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Green tea polyphenol epigallocatechin-3 gallate induces apoptosis of proliferating vascular smooth muscle cells via activation of p53¹

Department of Biochemistry, Boston University School of Medicine, Boston Massachusetts, USA

²Correspondence: Boston University School of Medicine, Department of Biochemistry, 715 Albany St., Boston MA 02118, USA. E-mail: gsonensh{at}bu.edu

SPECIFIC AIMS

Green tea polyphenols such as epigallocatechin-3 gallate (EGCG) have protective effects against cardiovascular disease. In the present study we sought to elucidate the molecular mechanism of any direct effects on vascular SMC proliferation and survival.

PRINCIPAL FINDINGS

1. EGCG treatment induces p53 tumor suppressor levels which promotes apoptosis
Treatment of bovine aortic SMCs in exponential growth with 40–50 µg/mL green tea polyphenol mixture or EGCG slowed proliferation whereas 80 µg/mL EGCG induced apoptosis, as judged by TUNEL assay (48% cell death after 24 h). Addition of catalase offered no protection, indicating death was not due to the extracellular generation of hydrogen peroxide. Death was not observed with confluent SMC cultures (3% cell death after 24 h), suggesting EGCG preferentially affects proliferating cells. Since induction of the tumor suppressor p53 has been implicated in control of growth and apoptosis of SMCs, the effects of 80 µg/mL EGCG on nuclear p53 protein levels were assessed. An increase was observed by 2 h and the level continued to increase over the 8 h time course (Fig. 1 A), reaching a 9.0 ± 2.7-fold increase at 8 h in this and a duplicate experiment. The expression of p21^CIPI cyclin-dependent kinase inhibitor (CKI), a major downstream target of p53, was assessed by Northern analysis (Fig. 1B ). The level of p21 mRNA was increased 15.5-fold after 4 h of treatment with 80 µg/mL EGCG, remaining elevated up to 12 h. Thus, EGCG treatment induces functional p53 protein.

View larger version (30K): [in this window] [in a new window]   Figure 1. EGCG induces p53 and p21CIP1 expression. SMCs were plated at a density of 1.5 x 106 cells/P150 dish. After overnight incubation, cells were either left untreated or treated with 0 or 80 µg/mL EGCG. Either nuclear RIPA extracts (A) or RNA (B) were isolated. A) Nuclear extracts (50 µg) were subjected to immunoblot analysis for p53 and ß-actin. (B) RNA samples (20 µg) were subjected to Northern blot analysis for p21 mRNA expression (upper panel). RNA integrity was verified by ethidium bromide staining of 18S and 28S rRNA, and loading appeared equal except for slight underloading of the 12 h control time point (bottom panel). (C) SMCs were plated at 2 x 103 cells/well in 96-well plates, transfected for 24 h, in triplicate, with 30 ng pSV40-ß-Gal and 45 ng of either pCMVp53–175P or pCMV (parental vector) in 0.23 µL FuGENE Reagent. Cultures were treated with 0 or 80 µg/mL EGCG for 24 h and subjected to the X-Gal viability assay. The number of viable cells staining blue was determined by examination under phase contrast microscopy; % of live cells ± standard deviation (SD) is presented. The results of 3 independent experiments are shown. D) SMC cultures (2x106 cells/P150 dish) were incubated overnight and treated with either 0 or 80 µg/mL EGCG for 2 h. Then 15 µg/mL cycloheximide (CHX) was added; total cellular extracts were prepared after 0, 1, 2, or 3 h and samples (50 µg) were subjected to immunoblot analysis for p53 and ß-actin.

To verify the role of p53 in cell death, a ß-Gal survival assay was performed using a dominant negative form of p53, pCMV-p53–175P with a His to Pro conversion at codon 175, which has been shown to block p53 function in apoptosis. (Fig. 1C ). Upon expression of the dominant negative p53, an average of 70.4% of SMCs remained viable vs. an average of 35.4% on transfection with the control vector. Thus, inhibition of the apoptotic activity of p53 substantially protected vascular SMCs from EGCG-mediated apoptosis, indicating a role for p53 in the death of these cells.

Since regulation of p53 is often mediated via control of protein stability, SMCs were treated with 0 or 80 µg/mL EGCG for 2 h, then 15 µg/mL cycloheximide was added for 1, 2, or 3 h. Whole cell extracts were subjected to immunoblot analysis (Fig. 1D ). Exposure of cells to EGCG caused an increase of the half-life of p53 from 20 min to > 3 h, which can account for the increase in p53 levels by EGCG treatment.

2. EGCG treatment induces NF-B activity which promotes SMC death
NF-B has been implicated in p53-mediated cell death. To determine whether EGCG treatment alters NF-B binding levels, EMSA analysis was performed using the NF-B element upstream of the c-myc promoter (URE), which binds all NF-B/Rel family members, as probe. Treatment with 40 or 80 µg/mL EGCG for 24 or 48 h caused an increase in binding of a slower migrating NF-B complex (Band 1). The increase in complex 1 binding occurred by 1 h of EGCG treatment. Supershift analysis identified Band 1 as a complex of p50/p65, indicating that EGCG treatment leads to induction of classical NF-B. Transfection analysis was performed with a two-copy NF-B URE element thymidine kinase promoter-driven CAT reporter construct (E8-CAT) (Fig. 2 A). Treatment with EGCG for 12 or 24 h resulted in a 3.3-fold and 6.4-fold increase in CAT activity compared with DMSO carrier-treated control. Thus, EGCG induces functional NF-B.

View larger version (18K): [in this window] [in a new window]   Figure 2. EGCG treatment of SMCs induces functional NF-B, which is essential for EGCG-mediated apoptosis. A) SMC cultures plated in duplicate at a density of 1.5 x 105 cells/P60 dish were transfected with 3 µg E8-CAT, 0.5 µg pSV40-ß-Gal, and 1.5 µg pBluescript in 18 µL FuGENE Reagent. After 24 h, cells were treated with 0 or 80 µg/mL EGCG. After 12 or 24 h, cells were harvested, assayed, and the values of normalized CAT activity ± SD are presented. B) SMCs were plated at 2000 cells/well in 96-well plates, transfected in triplicate with 30 ng pSV40-ß-Gal and 45 ng of either pSV-IB2N3C or pSVneo (parental vector) in 0.23 µL FuGENE Reagent. After 24 h, cells were treated with 0 or 80 µg/mL EGCG. After 24 h, cells were subjected to the X-Gal viability assay. The number of viable cells staining blue was determined by examination under phase contrast microscopy; the % of live cells ± SD is presented. The results of 3 independent experiments are shown.

To determine whether the NF-B activity is necessary for EGCG-mediated apoptosis, a super repressor form of the NF-B inhibitory protein IB (IB2N3C), which cannot be phosphorylated by the IKK or CK2 kinases, was used in a ß-Gal survival assay (Fig. 2B ). In three independent experiments, treatment with EGCG reduced viability of SMCs transfected with the empty vector to an average of 43.8% of untreated cells (Fig. 2B ). In contrast, transfection with the IB super repressor almost completely eliminated death, with an average of 95.6% live cells remaining compared with untreated cells. Thus, inhibition of NF-B activity by expression of the IB super repressor protects vascular SMCs from EGCG-mediated apoptosis.

CONCLUSIONS AND SIGNIFICANCE

Treatment of aortic SMCs with the green tea polyphenol EGCG at a dose of 80 µg/mL increased expression of p53 and its downstream target, the p21^CIP1 CKI, and caused cell death, whereas a lower dose of 40 to 50 µg/mL of EGCG or GTP mixture arrested proliferation (Fig. 3 ). The observed increase in p53 protein level was due to a slower rate of turnover, which is regulated by the proteasome and usually involves either phosphorylation of p53 and/or interaction with MDM2. Classical NF-B was induced with EGCG treatment, and this increase appeared essential for EGCG-mediated apoptosis (Fig. 3) . It is likely that NF-B was released on degradation of its cytoplasmic inhibitor IB, allowing for its translocation to the nucleus, where it induces expression of B element containing target genes. Thus, activation of the p53 and NF-B signaling pathways leads to apoptosis of proliferating SMCs.

View larger version (29K): [in this window] [in a new window]   Figure 3. EGCG inhibits growth and induces apoptosis of vascular SMCs. Treatment of aortic SMCs with EGCG increases p53 protein stability, possibly by destabilizing the interaction of p53 with MDM2 ubiquitin ligase, which targets p53 for degradation by the proteasome. The induction of p53 causes increased transcription of its target p21CIP1. EGCG treatment also induces classical NF-B activity, presumably by degradation of its cytoplasmic inhibitor IB and subsequent translocation to the nucleus, where it binds B target sequences. These events lead to growth arrest and apoptosis.

EGCG and green tea have been found to exert protective effects against cardiovascular disease in several animal models and in humans. Previously, this protection has been related to its affects on cholesterol levels. Our studies elucidate a new direct mechanism of inhibition of growth of proliferating SMCs and induction of apoptosis via p53 and p21. These two proteins have been shown to be important players in the regulation of growth of vascular SMCs. Inhibition of p53 in SMCs within the rat carotid artery, via introduction of antisense oligonucleotides, resulted in an increase in the neointima. Similarly, adenoviral gene transfer of the p21 CKI target gene prevented neointimal formation. In p53^(-/-) mice, neointimal hyperplasia of vein grafts was increased significantly, consisting mainly of SMCs, compared with wild-type mice, where mostly macrophages were observed. This and other findings suggest that strategies that induce the expression of p53 would be beneficial in the prevention of restenosis and atherosclerosis. Several in vivo studies have confirmed this approach, including low-dose irradiation. Since EGCG treatment specifically induced apoptosis in proliferating cells whereas nonproliferating cells remained viable, our results suggest the possibility that green tea polyphenols represent a noninvasive class of compounds that have the potential to induce endogenous p53 and p21 levels. Given the low toxicity of green tea, further studies are warranted to evaluate the potential of green tea in prevention of plaque formation and cardiovascular disease.

Proapoptotic roles have been described for NF-B in various systems. NF-B has been identified as an essential downstream factor in a p53-mediated apoptotic pathway. Murine embryonic fibroblasts derived from p65^-/- mice were resistant to p53-induced apoptosis. Here, a critical role for the induction of NF-B in EGCG-mediated death of SMCs was demonstrated. Transfection of an IB- super repressor protein of NF-B activity protected the vascular SMCs from apoptosis on EGCG treatment. Recent studies have implicated MEK-1 in activation of NF-B on induction of p53-mediated apoptosis, i.e., inhibition of MEK-1 blocked the activation of NF-B and abrogated p53-induced cell death. EGCG has been shown to up-regulate the MAP kinase signaling pathway in macrophage cell lines, and thus it is interesting to speculate that MAP kinase signaling leads to the activation of NF-B in our system (Fig. 3) . The effects of EGCG on NF-B activity appear cell type specific and likely reflect differences in intersecting signaling pathways. EGCG was shown to prevent the activation of NF-B by TNF, and we observed that EGCG reduced NF-B levels in NF639 breast cancer cells via blocking the activation pathway induced by Her-2/neu signaling. In contrast, EGCG had no apparent effect on NF-B expression in Hs578T breast cancer cells, which has been related to constitutive IKK and CK2 activities. Further work is required to elucidate the mechanism of activation of NF-B by EGCG and its role in p53-mediated death of vascular SMCs.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0665fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0665fje

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