Angiogenesis is target for antineoplastic and chemopreventive therapies. The natural phytoalexin resveratrol is found in grapes and red wine as cis and trans stereoisomers. trans-Resveratrol shows antiangiogenic activity, but its mechanism of action is not fully elucidated. Recently, trans-resveratrol has been shown to interact with the β3 integrin subunit, raising the possibility that inhibition of endothelial αvβ3 integrin function may concur to its angiosuppressive activity. To get novel insights about the antiangiogenic activity of resveratrol, we compared cis- and trans-resveratrol stereoisomers for their effect on the angiogenesis process and endothelial αvβ3 integrin function. trans-Resveratrol inhibits endothelial cell proliferation and the repair of mechanically wounded endothelial cell monolayers. Also, it prevents endothelial cell sprouting in fibrin gel, collagen gel invasion, and morphogenesis on Matrigel. In vivo, trans-resveratrol inhibits vascularization of the chick embryo area vasculosa and murine melanoma B16 tumor growth and neovascularization. In all the assays, cis-resveratrol exerts a limited, if any, effect. In keeping with these observations, trans-resveratrol, but not cis-resveratrol, inhibits αvβ3 integrin-dependent endothelial cell adhesion and the recruitment of enhanced green fluorescent protein-tagged β3 integrin in focal adhesion contacts. In conclusion, stereoisomery affects the antiangiogenic activity of resveratrol, the trans isomer being significantly more potent than the cis isoform. The different antiangiogenic potential of resveratrol stereoisomers is related, at least in part, to their different capacity to affect αvβ3 integrin function. This may have profound implications for the design of synthetic antiangiogenic/angiopreventive phytoalexin derivatives. [Mol Cancer Ther 2008;7(12):3761–70]
Angiogenesis is a multistep process that begins with the degradation of the basement membrane by activated endothelial cells that will migrate and proliferate, leading to the formation of solid endothelial cell sprouts into the stromal space. Then, vascular loops are formed and capillary tubes develop with formation of interendothelial tight junctions and deposition of new basement membrane (1). Uncontrolled neovascularization is observed in tumors, angioproliferative diseases, and angiogenesis-dependent diseases (2, 3). Thus, angiosuppressive molecules able to affect different steps of the angiogenesis process may have broad applicability for the therapy of a wide spectrum of diseases, including cancer (4). Also, antiangiogenic compounds may act as cancer chemopreventive drugs, the so-called “angiopreventive” agents (5, 6). To this respect, dietary phytochemicals derived from edible plants may represent a promising class of compounds (7).
The polyphenolic phytoalexin trans-resveratrol (3,5,4′-trihydroxy-trans-stilbene), abundant in grapevines and red wine, exists as cis and trans stereoisomers with trans to cis isomerization facilitated by UV exposure (8). trans-Resveratrol has received wide attention because of its possible role in the prevention of human pathologic processes, including cardiovascular diseases and cancer (9–11). Much less is known about the biological activity of cis-resveratrol that appears to exert antioxidant (12) and anti-inflammatory (13) activities similar to those exerted by the trans form.
Relevant to its putative antineoplastic and chemopreventive activity (5, 7), trans-resveratrol inhibits fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor-triggered neovascularization and tumor angiogenesis (14, 15). Although the molecular bases for its antiangiogenic activity are not fully elucidated (16), recent observations have shown that trans-resveratrol binds purified β3 integrin chain in vitro (17). Given the role of endothelial αvβ3 integrin receptors in angiogenesis (18) and the antiangiogenic potential of αvβ3 integrin antagonists (19–22), this observation raises the hypothesis that inhibition of αvβ3 integrin function may concur to the angiosuppressive activity of resveratrol.
On this basis, to get novel insights about the mechanisms responsible for the antiangiogenic activity of resveratrol, we have compared cis- and trans-resveratrol stereoisomers for their capacity to affect angiogenesis and endothelial αvβ3 integrin activity. The results show that trans-resveratrol is significantly more potent than the cis isomer in inhibiting different steps of the angiogenesis process in vitro and tumor growth and neovascularization in vivo. This is due, at least in part, to the previously unrecognized capacity of the two resveratrol stereoisomers to differently affect αvβ3 integrin function.
Materials and Methods
trans-Resveratrol and bibenzyl were from Sigma. cis-Resveratrol was synthesized as described (12). All the compounds were 95% pure or better. For in vitro and chick embryo studies, stock solutions of these compounds (whose chemical structure is shown in Fig. 1A) were prepared in DMSO and diluted directly in cell culture medium. Final concentration of DMSO (vehicle) was ≤0.5% (v/v) and control cells were treated with the same concentrations of vehicle that did not exert any affect in all the assays. Vascular endothelial growth factor was from Calbiochem. FGF2 was expressed and purified from Escherichia coli cell extract as described (23).
Cell Cultures and Enhanced Green Fluorescent Protein-Tagged β3 Integrin Chain Transfectants
Immortalized BALB/c murine aortic endothelial cells (MAEC) were obtained from R. Auerbach (University of Wisconsin) and grown in DMEM added with 10% FCS (Life Technologies, Invitrogen). FGF2-T-MAECs, a highly tumorigenic subclone of FGF2-transfected MAECs (24), were grown in DMEM supplemented with 4.0 mmol/L glutamine (Life Technologies) and 10% FCS. Bovine aortic endothelial cells (BAEC; provided by A. Vecchi, Istituto Clinico Humanitas) were cultured in DMEM supplemented with 10% heat-inactivated donor calf serum. Cultures were used between the 6th and the 10th cell passages. Human umbilical vein endothelial cells (HUVEC) were from Clonetics (Biowhittaker) and cultured in growth factor-enriched EGM-2 medium (Clonetics). Transformed fetal GM7373 BAEC cells (25) were transfected with the pcDNA3/enhanced green fluorescent protein (EGFP) vector harboring the full-length mouse β3 integrin cDNA (kindly provided by B. Wehrle-Haller, Centre Medical Universitaire; ref. 26). After G418 selection, cell clones (β3-EGFP-GM7373 cells) were screened for stable expression and correct subcellular localization of the β3-EGFP fusion protein by Western blotting and fluorescence microscopy of cells adherent to different substrata (see below). GM7373 cells transfected with EGFP devoid of the β3 integrin chain moiety (EGFP-GM7373 cells) were used as controls.
Cell Proliferation Assay
GM7373 cells were seeded at 20,000 per cm2. After overnight incubation, cells were incubated in fresh medium in the presence of the compound under test. At the indicated time points, cells were trypsinized and counted in a Burker chamber. MAECs, FGF2-T-MAECs, BAECs, and HUVECs were seeded at 25,000, 75,000, 13,000, and 6,250 cells/cm2, respectively, treated as above, and counted after 48 h.
Wounding of Endothelial Cell Monolayers
Confluent GM7373 and MAEC monolayers were wounded with a 1.0-mm-wide rubber policeman. After washing, monolayers were incubated in fresh medium added with 10% FCS and the compound under test (23). After 16 h, wounds were photographed at ×40 magnification using an inverted phase-contrast photomicroscope and endothelial cells invading the wound were quantified by computerized analysis of the digitalized images (27).
Three-Dimensional Gel Invasion Assays
Fibrin-embedded FGF2-T-MAEC aggregates were prepared as described (24). Then, culture medium containing the compound under test was added on the top of the gel in the presence of 10 μg/mL aprotinin to prevent the dissolution of the substrate. The formation of radially growing cell sprouts was observed during the next 48 h, photographed at ×40 magnification, and quantified by computerized analysis of the digitalized images (23). BAECs were seeded on the top of rat tail tendon type I collagen (100,000 cells per well; ref. 28) and allowed to reach confluence. Cell cultures were then treated with growing medium containing recombinant FGF2 plus vascular endothelial growth factor (both at 30 ng/mL) in the absence or presence of the compound under test. After 24 h, cells were photographed at ×100 magnification using an inverted phase-contrast photomicroscope, and endothelial cells invading the gel, in a plane of focus beneath the cell monolayer surface, were quantified by computerized analysis of the digitalized images (23). HUVECs were seeded onto Matrigel-coated dishes (Becton Dickinson) at 40,000 cells/cm2 in the absence or presence of the compound under test. Cells were photographed after 16 h at ×100 magnification using an inverted phase-contrast photomicroscope and scored by two investigators without knowledge of the samples tested and graded on an arbitrary scale of 0 to 4+, with 0 representing no morphogenic response and 4+ representing the strongest response.
Cell Adhesion Assay
The assay was done as described (25). Briefly, parental and transfected GM7373 cells (50,000 cells in 200 μL culture medium containing 1% FCS) were allowed to adhere at 37°C for 3 h to wells coated with 20 μg/mL of different substrata in the absence or presence of the compound under test. Then, adherent cells were fixed and stained with methylene blue/Azur II (1:1, v/v). Plates were read with a microplate reader at 595 nm. Focal adhesion contacts were visualized in β3-EGFP-GM7373 cells seeded in medium containing 10% FCS in the absence or presence of resveratrol stereoisomers on coverslips coated with different substrata. After 3 h of incubation, adherent cells were fixed in 3% paraformaldehyde/2% sucrose in PBS, permeabilized with 0.5% Triton X-100, and analyzed for β3-EGFP localization and paxillin immunostaining by fluorescence microscopy.
Chick Embryo Area Vasculosa Assay
Fertilized White Leghorn chicken eggs were incubated under conditions of constant humidity at a temperature of 37°C. At day 3, gelatin sponges (Gelfoam Upjohn) were cut to a size of 1 mm3, adsorbed with the compound under test (3 μL/implant of a 10 or 100 μmol/L solution, corresponding to 30 and 300 pmol/embryo, respectively), and placed on top of the area vasculosa. After 48 h, membranes were fixed in ovo for 2 h in Bouin's fluid, removed from the eggs, placed on a light box, and photographed under a stereomicroscope at ×8 magnification. Blood vessel area surrounding the gelatin implant was quantified in ovo by an image analyzer system connected to the microscope (23).
Tumor Growth Assay
Animal experiments were approved by the Animal Ethical Committee of the School of Medicine of the University of Brescia and conducted in conformity with the Italian and European guidelines for the protection of animals used for scientific experiments. Seven-week-old female C57BL/6N mice (Charles River Laboratories) were given trans- or cis-resveratrol in the drinking water (30 μg/mL in 0.15% ethanol) with a change every day throughout the whole experimental period starting 4 days before tumor cell transplantation. Control animals received the same concentration of ethanol. Murine B16 melanoma cells (1.5 × 105 cells suspended in 100 μL PBS) were injected s.c. in both flanks of the animals. The growth of the tumors was monitored every other day by measuring tumor diameters (mm) in three dimensions with a metric caliper and tumor volume (mm3) was calculated according to the formula: a × b × c × 0.5. Both stereoisomers had no effect on body weight of tumor-bearing mice when compared with controls.
At sacrifice, frozen tumor sections were immunostained with the rat monoclonal anti-mouse CD31 antibody MEC 13.3 (provided by A. Vecchi, Istituto Clinico Humanitas) for the evaluation of microvessel density (29). For each tumor section, the number of CD31+ vessels was counted in the five most vascularized areas at ×200 magnification.
Also, five tumors for each experimental group were snap-frozen in liquid nitrogen for the evaluation of steady-state CD31 mRNA levels by two-step quantitative real-time PCR analysis. Briefly, cDNA was generated from 2.0 μg total RNA using the MMLV-RT kit (Invitrogen). Real-time PCR was carried out on a iCycler Real-time PCR Detection System (Bio-Rad) using 25 μL reactions containing iQ SYBR Green Supermix, 150 to 300 nmol/L forward and reverse primers, and 5 μL cDNA diluted template. The PCR cycling profile was as follows: 3 min at 95°C and 40 cycles for 15 s at 95°C, 60°C for 1 min. After PCR amplification, melting curve analysis was done for each reaction. Each PCR was done in triplicate on one plate and fluorescence data were recorded using iCycler software (Bio-Rad). Relative expression ratios were calculated by use of Pfaffl equation and Relative Expression Software Tool.5
The significance of differences between means was assessed by Student's t test.
In vitro Effects of Resveratrol Stereoisomers on Different Steps of the Angiogenesis Process
Because of endothelial cell heterogeneity (30, 31), the antiangiogenic potential of trans- and cis-resveratrol was assessed using endothelial cells of bovine, murine, and human origin. Bibenzyl, a resveratrol analogue here used as negative control (23), did not exert any effect in all the assays.
As shown in Fig. 1B, trans-resveratrol is significantly more potent than the cis isoform in inhibiting the proliferation of bovine endothelial GM7373 cells. Accordingly, dose-response experiments showed that trans- and cis-resveratrol inhibit the proliferation of GM7373 after 24 h of treatment with an ID50 equal to 3.0 and 100 μmol/L, respectively (Fig. 1C). Similar results were obtained in MAECs and BAECs stimulated by serum, in FGF2-T-MAECs stimulated by endogenous overexpressed FGF2 (24), and in HUVECs stimulated by angiogenic growth factor-enriched medium (data not shown). The inhibitory effect exerted by the two stereoisomers was reversible as shown by the ability of both GM7373 cells and FGF2-T-MAECs to recover their proliferative capacity after 24 h of incubation with the compound followed by an extensive washing and incubation with fresh medium (data not shown). Also, in keeping with previous observations on different cell types (12), trans-resveratrol caused the reversible arrest of FGF2-T-MAECs in the S phase of the cell cycle (data not shown). Thus, trans-resveratrol is more potent than the cis isoform in exerting a cytostatic effect on endothelial cells of different origin activated by either exogenous or endogenous stimuli.
The capacity of resveratrol isomers to affect different steps of the angiogenesis process was investigated further in various in vitro assays. trans-Resveratrol significantly inhibits the ability of GM7373 cells (Fig. 2A) and MAECs (data not shown) to repair a wounded monolayer, a phenomenon that depends on the migratory and proliferative activity of the cells at the edge of the wound (32). Also, as shown in Fig. 2B, addition of trans-resveratrol to the cell culture medium prevents the capacity of endogenously activated FGF2-T-MAECs to invade a fibrin gel and to form radially growing sprouts (24). Similarly, trans-resveratrol inhibits the capacity of BAECs to invade a three-dimensional type I collagen gel and to organize capillary-like structures when stimulated by exogenously added FGF2 plus vascular endothelial growth factor (Fig. 2C). Finally, trans-resveratrol prevents vascular tube formation by HUVECs seeded on Matrigel (Fig. 2D), a morphogenic phenomenon known as “spontaneous angiogenesis” (33). No activity was instead exerted by cis-resveratrol in all these assays.
Effect of Resveratrol Stereoisomers on Endothelial αvβ3 Integrin Function
Endothelial GM7373 cells express αvβ3 integrin that specifically mediates their adhesion to the prototypic αvβ3 integrin-binding protein vitronectin (25). Recent observations have shown the capacity of trans-resveratrol to bind the β3 integrin chain in vitro (17), raising the possibility that inhibition of β3 integrin function may be responsible, at least in part, for the antiangiogenic potential of resveratrol. To assess this hypothesis, resveratrol stereoisomers were evaluated for their capacity to affect GM7373 cell adhesion to the αvβ3 integrin substratum vitronectin as well as to the β1 integrin substrata laminin and fibronectin (8). As shown in Fig. 3, trans-resveratrol inhibits the adhesion of GM7373 cells to immobilized vitronectin, without affecting their adhesion to fibronectin or laminin. No effect was instead exerted by cis-resveratrol or bibenzyl on any substratum.
Next, to follow the fate of β3 integrin in resveratrol-treated cells, we generated stable GM7373 cell transfectants (β3-EGFP-GM7373 cells) coexpressing fluorescent EGFP tagged to the COOH terminus of the cytoplasmic domain of the β3 integrin subunit (26). Cells transfected with EGFP devoid of the β3 chain moiety were used as controls (EGFP-GM7373 cells). As shown in Fig. 4A, β3-EGFP-GM7373 cells express significant levels of the β3-EGFP protein chimera and levels of the endogenous β3 chain were similar to those expressed by parental and EGFP-transfected cells. Also, β3-EGFP-GM7373 cells adhere to vitronectin, laminin, and fibronectin in a manner undistinguishable from parental cells (data not shown). Moreover, β3-EGFP chimera properly localizes in focal adhesion contacts when β3-EGFP-GM7373 cells are seeded on vitronectin but not when seeded on laminin (Fig. 4B). Finally, as observed for parental GM7373 cells, trans-resveratrol significantly inhibits β3-EGFP-GM7373 cell adhesion to vitronectin, but not to fibronectin or laminin, whereas cis-resveratrol was ineffective (Fig. 4C). Thus, β3-EGFP transduction does not affect the adhesive behavior of GM7373 cells and their response to resveratrol isomers. On this basis, cis- and trans-resveratrol were assessed for their capacity to affect β3-EGFP recruitment in focal adhesion contacts of vitronectin-adherent β3-EGFP-GM7373 cells. As shown in Fig. 4D, trans-resveratrol, but not the cis isoform, prevented the formation of paxillin-positive focal adhesion contacts in β3-EGFP-GM7373 cells adherent to vitronectin and the recruitment of the β3-EGFP chimera in these contacts.
Effect of Resveratrol Stereoisomers on In vivo Angiogenesis and Tumor Growth
trans- and cis-Resveratrol were assessed for their antiangiogenic activity on the chick embryo area vasculosa, a copiously vascularized extraembryonal adnexum (34). To this purpose, 30 or 300 pmol/embryo of the polyphenol under test were applied topically on the area vasculosa membrane via a gelatin sponge implant at day 3 of development. Control embryos were treated with the same doses of bibenzyl or vehicle. At day 5 of incubation, no effect on area vasculosa was observed in embryos treated with vehicle, with both doses of bibenzyl, or with low-dose cis- or trans-resveratrol (data not shown). In contrast, area vasculosa treated with 300 pmol trans-resveratrol were characterized by the lack of blood vessel growth and the disappearance of the majority of preexisting blood vessels (Fig. 5) in the absence of any effect on embryonic development and survival. This resulted in 50% reduction of the vascular density when compared with vehicle- and bibenzyl-treated embryos (Fig. 5C). The same dose of cis-resveratrol had only a limited effect on area vasculosa, resulting in ∼25% inhibition of vascular density (Fig. 5C).
Next, resveratrol isomers were assessed for their capacity to affect tumor growth in mice. Oral administration of trans-resveratrol at the concentration of 30 μg/mL in drinking water inhibits the growth of s.c. grafts of murine B16 melanoma cells in C57BL/6N mice. Inhibition of tumor growth was statistically significant from day 8 after grafting and onward when compared with animals given vehicle alone (P < 0.01, Student's t test). No significant inhibition was instead exerted by the cis isomer at all the time points investigated (Fig. 6A). Accordingly, analysis of tumor neovascularization done at day 18 after grafting (Fig. 6B; Supplementary Fig. S1)6
Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
In the present article, we compared the antiangiogenic activity of trans- and cis-resveratrol on endothelial cells of different origin activated by endogenous or exogenous stimuli. The results indicate that trans-resveratrol affects endothelial cell proliferation and various cell proliferation-independent aspects of the angiogenesis process, including endothelial cell sprout formation, collagen gel invasion, and morphogenesis. Thus, trans-resveratrol acts as an angiostatic molecule on multiple targets of the angiogenic process. In all the assays, the cis stereoisomer exerted only a limited effect, if any. In keeping with the in vitro observations, trans-resveratrol was more effective than the cis isomer in inhibiting angiogenesis and tumor growth in vivo.
The molecular bases of the antiangiogenic activity of resveratrol are poorly understood (16). trans-Resveratrol is endowed with a significant antioxidant and anti-inflammatory activity (8, 9). However, no direct relationship exists between the antiangiogenic and the antioxidant activities of various resveratrol derivatives (23). Accordingly, despite its antioxidant potential (12, 13), cis-resveratrol is characterized by a limited angiostatic activity when compared with the trans isomer.
Our results show that stereoisomery plays an important role in defining the antiangiogenic potential of resveratrol. This suggests that the inhibitory effects exerted by this phytoalexin on endothelial cells are mediated, at least in part, by a stereoselective receptor-dependent mechanism of action. Recent observations have shown the capacity of trans-resveratrol to bind αvβ3 integrin receptor in vitro near the RGD recognition site by interacting with the β3 integrin chain (17). Our results extend these observations by showing that trans-resveratrol, but not the cis stereoisomer, affects αvβ3 integrin function by inhibiting the adhesion of parental and β3-EGFP-transfected GM7373 cells to the prototypic αvβ3 substrate vitronectin. In contrast, trans-resveratrol does not affect β1 integrin-mediated GM7373 cell adhesion to fibronectin or laminin. This indicates that trans-resveratrol exerts its action by affecting β3 integrin function rather than by exerting a generic effect on cytoskeleton organization and/or integrin activity. Indeed, previous observations have shown that trans-resveratrol does not perturb cytoskeleton organization even when delivered to endothelial cells at doses as high as 300 μmol/L (23). In keeping with its αvβ3 antagonist function, trans-resveratrol selectively prevents the formation of paxillin-positive focal adhesion contacts and the recruitment of the EGFP-β3 integrin chimera in endothelial β3-EGFP-GM7373 cells seeded on vitronectin.
Similar to trans-resveratrol, RGD-derived αvβ3 integrin antagonists affect endothelial cell adhesion, proliferation, invasion, and morphogenesis when added to endothelial cell cultures in a micromolar range of concentrations (19, 21). Thus, our results support the hypothesis that interaction of trans-resveratrol with the β3 integrin chain may lead to inhibition of αvβ3 integrin activity. This may be responsible, at least in part, for the antiangiogenic potential of the phytoalexin. Nevertheless, the antiangiogenic activity of trans-resveratrol is likely the sum of several distinct mechanisms. Indeed, besides inhibiting αvβ3 integrin function, our results indicate that trans-resveratrol can exert an antiangiogenic effect in different in vitro assays where endothelial cells are adherent to various substrata distinct from vitronectin, including Matrigel, fibrin, and type I collagen. This may represent the consequence of the capacity of trans-resveratrol to affect cell cycle progression, cell survival, and intracellular signaling in endothelium (16, 35). Further studies are required to further dissect the complex mechanisms of action of trans-resveratrol on the angiogenesis process.
Dietary phytochemicals derived from edible plants may represent a promising class of tumor chemopreventive compounds by acting directly on tumor cells (7) and/or by inhibiting the angiogenesis process, thus acting as “angiopreventive” agents (5, 6). As confirmed in the present work, resveratrol is endowed with antiangiogenic and antineoplastic effects in vivo (14, 15) and exerts a significant cancer chemopreventive activity (10). However, results from pharmacokinetic studies indicate that resveratrol is rapidly metabolized in the body via glucuronidation, sulfation, and hydroxylation reactions (11, 36), casting some doubts about the physiologic relevance of the high concentrations that are effective in the in vitro experiments. This has led to the hypothesis that resveratrol metabolites may retain a significant biological activity (discussed in ref. 11). For instance, the resveratrol derivatives piceatannol and resveratrol-3-O-β-d-glucoside are endowed with significant anticancer and antiangiogenic activities (37, 38). Relevant to this point, resveratrol stereoisomers can undergo regioselective and stereospecific metabolic conversions (39, 40), thus suggesting that their bioavailability and metabolism may differ in vivo.
Previous observations from our laboratory have shown that subtle modifications in the chemical structure of trans-resveratrol deeply affect its antiangiogenic potency (23). Here, we have shown that stereoisomery is of importance in conferring to this phytoalexin a significant antiangiogenic and antitumor activity, possibly related to its αvβ3 antagonist activity. These data may provide the basis for the design of antiangiogenic/angiopreventive resveratrol derivatives endowed with a more potent efficacy in vitro and in vivo.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: Istituto Superiore di Sanità (Oncotechnological Program), Ministero dell'Istruzione, Università e Ricerca (Centro di Eccellenza per l'Innovazione Diagnostica e Terapeutica, Cofin projects), Associazione Italiana per la Ricerca sul Cancro, Fondazione Berlucchi, SPAFARM Project (Regione Lombardia), and NOBEL Project Cariplo (M. Presta).
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