Lycopene inhibits angiogenesis both in vitro and in vivo by inhibiting MMP-2/uPA system through VEGFR2-mediated PI3K-Akt and ERK/p38 signaling pathways

2.1 Lycopene solubilization

Lycopene was purchased from Wako (Kawagoe-shi, Saitama-ken, Japan) and the purity of commercial lycopene was approximately 98%, as claimed by the supplier (Wako). Lycopene was dissolved in tetrahydrofuran (THF)/butylated hydroxytoluene (BHT) to form a stock solution of 10-mM lycopene, which was diluted with THF at indicated ratios (1:1, 1:3, 1:7), and then diluted with fetal bovine serum (FBS) at the indicated ratio (1:9) [29]. THF/BHT-FBS-lycopene was added to the culture medium at a calculated final concentration of 2.5, 5, or 10 μM. THF at 0.2% (v/v) and FBS at 1.8% (v/v) served as the solvent control for lycopene, which did not significantly affect the assays described later.

2.2 Rat aortic ring assay

The ex vivo rat aortic ring assay was performed as described by Go and Owen [30] with minor modification. Twenty-four well tissue culture plates were precoated with 0.5 mL type I collagen gels mixed with Dulbecco's modified Eagle medium (DMEM) and allowed to gel for 1 h at 37°C. Thoracic aortas were isolated from 8-week-old male Sprague–Dawley rats. The thoracic aortas were quickly harvested and rinsed with phosphate-buffered saline (PBS) containing 4% gentamicin and sectioned into 1–1.5 mm aortic rings. The rings were embedded in a 24-well plate, and then 0.5 mL DMEM containing 2% FBS and various concentrations of lycopene (0–10 μM) was added and changed every day for 7 days. After incubation for 7 days, vessel sprouting around the rat aortic rings were photographed with an optical microscope at a 10× magnification and quantified by microvessel length using image software (Image-Pro Plus 5.0). Four fields were counted for each well and the mean number was expressed for each condition.

2.3 CAM assay

Eight-day-old fertilized chick eggs were used to examine the in vivo anti-angiogenic activity of lycopene as described by Ribatti et al. [31] with minor modification. Fertilized eggs (five eggs per group) were incubated at 37°C with 65% relatively humidity. On day 8, a 1-cm² window was carefully created on the air-cell side of the egg to assure the existence of embryonic blood vessels. Corn oils (20 μL) containing 1–15 μg lycopene were applied on a 0.5-cm² filter paper disk to the CAM, and the permeable sticky tape was immediately appended to the window. After incubation for 2 days, the eggshell was pushed aside around the window, and the blood vessels were photographed. The anti-angiogenic effect of lycopene on CAMs was quantified by counting the number of blood vessel branch points, which were marked using artistic software on the photos.

2.4 Matrigel plug assay

The in vivo matrigel plug assay was conducted according to the procedures described by Komi et al. [32] in which the lipid-soluble test compound, acyclic retinoid, was dissolved in dimethyl sulfoxide (DMSO) and mixed with matrigel before implantation subcutaneously (s.c.) to mice. In this study, lycopene (40 or 400 μg per plug or per mouse) was first solubilized in DMSO, of which 30 μL was added to the 0.5 mL matrigel. The final concentration of DMSO was 6% (v/v) in matrigel, which did not affect the plug formation. These doses of lycopene were based on those of our previous studies [20, 28], in which lycopene (1–20 mg/kg) was given orally and repeatedly to the nude mice twice a week for 12 weeks. C57BL/6 male mice (6-wk-old) were obtained from the Animal Center of the National Science Council. The mice were housed individually in cages with controlled temperature (25 ± 2°C), humidity (65 ± 5%) with 12-h light/dark cycles. After acclimatization for 1 week, the mice were randomly divided into four groups (n = 5 for each group) as follows. Group 1: control (implanted s.c. with 0.5 mL of matrigel containing 100 unit/mL of heparin and 6% DMSO); group 2: VEGF (implanted s.c. with 0.5 mL of matrigel containing 100 unit/mL of heparin, 100 ng/mL of VEGF, and 6% DMSO); group 3: low dose of lycopene (implanted s.c. with 0.5 mL of matrigel containing 100 unit/mL of heparin, 100 ng/mL of VEGF, and 40 μg lycopene); and group 4: high dose of lycopene (implanted s.c. with 0.5 mL of matrigel containing 100 unit/mL of heparin, 100 ng/mL of VEGF, and 400 μg lycopene). After implantation for 1 week, the matrigel formed a plug, and the mice were killed by CO₂ anesthetization; after which the plugs were removed carefully, fixed in 10% formalin, embedded in paraffin, and then sectioned for staining with hematoxylin and eosin. The vascular endothelial cells were quantified using image software (Image-Pro Plus 5.0), and the formula for calculation the anti-angiogenic effect of lycopene is (total vascular endothelial cells area ÷ total matrigel plug area) × 1000. The study protocol was approved by the Animal Research Committee of National Chung Hsing University (IAUCC Approval No: 99-71).

2.5 Cell culture and cell proliferation assay

HUVECs (BCRC H-UV001) with a passage of five were used in this study.

HUVECs were cultured in M199 medium (Gibco, Carlsbad, CA, USA), supplemented with 20% FBS, 30 μg/mL endothelial cell growth supplement (ECGS), 25 U/mL heparin, 2 mM l-glutamine, 1.5 g/L sodium bicarbonate, and 1% antibiotic–antimycotic (Gibco). The supplementation of ECGS to M199 medium [33] provides several essential growth factors such as acidic fibroblast growth factor (FGF) and endothelial cell growth factor (ECGF) for the growth of endothelial cells, as claimed by the supplier (Millipore, Billerica, MA). For cell proliferation assay, cells (5 × 10⁴ cells/mL) were seeded onto 24-well precoated 1% gelatin. HUVECs at 80% confluence were incubated with lycopene at the indicated concentration for 6, 12, and 24 h. The cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously [34].

2.6 Determination of tube formation in HUVECs

The effect of lycopene on angiogenesis in vitro was estimated by the tube formation assay, as described previously [35] with minor modification. Briefly, μ-slides were precoated with the 10 μL/well ECMatrix^TM (Chemicon, Darmstadt, Germany) and incubated for 2 h at 37°C. ECMatrix^TM, a soluble basement membrane isolated from Engelbreth-Holm-Swarm mouse sarcoma, has been used for in vitro assays of invasion and angiogenesis as well as for in vivo assays of angiogenesis [36, 37]. After pretreatment with lycopene for 6, 12, and 24 h, the number of HUVECs was adjusted to 8 × 10⁴ cells/mL, and 50 μL M199 containing 2% (v/v) FBS and 8 × 10⁴ cells was placed in the μ-slides followed by incubation for 6 h. The tube formation was examined microscopically. For each replicate, the neovascularization in five randomly selected fields was determined.

2.7 Cell invasion and migration assays

Cell invasion and migration were assayed using transwell chambers (Millipore, Darmstadt, Germany) according to the method reported by Repesh [38] with minor modification. The main difference between cell invasion and cell migration assays is that each filter was coated with 100 μL of a 1:20 diluted matrigel in cold DMEM to form a thin continuous film on the top of the filter for cell invasion assay. In both assays, transwell chambers with 6.5-mm polycarbonate filters of 8-μm pore size were used. After HUVECs were pretreated with lycopene for 6 h, the number of cells was adjusted to 1.25 × 10⁵/mL, and 400 μL of M199 serum-free medium containing 5 × 10⁴ cells was placed in the upper chamber. The lower chamber was loaded with 600 μL of M199 containing 20% FBS. After an incubation of 6 h (for the migration assay) or 24 h (for the invasion assay), the cells on the upper surface of the chamber were completely wiped away with a cotton swab. The cells on the lower surface of the filter were immobilized in methanol, stained with Giemsa, and counted under a microscope. For each replicate, the cells in five randomly selected fields were selected, and the counts were averaged.

2.8 Gelatin and plasminogen zymography

The activities of MMP-2 and uPA were assayed using the gelatin and plasminogen zymography, respectively, according to the methods reported by Yang et al. [39] with minor modifications. The cells (5 × 10⁴ cells/mL) were pretreated with lycopene for 6 h in M199 medium containing 20% (v/v) FBS and incubated for an additional 24 h in M199 serum-free medium, after which the culture medium was harvested and stored at –80°C until use. For the assay of gelatin and plasminogen zymography, the culture medium was electrophoresed in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing 0.1% (w/v) gelatin (for assay of MMP-2 activity) or 0.25% (v/v) plasminogen (for assay of uPA activity). After electrophoresis, gels were washed with 2.5% Triton X-100 and then incubated in reaction buffer (40 mM Tris–HCl, pH 8.0; 10 mM CaCl₂ and 0.01% NaN₃) for 12–15 h at 37°C. Then, gel was stained with Coomassie brilliant blue R-250 for 30 min and destained in 10% acetic acid (v/v) and 50% methanol (v/v). The relative MMP-2 anduPA activities were quantified using Matrox Inspector 2.1 software.

2.9 Western blotting

Protein expression of Rac1, TIMP-2, PAI-1, PI3K, Akt, p-Akt, ERK, p-ERK, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, and VEGFR2 was measured by Western blotting. Cells were lysed with cold radio-immuno-precipitation assay (RIPA) buffer containing protease inhibitor cocktail. The cell lysates were then centrifuged at 12 000 × g for 30 min at 4°C. An amount of protein (40 μg) from the supernatant was resolved by SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with Tris Buffered Saline buffer (20 mmol/L Tris–HCl, 150 mmol/L NaCl, pH 7.4) containing 5% nonfat milk, the membrane was incubated with monoclonal antibody followed by horseradish peroxidase-conjugated anti-mouse IgG, and then visualized using an Electrogenerated chemiluminescence (ECL) chemiluminescent detection kit (Amersham, Uppsala, Sweden).

2.10 Statistical analysis

Values are expressed as means ± SD and analyzed using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) for comparisons of group means. Student's t-test was used for comparisons of two groups. All statistical analyses were performed using SPSS for Windows, version 10. Unless specified otherwise, a p < 0.05 is consideredsignificant.

3.1 Lycopene inhibits angiogenesis both ex vivo and in vivo

We used both ex vivo and in vivo models to confirm the anti-angiogenic effect of lycopene. In the rat aortic ring assay (an ex vivo model), treatment of lycopene (2.5–10 μM) induced a significant and concentration-dependent inhibition of the sprouting of vessels from rat aortic rings, with an inhibition of 51% (p < 0.01) at 10 μM lycopene, as compared with control group (Fig. 1). In the in vivo CAM experiment, neovascularization was markedly increased during 8–10 days of incubation (Fig. 2). Treatment of fertilized eggs with lycopene (1–15 μg or 1.86–27.9 nmol/egg) significantly inhibited the number of blood vessel points, when the dose reached 2.5 μg or 4.65 nmol/egg. The inhibitory effect of lycopene continued to increase in a dose-dependent manner, reaching an inhibition of 38% (p < 0.05) at 15 μg/egg, as compared with the control group (Fig. 2).

In the in vivo matrigel plug assay, subcutaneous injection of mice with matrigel containing VEGF (100 ng/mL) induced a significant angiogenic response, as evidenced by increased vascular endothelial cells, as compared with the control group (Fig. 3). Lycopene treatment at the low dose (40 μg/plug or 74.5 nmol/plus for each mouse) did not significantly affect the number of vascular endothelial cells induced by VEGF, whereas lycopene at the high dose (400 μg/plug or 745 nmol/plug for each mouse) completely inhibited the formation of vascular endothelial cells (Fig. 3).

3.2 Lycopene inhibits tube formation in HUVECs

Pre-incubation of HUVECs with lycopene (2.5–10 μM) for 6, 12, and 24 h significantly inhibited tube formation in a concentration-dependent manner (Fig. 4), and the strongest inhibitory effect occurred at 6 h of incubation with an inhibition of 39% (p < 0.05) at 10 μM lycopene (Fig. 4). In addition, lycopene did not affect cell proliferation at either 6 or 12 h of incubation, but slightly inhibited cell proliferation at 24 h of incubation with an inhibition of 18% (p < 0.05) at 10 μM lycopene in HUVECs (Fig. 5). Based on these results, we excluded the possibility that the inhibitory effect of lycopene on tube formation is attributed to inhibition of cell proliferation. Therefore, we chose a pre-incubation time of 6 h or less than 6 h for the following studies.

3.3 Lycopene inhibits invasion and migration in HUVECs

Pretreatment of HUVECs with lycopene (2.5–10 μM) for 6 h markedly inhibited cell invasion and migration in a concentration-dependent manner, with an inhibition of 33% (p < 0.05) and 65% (p < 0.01), respectively, when added at 10 μM lycopene (Fig. 6).

3.4 Lycopene inhibits activities of proMMP-2, MMP-2, and uPA in HUVECs

Pre-incubation of HUVECs with lycopene (2.5–10 μM) for 6 h significantly inhibited activities of proMMP-2, MMP-2, and uPA in a concentration-dependent manner, with an inhibition of 26% (p < 0.05), 27% (p < 0.05), and 30% (p < 0.05) at 10 μM lycopene, respectively (Fig. 7).

3.5 Effect of lycopene on TIMP-2, PAI-1, and Rac1 protein expression in HUVECs

Pre-incubation of HUVECs with lycopene (2.5–10 μM) for 6 h significantly increased protein expression of TIMP-2 and PAI-1 in a concentration-dependent manner, with a promotion of 68% (p < 0.01) and 60% (p < 0.01), respectively, at 10 μM lycopene (Fig. 8). In contrast, lycopene significantly inhibited protein expression of Rac1 in a concentration-dependent, with an inhibition of 63% (p < 0.01) at 10 μM lycopene (Fig. 8).

3.6 Lycopene attenuates VEGFR2-mediated PI3K-Akt axis and ERK/p38 pathway in HUVECs

HUVECs were incubated with lycopene (10 μM) for 2, 4, and 6 h, and the protein expression of VEGFR2, PI3K-Akt, and MAPK family members (ERK1/2, JNK1/2, and p38) was determined by Western blotting. Lycopene (10 μM) significantly decreased the protein expression of VEGFR2 at 2 h of incubation, with an inhibition of 22% (p < 0.05; Fig. 9). In addition, lycopene (10 μM) significantly reduced the protein expression of PI3K and the phosphorylation of Akt at 6 h of incubation with inhibition of 17% (p < 0.05) and 40% (p < 0.05), respectively, without affecting total protein expression of Akt (Fig. 9). Moreover, lycopene (10 μM) significantly inhibited the phosphorylation of p38 and ERK1/2 at 2 h and 4 h of incubation, with an inhibition of 32% (P < 0.05) and 29% (P < 0.05), respectively, without changing their protein expression levels (Fig. 10). In contrast, lycopene did not affect the phosphorylation and total protein expression of JNK1/2 during 6 h of incubation (Fig. 10).