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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 12, 2001 as doi:10.1096/fj.00-0519fje. |
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action of epigallocatechin-3-gallate mediated by specific inhibition of STAT1 activation 1
Biochemistry Section, Department of Neuroscience and Vision, University of Verona, I-37134 Verona, Italy
2Correspondence: Biochemistry Section, Department of Neuroscience and Vision, University of Verona, Strada Le Grazie 8, I-37134 Verona, Italy. E-mail: aquila{at}borgoroma.univr.it
SPECIFIC AIMS
Epigallocatechin-3-gallate (EGCG), the main polyphenol component of green tea, exerts inhibitory action on urokinase, metalloproteinase, and inducible nitric oxide synthase (NOS II), accounting for its antitumor and anti-inflammatory action. The aim of the present study was to identify in human cell lines the molecular target of EGCG that modulates the expression of a number of genes involved in inflammatory and neoplastic processes.
PRINCIPAL FINDINGS
1. EGCG blocks interferon 
(IFN-)-elicited activation of
signal transducers and activators of transcription 1 (STAT1) by
interfering with tyrosine phosphorylation in human MDA MB 231 cell line
Treatment of the estrogen nonresponsive human mammary carcinoma
cell line MDA MB 231 with lipopolysaccharide (LPS)/IFN-/tumor
necrosis factor (TNF)-
/interleukin 1ß (IL-1ß) (MIX) promptly
induced the activation of a nuclear factor, NF-
B, that was not
affected by the pretreatment with 20 µM EGCG; 20 µM EGCG did not
inhibit the constitutively activated nuclear factor SP-1. However, EGCG
inhibited the IFN--elicited STAT1 activation dose dependently: the
estimated value of the concentration giving 50% inhibition (IC50) was
5 µM. Among the components of EGCG, EGC only slightly inhibited the
IFN--elicited STAT1 activation (IC50=100 µM), whereas gallate had
no suppressive effect.
Treatment of the MDA MB 231 cell line with IFN- rapidly induced the
appearance of heavily tyrosine-phosphorylated-STAT1, which completely
disappeared upon the addition of 10 µM EGCG (Fig. 1
). Western blot analyses of the same samples with antibodies anti-STAT1
showed no detectable change in STAT1 content (Fig. 1)
, indicating that
the inhibitory effect of EGCG on the IFN--elicited activation of
STAT1 was directed toward the tyrosine phosphorylation of STAT1
proteins.
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2. EGCG suppresses the IFN--elicited expression of NOS II as
well as interferon regulating factor 1 (IRF-1)
Treatment of MDA MB231 cell line with MIX induced the expression
of NOS II mRNA, which was strongly inhibited by pretreatment with 50
µM EGCG but not by 50 µM EGC and gallate; 50 µM EGCG also
inhibited the expression of IRF1 mRNA induced by IFN-. These data
indicate that inhibition of IFN--elicited STAT1 activation by EGCG
leads to a specific inhibition of STAT1-mediated expression of genes.
3. EGCG inhibits IFN--elicited STAT1 activation in human HeLa,
MCF7, MDA MB 468, and HepG2 cells
As observed in the MDA MB 231 cell line, IFN- induced a rapid
activation of STAT1 in other human cell lines such as MDA MB 468, the
estrogen-responsive mammary carcinoma cell line MCF7, hepatocarcinoma
cell line HepG2, and cervical carcinoma cell line HeLa (Fig. 2
). EGCG (50 µM) administered to the cell culture 30 min before IFN-
treatment inhibited the IFN--elicited STAT1 activation in all cell
lines examined. However, the estimated IC50 value of the inhibitory
action of EGCG on IFN--elicited STAT1 activation varied
significantly depending on cell type. It was determined to be 10 µM
in MDA MB 468 and HeLa cells, 20 µM in MCF7, and 50 µM in
HepG2.
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4. EGCG has no effect on IL-6-elicited STAT3 activation in
IL-6-responsive cells
To further examine whether EGCG exerts its action only on STAT1
signaling or whether it might also affect other components of the STAT
family, we treated all cell lines examined with IL-6, which in
different cell types induces the activation of STAT3. After 30 min
treatment, IL-6 induced the activation of STAT3 in HeLa, HepG2, and
MCF7 cell lines, whereas MDA MB 231 and 468 cells were unresponsive.
When added to the cell culture 30 min before IL-6 administration, 50
µM EGCG was not able to suppress the IL-6-elicited STAT3 activation
in the cell lines mentioned above.
CONCLUSIONS
Besides the reported inhibitory effect of EGCG on angiogenesis and
the catalytic activity of correlated enzymes, such as urokinase and
matrix metalloproteinases, EGCG elicited a strong inhibitory action
against IFN--induced STAT1 activation in MDA MB 231/468, MCF7,
HepG2, and HeLa cell lines. The preferential action of EGCG toward
STAT1 was supported by the lack of EGCG inhibitory action against
IL-6-induced STAT3 activation in HeLa, MCF7, and HepG2 cell lines. The
inhibitory effects of EGC and gallate, two antioxidative polyphenol
components of EGCG, on IFN--elicited STAT1 activation were very weak
and null, respectively. Furthermore, in the MDA MB 231 cell line, EGCG
was ineffective in inhibiting the MIX-elicited activation of the
oxidative stress-sensitive nuclear factor NF-B. Therefore, the
antioxidative action of EGCG may not account for the inhibition of
STAT1 activation. As shown in Fig. 1
, EGCG exerts its inhibitory effect
to inhibit IFN--elicited tyrosine-phosphorylation of STAT1 rather
than to modulate the amounts of STAT1 protein.
Of the cell lines examined, the strongest inhibitory action of EGCG
against STAT1 activation was observed in MDA MB 231/468 cells and HeLa
cells. The estimated IC50 value of EGCG in its action in these cell
lines (5–10 µM) is near to the reported value of the blood and
tissue level of EGCG after consumption of several cups of green tea.
Different IC50 values estimated in other cell lines (20 and 50 µM for
MCF7 and HepG2 cell lines, respectively) indicate that the degree of
the inhibitory effect of EGCG against IFN--elicited STAT1 activation
depends on the cell type.
The strong inhibition of IFN--elicited activation of STAT1 by EGCG
arises the following question: Could the well-documented properties of
green tea be reconciled with the inhibitory effect of EGCG against
IFN--elicited STAT1 activation?
An increasing body of evidence indicates that STATs may play an
important role in the biology of both hematopoietic and
nonhematopoietic tumors. Since STATs are activated by the widest array
of cytokines and may have relatively broad effects on cell growth
and/or survival, the down-regulation of STATs signaling was considered
a promising approach to the possible therapy of some types of tumor.
Nevertheless, few molecules without a harmful secondary effect have
been identified as specific inhibitors of STATs signaling. Fludarabine,
a nucleoside analog used to treat hematologic malignancies, has
recently been reported to inhibit STAT1 signaling in human lymphocytes
by acting on the synthesis of STAT1 protein. This could suggest that
the antitumor action of fludarabine might be correlated to its
inhibitory action on STAT1 activation. As the inhibitor of STAT1
signaling, EGCG, which inhibits STAT1 phosphorylation, acts more
rapidly (in 30 min) than fludarabine. Therefore, the physiologically
relevant IC50 value of EGCG in inhibiting IFN--elicited
STAT1 activation in MDA and HeLa cells should make EGCG a promising
candidate as a STAT1 signaling-modulating drug.
Evidence in the literature favors the idea that among all known STATs,
STAT1 has a predominant role in growth inhibition, consistent with the
well-documented antiproliferative action of IFN-, which uses STAT1
almost exclusively as a mediator for its action. Inhibition of the
IFN--elicited STAT1 activation by EGCG may enhance rather than
reduce tumor development and may not provide clear molecular evidence
for the general antitumor action of green tea. At any rate, no toxic
effect reported on the drinking of more than 10 cups of green tea per
day suggests that this aspect may not be pathophysiologically relevant.
Moreover, since IFN--elicited STAT1 activation enhances the response
to growth factor in mesangial cells and wild-type p53-induced apoptosis
is inhibited by IFN- in a Burkitt lymphoma cell line, the
possibility that EGCG, by blocking IFN--elicited STAT1 activation,
could exert a beneficial effect in the surveillance in limited tumor
types other than CLL is not conclusive.
On the other hand, IFN--elicited activation of STAT1 is correlated
with the pathogenesis of inflammatory diseases such as asthma and
extracapillary proliferative glomerulonephritis. In the mouse model,
green tea prevents collagen-induced arthritis because of the presence
of the antioxidant polyphenols that reduce the incidence and severity
of the disease. According to the present study, inhibition of
IFN--elicited STAT1 activation by the EGCG present in green tea may
further account for the preventive effect of green tea against the
formation of experimental arthritis. Furthermore, the inhibitory action
of EGCG against IFN--induced STAT1 activation leads to a block of
the expression of both NOS II and IRF-1, two proteins deeply involved
in mediating inflammatory processes. Since the expression of many other
genes involved in inflammatory and immune responses are also
STAT1 dependent, EGCG, by inhibiting STAT1 activation, may play an
important role in modulating the entire process of inflammation. Thus,
the down-regulation of IFN- action by EGCG provides a molecular
basis for the anti-inflammatory effect of green tea or EGCG.
In conclusion, EGCG appears to be a specific and strong inhibitor of
IFN--elicited activation of STAT1. This property of EGCG may account
for the well-documented beneficial effect of green tea or EGCG toward
some types of tumors. The anti-inflammatory action of green tea may
well account for the ability of EGCG to efficiently inhibit the
IFN--elicited activation of STAT1. EGCG may be a promising candidate
as a drug modulating STAT1 signal devoid of undesirable secondary
effects.FIGURE 3
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0519fje ;
to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0519fje 
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