Showa Univ J Med Sci 19(2). 59•`72, June 2007 Original Theaflavin-3,3'-digallate Inhibits Tube Formation in Cocultured Endothelial Cells with Fibroblasts Set KOBAYASHI1), Shlnlchl IWAI2), Kazuko TSUJIYAMA2) , Chika KURAHASHI2),Yuko UDAKA2),Takeyuki SANBEl), Harumi SUZAKI1)and Katsuji OGUCHI2) Abstract : Several tea polyphenols, particularly those containing galloyl, have antitumor affects via strong antioxidant and antiangiogenic activi- ties. Theaflavin-3,3'-digallate (TF3), a theaflavin derivative in black tea, has 2 galloyl groups. Matrix metalloproteinases (MMPs) are associated with extracellular matrix degradation, cellular migration, and angiogenesis, and (-) -epigallocatechin-3-gallate (EGCG) is an inhibitor of MMP activity and secre- tion; thus one of its major actions is the inhibition of angiogenesis. However, there are few studies of angiogenesis in theaflavin derivatives. We investigated the effects of TF3 on angiogenesis in vitro. Angiogenesis was assayed using cocultured human umbilical vein endothelial cells with fibroblasts. Cells were cultivated in various concentrations of TF3 and EGCG in the presence or absence of vascular endothelial growth factor-A. After 11 days, MMP-2 and MMP-9 activities and the pro-MMP-2 protein in the medium were measured by gelatin zymography and immunoassay, respectively. Tube formation was markedly inhibited by 100 umol/L TF3 or EGCG. Even at 10 iumolIL, TF3 or EGCG inhibited tube formation. The MMP-2 and MMP-9 activities were inhibited and pro-MMP-2 protein concentrations were reduced by TF3 or EGCG in a concentration-dependent manner, regardless of the presence of vascular endothelial growth factor. The effect of TF3 was similar to that of EGCG, indicating that the tube formation of endothelial cells was suppressed via decreased both MMP-2 and MMP-9 activities in vitro. Our results dem- onstrate antiangiogenic activity of TF3 in vitro, and suggest possible anti-tumor effects of TF3. Key words : theaflavin-3,3'-digallate,(-) -epigallocatechingallate, tube formation, matrix metalloproteinase Introduction Green tea is extracted from the leaves of Camellia sinensis. It has been a popular bever- age in east Asia for 3000 to 4000 years. Catechins are important ingredients of green tea and include (-) -epigallocatechin-3-gallate (EGCG), (-) -epigallocatechin, (-) -epicatechin-3- 1) Department of Otorhinolaryngology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan.2 ) Department of Pharmacology, Showa University School of Medicine. 60 Set KOBAYASHI, et al gallate (ECG), and (-) -epicatechin (EC) 1). Black tea contains catechins and also theafla- vins, including theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate (TF3) 2). Catechins, especially EGCG, have antitumor activity via antioxidation, antiangio- genesis, and other mechanisms 3,4-7). TF3 has 2 galloyl groups, which confer antioxidant potencies similar to or greater than those of the catechins present in green tea 8). Theafla- vins also have an antitumor effect 9). However, there have been few studies on the role of theaflavin derivatives in antiangiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases associated with extracellular matrix (ECM) degradation, cellular migration, tissue remodeling, and angiogenesis 10). MMPs are secreted by many types of cells as proenzymes. After activation by proteolytic cleavage, the enzymes are capable of degrading many ECM components 11) MMPs are believed to be the major enzymes responsible for ECM degradation 12). M1VIP-2 and MMP-9, in particular, are involved in tumor angiogenesis 13,14) The activities of MMPs are regulated by the activation of precursor zymogens. They are inhibited by endogenous inhibitors, or by tissue inhibitors of metalloproteinases (TIMPs). Thus, the balance between MMPs and TIMPs is critical for the eventual remodeling of the ECM in tissue 15) EGCG is a potent inhibitor of M1VIP activity and secretion and such inhibition is a major contributor to its antitumor and antiangiogenic activity. Theaflavins can also inhibit MMPs 19),suggesting that they may also play a role in antiangiogenesis. However, few stud- ies have examined this possibility. In the present study we examined the effect of EGCG and TF3 on the tube formation of endothelial cells in vitro. We found that EGCG and TF3 inhibited tube formation and this was associated with the suppression of MMP-2 and MMP-9 activity. Materials and Methods Reagents The reagents used were EGCG and TF3 (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and vascular endothelial growth factor-A (VEGF) (Kurabo Industries, Ltd., Osaka, Japan). Quantitative analysis of tube formation by human umbilical vein endothelial cells Experiments on tube formation were conducted in triplicate in 24-multiwell dishes using the Kurabo Angiogenesis Kit according to the manufacturer's instructions. Human umbilical vein endothelial cells (HUVECs) were cocultured with human fibroblasts in these media for 10 days. The medium was changed every 3 days for 6 days, and then every 2 days. EGCG or TF3 were then added in amounts of 10, 50, or 100 imol/L into the medium at each change. The dishes were then washed with phosphate-buffered saline (PBS) and fixed with 70% ethanol at 4•Ž. After the fixed cells were rinsed 3 times with PBS, they were incubated with mouse antihuman CD31 (Kurabo) in PBS containing 1 o bovine serum albumin for 60 minutes. After washing with 1 % bovine serum albumin in PBS 3 times, the cells were incubated with goat antimouse IgG conjugated with alkaline phosphatase for 60 minutes. The substrate was 5-bromo-4-chloro-3- indolyl phosphate /nitro blue tetrazolium (Kurabo), and the reaction yielded a dark reddish-brown precipitate. Finally, the cells were washed with PBS 3 times and viewed under a microscope (Nikon Co, Tokyo, Japan). Tube area, length, and joints were measured with the Kurabo Angiogenesis Image Analyzer Theaflavin Inhibits Tube Formation 61 (version 2) in 5 different fields for each well20). Zymography MMP-2 and MMP-9 activities were measured by gelatin zymography on 10% polyacryl- amide gels containing 1 mg/mL gelatin in a polymerization mixture of sodium dodecyl sulfate (SDS). Samples were collected from the precultured medium and from the postcultured medium at 11 days and 10 iL of samples was mixed with 5 iiL of sample buffer. After electrophoresis for the detection of proteinase activity, SDS was washed out of the gels with 2.5% Triton X-100 for 30 minutes at room temperature , and the gels were then incubated at 37°C for 1 day in a solution containing 50 mmol!L Tris-HCL (pH 7.5), 5 mmol/L CaCl2 and 0.02% Na azide. The gels were then stained with 0.1% Coomassie blue in 10% acetic acid and 50% methanol for 30 minutes and destained in 10% acetic acid and 40% methanol. Clear areas indicated proteolytic activity. Molecular weight standards (Bio-Rad Laboratories, Hercules, CA) were included to calibrate the gels. The 72-kDa purified proenzyme MMP-2 (0.8 ng protein per lane) and the 92-kDa purified proenzyme MMP-9 (0.1 ng protein per lane) were used as positive controls and standards for the gels (MMP-2 : PF037 ; MMP-9 : PF038 ; EMD Biosciences Inc., San Diego, CA). The zymography bands were digitized using the ATTO imaging system (ATTO Co ., Tokyo, Japan), and analyzed using NIH Image software 21) Assay of pro-MMP-2 in the postcultured medium After the cells had been cultured for 11 days in complete growth medium, the postcul- tured medium was collected and the MMP-2 concentrations were measured using a one- step sandwich enzyme immunoassay (EIA) kit (Daiichi Fine Chemical Co., Ltd., Toyama , Japan), following the manufacturer's protocol. Pro-MMP-2 in the postcultured medium was measured by absorbance at 492 nm using a spectrophotometer (SpectraMax 340PC ; Molecular Devices Co., Ltd., Tokyo, Japan) 22.23), Statistical Analysis Results are expressed as the mean•}SEM. Data were analyzed by one-way analysis of variance. P<.05 was considered significant. Results At 100 jcmol/L EGCG and TF3, there were no changes in cell death or morphology in the angiogenesis kit ; there was a similar result in HUVEC single culture (data not shown) . Therefore, EGCG and TF3 showed little toxicity at concentrations <100 ƒÊmol/L . Precultured medium containing 10, 50 or 100 ,umollL of EGCG or TF3 was applied to HUVECs from the second medium change (at 4 days) to study tubular morphogenesis . When endothelial cells were cultured in the normal medium alone, tube-like structures were apparent (Fig. 1A) ; in medium with the addition of VEGF, tube-like structures were more prominent (Fig. IE). However, with the addition of EGCG or TF3, the tube- like structures decreased in a dose-dependent manner (Fig. lB to D, F to H). Tube-like structures were rare when 100 imol/L of EGCG or TF3 was added to media . Total area, length, and joints of the tubular formations are shown in Fig. 2A to C, respectively ; these measurements were similar for the same EGCG and TF3 concentrations . Concentrations . 62 Sei KOBAVASHI, et al Fig. 1. Development of tube-like structures in HUVECs cocultured with fibroblasts. A : Conditioned medium was tested alone as the control. B-D : Conditioned medium was tested with 10, 50 and 100 ƒÊmoUL of EGCG. E : Conditioned medium was tested with VEGF. F-H : Conditioned medium was tested with 10. 50 and 100 timoUL of TF3. of 10 ƒÊmol/L EGCG or TF3 inhibited tube formation by 36% to 78% ; and 100 ƒÊmol/L completely inhibited tube formation. To investigate the effects of EGCG and TF3 in combination with VEGF treatment, precultured medium (containing 10, 50, or 100 ƒÊmol/L of EGCG or TF3, with or without 10 ng/mL VEGF) was applied to HUVECs from the first medium change. Tube-like structures decreased in a dose-dependent manner with increased concentrations of EGCG or TF3 with VEGF (Fig.