Digallate Inhibits Tube Formation in Cocultured Endothelial Cells with Fibroblasts

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 activities. 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 secretion; 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 theaflavins, including theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate (TF3) 2). Catechins, especially EGCG, have antitumor activity via antioxidation, antiangiogenesis, 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 M1VIPactivity 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 studies 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 postcultured 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. 3A to H) ; and 100 ƒÊmol/L of EGCG or TF3 almost completely inhibited the formation of tube-like structures. The total area, length, and joints of the tubular formations are shown in Fig. 4A, 4B, and 4C, respectively. In particular, 10 ftmol/L

TF3 clearly inhibited tubular formation as much as levels in the absence VEGF. EGCG or TF3 at 100 ƒÊmot/L entirely inhibited tube-like structures, although VEGF stimulated

HUVECs.

Fig. 5A and 6A show the results of gelatin zymography of the samples after 11 days in the medium. Pro-MMP-2 (72 kDa) and pro-MMP-9 (92 kDa) were detected in all of

the 10-jiL samples of medium, but pro-MMP-9 was barely expressed at concentrations of

100 timoUL TF3 or EGCG. There were small amounts of active MMP-2 (62 kDa) detected

in controls, VEGF controls, 10 i.tmol/L EGCG, and 10 umol/L TF3. The expression of

MMP-2 and MMP-9 tended to be the same regardless of the presence of VEGF. MMP-2

activity was directly inhibited in a concentration-dependent manner by TF3 or EGCG in

the precultured medium (Fig. 5B and 6B). MMP-2 activity was inhibited significantly in

the postcultured medium, especially at 100 ƒÊmol/L TF3 or EGCG. MMP-9 activity was

inhibited in a concentration-dependent manner by TF3 or EGCG in the precultured and

postcultured medium (Fig. 5C and 6C). There was very little MMP-9 activity in the post- Theaflavin Inhibits Tube Formation 63

Fig. 2. The tube-like structures were analyzed at 11 days of culture. Values represent the percent increases in tube-like structures relative to the control. A : Total area of tube-like structures in HUVECs. B : Total length of tube-like structures in HUVECs. C :

The number of joints of the tube-like structures in HUVECs. Tube length was measured quantitatively with an image analyzer. Cont : n=8 ; EGCG 10 and 100 ƒÊmol/L, TF3 10 and 100 ƒÊmol/L, and+VEGF : n=6 ;

EGCG 50 ƒÊmol/L and TF3 50 ƒÊmol/L : n= 4. * p<.05 ; **p< .01. 64 Sei KOBAYASHI, et al

Fig. 3. Development of tube-like structures in HUVECs cocultured with fibroblasts, in the presence or absence of VEGF. A: Conditioned medium was treated with VEGF as the control (+ VEGF). B-D: Conditioned medium with the addition of 10, 50 or 100 umol/L of EGCG, plus VEGF. E : Conditioned medium alone (- VEGF). F-H : Conditioned medium with the addition of 10, 50 or 100 umoUL of TF3, plus VEGF.

cultured medium containing 100 ttmol/L TF3 or EGCG. The inhibiting effect of EGCG and TF3 on MMP-9 activity was stronger than on MMP-2 activity. The precultured medium contained bovine serum that included bovine pro-MMP-2. Unlike zymography, the EIA kit detects only human pro-MMP-2, because bovine pro-MMP-2 was not detected in the precultured medium. Fig. 7 and 8 show pro-MMP-2 secretion from HUVECs and fibroblasts. The secretion of MMP-2 proteins was inhibited in a concentration-dependent manner by TF3 or EGCG in the postcultured medium, regardless of the presence of VEGF.

Discussion In the present study, the ettects of I F3 and EGCG were similar, snowing tnat tube formation by endothelial cells was suppressed in vitro. The inhibitory effect of TF3 and EGCG occurred at concentrations as low as 10 itmol/L. Previous studies have indicated that consumption of. green tea extract (1.5-4.5g, or 1-3 cups) by healthy individuals is associated with the appearance of EGCG, ECG, and EC in the plasma and that the plasma concentration of EGCG may reach 4 timol/L24-26). This conclusion is strengthened by epidemiological studies indicating that the consumption of large amounts of green tea, especially more than 5 cups daily (in which case the plasma concentration of EGCG may reach 6.7-20 timol/L), is associated with a decreased risk for several types of cancer, or a decreased risk of their progression 2729). The results of the present study are in accordance with these studies. It has been reported that locally high concentrations (up to 131.0 itmol/ L) of tea polyphenols occur when holding green tea leaves in the oral cavity30). Theaflavin Inhibits Tube Formation 65

Fig. 4. The tube-like structures were analyzed at 11 days of culture. Values represent the percent increases in tube-like structures relative to the control (+ VEGF).

A : Total area of tube-like structures in HUVECs. B : Total length of tube-like structures in HUVECs . C : The number of joints of the tube-like structures in HUVECs. Tube length was measured quantitatively with an image analyzer. Cont (+VEGF), TF3 10 and 100 ,umoUL : n=8 ; -VEGF : n=6 ; EGCG 10, 50 and 100 ƒÊmol/L and TF3 50 ƒÊmol/L : n= 4. * p<.05 ; ** p<.01. 66 Sei KOBAYASHI, et al

Fig. 5. A : Representative example of gelatin zymogram in spent medium in the control, 10, 50 and 100 ƒÊmol/L of EGCG or TF3, and 10 ng/mL VEGF. The gelatinolytic activities are recognized as

clear bands against a black background. The 2 bands in the last lane correspond to pro-MMP-2 and

pro-MMP-9 standards. Active-MMP-2, pro-MMP-2 and pro-MMP-9 are recognized at 62, 72 and 92 kDa, respectively. Active MMP-9 (83 kDa) was not detected clearly. B : Values represent increases in activity of pro-MMP-2 in the medium relative to the control in spent medium at 11 days, determined by gelatin zymography. The left and right columns represent MMP-2 activities in spent and

precultured medium, respectively. Cont : n=8 ; EGCG 10 and 100 ttmol/L, TF3 10 and 100 ftmoVL and + V EGF : n=6 : EGCG 50 f-tmoUL and TF3 50 f tmoUL : n=4. * P<.05 ; ** P<.01. C : Values represent increases in activity of pro-MMP-9 in the medium relative to the control in spent medium at 11 days determined by gelatin zymography. The left and right columns represent MMP-9

activities in spent and precultured medium, respectively. Cont : n = 8 ; EGCG 10 and 100 ƒÊmol/L, TF3 10 and 100 ltmoUL and + VEGF : n=6 ; EGCG 50 ƒÊmol/L and TF3 50 ƒÊmol/L : n=4. *P< .05. **P<.01. Theaflavin Inhibits Tube Formation 67

Fig. 6. 68 Set KOBAYASHI, et al

MMP-2 and MMP-9 have important roles in angiogenesis by degrading ECM to make room for migrating endothelial cells 31). Both HUVECs and fibroblasts secrete MMP-2 and MMP-9 32,33) MMP-2 is not responsive to phorbol esters or most cytokines or growth factors 34-36). It is constitutively expressed by many cell types, and its promoter lacks the TATA sequence, as for many so-called housekeeping genes37). In the present study, the activity of MMP-2 was not significantly changed by VEGF stimulation. Pro-MMP-2 protein (72 kDa) is secreted by the cell when it is in a latent state. Activation of M1VIP-2requires the formation of a ternary complex ; with the interaction between membrane type 1-MMP (MT1-MMP) and MMP-2 mediated by TIMP-238). TIMP-2 forms a bridge through simul- taneous interactions at one domain with the active site of MT1-MMP and through another domain with the carboxy-terminal region of MMP-2. Following formation of the ternary complex, a neighboring unoccupied MT1-MMP cleaves the MMP-2 propeptide domain 39,40)

Fig. 7. Values represent pro-MMP-2 (ng/mL) concentration in the medium at 11 days, determined by EIA. Cont : n=8 ; EGCG 10 and 100 ƒÊmol/L, TF3 10 and 100 ƒÊmol/L and + VEGF : n=6 ; EGCG 50 jtmol/L and

TF3 50 ƒÊmol/L : n=4. ** P<.01.

Fig. 6. A : Representative example of gelatin zymogram in spent medium in control+VEGF, 10, 50 and 100 ƒÊmol/L of EGCG or TF3 with 10 ng/mL VEGF, and without VEGF (-VEGF). The gelatinolytic activities are recognized as clear bands against a black background. The 2 bands in the last lane correspond to pro-MMP-2 and pro-MMP-9 standards. Active-MMP-2, pro-MMP-2 and pro-MMP-9 are recognized at 62, 72 and 92 kDa, respectively. Active MMP-9 (83 kDa) was not clearly detected. B : Values represent the increases in activity of pro-MMP-2 in the medium relative to the control (+ VEGF) spent medium at 11 days, determined by gelatin zymography. The left and right columns represent MMP-2 activities in spent and precultured medium, respectively. Cont : n=8 ; EGCG 10 and 100 ƒÊmol/L, TF3 10 and 100 umol/L and +VEGF : n=6 ; EGCG 50 umo1JL and TF3 50 imol/L : n = 4. * P<.05; ** P<.01. C : Values represent the increases in activity of pro-MMP-9 in medium relative to control (+ VEGF) spent medium at 11 days, determined by gelatin zymography. The left and right columns represent MMP-9 activities in spent and precultured medium, respectively. Cont : n =8 ; EGCG 10 and 100 ƒÊmol/L , TF3 10 and 100 ƒÊmol/L and +VEGF : n=6 ; EGCG 50 ƒÊmol/L and TF3 50 ƒÊmoI/L : n = 4. * P<.05; ** P<.01. Theaflavin Inhibits Tube Formation 69

Fig. 8. Values represent the pro-MMP-2 concentration (ng/mL) in the medium at 11 days, determined by ETA. Cont : n=8 ; EGCG 10 and 100 ƒÊmol/L, TF3 10 and 100 ƒÊmol/L and + VEGF : n=6 ; EGCG

50 ƒÊmol/L and TF3 50 ƒÊmol/L : n=4. * P<.05, ** P<.01.

In contrast, in most cell types, gene transcription of MMP-9 is inducible ; after translation, the enzyme is immediately secreted via the normal secretory pathway. In specific cell types, human MMP-9 is induced by multiple polypeptide factors, including epidermal growth factor, platelet-derived growth factor, hepatocyte growth factor/scatter factor, basic fibroblast growth factor, transforming growth factor (TGF) -a, amphiregulin, tumor necrosis factor-a, IL-1 a, IL-1a, interferon-a, interferon-y, and TGF-,3, and phorbol ester stimulation41). MMP-9 is a component of the angiogenic switch, as it has a role in the release of biologically active VEGF 14,42),to a greater extent than other MMPs. EGCG and TF3 inhibited MMP activity in the medium containing HUVECs and fibroblast cells, and the effective concentration range paralleled the inhibitory effects on tube formation. EGCG and TF3 have two ways of inhibiting MMP-2 and MMP-9 ; one is by direct inhibition of activity. In the zymography experiments, MMP-2 activity was reduced in medium containing EGCG or TF3, before culturing. Polyphenols can form complexes with various substances, including proteins and metal ions. Inhibition of MMP-2 and MMP-9 activity by ester-type catechins and theaflavin may have been caused by the polyphenols chelating zinc, which is essential for enzymatic activity 19). The other way in which EGCG or TF3 can influence MMPs is by inhibiting secretion )8,43) (via antioxidant or other actions) The MMP-9 promoter has several transcription factor binding motifs, including activator protein-1 (AP-1) sites (TRE-1, -2, and -3) and a nuclear factor-kappa B (NF-ƒÈB) site 21) EGCG and TF3 suppress signal transduction pathways leading to AP-1 and NF-ƒÈB 44), thus decreasing MMP-9 activity. In the present study, the secretion of MMP-2 protein was inhibited by EGCG and TF3. The MMP-2 promoter has no AP-1 site within the published 1.6-kilobase sequence and is not responsive to phorbol esters, most cytokines, or growth factors 21) However, zymographic analysis has 70 Set KOBAYASHI, et al shown increased activity of the gelatinolytic enzyme MMP-2 in the culture supernatant from cells exposed to modified lamnin-145). Both MMP-2 mRNA expression and activity were significantly reduced in Mr 67,000 laminin receptor (67LR) antisense-transfected cells 46). EGCG-induced reduction of the myosin II regulatory light chain phosphorylation is involved in signal transduction pathways that mediate post-EGCG binding to 67LR 47.E. TF3 and EGCG may be able to bind to 67LR. The control of angiogenesis is important for the treatment of cancer and retinopathy. We have demonstrated that TF3 and EGCG inhibit the formation of tubes by endothelial cells and this suggests that inhibition of MMPs is one of the causes of the antiangiogenic effects of TF3 and EGCG. Our results show that TF3 has similar antiangiogemc effects as EGCG. The antiangiogenic effects of black tea may not be equal to green tea, since TF3 and EGCG concentrations in black tea are lower 2,49). The antiangiogenic effects of TF3 may give it a role as a natural antitumor medication. Both TF3 and EGCG may be useful agents or lead compounds for the treatment of cancer and retinopathy.

Acknowledgements

This work was supported in part by the Showa University Medical Foundation.

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[Received December 14, 2006: Accepted January 12, 2007]