracts blood capillary/vessel growth into the Matrigel plugs when implanted in mice, both plug-weight as well as Hb content of plugs increase with VEGF treatment compared with control group. Plug-weight (in mg) and Hb content (in g/dL) were measured to quantitate angiogenesis. We observed that acacetin strongly suppressed these VEGF-induced angiogenic parameters after 2 weeks of treatment (Fig. 5E). Acacetin treatment decreased plug-weight by 36% and 32% (P < 0.05) and Hb content of plugs by 80% and 95% (P < 0.001) compared with VEGF only control, respectively, at 25 and 50 mg/kg body weight doses (Fig. 5F and G). These results suggest that acacetin possesses strong in vivo antiangiogenic efficacy.

Cancer cells secrete various angiogenic factors that change behavior of neighboring endothelial cells and promote growth of blood vessels/capillaries to supply tumors with nutrients, support their gaseous exchange, and metabolic waste removal, and hence, promote tumor growth and progression (11, 32, 33, 34). Expression of proangiogenic factors such as VEGF in solid tumors such as prostate and lung cancers is very high owing to the activation of various signaling pathway including Stat signaling (35–37). These factors play important role in vessel remodeling and tumor angiogenesis. Therefore, we examined whether acacetin could suppress Stat activation and expression of VEGF in different human prostate (DU145, PC-3, and 22Rv1) and lung (A549) cancer cell lines. Acacetin suppressed phosphorylation of Stat-1 (Tyr701) and Stat-3 (Tyr705) in prostate carcinoma DU145 cells, and strongly downregulated VEGF protein expression in prostate and lung cancer cells (Fig. 6A–D). Therefore, acacetin has potential to suppress Stat–VEGF axis in tumors as observed in endothelial cells.

Solid tumors grow up to approximately 2 to 3 mm in diameter and stop growing further when entering the phase of tumor dormancy, which is broken down by the onset of angiogenesis (2, 3). Angiogenesis plays a vital role in tumor growth and their metastatic progression, and is a potential target in cancer prevention and control (1, 11, 38) Angioprevention with phytochemicals, shown to suppress tumor growth and metastatic progression in animal models, is considered as an effective strategy in prevention, control, and treatment of cancer (1, 11). Herein, we evaluated the angiopreventive efficacy and associated mechanisms of acacetin, which has been shown to inhibit the growth of cancer cells (19–25), in in vitro, ex vivo, and in vivo models of angiogenesis. The major findings are that acacetin inhibits various attributes of angiogenesis, including (i) growth and survival, (ii) invasion, (iii) migration, and (iv) capillary tube formation by HUVEC under various experimental conditions; ex vivo angiogenesis in (v) rat aortic ring and (vi) CAM models; (vii) in vivo angiogenesis in Matrigel plugs implanted in Swiss albino mice; and (viii) these effects could be due to acacetin-mediated suppression of Stat–VEGF axis in endothelial as well as cancer cells. These findings suggest that antiangiogenic activity of acacetin could be a major contributor to its overall antitumor effects.

Angiogenesis is a multistep process involving endothelial cell growth and survival, invasion, migration, capillary tube formation, organization, and maturation into blood vessels, and VEGF plays a very important role during this process. Any agent inhibiting one or more of the above processes can impair angiogenesis, and hence, halt tumor growth and metastasis (11). In the present study, acacetin strongly suppressed HUVEC proliferation and survival in regular growth conditions along with the induction of significant cell death. It also strongly inhibited HUVEC growth and survival, and induced potent cell death under hypoxic conditions (data not shown). Furthermore, acacetin strongly inhibited VEGF-induced HUVEC proliferation and survival. These results suggest that acacetin has the potential to suppress angiogenesis in response to angiogenic stimuli.

During tumor angiogenesis, the formation of new blood capillaries is an important event and its inhibition halts vessel recruitment to tumors. We observed that acacetin robustly (up to 98%) inhibited HUVEC capillary tube formation in a dose- and time-dependent manner under regular as well as VEGF-stimulated conditions. Because aggressive or established solid tumors are angiogenic and harbor blood capillaries supplying them nutrients and other factors, therefore, disruption of established vessel/capillary growth in tumors could be an important strategy in controlling the growth of solid tumors. In this regard, acacetin strongly (∼91%) inhibited the growth of preformed or rudimentary capillary network on Matrigel. VEGF stimulates blood vessel formation both in vitro and in vivo (35, 36, 39, 40). Acacetin also suppressed VEGF-induced capillary tube formation by HUVEC. Because tumor cells secrete various proangiogenic factors that attract and stimulate blood capillary growth toward tumor mass leading to tumor growth and progression, therefore, targeting tumor-induced angiogenesis would be a desired strategy in angioprevention of cancer. We observed that acacetin strongly inhibited A549 TCM-induced capillary formation by HUVEC. These findings suggest that acacetin can abrogate angiogenesis, and hence, tumor growth by suppressing both de novo blood capillary formation as well as growth of preformed rudimentary capillary networks.

Endothelial cell migration and invasion are essential and rate-limiting events during capillary growth and organization, and hence, inhibition of such events is a promising angiopreventive strategy. Acacetin potently inhibited HUVEC motility in wound-healing assay, which was almost completely inhibited at higher dose. This effect was further examined using Matrigel invasion and migration that showed a similar trend in inhibiting these biologic events. Altogether, acacetin showing inhibition of endothelial cell proliferation, invasion, migration, and capillary tube formation emphasize its importance in targeting various key attributes of angiogenesis process.

VEGF is a prominent proangiogenic factor that stimulates angiogenesis and has been identified as a potential antiangiogenic target (39, 40). Stat signaling that regulates the expression of angiogenic molecules is implicated in tumor angiogenesis as well as tumor growth and progression (30, 31, 41) Inhibiting Stat activation by suppressing its phosphorylations at key tyrosine residues inhibits tumor angiogenesis (21, 30, 37, 41–43). Phosphorylations at Tyr 701 in Stat-1 and Tyr 705 in Stat-3 leading to their transcriptional activities are important events in activating the expression of VEGF, iNOS, eNOS, surviving, and MMP-2 (42, 44). We found that acacetin inhibits these Tyr phosphorylations of Stat-1 and -3 with concomitant suppression of expression of the targets. bFGF, another important molecule involved in promoting angiogenesis, was also downregulated by acacetin in endothelial cells. Stat-3 activation and its subsequent translocation into nucleus are important for its transcriptional activity. In immunofluorescence study, acacetin decreased Stat-3 (Tyr 705) phosphorylation as well as its nuclear translocation. Therefore, acacetin-mediated downregulation of VEGF as well as survivin, MMP-2, bFGF, iNOS, and eNOS in HUVEC could, in part, occur through inhibition of Stat signaling.

To test whether acacetin could inhibit angiogenesis in relevant ex vivo models where more complex angiogenic system works involving more cell types, we used well-accepted aortic ring and CAM angiogenesis assays. Aortic rings cultured on Matrigel give rise to microvascular networks composed of branched endothelial channels, and it recapitulates more accurately the environment in which angiogenesis takes place than those with isolated endothelial cells. Acacetin showed strong to almost complete inhibition of capillary sprouting from the rat aortic ring explants. CAM is a specialized vascular tissue of avian embryo and is used to test pro- or antiangiogenic activities of various agents. Blood vessel formation in CAMs mimics closely in vivo angiogenesis as they follow same pattern involving many cell types including pericytes. Acacetin inhibited vessel growth and networking from the established blood vessels in CAMs. These findings suggest that acacetin may also inhibit in vivo angiogenesis, which was examined using in vivo model of mouse Matrigel plug assay. Acacetin strongly inhibited VEGF-induced angiogenic parameters including plug-weight and Hb content of plugs. These results together advocate that acacetin has the potential to suppress ex vivo as well as in vivo angiogenesis.

Solid tumors express and secrete diffusible angiogenic factors, such as VEGF, that change the behavior of neighboring blood vessels and encourage the growth of new capillaries toward tumor mass (11, 45, 46). It is known that Stat activation regulates VEGF expression in cancer cells in response to multiple oncogenic growth signaling (37, 43). Therefore, suppression of Stat signaling and angiogenic factors in tumors can inhibit tumor angiogenesis and hence tumor growth. To examine such possibility, we used human prostate carcinoma DU145, PC-3, and 22Rv1 cells. Acacetin decreased the phosphorylation of Stat-1 (Tyr 701) and Stat-3 (Tyr 705) in DU145 cells concomitant with decrease in VEGF protein level. Acacetin also decreased the VEGF protein level in PC-3, 22Rv1, and lung carcinoma A549 cells. Thus acacetin has capability to target Stat–VEGF axis in cancers cells to suppress tumor angiogenesis.

Overall, the present study shows that acacetin is a potent and promising small-molecule antiangiogenic agent that inhibits various attributes of angiogenesis including HUVEC growth, invasion, and migration, and strongly suppresses in vitro, ex vivo, and in vivo angiogenesis. The antiangiogenic effects of acacetin could be mediated via inhibition of Stat–VEGF axis in endothelial as well as cancer cells. Further studies using animal tumor models are warranted to support clinical usefulness of acacetin in cancer prevention and control.

No potential conflicts of interest were disclosed.

Conception and design: T.A. Bhat, R. Agarwal, R.P. Singh

Development of methodology: T.A. Bhat, D. Nambiar, R.P. Singh

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T.A. Bhat, D. Nambiar, D. Tailor, R.P. Singh

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T.A. Bhat, D. Nambiar, D. Tailor, A. Pal, R.P. Singh

Writing, review, and/or revision of the manuscript: T.A. Bhat, D. Nambiar, R. Agarwal, R.P. Singh

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T.A. Bhat, A. Pal, R.P. Singh

Study supervision: T.A. Bhat, R.P. Singh

The authors thank assistance provided by Vijay Mohan (Central University of Gujarat) for confocal microscopy.

The study was supported by grants from Council of Scientific and Industrial Research (CSIR; 37/1391-09/EMR-II) India, University Grants Commission (UGC) Resource Networking fund, DST-PURSE, India, Capacity Build-up fund from Jawaharlal Nehru University, Central University of Gujarat, Gandhinagar and National Cancer Institute (NCI) R01 CA102514. T.A. Bhat and D. Nambiar are supported by fellowships from CSIR, India.

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