ARTICLE IN PRESS Phytomedicine 17 (2010) 42–46
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Downregulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 by grape seed proanthocyanidin extract Dhungana Sandra, Madhyastha Radha, Madhyastha Harishkumar, Nakajima Yuichi, Omura Sayuri, Maruyama Masugi Department of Applied Physiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
a r t i c l e in f o
Keywords: Urokinase-type Plasminogen activator and plasminogen activator inhibitor-1 Downregulation Grape seed proanthocyanidin extract
a b s t r a c t Urokinase plasminogen activator (uPA) system, comprising of uPA, its receptor uPAR and inhibitor, type 1 plasminogen activator inhibitor (PAI-1), plays a vital role in various biological processes involving extracellular proteolysis, ﬁbrinolysis, cell migration and proliferation. The timely occurence of these processes are essential for normal wound healing. This study examines the regulation of uPA and PAI-1 by a natural polyphenol-rich compound, grape seed extract (GSE). GSE is reported to have beneﬁcial effects in promoting wound healing. Fibroblast cells exposed to different doses of GSE for 18 hours were processed for further studies such as ELISA, RT-PCR, western blotting, ﬁbrinolytic assay, cell surface plasmin activity assay and in vitro wound healing assay. GSE treatment caused a signiﬁcant downregulation of uPA and PAI-1 expression, both at the RNA and protein levels. ELISA also revealed a dose-dependent decrease in uPA and PAI-1 activities. Functional signiﬁcance of the downregulation was evident in decreased ﬁbrinolytic activity, concomittant with decreased cell-surface plasmin activity. In vitro wound healing studies showed that GSE also retarded the migration of cells towards the wounded region. & 2009 Elsevier GmbH. All rights reserved.
Introduction The serine protease, urokinase-type plasminogen activator (uPA) plays an important role in tissue remodeling, cell migration and wound healing (Toriseva and Kahari, 2008; Watanabe et al., 2006). It catalyzes the conversion of inactive zymogen plasminogen to the enzymatically active plasmin. Plasmin participates in ﬁbrinolysis by degrading ﬁbrin and in tissue remodeling by degrading extracellular matrix (ECM) and activating other matrix degrading proteases. The main physiological inhibitor of uPA is plasminogen activator inhibitor-1 (PAI-1). Besides inhibiting plasminogen activation, PAI-1 has multi-faceted roles with relation to cell adhesion/ migration and thus is involved in wound healing (Lijnen, 2005). uPA-PAI-1 system is implicated in the early phase of wound healing that requires dissolution of ﬁbrin clot and cellular migration into the wound region. Proanthocyanidins are a group of biologically active polyphenolic bioﬂavonoids synthesized by many plants, and are known to facilitate wound healing (Hupkens et al., 1995). Grape seed extract (GSE) is a rich source of proanthocyanidins. Recent studies suggest that GSE facilitates wound healing by regulating oxidant-induced changes in keratinocytes and improving wound closure (Khanna Corresponding author. Tel.: +81 985 85 1785; fax: +81 985 85 7932.
E-mail address: [email protected]
(M. Masugi). 0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.06.010
et al., 2001, 2002). To date, the potential inﬂuence of GSE on uPA system has not been evaluated. The aim of the present study was to examine the effects of GSE on uPA and PAI-1 in cultured human ﬁbroblast cells. We report that GSE downregulates uPA and PAI-1, with functional consequences of decrease in ﬁbrinolytic activity as well as cellular migration towards wounded region.
Materials and Methods Cell culture and reagents Human ﬁbroblast cells TIG 3-20 (HSRBB Cell bank, Osaka, Japan) were cultured in Modiﬁed Eagles Medium. Semi-conﬂuent cells between passages 3 and 5 were used for the studies. GSE (VinofelonTM) was a kind gift from Tokiwa Phytochemical Co., Ltd, Chiba, Japan. Dried ground seeds (500 kg) of Grape (Vitis spp.) were reﬂuxed for 1 hr in aqueous ethanol (80% v/v, 5000 l) twice and the combined alcoholic extract was evaporated and ﬁltered. The ﬁltrate was adsorbed onto porous synthetic resin (DIAION HP20, 500 l) and eluted with water (1000 l). Proanthocyanidin fraction was then collected by elution with aqueous ethanol (70% v/v, 1000 l). The fraction was evaporated and dried by spray dryer. The obtained solid was crushed, mixed and passed through a sieve to obtain powder of proanthocyanidin-rich extract (15 kg).
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Western blotting Total protein extracts from cells were obtained by lysing cells in mammalian protein extraction reagent (M-PER, Pierce Biotechnology Inc, IL, USA). Twenty microgram proteins were resolved over 10% SDS–PAGE gels and electroblotted onto nitrocellulose membrane using Trans-blot SD, semi dry transfer cell (Bio-Rad Laboratories, CA, USA). The membrane was subjected to western blotting using anti-uPA, anti-PAI-1 and anti-b-actin antibodies, as the case may be. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Expression of proteins was detected by chemiluminescence using ECL Plus Western Blotting Detection System (Amersham Life Science, Inc., Buckinghamshire,
120 100 80 % cell viability
Although the chemical composition of GSE has not been speciﬁed completely, the product analysis sheet provided by Tokiwa Phytochemical Co., Ltd. shows proanthocyanidin content as not less than 90% of total ﬂavanol content on dry matter basis. Following is the procedure provided by Tokiwa Phytochemical for standardization of puriﬁed proanthocyanidins. Total ﬂavanols in the GSE were determined by the standard vanillin-HCl method (Broadhurst and Jones, 1978) using (+)-catechin (Kurita Kogyo Co., Ltd, Japan) as standard. Brieﬂy, 20 mg of GSE was weighed and dissolved in water to get a sample solution of 100 ml. To 1 ml of the sample solution in a brown tube, 9 ml of 2% vanillin/HClmethanol reagent (2 g vanillin dissolved in 12 N HCl-methanol (1:2) solution to get ﬁnal volume of 100 ml) was added, immediately capped, mixed for 10 seconds and incubated at 18–221C for 20 min. Absorbance of this solution was measured by spectrophotometer at 500 nm (reference: water) (ABS-S). To correct inﬂuences of anthocyanidins in sample, HCl-methanol (1:2) was added instead of 2% vanillin/HCl-methanol reagent to each sample solution (ABS-C). Proanthocyanidins (as total ﬂavanol) contents was calculated from the value of (ABS-S) – (ABS-C) – (ABS-BLANK) by using working curve. Working curve was obtained as follows: 1, 2, 3 mg of (+)-catechin was dissolved in water to a ﬁnal volume of 10 ml (the standard solution). One ml of each standard solution was taken in a brown tube and 9.0 ml of 2% vanillin/HCl-methanol reagent was added, immediately capped, mixed for 10 seconds and incubated at 18–22 1C for 20 min. Absorbance of this solution was measured at 500 nm by spectrophotometer (reference: water) (ABS-CAL). In case of blank, water was used instead of standard solution (ABS-BLANK). Working curve was obtained with correcting values; (ABS-CAL)-(ABSBLANK). GSE was dissolved in double distilled water and ﬁltered prior to experiments. Cells were incubated with different doses of GSE in serum free medium for 18 hours. Conditioned medium was collected, centrifuged at 8000 g for 10 min to remove cell debris and stored at 70 1C until further assays.
60 40 20 0 0
20 30 GSE g/ml
Fig. 1. Cytotoxic proﬁle of GSE on TIG 3–20 ﬁbroblasts: Semi-conﬂuent cells were treated with various doses of GSE for 18 hours. Cell viability was analyzed using Cell titer Glo Luminescent Assay. Values are mean7SD of six independent experiments.
Cell viability assay Effect of GSE on cell viability was analyzed by CellTiter Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) following manufacturer’s protocol.
Effect of GSE on generation of intracellular reactive oxygen species was estimated using dichloroﬂuorescein diacetate (DCFHDA) (Balasubramanyam et al., 2003). Following incubation of ﬁbroblasts with different doses of GSE for 18 hours, 10 mM DCFHDA was added to the cells. Non-ﬂuorescent DCFH-DA is converted to ﬂuorescent DCF, in proportion to the amount of ROS generated in the cells. The ﬂuorescent signal was measured using FP-6200 spectroﬂuorometer (Excitation 485 nm; Emission 530 nm).
Quantiﬁcation of uPA and PAI-1 antigen levels by ELISA
% ROS generation
Reactive Oxygen species (ROS) assay 80
The levels of uPA and PAI-1 antigens in the conditioned medium from cells incubated with GSE were measured by ELISA using scuPA ELISA reagent kit (Technoclone, Mullnergasse, Vienna, Austria) for uPA and Assay Max Human PAI-1 ELISA kit (AssayPro, St. Charles, MO, USA) as per manufacturers’ protocols. The results of antigen assays are expressed as mean7SD.
GSE g/ml Fig. 2. Anti-oxidant property of GSE: TIG 3-20 cells were treated with different doses of GSE for 18 hours. Cells were treated with 10 mM DCFH-DA for 45 minutes. Intracellular ROS generation was measured using spectrophotometer (Exc 485 nm; Emi 530 nm). Data is represented as percentage ROS generation. Values are mean7SD of six independent experiments.
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50 PAI-1 ng/ml
40 30 20
20 30 GSE g/ml
20 30 GSE g/ml
Fig. 3. ELISA to assess uPA and PAI-1 antigen levels: Cells were treated with different doses of GSE for 18 hours. The conditioned medium was used to analyze uPA and PAI-1 antigen levels by ELISA using standard commercial kits. Data represent mean7SD of quadruplicate experiments.
UK). Intensities of the bands were quantiﬁed with a Luminescent Image Analyzer LAS-3000 (Fuji, Tokyo, Japan). To ensure equal protein loading, membranes were stripped and re probed with anti-b-actin antibodies.
RT-PCR analysis Total RNA was isolated using RNAiso (Takara, Tokyo, Japan) as per manufacturer’s protocol. Reverse-transcriptase-polymerase chain reaction (RT-PCR) was carried out as per standard procedures using ReverTra Ace enzyme (Toyobo, Tokyo, Japan) for ﬁrst strand cDNA synthesis and GoTaq Green master mix (Promega) for PCR ampliﬁcation with primers speciﬁc for uPA, PAI-1 or 18S rRNA. Sequences targeting the coding region of the genes were selected for primer designing and are published elsewhere (Radha et al., 2008). PCR-ampliﬁed products were run on a 1% agarose gel and visualized by ethidium bromide staining. The expression intensities of optimized bands were quantiﬁed with a Luminescent Image Analyzer LAS-3000 (Fuji).
Fig. 4. RT-PCR to study mRNA expressions of uPA and PAI-1: Total RNA was isolated from cells treated with or without GSE (20 mg/ml) and subjected to RT-PCR as described in Materials and Methods. 18S rRNA gene expression was used as internal control. Figure is representative of three independent experiments.
Fibrin autography Fibrinolytic activity was assayed employing ﬁbrin gel plates containing 6 mg/ml bovine ﬁbrinogen (Miles, Kankakee, IL, USA) and 1 U/ml bovine thrombin (Mochida Pharmaceutical, Tokyo, Japan). Thirty microlitres of conditioned medium from cells incubated with different doses of GSE was loaded on to the ﬁbrin plate and incubated at 37 1C for 16 hours. Fibrinolytic activity was estimated by the lysis area obtained.
Cell-surface plasmin activity assay Cell-surface plasmin activity assay was conducted as per standard protocol (Stephens et al., 1989). Brieﬂy, cells conditioned by GSE were incubated in serum free medium containing 20 mg/ml plasminogen for 3 hours. Cells were washed and incubated in 100 ml of 1 mM tranexamic acid in PBS, pH 7.4 for 15 min, to facilitate dissociation of cell surface plasmin. Fifty microliters of the samples were incubated with chromogenic substrate S-2251 (1 mM in 75 ml of 0.1 M Tris, pH 8.0) at 37 1C for 2 hours. The amount of p-nitroaniline released was measured at 405 nm and referenced to a plasmin standard curve that was run parallel to the samples.
Fig. 5. Western blot analyses of uPA and PAI-1 expressions: Lysates from cells treated with or without GSE (20 mg/ml) were analyzed by western blot technique using primary antibodies for uPA or PAI-1. The membranes were stripped and reprobed with antibody for b–actin to ensure even loading of samples. Figure is representative of three independent experiments.
In vitro wound healing assay Fibroblasts were grown in 6-well plates at a density of approximately 4 104 and a small linear wound was created in the conﬂuent monolayer by gently scraping with sterile cell scrapper as per standard methods (Liang et al., 2007). Cells were extensively rinsed with medium to remove cellular debris before treating with 20 mg/ml GSE. Twenty four hours later, images of the cells were obtained using digital camera (Nikon, Japan) connected
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was analyzed by Student’s t-test. po0.05 was considered as statistically signiﬁcant.
Results and Discussion
Fig. 6. Fibrin autography using conditioned medium from treated cells: TIG 3–20 cells were treated with different doses of GSE (0–50 mg/ml) for 18 hours. Thirty microlitres of conditioned medium from treated cells was loaded on ﬁbrin plate and incubated at 37 1C for 16 hours. Fibrinolytic activity was measured by the lysis area obtained. Figure is representative of three independent experiments.
Cell surface plasmin activity (mU/well)
20 30 GSE g/ml
Fig. 7. Effect of GSE on cell-surface plasmin activity: Cells were treated with different doses of GSE (0–50 mg/ml) for 18 hours. Plasmin activity was analyzed as described in Materials and Methods. Data is representative of three independent experiments.
to the inverted microscope (Nikon TMS-F, Japan) and analyzed by image analysis software (Image J 1.32e, National Institutes of Health, Maryland, USA). Extent of wound healing was determined by the distance traversed by cells migrating into the denuded area. Data shown is representative of three independent experiments.
Statistical analysis All experiments were performed in triplicates. Data were expressed as mean7SD and the difference between the groups
Effect of GSE on viability of ﬁbroblast cells was analyzed by Cell titer Glo Luminescent Cell Viability Assay (Promega, USA). Lower doses of GSE (upto 30 mg/ml) was less cytotoxic whereas higher concentrations of 40 and 50 mg/ml GSE drastically reduced the percentage of viable cells, to as low as 20% (Fig. 1). We next checked the anti-oxidant property of GSE by measuring the amount of intracellular ROS. Cells incubated with GSE demonstrated lower levels of ROS generation (Fig. 2). GSE reduced the amount of intracellular ROS generation, thus exhibiting anti-oxidant property, in a dose dependent manner. GSE is a strong anti-oxidant and is more potent than vitamin C in scavenging oxygen free radicals (Bagchi et al., 1997). Since both uPA and PAI-1 genes respond positively to oxidative stress (Bonello et al., 2007; Crippa, 2007), we proceeded to study the effect of GSE on their expressions. ELISA revealed that GSE caused a signiﬁcant reduction in antigen levels of both uPA and PAI-1 (Fig. 3). uPA antigen level was undetectable in conditioned medium obtained from cells incubated with higher concentrations of 30–50 mg/ml. Similarly PAI-1 antigen level was very low in these higher concentrations. Twenty mg/ml GSE was the optimal concentration since it was non-cytotoxic, reduced ROS generation by 80% besides signiﬁcantly reducing the antigen levels. Hence, further tests on RNA and protein levels were conducted using 20 mg/ml GSE. Incubation of cells with 20 mg/ml GSE decreased expressions of both uPA and PAI-1, at the mRNA (Fig. 4) and protein (Fig. 5) levels. Although the molecular basis for this modality is currently not known, downregulation of uPA system has been reported by other polyphenols such as epigallocatechin-3-gallate (EGCG) (Ho et al., 2007; Siddiqui et al., 2008) and curcumin (Aggarwal et al., 2003) through regulation of multiple signaling pathways. The functional signiﬁcance of downregulation of uPA system was evident in ﬁbrinolytic assay (Fig. 6), where cells incubated with GSE demonstrated decreased ﬁbrinolytic activity. This ﬁnding is in accordance to other studies where gene expression of uPA was suggested to play a critical role in local ﬁbrin deposition/ dissolution (Yamamoto and Loskutoff, 1996). Reduced uPA-mediated proteolysis correlated with excessive ﬁbrin deposition (Mondino and Blasi, 2004). Activation of plasmin by plasminogen activators is primary for ﬁbrinolysis to occur. Plasmin degrades ﬁbrin and prevents its extracellular deposition. GSE caused a reduction in cell surface plasmin activity as evidenced by plasmin activity assay (Fig. 7). The importance of ﬁbrinolytic system in wound healing was demonstrated in plasminogen-deﬁcient mice models, where healing was impaired primarily due to impaired ﬁbrinolysis, a consequence of insufﬁcient plasmin generation (Lund et al., 2006). Besides ﬁbrinolysis, uPA and PAI-1 also play prominent roles in cellular migration and are vital during the initial phases of wound healing (Madhyastha et al., 2008; Maquerlot et al., 2006; Radha et al., 2008). We used a standard in vitro wound healing assay (Liang et al., 2007) to observe the effect of GSE on cellular migration in a wound environment. GSE signiﬁcantly retarded the migration of cells towards wounded area (Fig. 8). Our current study revealed that GSE inhibited two important mechanisms that are vital during the early phase of wound healing; ﬁbrinolysis and cell migration. On contrary, GSE was reported to promote wound healing by increasing H2O2-induced VEGF and tenascin expressions at the wound edge (Khanna et al., 2001, 2002). In murine model of dermal wound, GSE also enhanced the oxidizing
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Fig. 8. Effect of GSE on cell migration towards wound area: Semi-conﬂuent cells on 6-well plates were wounded by gently scratching using cell scraper. The arrow mark represents the wound edge. Cells were incubated in serum-free medium with or without GSE (20 mg/ml) for 24 hours. 0 h denotes the condition immediately after wounding; 24 h denotes 24 hours post wounding. Image is representative of three independent experiments.
environment at the wound site and markedly accelerated wound healing (Khanna et al., 2002). ROS or oxidants are a vital part of healing (Hunt et al., 2001) and promote ﬁbroblast migration and proliferation (Papa et al., 1998). Given that under certain conditions, some anti-oxidants may assume the characteristics of a pro-oxidant (Ohshima et al., 1998), it is plausible that GSE that is highly rich in anti-oxidants, assumes a pro-oxidant property in the oxidant-rich wound environment. Since oxidative stress positively regulates the uPA system at transcriptional level (Kiguchi et al., 2001; Pawlak et al., 2006; Radha et al., 2008), it is interesting to speculate the effect of GSE on uPA system in an oxidant-rich wound environment. In vivo wound healing studies using murine models will throw more light into this speculation. References Aggarwal, B.B., Kumar, A., Bharti, A.C., 2003. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res. 23 (1A), 363–398. Bagchi, D., Garg, A., Krohn, R.L., Bagchi, M., Tran, M.X., Stohs, S.J., 1997. Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro. Res. Commun. Mol. Pathol. Pharmacol. 95 (2), 179–189. Balasubramanyam, M., Koteswari, A.A., Kumar, R.S., Monickaraj, S.F., Maheswari, J.U., Mohan, V., 2003. Curcumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications. J. Biosci. 28 (6), 715–721. Bonello, S., Zahringer, C., BelAiba, R.S., Djordjevic, T., Hess, J., Michiels, C., Kietzmann, T., Gorlach, A., 2007. Reactive oxygen species activate the HIF1alpha promoter via a functional NFkappaB site. Arterioscler. Thromb. Vasc. Biol. 27 (4), 755–761. Broadhurst, R.B., Jones, W.T., 1978. Analysis of condensed tannins using acidiﬁed vanillin. J. Sci. Food Agric. 29 (9), 788–794. Crippa, M.P., 2007. Urokinase-type plasminogen activator. Int. J. Biochem. Cell Biol. 39 (4), 690–694. Ho, Y.C., Yang, S.F., Peng, C.Y., Chou, M.Y., Chang, Y.C., 2007. Epigallocatechin-3gallate inhibits the invasion of human oral cancer cells and decreases the productions of matrix metalloproteinases and urokinase-plasminogen activator. J. Oral Pathol. Med. 36 (10), 588–593. Hunt, T.K., Hussain, Z., Sen, C.K., 2001. Give me ROS or give me death. Pressure 30, 10–11. Hupkens, P., Boxma, H., Dokter, J., 1995. Tannic acid as a topical agent in burns: historical considerations and implications for new developments. Burns 21, 57–61. Khanna, S., Roy, S., Bagchi, D., Bagchi, M., Sen, C.K., 2001. Upregulation of oxidantinduced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract. Free Radical Biol. Med. 31 (1), 38–42. Khanna, S., Venojarvi, M., Roy, S., Sharma, N., Trikha, P., Bagchi, D., Bagchi, M., Sen, C.K., 2002. Dermal wound healing properties of redox-active grape seed proanthocyanidins. Free Radical Biol. Med. 33 (8), 1089–1096.
Kiguchi, T., Niiya, K., Shibakura, M., Miyazono, T., Shinagawa, K., Ishimaru, F., Kiura, K., Ikeda, K., Nakata, Y., Harada, M., 2001. Induction of urokinase-type plasminogen activator by the anthracycline antibiotic in human RC-K8 lymphoma and H69 lung-carcinoma cells. Int. J. Cancer 93 (6), 792–797. Liang, C.C., Park, A.Y., Guan, J.L., 2007. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2 (2), 329–333. Lijnen, H.R., 2005. Pleiotropic functions of plasminogen activator inhibitor-1. J. Thromb. Haemostasis 3 (1), 35–45. Lund, L.R., Green, K.A., Stoop, A.A., Ploug, M., Almholt, K., Lilla, J., Nielsen, B.S., Christensen, I.J., Craik, C.S., Werb, Z., Dano, K., Romer, J., 2006. Plasminogen activation independent of uPA and tPA maintains wound healing in genedeﬁcient mice. Embo J. 25 (12), 2686–2697. Madhyastha, H.K., Radha, K.S., Nakajima, Y., Omura, S., Maruyama, M., 2008. uPA dependent and independent mechanisms of wound healing by C-phycocyanin. J. Cell. Mol. Med 12 (6B), 2691–2703. Maquerlot, F., Galiacy, S., Malo, M., Guignabert, C., Lawrence, D.A., d’Ortho, M.P., Barlovatz-Meimon, G., 2006. Dual role for plasminogen activator inhibitor type 1 as soluble and as matricellular regulator of epithelial alveolar cell wound healing. Am. J. Pathol. 169 (5), 1624–1632. Mondino, A., Blasi, F., 2004. uPA and uPAR in ﬁbrinolysis, immunity and pathology. Trends Immunol. 25 (8), 450–455. Ohshima, H., Yoshie, Y., Auriol, S., Gilibert, I., 1998. Antioxidant and pro-oxidant actions of ﬂavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite and nitroxyl anion. Free Radical Biol. Med. 25 (9), 1057–1065. Papa, F., Scacco, S., Vergari, R., Bucaria, V., Dioguardi, D., Papa, S., 1998. Respiratory activity and growth of human skin derma ﬁbroblasts. Ital. J. Biochem. 47 (3), 171–178. Pawlak, K., Pawlak, D., Mysliwiec, M., 2006. Oxidative stress effects ﬁbrinolytic system in dialysis uraemic patients. Thromb. Res. 117 (5), 517–522. Radha, K.S., Madhyastha, H.K., Nakajima, Y., Omura, S., Maruyama, M., 2008. Emodin upregulates urokinase plasminogen activator, plasminogen activator inhibitor-1 and promotes wound healing in human ﬁbroblasts. Vasc. Pharmacol. 48 (4–6), 184–190. Siddiqui, I.A., Malik, A., Adhami, V.M., Asim, M., Hafeez, B.B., Sarfaraz, S., Mukhtar, H., 2008. Green tea polyphenol EGCG sensitizes human prostate carcinoma LNCaP cells to TRAIL-mediated apoptosis and synergistically inhibits biomarkers associated with angiogenesis and metastasis. Oncogene 27 (14), 2055–2063. Stephens, R.W., Pollanen, J., Tapiovaara, H., Leung, K.C., Sim, P.S., Salonen, E.M., Ronne, E., Behrendt, N., Dano, K., Vaheri, A., 1989. Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surfacebound reactants. J. Cell Biol. 108 (5), 1987–1995. Toriseva, M., Kahari, V.M., 2008. Proteinases in cutaneous wound healing. Cell. Mol. Life Sci. 66 (2), 203–224. Watanabe, M., Kondo, S., Mizuno, K., Yano, W., Nakao, H., Hattori, Y., Kimura, K., Nishida, T., 2006. Promotion of corneal epithelial wound healing in vitro and in vivo by annexin A5. Invest. Ophthalmol. Visual Sci. 47 (5), 1862–1868. Yamamoto, K., Loskutoff, D.J., 1996. Fibrin deposition in tissues from endotoxintreated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator. J. Clin. Invest. 97 (11), 2440–2451.