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Vol. 10, Issue 4, 1093-1104, April 1999
*Medizinische Universitäts-Poliklinik, 53111 Bonn, Germany;
and Enhanced activity of receptor tyrosine
kinases such as the PDGF Department of Pharmacology,College of Pharmacy, Chungbuk National University,
Cheongju 361-763, South Korea
-receptor and EGF receptor has been
implicated as a contributing factor in the development of malignant and
nonmalignant proliferative diseases such as cancer and atherosclerosis.
Several epidemiological studies suggest that green tea may prevent the
development of cancer and atherosclerosis. One of the major
constituents of green tea is the polyphenol epigallocathechin-3 gallate
(EGCG). In an attempt to offer a possible explanation for the
anti-cancer and anti-atherosclerotic activity of EGCG, we examined the
effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and
FCS-induced activation of the 44 kDa and 42 kDa
mitogen-activated protein (MAP) kinase isoforms
(p44mapk/p42mapk) in cultured vascular smooth
muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG
(1-100 µM) for 24 h and stimulated with the above mentioned
agonists for different time periods. Stimulation of the
p44mapk/p42mapk was detected by the enhanced
Western blotting method using phospho-specific MAP kinase antibodies
that recognized the Tyr204-phosphorylated (active) isoforms. Treatment
of VSMCs with 10 and 50 µM EGCG resulted in an 80% and a complete
inhibition of the PDGF-BB-induced activation of MAP kinase isoforms,
respectively. In striking contrast, EGCG (1-100 µM) did not
influence MAP kinase activation by EGF, angiotensin II, and FCS.
Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA
expression as well as on intracellular free Ca2+
concentration was completely inhibited in EGCG-treated VSMCs, whereas
the effect of EGF was not affected. Quantification of the
immunoprecipitated tyrosine-phosphorylated PDGF-R,
phosphatidylinositol 3'-kinase, and phospholipase C-
1 by the
enhanced Western blotting method revealed that EGCG treatment
effectively inhibits tyrosine phosphorylation of these kinases in
VSMCs. Furthermore, we show that spheroid formation of human
glioblastoma cells (A172) and colony formation of
sis-transfected NIH 3T3 cells in semisolid agar are
completely inhibited by 20-50 µM EGCG. Our findings demonstrate that
EGCG is a selective inhibitor of the tyrosine phosphorylation of
PDGF-R and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of
green tea.
§
Corresponding author. E-mail address:
umm501{at}uni-bonn.de.
¶
Contributed equally to this work.
Molecular Biology of the Cell
Vol. 10, 1093-1104, April 1999
Copyright © 1999 by The American Society for Cell Biology
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