Cellular Sensing System for Green Tea Polyphenol Epigallocatechin Gallate Hirofumi Tachibana

AGri-Bioscience Monographs, Vol. 4, No. 2, pp. 19–35 (2014)
www.terrapub.co.jp/onlinemonographs/agbm/
Cellular Sensing System for Green Tea Polyphenol
Epigallocatechin Gallate
Hirofumi Tachibana1,2,3,4
1
Department of Bioscience and Biotechnology, Faculty of Agriculture,
Food Functional Design Research Center,
3
Bio-Architecture Center,
4
Innovation Center for Medical Redox Navigation,
Kyushu University
Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan
e-mail: [email protected]
2
Abstract
The green tea catechin (–)-epigallocatechin-3-O-gallate (EGCG) is known to exhibit various biological and pharmacological properties. We have identified 67-kDa laminin receptor
(67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. Here we provide an overview of several pathways that sense and manage the activities of EGCG.
EGCG has been shown to rescue mice from lipopolysaccharide (LPS)-induced lethal
endotoxemia and downregulate inflammatory responses in macrophages. LPS is one of
the most powerful activators of toll-like receptor (TLR)4 signaling and is also well known
to induce production of inflammatory mediators. A negative regulator of TLR signaling,
Toll-interacting protein (Tollip), is indispensable for mediating the anti-inflammatory
action of EGCG, and its protein expression level is upregulated by EGCG through 67LR.
Additionally, EGCG can reduce the expression of TLR4 via 67LR.
Using a direct genetic screen, eukaryotic translation elongation factor 1A (eEF1A) is
identified as a component responsible for the anti-melanoma activity of EGCG. EGCG
induces the dephosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) at
Thr-696 and activates myosin phosphatase through both eEF1A and 67LR. Silencing of
67LR, eEF1A, or MYPT1 in tumor cells results in abrogation of EGCG-induced tumor
growth inhibition in vivo. Additionally, we found that eEF1A is up-regulated by EGCG
through 67LR.
EGCG has been shown to be able to induce apoptotic cell death in multiple myeloma
cells through the 67LR, while having no significant effect on peripheral blood mononuclear cells (PBMCs). The expression of 67LR was significantly elevated in myeloma
cells compared to PBMCs. We found that the apoptosis-inducing activity of EGCG in
multiple myeloma cells is attributable to the activation of acid sphingomyelinase (ASM)
that hydrolyzes sphingomyelin into ceramide. EGCG induces ASM translocation to the
plasma membrane and protein kinase C delta (PKCδ) phosphorylation at Ser 664, which
was necessary for ASM/ceramide signaling, via 67LR. Furthermore, EGCG activates
PKC δ /ASM/ceramide pathway by activating Akt/eNOS/NO/cGMP signaling through
67LR. We also found that the upregulation of cGMP is a rate-determining process of this
cell death pathway, and cancer-overexpressed negative regulator of cGMP, PDE5 attenuates the cell death induced by EGCG. Vardenafil, one of the PDE5 selective inhibitors
used for treating erectile dysfunction potentiates the anti-cancer effect of EGCG. These
results demonstrate that cGMP elevation caused by targeting the overexpressed 67LR
and PDE5 in cancer cells may be a useful approach for cancer-specific chemotherapy.
© 2014 TERRAPUB, Tokyo. All rights reserved.
doi:10.5047/agbm.2014.00402.0019
Received on October 3, 2013
Accepted on February 24, 2014
Online published on
July 30, 2014
Keywords
• EGCG
• green tea
• catechins
• sensing
• receptor
• 67LR
• food factors
• cGMP
20
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
Fig. 1. Chemical structures of green tea catechins. Reprinted with permission from ILSI, 116, Tachibana, Green tea catechin
sensing system, 6–13, Fig. 1,  2014, ILSI Japan.
1. Introduction
Green tea is one of the most widely consumed beverages in the world after water. It has been demonstrated that tea constituents exhibit various beneficial
health effects such as anti-carcinogenic, anti-oxidative,
anti-allergic, anti-virus, anti-hypertensive, antiatherosclerosis, anti-cardiovascular disease and antihypercholesterolemic activities (Bors and Saran 1987;
Sano et al. 1995; Sazuka et al. 1995; Cao and Cao 1999;
Hodgson et al. 1999; Lambert and Yang 2003a;
Kuriyama et al. 2006). Principles for these activities
were shown to be a group of polyphenols, catechin.
The major green tea catechins are (–)-epigallocatechin3-gallate (EGCG), (–)-epigallocatechin (EGC), (–)epicatechin-3-gallate (ECG) and (–)-epicatechin (Fig.
1). Among the green tea catechins, EGCG is the most
abundant, representing ~16.5 wt% of the water extractable fraction of green tea leaves, and most active catechin in various kinds of physiological activities. Because EGCG is not found to a plant except tea, EGCG
is regarded as a constituent characterizing green tea.
The bioavailability and biotransformation of tea
catechins following tea ingestion has been investigated
in human volunteers, and a time to reach maximal concentration in the plasma of 1.5 to 2.5 h after consumption of decaffeinated green tea solids (1.5, 3.0, a and
were not detectable by 24 h (Lambert and Yang 2003b).
Whereas EGCG and ECG were not detected in the
urine, 90% of the urinary EC and EGC were excreted
by 8 h. Most of the ingested EGCG apparently does
not get into the blood, and absolute EGCG is preferentially excreted through the bile to the colon.
Glucuronidation, sulfation, methylation, and ringfission metabolism represent the major metabolic pathways for green tea catechins. Plasma EC and EGC were
present mainly in the conjugated form such as glucuronide and sulfate conjugates, whereas 77% of the EGCG
was in the free form. EGCG has also been shown to
undergo methylation (Lambert and Yang 2003). Although most of published studies in cell culture systems used 20–100 µM of EGCG, the blood level of
EGCG after consuming the equivalent of 2–3 cups of
green tea was 0.1–0.6 µM and for an equivalent of 7–
9 cups was still lower than 1 µM (Lee et al. 1995). The
rather poor bioavailability of tea catechins needs to be
considered when we extrapolate results obtained in
vitro to situations in vivo.
The polyphenolic structure of tea polyphenols makes
them good donors for hydrogen bonding. This hydrogen bonding capacity enables tea polyphenols to bind
strongly to proteins and nucleic acids. The difference
between in vitro and in vivo systems should be considered in studies attempting to elucidate the mechanisms
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
of action of EGCG.
In this review we focused on the current understanding of EGCG sensing mechanisms by which EGCG
exerts biological and pharmacological properties.
2. EGCG sensing receptor
Pharmacokinetic studies in humans indicate that the
peak plasma concentration after a dose of EGCG is
<1.0 µM. It should be noted that most of the effects of
EGCG in cell culture systems and cell-free systems
have been obtained with considerably high concentrations than those observed in the plasma or tissues of
animals or in human plasma after administration of
green tea or EGCG. Furthermore, the intracellular levels of EGCG are much lower than the concentrations
observed in the extracellular levels. Searching for highaffinity proteins that bind to EGCG is the first step to
understanding the molecular and biochemical mechanisms of the anti-cancer effects of tea polyphenols.
Several proteins that can directly bind with EGCG have
been identified with in vitro models (Yang et al. 2009).
All of these proteins were demonstrated to be important for the inhibitory activity of EGCG in cell lines,
but higher EGCG concentrations than the Kd values
were needed. For example, vimentin binds to EGCG
with a Kd of 3.3 nM, and functional studies showed
that EGCG inhibited the phosphorylation of vimentin
at Ser55 and Ser50 by Cdc2 (IC 50 = 17 µM) (Ermakova
et al. 2005). The difference in effective concentrations
is probably due to the nonspecific binding of EGCG to
other proteins, which compete with the target proteins.
Therefore, it is not clear whether the activities observed
with high EGCG concentrations in vitro can be observed in vivo and the proposed EGCG-binding molecules as mentioned above are responsible for in vivo
physiological activities of EGCG.
To elucidate the detailed molecular basis for the action of EGCG, it is necessary to identify the molecular
target triggering a specific signaling of EGCG. Studies of Toll-like receptors teach us the principal role in
the pathogens sensing and the necessity of identification of the specific receptor as a signal initiator for
generating cellular responses for understanding the
specific cellular signaling of foreign or functional substances. However, the molecular target for physiologically relevant EGCG that can mediate its anti-cancer
effect still remained unknown.
We found that all-trans-retinoic acid (ATRA) enhances the binding of EGCG to the cell surface of cancer cells when the binding was monitored on the basis
of the increase in response units in a surface plasmon
resonance (SPR) assay. To identify candidates through
which EGCG inhibits cell growth, we used a subtraction cloning strategy involving cDNA libraries constructed from cells treated or untreated with ATRA.
We isolated a single target that allows EGCG to bind
21
to the cell surface. An analysis of the DNA sequence
identified this unknown cell surface candidate as the
67-kDa laminin receptor (67LR) (Tachibana et al.
2004). In fact, the expression of this 67LR was enhanced by ATRA treatment.
Human lung cancer A549 cells were used to assess
how effectively the 67LR mediates EGCG-mediated
growth inhibition. Cells transfected with empty vector
and treated with EGCG showed no growth inhibition.
However, cells transfected with the gene encoding
67LR and treated with EGCG demonstrated considerable inhibition as compared with the control cells. We
next tested whether the growth inhibitory activity of
EGCG correlates with the binding strength of EGCG
to the cell surface. We found increased binding of
EGCG to the cell surface of cells transfected with
67LR. EGCG binding to the 67LR-transfected cells was
inhibited by treatment with an antibody to 67LR. The
predicted Kd value for the binding of EGCG to the
67LR protein is 39.9 nM. Most of the 67LR protein
was found to exist in the raft fraction rather than the
non-raft fraction (Fujimura et al. 2005), and this distribution pattern correlated well with the plasma
membrane-associated EGCG level after treating the
cells with EGCG (Fujimura et al. 2004).
To investigate whether the 67LR can confer a sensitivity to EGCG at physiologically relevant concentrations, we treated the 67LR-transfected A549 cells with
two concentrations of EGCG (0.1 and 1.0 µM); these
concentrations are similar to the amount of EGCG
found in human plasma after drinking more than two
or three cups of tea. The growth of the transfected cells
was inhibited at both of these concentrations
(Tachibana et al. 2004). In addition, this growthsuppressive effect was completely eliminated upon
treatment with anti-67LR antibody before the addition
of EGCG.
Next, we investigated the effect of oral administration of EGCG on subcutaneous tumor growth in
C57BL/6N mice challenged with 67LR-ablated B16
cells (Umeda et al. 2008a). We confirmed both silencing of 67LR by stable RNAi in B16 cells and attenuation of the inhibitory effect of 1 µ M EGCG on cell
growth in 67LR-ablated B16 cells in vitro. Tumor
growth was significantly retarded in EGCGadministered mice implanted with the B16 cells
harboring a control shRNA, whereas tumor growth was
not affected by EGCG in the mice implanted with
67LR-ablated B16 cells, suggesting that 67LR functions as an EGCG receptor not only in vitro but also in
vivo. Together, these observations demonstrate that the
cell-surface 67LR is the receptor for antitumor action
of EGCG at the physiologically relevant concentration.
The discovery of EGCG receptor as 67LR has solved
some of the discrepancies of the cancer-preventing
activity of EGCG between in vitro data and in vivo data.
Recently, we identified the 67LR extracellular do-
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
22
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
main corresponding to the 161–170 region as the EGCG
binding site (Fujimura et al. 2012). Additionally, mass
spectrometric analysis revealed that intact (chemically
non-altered) EGCG forms the non-covalent complex
with 67LR161–170 peptide, and there is no ion peak corresponding to the covalent complex of chemically altered EGCG with the 67LR peptide, indicating that
EGCG activates 67LR by forming a non-covalent complex with the receptor (Fujimura et al. 2012).
67LR was first discovered by three independent laboratories in 1983 (Rao et al. 1983; Malinoff and Wicha
1983; Lesot et al. 1983) through its ability to bind to
and be isolated by laminin sepharose. The receptor
bound laminin with high affinity with a Kd of 2 nM
(Rao et al. 1983). Consequently, this receptor was
named 67LR and, more recently, LAMR1 (laminin
receptor-1). Its gene, however, was found to encode a
protein of only 37-kDa. The discrepancy between these
two molecular masses was later resolved by showing
that the 37-kDa gene product serves as a monomeric
precursor to a 67-kDa dimmer (Rao et al. 1989). The
exact composition of the 67-kDa dimer and the process by which it is formed remains obscure as evidence
supports both a homo (Landowski et al. 1995) and a
heterodimer (Butò et al. 1998). 37/67-kDa laminin
receptor was shown to be acylated by three fatty acids
(palmitate, stearate, and oleate) (Landowski et al. 1995)
and fatty acid synthesis is required for 67-kDa laminin
receptor formation (Butò et al. 1998). Beyond this not
much is known about what regulates the dimerization
process.
Expression of the 67LR has been shown to be
upregulated in neoplastic cells compared with their
normal counterparts and directly correlate with an enhanced invasive and metastatic potential in many malignancies (Sanjuán et al. 1996; Menard et al. 1998).
The receptor has been implicated in laminin-induced
tumor cell attachment and migration, as well as in
tumor angiogenesis, invasion, and metastasis (Mafune
and Ravikumar 1992; Tanaka et al. 2000). Surface expression of the 67LR has also been reported to be a
dominant laminin-binding protein expressed in
neutrophils, macrophages, and monocytes, which suggests that the receptor may play an important role in
the regulation of cell adherence via the basement membrane laminin (Huard et al. 1986; Yoon et al. 1987). It
has been reported that the expression of 67LR in mast
cells is related to the adhesion to laminin and may contribute to the tissue distribution of mast cells and to
mast cell accumulation at sites of tissue injury and inflammation (Thompson et al. 1989). 67LR has also
been shown to be able to modulate granulocytemacrophage colony-stimulating factor (GM-CSF)
signaling by inhibiting complex formation of GM-CSFreceptor (GMR) through the interaction with GMR
(Chen et al. 2003). Furthermore, it has been suggested
that the 67LR expression in human T cells is
upregulated by stimulating the neuropeptides GnRHII and GnRH-I which trigger T cells homing to specific organs (Chen et al. 2002).
67LR also acts as a receptor for pathogenic prion
protein (Gauczynski et al. 2001), cytotoxic necrotizing factor 1 from E. coli (Kim et al. 2005), Sindbis
virus (Wang et al. 1992), Dengue virus (Thepparit and
Smith 2004), and adeno-associated virus subtypes 2,
3, 8, and 9 (Ludwig et al. 1996). Interestingly, unmodified 37-kDa laminin receptor precursor (37LRP) is a
ribosomal component and homologues of this protein
are found in all five kingdoms. 37/67-kDa laminin
receptor has been shown to localize to the nucleus and
interact with histones in the eukaryotic cell nucleus
(Akache et al. 2006).
Mice that are heterozygous for the Rpsa tm1Ells ,
LAMR1, targeted mutation are viable, fertile, and do
not display any gross behavioral abnormalities. Homozygous null mice have an embryonic lethal phenotype, failing to develop past embryonic days 3.5.
Heterozygotes exhibit delayed embryonic growth that
normalizes postnatally (unpublished data). Isolated
congenital asplenia (ICA) is characterized by the absence of a spleen at birth in individuals with no other
developmental defects. Sequence analysis of familial
and sporadic cases revealed that ICA patients carry
mutations in the gene encoding LAMR1 and as a result express about half the normal amount of this protein (Bolze et al. 2013). This discovery establishes an
essential role for LAMR1 in human spleen development. The mechanism by which reduced expression of
a housekeeping protein causes an organ-specific defect remains unclear.
3. Anti-cancer actions of EGCG through EGCG
sensing receptor 67LR
Phosphorylation of the myosin regulatory light chain
(MRLC) at Thr18/Ser19 was shown to regulate the
association between myosin II and filamentous actin
(F-actin). The association of myosin II with F-actin
results in the formation of stress fibers in interphase
cells and the contractile ring in dividing cells. When
human cervical carcinoma HeLa cells were incubated
with EGCG, the cells retracted and left intercellular
gaps. In addition, disappearance of the stress fibers in
the central cell body was observed upon treatment with
EGCG and the MRLC phosphorylation was reduced
by EGCG treatment. The phosphorylation of MRLC
at Thr18/Ser19 has been shown to be necessary for
formation of the contractile ring in dividing cells.
EGCG treatment significantly increased the percentage of cells in the G2/M phase.
To analyze whether the suppressive effect of EGCG
on the MRLC phosphorylation is mediated by the
67LR, RNAi-mediated gene silencing was utilized to
knock down the expression of the 67LR (Umeda et al.
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
2005). EGCG significantly reduced the phosphorylation of MRLC in the HeLa cells, however in the 67LRablated cells, EGCG only slightly reduced the phosphorylation, suggesting that EGCG inhibits the cancer
cell growth by reducing the MRLC phosphorylation
and this effect is mediated by the 67LR. Epidemiological studies have suggested that the consumption
of green tea may decrease colon cancer risk in woman
(Yang et al. 2007). We also found that a physiologically achievable concentration of EGCG inhibited cell
cycle progression of human colon adenocarcinoma
Caco-2 cells through 67LR (Umeda et al. 2008b).
EGCG has been shown to be able to induce growth
arrest and subsequent apoptotic cell death in multiple
myeloma (MM) cells and primary patient MM cells in
vitro, while having no significant effect on growth of
normal cells such as peripheral blood mononuclear
cells (PBMCs) and fibroblasts (Shammas et al. 2006).
Treatment with EGCG (33 mg/kg/d) also led to significant apoptosis in human myeloma cells grown as
tumors in SCID mice. The expression of 67LR was significantly elevated in myeloma cell lines and patient
samples compared to normal PBMCs. RNAi-mediated
inhibition of 67LR expression resulted in abrogation
of EGCG-induced apoptosis in myeloma cells, indicating that 67LR plays an important role in mediating
EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that
EGCG treatment activates distinct pathways of growth
arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent
apoptosis (Fas ligand, Fas, and caspase 4), p53-like
proteins (p73, p63), positive regulators of apoptosis
and NF- κ B activation (CARD10, CARD14), and
cyclin-dependent kinase inhibitors (p16 and p18).
These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale
for its clinical evaluation.
EGCG also induces cell death in acute myeloid leukaemia (AML) patient samples while granulocytes and
PBMC from healthy donors were not affected by
EGCG. AML cells express the 67LR while normal controls do not express the receptor. Moreover, the susceptibility of AML patient samples has been shown to
be closely associated with the level of 67LR expression (Britschgi et al. 2010).
Polyphenon E is a clinical grade catechin mixture
containing about 50% EGCG that is currently under
investigation in multiple cancer trials run by the National Cancer Institute. Recently, Polyphenon E has
been shown to exert anti-neoplastic effects by antagonizing tumor-induced myeloid derived suppressor cells
(defined as HLADR – CD11b +CD66b + cells) though
67LR (Santilli et al. 2013).
On the other hand, EGCG has been shown to inhibit
cell death of muscle cells. Duchenne muscular dystro-
23
phy is a fatal muscle wasting disease caused by the
absence of the protein dystrophin. 67LR was seven
times more abundant in dystrophic muscle cells compared with normal cells. EGCG protected primary dystrophic muscle cells from oxidative damage induced
by hydrogen peroxide in the widely used mdx mouse
model but could not protect normal cells (Dorchies et
al. 2009).
4. Anti-allergic actions of EGCG through EGCG
sensing receptor 67LR
Mast cells and basophils play a central role in immediate allergic reactions mediated by IgE. Binding
of multivalent allergens to specific IgE attached to the
FcεRI on the surface of mast cells or basophils leads
to the release of both preformed and newly generated
inflammatory mediators such as histamine. These mediators ultimately cause various symptoms including
atopic dermatitis, bronchial asthma, and food allergy
(Ravetch and Kinet 1991; Metzger 1992). The early
phase of cell activation of mast cells and basophils includes the phosphorylation and activation of protein
tyrosine kinases and their substrates, generation of the
second messengers such as inositol trisphosphate and
diacylglycerol, and elevation of intracellular Ca2+ levels (Turner and Kinet 1999; Rivera 2002). The late
phase of the activation, which occurs after the influx
of Ca2+, includes the fusion of secretory granules with
the membrane and dramatic morphological changes due
to remodeling of actin cytoskeleton, which undergo
extensive membrane ruffling (Pfeiffer et al. 1985; Choi
et al. 1994; Edgar and Bennett 1997). We found that
EGCG inhibited the calcium ionophore A23187induced histamine release from the human basophilic
KU812 cells and could not inhibit the increase of the
intracellular Ca2+ level after stimulation with A23187
(Fujimura et al. 2006). This result suggested that the
effect of EGCG on histamine release occurs after the
elevation of the intracellular Ca2+ concentration. Thr18/
Ser19 phosphorylation of MRLC has been reported to
be temporally correlated with degranulation in the rat
basophilic RBL-2H3 cells, and the inhibition of MRLC
phosphorylation has been shown to impair the degranulation (Ludowyke et al. 1989). Although EGC, having
no ability to inhibit histamine release, showed no inhibitory effect on MRLC phosphorylation, EGCG
clearly reduced the level of phosphorylated MRLC
(Fujimura et al. 2006). After treatment of KU812 cells
with the anti-67LR antibody, cells were incubated with
EGCG, and further challenged with A23187. The
reductive effect of EGCG on the histamine release was
almost completely inhibited in cells treated with the
anti-67LR antibody (Fujimura et al. 2006). An experiment using such 67LR-downregulated cells revealed a
significant abrogation of the inhibitory effect of EGCG
on degranulation. Furthermore, the lowering effect of
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
24
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
Fig. 2. EGCG sensing pathway for the anti-allergic and anti-inflammatory actions through 67-kDa laminin receptor (67LR).
The suppression of MRLC phosphorylation through the cell-surface binding to the 67LR contributes to the inhibitory effect of
EGCG on the histamine release from basophils. The 67LR also mediates the EGCG-induced suppression of FcεRI expression
in basophils by reducing ERK1/2 phosphorylation. 67LR and Tollip are indispensable for mediating anti-inflammatory action
of EGCG on TLRs signaling induced by LPS and PGN.
EGCG on the phosphorylation of MRLC was also inhibited either by treatment with the anti-67LR antibody or 67LR-knockdown. These findings indicate that
the inhibitory effect of EGCG on degranulation was
caused by a modification of myosin cytoskeleton
through the binding of EGCG to 67LR on the cell surface. When the basophilic cells were stimulated with
A23187 in the presence of EGCG, membrane ruffling
was inhibited and a biased F-actin accumulation was
observed. Furthermore, this EGCG-induced actin
remodeling was abolished in both anti-67LR antibodytreated cells and 67LR-knockdowned cells (Fujimura
et al. 2006). Our findings indicated that EGCG-induced
actin remodeling is caused by lowering MRLC phosphorylation mediated through the binding of EGCG to
the 67LR. Thus, these cytoskeletal modifications may
have an important role in the inhibition of histamine
release by EGCG.
FcεRI plays a central role in the induction and maintenance of IgE mediated allergic responses such as atopic dermatitis, bronchial asthma, and food allergy.
Analysis of FcεRIα chain-deficient mice demonstrated
that IgE was unable to bind to the cell surface of mast
cells, thereby inabling the induction of degranulation
through IgE binding (Dombrowicz et al. 1993). Thus,
it is expected that the downregulation of FcεRI expression in mast cells and basophils may lead to the attenuation of the IgE-mediated allergic symptoms. We
found that EGCG was able to decrease the cellsurface expression of Fc εRI in human basophilic
KU812 cells. Total cellular expression of the FcεRIα
chain decreased upon treatment with EGCG. We also
found that EGCG has an ability to inhibit the phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2) (Fujimura et al. 2004). This inhibition was involved in downregulation of FcεRI expression by EGCG. Moreover, the inhibitory effect elicited by EGCG on ERK1/2 was prevented by disruption of lipid rafts. Accordingly, the interaction between
EGCG and the lipid rafts is important for EGCG’s ability to downregulate FcεRI expression, and ERK1/2 may
be involved in this suppression signal. We also demonstrated that the suppressive effect of EGCG was inhibited by the knockdown of 67LR. Furthermore, the
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
ability of EGCG to decrease the phosphorylation of
ERK1/2 was reduced in the 67LR-knocked down cells.
These results indicate that the effect of EGCG on
ERK1/2 phosphorylation correlates with the expression of 67LR, which implies that the 67LR is the molecule responsible for transducing the EGCG’s
downregulatory signaling of the Fc εRI (Fig. 2).
The O-methylated derivatives of EGCG, (–)epigallpcatechin-3-O-(3-O-methyl)-gallate
(EGCG3″Me) (Fig. 1) and (–)-epigallpcatechin-3-O(4-O-methyl)-gallate (EGCG4″Me), which were isolated from tea leaves such as Tong-ting oolong tea or
cultivars ‘Benifuuki’ have been shown to inhibit allergic reactions in vitro (Sano et al. 1999; Tachibana et
al. 2000). The inhibitory effects of O-methylated
EGCG on mouse type I and IV allergies in vivo were
more potent than that of EGCG (Sano et al. 1999;
Suzuki et al. 2000). These catechins also strongly inhibited mast cell activation through the prevention of
tyrosine phophorylation of cellular protein, histamin/
leukotriene release, and interleukin-2 secretion after
Fc εRI cross-linking (Maeda-Yamamoto et al. 2004).
A double-blind clinical trial to treat allergic cedar pollinosis patients with ‘Benifuuki’ green tea rich in
EGCG3″Me was carried out, and promising results
have been obtained by using a protocol of drinking of
1.5 g of tea powder with water twice a day for 13 weeks
(Maeda-Yamamoto et al. 2007). We have found that
EGCG3″Me can inhibit histamine release and suppress
the FcεRI expression in human basophilic KU812 cells
the same as EGCG (Tachibana et al. 2000; Fujimura et
al. 2002). RNAi-mediated knockdown of 67LR expression resulted in a decreased activity of EGCG3″Me
(Fujimura et al. 2007). The suppression of MRLC phosphorylation through the cell-surface binding to the
67LR contributes to the inhibitory effect of
EGCG3″Me on histamine release. The 67LR also mediated the EGCG3″Me-induced suppression of FcεRI
expression by reducing ERK1/2 phosphorylation.
EGCG is known to be unstable and is easily degraded
in animal bodies. On the other hand, EGCG3″Me and
EGCG4″Me are absorbed efficiently and are more stable than EGCG in animal and human plasma, suggesting that this is the reason for the methylated derivatives of EGCG having potent inhibitory activities to
allergies in vivo. EGCG has been reported to undergo
methylation, and (–)-4′-O-methyl-epigallocatechin-3O-(4-O-methyl) gallate (EGCG4′4″diMe) has been
shown to be a major metabolite of EGCG in plasma
(Lambert and Yang 2003b), and EGCG4′4″diMe did
not demonstrate a suppressive effect in KU812 cells
(Yano et al. 2007). Although our studies on methylated EGCGs may contribute to the elucidation of physiological activities of EGCG in vivo, further investigation of the relationship between metabolites of EGCG
and 67LR is necessary for a better understanding of
the molecular basis of EGCG activities in vivo.
25
5. Anti-inflammatory actions of EGCG through
EGCG sensing receptor 67LR
Toll-like receptors (TLRs) are important in the activation of the innate immune response and are pathogen recognition proteins that have important roles in
detecting microbes and initiating inflammatory responses (Takeda and Akira 2005). Recognition of microbial components by TLRs plays a central role in
the immune system’s decision to respond or not to a
particular microbial infection. Lipopolysaccharide
(LPS), a major component of the outer membrane of
Gram-negative bacteria, is one of the most powerful
activators of TLR4 signaling and is also well known
to induce production of inflammatory mediators, such
as TNF- α, IL-6, and NO, and activation of the MAPK
signaling pathway and NF-κB, leading to death from
endotoxic shock in animal models (Takeuchi and Akira
2001; Cohen 2002; Fujihara et al. 2003). EGCG has
been shown to rescue mice from LPS-induced lethal
endotoxemia and downregulate inflammatory responses in macrophages (Li et al. 2007).
Recently, we found the molecular basis for the
downregulation of TLR4 signal transduction by EGCG
at 1 µM in macrophages (Byun et al. 2010). Anti-67LR
antibody treatment or RNAi-mediated silencing of
67LR resulted in abrogation of the inhibitory action of
EGCG on LPS-induced activation of downstream
signaling pathways and target gene expressions in
murine macrophages. Additionally, we found that
EGCG reduced the TLR4 expression through 67LR.
Interestingly, EGCG induced a rapid upregulation of
Tollip protein, a nagative regulator of TLR-signaling,
and this EGCG action was prevented by 67LR silencing or anti-67LR antibody treatment. RNAi-mediated
silencing of Tollip impaired the TLR4 signaling inhibitory activity of EGCG. Taken together, these findings
demonstrate that 67LR plays a critical role in mediating anti-inflammatory action of a physiologically relevant EGCG and Tollip expression could be modulated
through 67LR in macrophages (Byun et al. 2010) (Fig.
2).
Peptidoglycan (PGN), a major component of the cell
wall of Gram-positive bacteria, is one of the most powerful activators of TLR2 signaling (Fournier and
Philpott 2005; Uckay et al. 2007). PGN induces most
of the clinical manifestations of bacterial infections,
including inflammation, fever and septic shock. We
also reported that 67LR and Tollip are indispensable
for mediating anti-inflammatory action of EGCG on
TLR2 signaling (Byun et al. 2011).
6. EGCG sensing-relating molecules
As previously shown, 67LR mediates anti-cancer
action of EGCG as the cell surface receptor. Therefore, we tried to illuminate the cell signaling pathway
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
26
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
mediated after the binding of EGCG to 67LR and its
biological and physiological significance for the
cancer-preventive activity of EGCG in vivo.
6-1. eEF1A
In an attempt to elucidate the pathways involved in
the anticancer action of EGCG, we applied genetic
suppressor element (GSE) methodology (Umeda et al.
2008a). GSEs are short cDNA fragments encoding
peptides acting as dominant inhibitors of protein function or antisense RNAs inhibiting gene expression.
GSEs behave as dominant selectable markers for the
phenotype associated with the repression of the gene
from which they derived, thus allowing identification
of this gene. For identifying genes mediating cell sensitivity to EGCG, we selected GSEs conferring resistance to EGCG. To search for the mediators of EGCGinduced cell growth inhibition in B16 mouse melanoma
cells, we utilized a targeted genetic screen with a GSE
complementary DNA library. Among genetic elements
protecting cells from EGCG-induced cell growth inhibition, we isolated a GSE that encoded the N terminus
of eukaryotic translation elongation factor 1A (eEF1A).
eEF1A is an important component of the eukaryotic
translation apparatus and is also known as a
multifunctional protein that is involved in a large
number of cellular processes (Negrutskii and El’skaya
1998).
To investigate the role of eEF1A in EGCG-induced
cell growth inhibition, we used stable RNAi to silence
eEF1A expression in B16 cells (Umeda et al. 2008a).
Remarkably, silencing of eEF1A attenuated the inhibitory effect of 1 µM EGCG on cell growth. In contrast,
overexpression of eEF1A enhanced the inhibitory effects of 1 µM EGCG on cell growth. This concentration is similar to the amount of EGCG found in human
plasma after drinking more than two or three cups of
green tea. Given this, we investigated the effect of oral
administration of EGCG on subcutaneous tumor
growth in C57BL/6N mice challenged with eEF1Aablated B16 cells. Tumor growth was significantly retarded in EGCG-administered mice implanted with the
B16 cells harboring a control shRNA, whereas tumor
growth was not affected by EGCG in the mice implanted with eEF1A-ablated B16 cells, indicating that
eEF1A is involved in EGCG-induced cancer prevention. These results support our conclusion that eEF1A
serves as a mediator for EGCG-induced cancer prevention.
6-2. MYPT1
As described previously, EGCG-induced cell growth
inhibition may result from the reduction of the phosphorylation of MRLC at Thr-18/Ser-19 through 67LR
(Umeda et al. 2005). The activity of myosin phos-
Fig. 3. EGCG sensing pathway for eliciting cancer cell
growth inhibition through 67LR. After EGCG binding to
67LR, through eEF1A, the phosphorylation of MYPT1 at
Thr-696 which leads to the activation of myosin phosphatase.
The activated myosin phosphatase dephosphorylates its
substrates (e.g. MRLC), and actin cytoskeleton rearrangement is induced. The alteration of actin cytoskeleton might
lead to cell growth inhibition.
phatase is known to be inhibited by phosphorylation
of its targeting subunit MYPT1 at Thr-696 and Thr853. We tested the effect of EGCG on the phosphorylation of MYPT1 at Thr-696 and Thr-853. Intriguingly, although the phosphorylation level at Thr-853
was unaffected by EGCG, EGCG induced the dephosphorylation of MYPT1 at Thr-696. Further, this effect
correlated with EGCG-induced reduction of the MRLC
phosphorylation, suggesting that EGCG activates
myosin phosphatase by reducing the MYPT1 phosphorylation level at Thr-696. Next, we investigated whether
MYPT1 is involved in anticancer action of EGCG in
vivo (Umeda et al. 2008a). In B16 cells, physiological
concentrations of EGCG reduced the MYPT1 phosphorylation at Thr-696 and the MRLC phosphorylation.
We confirmed both the silencing of MYPT1 by stable
RNAi in B16 cells and the attenuation of the inhibitory effect of 1 µM EGCG on cell growth in MYPT1ablated B16 cells in vitro. We tested the effect of oral
administration of EGCG on subcutaneous tumor
growth in C57BL/6N mice challenged with MYPT1ablated B16 cells (Umeda et al. 2008a). Tumor growth
was significantly retarded in EGCG-administered mice
implanted with the B16 cells harboring a control
shRNA, whereas tumor growth was not affected by
EGCG in the mice implanted with MYPT-1-ablated
B16 cells, suggesting that MYPT1 is indispensable for
EGCG-induced cancer prevention.
In both 67LR-ablated HeLa cells and eEF1A-ablated
HeLa cells, the inhibitory effect of EGCG on both the
phosphorylation of MYPT1 at Thr-696 and the phos-
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
phorylation of MRLC was attenuated. In addition,
EGCG-induced actin cytoskeleton rearrangement was
no longer observed in MYPT1-, eEF1A-, or 67LRablated HeLa cells. The involvement of MYPT1 in
downstream EGCG-triggered signaling from both
67LR and eEF1A was further documented by confirming abrogation of 1 µ M EGCG-induced reduction of
the MYPT1 phosphorylation level at Thr-696 and the
MRLC phosphorylation in 67LR- or eEF1A-ablated
B16 cells. These results suggest that MYPT1 is involved in downstream EGCG signaling from both
67LR and eEF1A (Fig. 3). It has been reported that
MYPT1 binds to eEF1A (Izawa et al. 2000), and more
than half of the total eEF1A (>60%) binds to the actin
cytoskeleton (Edomonds et al. 1996). Characterizing
the mechanisms by which EGCG induces reduction of
the MYPT1 phosphorylation at Thr-696 and reorganization of actin cytoskeleton through eEF1A should help
in more precise understanding of cytoskeleton organization.
6-3. Protein
kinase
Cδ
sphingomyelinase (ASM)
(PKC δ )/acid
EGCG induces apoptosis in several cancers, and the
EGCG-induced cell killing requires 67LR expression
in human AML (Britschgi et al. 2010) and MM patient
cells (Shammas et al. 2006) as described. However,
the downstream mechanisms are still unclear.
EGCG inhibits cell proliferation of primary myeloma
and U266 cells, as well as cervical carcinoma HeLa
cells, but does not affect PBMCs (Tsukamoto et al.
2012). EGCG also reduced the survival of U266 and
primary MM cells, but did not affect the viability of
HeLa cells. We have shown that dephosphorylation of
MYPT1 at Thr696 mediates the EGCG-induced growth
inhibition of B16 melanoma cells and HeLa cells
(Umeda et al. 2008a). In immunoblotting, EGCG dosedependently reduced phosphorylation of MYPT1 at
Thr696 in HeLa cells, but did not affect phosphorylation in U266 cells, indicating that this phosphorylation is not required for apoptosis induction in MM cells.
Taken together, these results suggest that EGCG induces apoptotic cell death through 67LR, but not via
the MYPT1 pathway, in MM cells.
Increases in membrane fluidity and clustering of lipid rafts play crucial roles in apoptosis (Chen et al.
1996). To examine the effect of EGCG on lipid-raft
clustering, we evaluated FRET signalling after staining with CTx-Alexa Fluor® 488 and CTx-Alexa
Fluor® 594 before stimulation with EGCG. EGCG
dose- and time-dependently increased lipid-raft clustering, but treatment with EC, which lacks biological
activity, did not induce lipid-raft clustering in U266
cells. We examined whether EGCG induced lipid clustering was mediated through 67LR. To block the interaction of EGCG and 67LR, U266 cells were treated
27
with either an anti-67LR antibody or control antibody.
Pretreatment with the anti-67LR antibody could block
EGCG-induced lipid raft clustering, whereas pretreatment with the control antibody did not. We next investigated the effect of cholesterol, a membranestabilizing agent, on EGCG-induced apoptosis. Exposure of myeloma cells to cholesterol inhibited lipidraft clustering and apoptosis, suggesting that the
apoptotic activity of EGCG is caused by lipid-raft clustering.
Lipid-raft clustering occurs after generation of
ceramide by acid sphingomyelinase (ASM) (Lacour et
al. 2004; Rebillard et al. 2007). ASM acts on membrane sphingomyelin to generate ceramide, which mediates cell death induced by diverse stimuli, such as
ionizing radiation, chemotherapeutic agents and UVlight. The expression of ASM was abnormally elevated
in all myeloma cell lines relative to normal PBMCs.
EGCG dose-dependently activated ASM in U266 cells,
human MM cell lines and primary MM cells, but did
not affect normal PBMCs. Moreover, pretreatment with
an anti-67LR antibody blocked EGCG-induced activation of ASM, suggesting that 67LR mediates ASM
activation by EGCG. ASM hydrolyses cell-surface
sphingomyelin after directly translocating to the intracellular membrane during cellular stress responses such
as ligation of death receptors (CD95), cisplatin or UV
radiation (Lacour et al. 2004; Mollinedo and Gajate
2006). EGCG increased ASM in the cell-membrane
fraction, and pretreatment with the anti-67LR antibody
blocked this translocation (Tsukamoto et al. 2012).
Moreover, an ASM-specific inhibitor, desipramine,
blocked EGCG-induced ceramide production. Taken
together, these observations show that EGCG modulates the sphingolipid pathway through activating ASM
via 67LR.
Desipramine, an ASM inhibitor, blocked EGCGinduced cell death in U266, MPC-11 and primary MM
cells, indicating that ASM activity mediated this activity. Desipramine also blocked EGCG-induced
apoptotic cell death, as well as normalizing EGCGinduced lipid-raft clustering in U266 cells. Transfection
of U266 cells with an shRNA expression vector to reduce ASM expression abolished EGCG-induced ASM
activation and apoptosis. Collectively, these results
suggest that ASM is necessary for EGCG-induced
lipid-raft clustering, leading to apoptotic cell death in
MM cells.
Protein kinase C δ (PKCδ) is critical for the induction of apoptosis (Frasch et al. 2000; Matassa et al.
2001; Anantharam et al. 2002). Therefore we tested
whether PKCδ was involved in EGCG induced activation of the ASM/ceramide pathway. EGCG dosedependently enhanced generic PKC activity. EGCG
treatment of U266 cells for 5 min led to phosphorylation at Ser664 that was dose-dependent, but did not
affect phosphorylation of Tyr155 and Thr507. More-
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
28
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
Fig. 4. EGCG sensing pathway for inducing cancer cell death through 67LR. 67LR activates the peculiar apoptotic signaling
Akt/eNOS/NO/sGC/cGMP/PKC δ/ASM pathway. Furthermore, upregulation of cGMP could be a rate-determining process of
67LR-dependent cell death. The combination of EGCG as a cancer-specific cGMP inducer with an inhibitor targeting canceroverexpressed PDE5 could be a useful strategy for cancer-selective chemotherapy. Reprinted with permission from ILSI, 116,
Tachibana, Green tea catechin sensing system, 6–13, Fig. 4,  2014, ILSI Japan.
over, EGCG increased Ser664 phosphorylation in three
MM cell lines and primary MM cells, but not in normal PBMCs.
Next, we examined whether EGCG induced activation of PKCδ was mediated through 67LR. To block
the interaction of EGCG and 67LR, U266 cells were
treated with either an anti-67LR antibody or control
antibody. EGCG-induced PKC δ phosphorylation at
Ser664 was not observed in cells pretreated with the
anti-67LR antibody, suggesting that 67LR mediates
EGCG-induced phosphorylation of PKCδ at Ser664.
Furthermore, treatment with the PKCδ-specific inhibitor rottlerin abolished the EGCG-induced activation
of ASM. Silencing of ASM in MM cells did not affect
EGCG-induced PKC δ phosphorylation at Ser664.
Overall, these results suggest that EGCG-induced ASM
activation is a secondary event that occurs after activation of PKCδ.
To evaluate the involvement of the PKCδ/ASM pathway in the killing activity of EGCG on MM cells in
vivo, we examined the effect of EGCG on activation
of caspase 3, PKCδ and ASM in tumor cells. ARH-77
cells were injected subcutaneously into SCID mice.
Oral administration of EGCG promoted the cleavage
of caspase 3, a key mediator of apoptosis, in tumor
cells. Moreover, EGCG induced PKCδ phosphorylation at Ser664 in tumors, indicating the in vivo antimyeloma activity of EGCG. We also tested the effects
of oral or intraperitoneal EGCG in an MPC-11 tumour
xenograft model. The intraperitoneal injection of
EGCG increased levels of cleaved caspase 3 in tumor
cells, as well as PKCδ phosphorylation at Ser664 and
ASM activation. Orally administered EGCG produced
similar effects on caspase 3, PKCδ and ASM activation (Tsukamoto et al. 2012). Consistent with the in
vitro results, these results demonstrate that EGCG activates PKCδ and ASM in MM cells in vivo.
6-4. NO/cGMP
Activation of the PKCδ/ASM pathway is involved
in downstream effectors in EGCG-induced apoptosis
(Tsukamoto et al. 2012). Next, we investigated the
mechanisms by which cancer-overexpressed 67LR activates PKCδ /ASM pathway as a novel death receptor.
67LR has been shown to be involved in shear stress-
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
induced eNOS expression in normal endothelial cells
(Gloe et al. 1999). Therefore, we investigated the role
of NO in 67LR-dependent cell death (Kumazoe et al.
2013a). Primary MM cells derived from a MM patient,
MM cell lines U266 and RPMI8226, and normal
PBMCs were treated with EGCG for 3 hours, and NO
production was evaluated. EGCG induced NO production in MM cell lines and primary MM cells, but had
no effect on PBMCs from healthy donors. We also
found that EGCG elicited eNOS phosphorylation at
Ser1177, which is involved in eNOS activation
(Kumazoe et al. 2013a), and that this phosphorylation
was attenuated by pretreatment of MM cells with an
anti-67LR monoclonal antibody. These results suggested that EGCG activates eNOS and induces NO
production via 67LR. To assess the role of NO in
EGCG-induced cell death, we examined the effect of
the NOS inhibitor L-NAME, which abrogated the cell
death elicited by EGCG. Remarkably, silencing of
eNOS abrogated the inhibitory effect of EGCG on cell
viability. Akt is activated in endothelial cells in response to shear stress and mediates the activation of
eNOS by phosphorylation at Ser1177, leading to an
increase in NO production. Hence, we hypothesized
that EGCG induces eNOS activation through Akt.
EGCG increased Akt kinase activity, which was attenuated by pretreatment of MM cells with an anti-67LR
monoclonal antibody. Importantly, an AKT1/2 kinase
inhibitor attenuated EGCG-induced phosphorylation of
eNOS at Ser1177. Taken together, our findings indicate that EGCG induces NO production through 67LRdependent activation of Akt and eNOS.
Next, we investigated the effect of EGCG on intracellular cGMP, a crucial mediator in NO-induced
signaling (Arnold et al. 1977). EGCG elevated the
amount of cGMP in a dose-dependent manner in primary MM cells and U266 cells, but had no effect on
normal PBMC. To investigate whether cGMP activates
the 67LR-dependent apoptotic pathway, we examined
the effects of the cell-permeable cGMP analog
dibutyryl-cGMP on the activation of ASM. DibutyrylcGMP induced ASM activation in a dose-dependent
manner. Pretreatment of U266 cells with anti-67LR
antibody inhibited EGCG-induced cGMP elevation.
NO increased the intercellular cGMP level by activating soluble guanylate cyclase (sGC). The sGC inhibitor NS-2028 prevented the cGMP upregulation induced
by EGCG. Furthermore, NS-2028 pretreatment also
attenuated EGCG-induced cell death and ASM activation. Taken together, these results suggested that the
67LR/Akt/eNOS/NO/sGC/cGMP pathway mediates
EGCG-induced cell death (Fig. 4). Other tea catechins
and their structurally related compounds have little
affinity for 67LR (Tachibana et al. 2004) and did not
affect the intracellular cGMP level. To our knowledge,
this is the first study to demonstrate that cGMP initiates an apoptotic pathway by activating ASM.
29
6-5. PDE5
cGMP has a crucial role in EGCG-induced MMspecific cell death. EGCG inhibited U266 cell growth
with an IC50 of 23.2 µM. This concentration was much
higher than the plasma concentration previously observed in clinical trials (Shanafelt et al. 2009). EGCG
at physiologically achievable levels could induce NO
production but could not upregulate the level of cGMP
sufficiently to induce MM cell death. These results
suggested that upregulation of cGMP may be a “choke
point” of the EGCG induced apoptotic signaling pathway. PDEs are enzymes that inactivate cGMP signaling
by hydrolyzing the 3,5′-phosphodiester bond. We hypothesized that the PDEs may protect MM cells from
EGCG-induced cell death by downregulating the cGMP
level. To determine the effect of various PDEs on the
anti-MM effect of EGCG, myeloid cell lines were
pretreated with inhibitors of different PDEs (Kumazoe
et al. 2013a). Significant inhibition of cell proliferation was observed when EGCG was combined with the
PDE5-selective
inhibitors
zaprinast,
methoxyquinazoline (MQZ), sildenafil, and vardenafil.
PDE5 is one of the major negative regulators of cGMP
signaling. However, the expression of PDE5 in MM
cells is not known. The protein levels of PDE5 and
67LR increased substantially in the MM cells of 10
patients as well as all human MM cell lines compared
with those in normal PBMCs of 10 healthy donors.
Surprisingly, a significant correlation was observed
between expression of 67LR and PDE5. These data may
provide a rational explanation for the insensitivity of
MM cells to low concentrations of EGCG despite the
high expression of 67LR. To confirm the role of PDE5
in EGCG resistance, we investigated the effect of PDE5
silencing. Reduction in the PDE5 protein expression
markedly potentiated the anti-MM effect of EGCG.
Furthermore, the PDE5 inhibitor vardenafil, which is
used for treating erectile dysfunction (Porst et al. 2001),
had no effect on the number of viable normal PBMCs
from healthy donors, but significantly enhanced the
killing activity of EGCG on primary MM cells from
patients and from the MM cell lines U266, RPMI8226,
and ARH-77. Treatment with EGCG and vardenafil
combined resulted in greater inhibition of the growth
of U266 cells, with an IC50 of 1.4 µM compared with
23.2 µ M for EGCG alone. Isobologram analyses
showed that the growth-inhibitory effects of combined
treatment with EGCG and vardenafil on the growth of
U266 cells and RPMI8226 cells were synergistic. We
also found that vardenafil sensitized U266 cells to
EGCG3″Me. Conversely, vardenafil in combination
with the other catechins did not induce cell death in
MM cells.
To evaluate the in vivo activity of EGCG and
vardenafil in combination, female BALB/c mice were
inoculated subcutaneously (Kumazoe et al. 2013a). Af-
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
30
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
ter the appearance of palpable tumors, mice were then
given a single i.p. injection of 15 mg/kg EGCG and/or
5 mg/kg vardenafil dissolved in physiological saline
(0.9%). After 6 hours, tumors were excised to evaluate the effect on the activity of PKC δ and ASM. Injection i.p. of EGCG and vardenafil in combination increased PKCδ phosphorylation at Ser662 (corresponding to human p-PKCδ Ser664) as well as ASM activity. Moreover, this combination significantly
upregulated the cleavage of caspase-3, a key mediator
of apoptosis in tumor cells. Collectively, these results
suggested that vardenafil potentiates the anticancer
effect of EGCG through amplification of the downstream effectors PKCδ and ASM. To evaluate the longterm effect of EGCG and vardenafil in combination
on tumor growth, female BALB/c mice were given i.p.
injection of 15 mg/kg EGCG and/or 5 mg/kg vardenafil
every 2 days. The combination of EGCG and vardenafil
significantly suppressed tumor growth in the mice.
Furthermore, log-rank analyses of the Kaplan-Meier
survival curves showed a significant increase in the
survival of mice treated with EGCG and vardenafil in
combination compared with mice treated with saline,
EGCG alone, or vardenafil alone. To confirm whether
PDE5 protects MM cells from EGCG-induced cell
death in vivo, BALB/c mice were inoculated subcutaneously MPC-11 cells stably transfected with control
shRNA or the PDE5 shRNA expression vector; after
the appearance of palpable tumors, mice were then
given i.p. injection of 15 mg/kg EGCG every 2 days.
A reduction in the expression of PDE5 protein markedly potentiated the anti-MM effect of EGCG in vivo.
To determine whether the combination of EGCG and
PDE5 inhibitor induces 67LR-dependent cell death, we
undertook antibody-blocking experiments, which demonstrated that 67LR mediated the apoptosis-inducing
effect of this combination (Kumazoe et al. 2013a). We
also found that vardenafil potentiated EGCG-induced
upregulation of cGMP in MM cells and enhanced the
anti-MM effect of the sGC activator BAY 41-2272. To
confirm the role of ASM in the anticancer effect of
EGCG and vardenafil combined, we transfected U266
myeloma cells with a lentivirus encoding scrambled
control shRNA or shRNA against ASM. Remarkably,
silencing of ASM abrogated the inhibitory effect of the
combination on U266 cells. Collectively, these results
demonstrated that the PDE5 inhibitor potentiated
EGCG-induced apoptosis by enhancing the 67LR/
cGMP/ASM-dependent signaling pathway in MM cells
(Fig. 4).
Next, we investigated the effect of EGCG and
vardenafil in various types of cancer (Kumazoe et al.
2013b). The combination of EGCG and vardenafil inhibited the proliferation of the gastric cancer cell line
MKN45, the pancreatic cancer cell line PANC-1, the
prostate cancer cell line PC3, and acute myeloid
leukemia cells but did not affect normal human dip-
loid fibroblasts (NHDFs) or normal HUVECs. Immunohistochemical analyses of paraffin-embedded tissue
sections showed that levels of 67LR and PDE5 were
elevated in various types of human cancers (gastric,
pancreatic, prostate, and breast) compared with their
normal counterparts. The combination of EGCG and
vardenafil inhibited proliferation of the human breast
cancer cell line MDAMB-231-RFP in vitro. To evaluate the in vivo activity of EGCG and vardenafil in combination on MDA-MB-231 cells, the cells were injected
subcutaneously into female nude mice; after the appearance of palpable tumors, mice were given i.p. injections of 15 mg/kg EGCG and/or 5 mg/kg vardenafil
every 2 days. EGCG and vardenafil in combination
significantly suppressed tumor growth, and did not
increase serum levels of ALT/AST. In addition, the
combination induced apoptosis in the pancreatic cancer cell line PANC-1. A prior study showed that a
PANC-1 cell subpopulation propagates colony formation and has the properties of stem cells (Gou et al.
2007). The combination of EGCG and vardenafil treatment inhibited colony formation in PANC-1 cells.
7. Fac tors that modulate the EGCG sensing
receptor 67LR
7-1. Vitamin A
As previously shown, the concentrations of EGCG
that are required to elicit the anticancer effects in a
variety of cancer cell types are much higher than the
peak plasma concentration that occurs after drinking
an equivalent of 10 cups of green tea. To obtain the
anticancer effects of EGCG when consumed at a reasonable concentration in daily life, we investigated the
combination effect of EGCG and food ingredient that
may enhance the anticancer activity of EGCG.
Vitamin A, also known as retinol, participates in
physiological activities related to the immune system,
maintenance of epithelial and mucosa tissues, growth,
reproduction, and bone development. It comes from
animal sources, such as eggs, meat, milk, cheese,
cream, liver, kidney, cod and halibut fish oil. In vitro
and in animal models, it has been demonstrated that
vitamin A is involved in the regulation and promotion
of growth and differentiation of many cells (Ozer et
al. 2005). All-trans-retinoic acid (ATRA), the active
derivative of vitamin A, has been well documented as
a growth and differentiation factor in many tissues and
cells, and proved to be an effective treatment to many
diseases including cancers (Xia et al. 2006; Haque et
al. 2007). Retinoids exert their physiological activities through retinoid receptor nuclear proteins that belong to the superfamily of steroid/thyroid hormone
receptors, of which there are two classes, retinoic acid
receptors (RARs) and the retinoic-X receptors (RXRs),
each of which has three subtypes, a, b, and c. The natu-
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
31
Fig. 5. 67LR is a critical sensor molecule to respond to EGCG and mediates the beneficial activities of this phyrochemical.
Reprinted with permission from ILSI, 116, Tachibana, Green tea catechin sensing system, 6–13, Fig. 5,  2014, ILSI Japan.
ral ligands for the RARs are ATRA and its
stereoisomers 9-cis-RA and 13-cis-RA.
We previously showed that ATRA enhances the expression of 67LR on MCF-7 cells (Tachibana et al.
2004). To determine whether ATRA enhances antitumor effect of EGCG in vivo, we examined the 67LR
expression on B16 melanoma cells by using Western
blot analysis after treatment with different concentrations of ATRA (Lee et al. 2010). ATRA enhanced the
expression of 67LR in a dose dependent manner. We
also found that ATRA treatment increased the cell surface expression of the 67LR as compared with the expression in the control cells. We next examined the
effects of combined EGCG and ATRA treatment on cell
growth of B16 cells. Combination treatment with ATRA
(0.1 µM) and EGCG at a physiological concentration
(0.5 µM) significantly suppressed the number of B16
cells to 52.4% of the control, whereas treatment with
EGCG or ATRA alone did not inhibit cell growth. These
results suggest that ATRA enhances EGCG-induced
cell growth inhibition through 67LR upregulation in
B16 cells. Cell surface binding of EGCG was assessed
using SPR biosensor assay. We found that ATRA significantly enhances the binding of EGCG to the surface of B16 cells. To investigate if the participation of
67LR in ATRA-induced the cell growth inhibitory activity of EGCG, B16 cells were treated with an anti67LR antibody. The growth of the cells treated with a
control antibody was inhibited by the combined EGCG
and ATRA treatment. This growth-suppressive effect
was eliminated upon treatment with an anti-67LR antibody. Together, these observations show that ATRA
action for the cell growth inhibitory activity of EGCG
is attributable to the enhancement of cell surface binding of EGCG via 67LR.
To determine the in vivo efficacy and safety of the
combined treatment, mice were implanted with B16
cells and treated with EGCG and/or ATRA (Lee et al.
2010). Compared to treatment with a vehicle control,
combined treatment significantly reduced the tumor
volume over the duration of the study. The tumor volume and weight in mice treated with EGCG or ATRA
alone did not differ from those in mice treated with the
vehicle control. On the other hand, the mean tumor
weight in the combination-treatment group was, 40%
less than that in the control group, indicating that ATRA
intensifies the anti-tumor activity of EGCG. To examine whether 67LR are involved in the inhibition of
tumor growth, we measured the expression of 67LR in
the tumor cells. The 67LR levels in the tumor were
increased upon oral administration ATRA, or combination of EGCG and ATRA.
RAR that binds to ligand ATRA form a heterodimer
with RXRs and regulate the expression of specific
genes (Martin et al. 2005). To investigate whether the
ATRA-induced enhancement of 67LR expression is
mediated through RAR α , B16 cells were stably
transfected with RARα shRNA expression vector that
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
32
allows knockdown of RARα (Lee et al. 2010). Knockdown of RARα attenuated the ATRA-induced enhancement of 67LR expression. These results suggest that
ATRA enhances 67LR expression through RARα.
To examine the participation of RAR in ATRAmediated enhancement of EGCG-induced cell growth
inhibition through 67LR, B16 cells were treated with
the pan-RAR agonist TTNPB (Lee et al. 2010). TTNPB
enhanced the protein levels and cell-surface levels of
67LR. Moreover, treatment with TTNPB enhanced
EGCG-induced cell growth inhibition. The growthsuppressive effect by combination treatment with
EGCG and TTNPB was obviously reduced upon treatment with anti-67LR antibody. Collectively, these findings indicate that any compounds which activate RAR
may be a candidate to enhance the antitumor activity
of EGCG.
7-2. Low oxygen pressure
The microenvironment of malignant solid tumors is
vastly different from normal tissues, and is characterized by extreme diversity in ionic strength, pH, the
distribution of nutrients and O2 concentration. Increasing experimental evidence indicates that low O2 conditions have a profound impact on malignant progression and response to therapy. O2 conditions can be categorized as “low O2 (not hypoxia)”, “chronic hypoxia”,
“acute hypoxia” and “cycling hypoxia”, according to
causative factors and the duration of cancer cell exposure to hypoxic conditions. Activation of hypoxiainducible factor 1 alpha (HIF-1α) in cancer cells induces the expression of various genes responsible for
adaptation to hypoxic conditions and resistance to
chemotherapy and radiation therapy. Pre-hypoxic regions (HIF-1-independent) are also known to exist
within tumors. However, to date there are no studies
regarding the effects of EGCG on cancer cells under
hypoxic conditions. Therefore, we investigated the effect of O 2 partial pressure on EGCG activity
(Tsukamoto et al. 2012). We found that 67LR protein
levels are reduced by exposure to low O2 levels (5%),
without affecting the expression of HIF-1α. We also
found that EGCG-induced anticancer activity is abrogated under low O2 levels (5%) in various cancer cells.
Notably, treatment with the proteasome inhibitor, prevented down-regulation of 67LR and restored sensitivity to EGCG under 5% O2. In summary, 67LR expression is highly sensitive to O2 partial pressure, and
the activity of EGCG can be regulated in cancer cells
by O2 partial pressure.
8. Conclusions
Beneficial health effects by edible phytochemicals
are now considered to be an inexpensive, readily applicable, acceptable, and accessible approach to can-
cer control and management (Surh 2003), however, little is known about the mechanism of the
chemopreventive action of most phytochemicals. The
essence is the identification of the primary target and
the demonstration of specific mechanisms of action in
animal models and human tissues. Here we described
that 67LR is a critical sensor molecule to respond to
EGCG and mediates the beneficial activities of this
phyrochemical (Fig. 5). We also described that eEF1A,
MYPT1, cGMP, and ASM are EGCG-sensing relating
molecules for EGCG-induced cancer prevention in
vivo, and these factors mediate unique signaling for
cancer prevention triggered by physiological concentrations of EGCG. Our findings suggest that these are
“master factors,” which determine the efficacy of
cancer-preventive activity of EGCG and have important implications for development and use of EGCG
as a cancer-chemopreventive agent. Probably, only a
tumor with a high expression level of these “master
factors” has sensitivity to physiological concentrations
of EGCG, while lower expression of those molecules
causes “EGCG-resistance”. Our results not only illuminate the mechanisms for the cancer-preventive activity of EGCG but should help in the design of new
strategies to prevent cancer and underscore the importance of tailoring cancer therapy on the basis of tumor
genotype.
More definitive information on the relationship between EGCG sensing-pathways and beneficial effects
of EGCG ingestion will emerge from cohort studies
and human intervention trials. We hope that this review will have wide-ranging implications, as many of
the issues discussed here might also be applicable to
studies of other dietary ingredients.
References
Akache B, Grimm D, Pandey K, Yant SR, Xu H, Kay MA.
The 37/67-kilodalton laminin receptor is a receptor for
adeno-associated virus serotypes 8, 2, 3, and 9. J. Virol.
2006; 80: 9831–9836.
Anantharam V, Kitazawa M, Wagner J, Kaul S, Kanthasamy
AG. Caspase-3-dependent proteolytic cleavage of protein
kinase C δ is essential for oxidative stress-mediated
dopaminergic cell death after exposure to
methylcyclopentadienyl manganese tricarbonyl. J.
Neurosci. 2002; 22: 1738–1751.
Arnold WP, Mittal CK, Katsuki S, Murad F. Nitric oxide
activates guanylate cyclase and increases guanosine 3′:5′cyclic monophosphate levels in various tissue preparations. Proc. Natl. Acad. Sci. USA 1977; 74: 3203–3207.
Bolze A, Mahlaoui N, Byun M, Turner B, Trede N, Ellis SR,
Abhyankar A, Itan Y, Patin E, Brebner S, Sackstein P, Puel
A, Picard C, Abel L, Quintana-Murci L, Faust SN,
Williams AP, Baretto R, Duddridge M, Kini U, Pollard
AJ, Gaud C, Frange P, Orbach D, Emile JF, Stephan JL,
Sorensen R, Plebani A, Hammarstrom L, Conley ME,
Selleri L, Casanova JL. Ribosomal protein SA
haploinsufficiency in humans with isolated congenital
asplenia. Science 2013; 340: 976–978.
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
Bors W, Saran M. Radical scavenging by flavonoid antioxidants. Free Radic. Res. Commun. 1987; 2: 289–294.
Britschgi A, Simon HU, Tobler A, Fey MF, Tschan, MP.
Epigallocatechin-3-gallate induces cell death in acute
myeloid leukaemia cells and supports all-trans retinoic
acid-induced neutrophil differentiation via deathassociated protein kinase 2. Br. J. Haematol. 2010; 149:
55–64.
Butò S, Tagliabue E, Ardini E, Magnifico A, Ghirelli C, van
den Brûle F, Castronovo V, Colnaghi MI, Sobel ME,
Ménard S. Formation of the 67-kDa laminin receptor by
acylation of the precursor. J. Cell Biochem. 1998; 69: 244–
251.
Byun EH, Fujimura Y, Yamada K, Tachibana H. TLR 4
signaling inhibitory pathway induced by green tea
polyphenol epigallocatechin-3-gallate through 67-kDa
laminin receptor. J. Immunol. 2010; 185: 33–45.
Byun EH, Omura T, Yamada K, Tachibana H. Green tea
polyphenol epigallocatechin-3-gallate inhibites TLR2
signaling induced by peptidoglycan through the
polyphenol sensing molecule 67-kDa laminin receptor.
FEBS Lett. 2011; 585: 814–820.
Cao Y, Cao R. Angiogenesis inhibited by drinking tea. Nature 1999; 398: 381.
Chen A, Ganor Y, Rahimipour S, Ben-Aroya N, Koch Y,
Levite M. The neuropeptides GnRH-II and GnRH-I are
produced by human T cells and trigger laminin receptor
gene expression, adhesion, chemotaxis and homing to specific organs. Nat. Med. 2002; 8: 1421–1426.
Chen J, Carcamo JM, Borquez-Ojeda O, Erdjument-Bromage
H, Tempst P, Golde DW. The laminin receptor modulates
granulocyte-macrophage colony-stimulating factor
receptor complex formation and modulates its signaling.
Proc. Natl. Acad. Sci. USA 2003; 100: 14000–14005.
Chen SY, Yang B, Jacobson K, Sulik KK. The membrane
disordering effect of ethanol on neural crest cells in vitro
and the protective role of GM1 ganglioside. Alcohol 1996;
13: 589–595.
Choi OH, Adelstein RS, Beaven MA. Secretion from rat
basophilic RBL-2H3 cells is associated with
diphosphorylation of myosin light chains by myosin light
chain kinase as well as phosphorylation by protein kinase
C. J. Biol. Chem. 1994; 269: 536–541.
Cohen J. The immunopathogenesis of sepsis. Nature 2002;
420: 885–891.
Dombrowicz D, Flamand V, Brigman KK, Koller BH, Kinet
JP. Abolition of anaphylaxis by targeted disruption of the
high affinity immunoglobulin E receptor alpha chain gene.
Cell 1993; 75: 969–976.
Dorchies OM, Wagner S, Buetler TM, Ruegg UT. Protection of dystrophic muscle cells with polyphenols from
green tea correlates with improved glutathione balance and
increased expression of 67LR, a receptor for (–)epigallocatechin gallate. Biofactors 2009; 35: 279–294.
Edgar AJ, Bennett JP. Circular ruffle formation in rat basophilic leukemia cells in response to antigen stimulation.
Eur. J. Cell Biol. 1997; 73: 132–140.
Ermakova S, Choi BY, Choi HS, Kang BS, Bode AM, Dong
Z. The intermediate filament protein vimentin is a new
target for epigallocatechin gallate. J. Biol. Chem. 2005;
280: 16882–16890.
Fournier B, Philpott DJ. Recognition of Staphylococcus
33
aureus by the innate immune system. Clin. Microbiol. Rev.
2005; 18: 521–540.
Frasch SC, Henson PM, Kailey JM, Richter DA, Janes MS,
Fadok VA, Bratton DL. Regulation of phospholipid
scramblase activity during apoptosis and cell activation
by protein kinase Cδ . J. Biol. Chem. 2000; 275: 23065–
23073.
Fujihara M, Muroi M, Tanamoto K, Suzuki T, Azuma H,
Ikeda H. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the
receptor complex. Pharmacol. Ther. 2003; 100: 171–194.
Fujimura Y, Tachibana H, Maeda-Yamamoto M, Miyase T,
Sano M, Yamada K. Antiallergic tea catechin, (–)epigallocatechin-3-O-(3-O-methyl)-gallate, suppresses
FcepsilonRI expression in human basophilic KU812 cells.
J. Agric. Food Chem. 2002; 50: 5729–5734.
Fujimura Y, Tachibana H, Yamada K. Lipid raft-associated
catechin suppresses the FcepsilonRI expression by inhibiting phosphorylation of the extracellular signal-regulated
kinase1/2. FEBS Lett. 2004; 556: 204–210.
Fujimura Y, Yamada K, Tachibana H. A lipid raft-associated
67kDa laminin receptor mediates suppressive effect of
epigallocatechin-3-O-gallate on FcepsilonRI expression.
Biochem. Biophys. Res. Commun. 2005; 336: 674–681.
Fujimura Y, Umeda D, Kiyohara Y, Sunada Y, Yamada K,
Tachibana H. The involvement of the 67 kDa laminin
receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-Ogallate. Biochem. Biophys. Res. Commun. 2006; 348: 524–
531.
Fujimura Y, Umeda D, Yano S, Maeda-Yamamoto M, Yamada
K, Tachibana H. The 67 kDa laminin receptor as a primary determinant of anti-allergic effects of O-methylated
EGCG. Biochem. Biophys. Res. Commun. 2007; 364: 79–
85.
Fujimura Y, Sumida M, Sugihara K, Tsukamoto S, Yamada
K, Tachibana H. Green tea polyphenol EGCG sensing
motif on the 67-kDa laminin receptor. PLoS ONE 2012;
7: e37942.
Gauczynski S, Peyrin JM, Haïk S, Leucht C, Hundt C, Rieger
R, Krasemann S, Deslys JP, Dormont D, Lasmézas CI,
Weiss S. The 37-kDa/67-kDa laminin receptor acts as the
cell-surface receptor for the cellular prion protein. EMBO
J. 2001; 20: 5863–5875.
Gloe T, Riedmayr S, Sohn HY, Pohl U. The 67-kDa lamininbinding protein is involved in shear stressdependent endothelial NO synthase expression. J. Biol. Chem. 1999;
274: 5996–16002.
Gou S, Liu T, Wang C, Yin T, Li K, Yang M, Zhou J. Establishment of clonal colony-forming assay for propagation
of pancreatic cancer cells with stem cell properties. Pancreas 2007; 34: 429–435.
Haque A, Banik NL, Ray SK. Emerging role of combination
of all-transretinoic acid and interferon-gamma as
chemoimmunotherapy in the management of human glioblastoma. Neurochem. Res. 2007; 32: 2203–2209.
Hodgson JM, Puddey IB, Burke V, Beilin LJ, Jordan N. Effects on blood pressure of drinking green and black tea. J.
Hypertens. 1999; 17: 457–463.
Huard TK, Malinoff HL, Wicha MS. Macrophages express
a plasma membrane receptor for basement membrane
laminin. Am. J. Pathol. 1986; 123: 365–370.
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
34
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
Izawa T, Fukata Y, Kimura T, Iwamatsu A, Dohi K, Kaibuchi
K. Elongation factor-1 alpha is a novel substrate of rhoassociated kinase. Biochem. Biophys. Res. Commun. 2000;
278: 72–78.
Kim KJ, Chung JW, Kim KS. 67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1expressing Escherichia coli K1 into human brain microvascular endothelial cells. J. Biol. Chem. 2005; 280: 1360–
1368.
Kumazoe M, Sugihara K, Tsukamoto S, Huang Y, Tsurudome
Y, Suzuki T, Suemasu Y, Ueda N, Yamashita S, Kim YH,
Yamada K, Tachibana H. 67-kDa laminin receptor increases cGMP to induce cancer-selective apoptosis. J. Clin.
Invest. 2013a; 123: 787–799.
Kumazoe M, Kim Y, Bae JH, Takai M, Murata M, Suemasu
Y, Sugihara K, Yamashita S, Tsukamoto S, Huang Y,
Nakahara K, Yamada K, Tachibana H. Phosphodiesterase
5 inhibitor acts as a potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin receptordependent apoptosis. FEBS Lett. 2013b; 587: 3052–3057.
Kuriyama S, Shimazu T, Ohmori K, Kikuchi N, Nakaya N,
Nishino Y, Tsubono Y, Tsuji I. Green tea consumption and
mortality due to cardiovascular disease, cancer, and all
causes in Japan: the Ohsaki study. JAMA 2006; 296: 1255–
1265.
Lacour S, Hammann A, Grazide S, Lagadic-Gossmann D,
Athias A, Sergent O, Laurent G, Gambert P, Solary E,
Dimanche-Boitrel MT. Cisplatin-induced CD95 redistribution into membrane lipid rafts of HT29 human colon
cancer cells. Cancer Res. 2004: 64: 3593–3598.
Lambert JD, Yang CS. Mechanisms of cancer prevention by
tea constituents. J. Nutr. 2003a; 133: 3262S–3267S.
Lambert JD, Yang CS. Cancer chemopreventive activity and
bioavailability of tea and tea polyphenols. Mutat. Res.
2003b; 523–524: 201–208.
Landowski TH, Dratz EA, Starkey JR. Studies of the structure of the metastasis-associated 67 kDa laminin binding
protein: fatty acid acylation and evidence supporting
dimerization of the 32 kDa gene product to form the mature protein. Biochemistry 1995; 34: 11276–11287.
Lee JH, Kishikawa M, Kumazoe M, Yamada K, Tachibana
H. Vitamin A enhances antitumor effect of a green tea
polyphenol on melanoma by upregulating the polyphenol
sensing molecule 67-kDa laminin receptor. PLoS ONE
2010; 5: e11051.
Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, Balentine
DA, Yang CS. Analysis of plasma and urinary tea
polyphenols in human subjects. Cancer Epidemiol.
Biomarkers Prev. 1995; 4: 393–399.
Lesot H, Kuhl U, Mark KV. Isolation of a laminin-binding
protein from muscle cell membranes. EMBO J. 1983; 2:
861–865.
Li W, Ashok M, Li J, Yang H, Sama AE, Wang H. A major
ingredient of green tea rescues mice from lethal sepsis
partly by inhibiting HMGB1. PLoS ONE 2007; 2: e1153.
Ludowyke RI, Peleg I, Beaven MA, Adelstein RS. Antigeninduced secretion of histamine and the phosphorylation
of myosin by protein kinase C in rat basophilic leukemia
cells. J. Biol. Chem. 1989; 264: 12492–12501.
Ludwig GV, Kondig JP, Smith JF. A putative receptor for
Venezuelan equine encephalitis virus from mosquito cells.
J. Virol. 1996; 70: 5592–5599.
Maeda-Yamamoto M, Inagaki N, Kitaura J, Chikumoto T,
Kawahara H, Kawakami Y, Sano M, Miyase T, Tachibana
H, Nagai H, Kawakami T. O-methylated catechins from
tea leaves inhibit multiple protein kinases in mast cells. J.
Immunol. 2004; 172: 4486–4492.
Maeda-Yamamoto M, Ema K, Shibuichi I. In vitro and in
vivo anti-allergic effects of ‘benifuuki’ green tea containing O-methylated catechin and ginger extract enhancement. Cytotechnology 2007; 55: 135–142.
Mafune K, Ravikumar TS. Anti-sense RNA of 32-kDa
laminin-binding protein inhibits attachment and invasion
of a human colon carcinoma cell line. J. Surg. Res. 1992;
52: 340–346.
Malinoff HL, Wicha MS. Isolation of a cell surface receptor
protein for laminin from murine fibrosarcoma cells. J. Cell
Biol. 1983; 96: 1475–1479.
Martin PJ, Lardeux V, Lefebvre P. The proliferating cell
nuclear antigen regulates retinoic acid receptor transcriptional activity through direct proteinprotein nteraction.
Nucleic Acids Res. 2005; 33: 4311–4321.
Matassa AA, Carpenter L, Biden TJ, Humphries MJ, Reyland
ME. PKC δ is required for mitochondrial-dependent
apoptosis in salivary epithelial cells. J. Biol. Chem. 2001;
276: 29719–29728.
Menard S, Tagliabue E, Colnaghi MI. The 67 kDa laminin
receptor as a prognostic factor in human cancer. Breast
Cancer Res. Treat. 1998; 52: 137–145.
Metzger H. The receptor with high affinity for IgE. Immunol.
Rev. 1992; 125: 37–48.
Mollinedo F, Gajate C. Fas/CD95 death receptor and lipid
rafts: new targets for apoptosis-directed cancer therapy.
Drug Resist. Updates 2006; 9: 51–73.
Negrutskii BS, El’skaya AV. Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and
possible role in aminoacyl-tRNA channeling. Prog. Nucleic Acid Res. Mol. Biol. 1998; 60: 47–78.
Ozer EA, Kumral A, Ozer E. Effect of retinoic acid on oxygen induced lung injury in the newborn rat. Pediatr.
Pulmonol. 2005; 39: 35–40.
Pfeiffer JR, Seagrave JC, Davis BH, Deanin GG, Oliver JM.
Membrane and cytoskeletal changes associated with IgEmediated serotonin release from rat basophilic leukemia
cells. J. Cell Biol. 1985; 101: 2145–2155.
Porst H, Rosen R, Padma-Nathan H, Goldstein I, Giuliano
F, Ulbrich E, Bandel T. The efficacy and tolerability of
vardenafil, a new, oral, selective phosphodiesterase type
5 inhibitor, in patients with erectile dysfunction: the first
at-home clinical trial. Int. J. Impotence. Res. 2001; 13:
192–199.
Rao CN, Castronovo V, Schmitt MC, Wewer UM, Claysmith
AP, Liotta LA, Sobel ME. Evidence for a precursor of the
high-affinity metastasis-associated murine laminin
receptor. Biochemistry 1989; 28: 7476–7486.
Rao NC, Barsky SH, Terranova VP, Liotta, LA. Isolation of
a tumor cell laminin receptor. Biochem. Biophys. Res.
Commun. 1983; 111: 804–808.
Ravetch JV, Kinet JP. Fc receptors. Annu. Rev. Immunol.
1991; 9: 457–492.
Rebillard A, Tekpli X, Meurette O, Sergent O, LeMoigneMuller G, Vernhet L, Gorria M, Chevanne M, Christmann
M, Kaina B, Counillon L, Gulbins E, Lagadic-Gossmann
D, Dimanche-Boitrel MT. Cisplatin-induced apoptosis
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.
H. Tachibana / AGri-Biosci. Monogr. 4: 19–35, 2014
involves membrane fluidification via inhibition of NHE1
in human colon cancer cells. Cancer Res. 2007; 67: 7865–
7874.
Rivera J. Molecular adapters in Fc(epsilon)RI signaling and
the allergic response. Curr. Opin. Immunol. 2002; 14: 688–
693.
Sanjuán X, Fernández PL, Miquel R, Muñoz J, Castronovo
V, Ménard S, Palacín A, Cardesa A, Campo E.
Overexpression of the 67-kD laminin receptor correlates
with tumour progression in human colorectal carcinoma.
J. Pathol. 1996: 179: 376–380.
Sano M, Takahashi Y, Yoshino K, Shimoi K, NakamuraY,
Tomita I, Oguni I, Konomoto H. Effect of tea (Camellia
sinensis L.) on lipid peroxidation in rat liver and kidney:
a comparison of green and black tea feeding. Biol. Pharm.
Bull. 1995; 18: 1006–1008.
Sano M, Suzuki M, Miyase T, Yoshino K, Maeda-Yamamoto
M. Novel antiallergic catechin derivatives isolated from
oolong tea. J. Agric. Food Chem. 1999; 47: 1906–1910.
Santilli G, Piotrowska I, Cantilena S, Chayka O,
D’Alicarnasso M, Morgenstern DA, Himoudi N, Pearson
K, Anderson J, Thrasher AJ, Sala A. Polyphenon E enhances the antitumor immune response in neuroblastoma
by inactivating myeloid suppressor cells. Clin. Cancer Res.
2013; 19: 1116–1125.
Sazuka M, Murakami S, Isemura M, Satoh K, Nukiwa T.
Inhibitory effects of green tea infusion on in vitro invasion and in vivo metastasis of mouse lung carcinoma cells.
Cancer Lett. 1995; 98: 27–31.
Shammas MA, Neri P, Koley H, Batchu RB, Bertheau RC,
Munshi V, Prabhala R, Fulciniti M, Tai YT, Treon SP,
Goyal RK, Anderson KC, Munshi NC. Specific killing of
multiple myeloma cells by (–)-epigallocatechin-3-gallate
extracted from green tea: biologic activity and therapeutic implications. Blood 2006; 108: 2804–2810.
Shanafelt TD, Call TG, Zent CS, LaPlant B, Bowen DA,
Roos M, Secreto CR, Ghosh AK, Kabat BF, Lee MJ, Yang
CS, Jelinek DF, Erlichman C, Kay NE. Phase I trial of
daily oral polyphenon E in patients with asymptomatic Rai
stage 0 to II chronic lymphocytic leukemia. J. Clin. Oncol.
2009; 27: 3808–3814.
Surh YJ. Cancer chemoprevention with dietary
phytochemicals. Nat. Rev. Cancer 2003; 3: 768–780.
Suzuki M, Yoshino K, Maeda-Yamamoto M, Miyase T, Sano
M. Inhibitory effects of tea catechins and O-methylated
derivatives of (–)-epigallocatechin-3-O-gallate on mouse
type IV allergy. J. Agric. Food Chem. 2000; 48: 5649–
5653.
Tachibana H, Sunada Y, Miyase T, Sano M, MaedaYamamoto M, Yamada K. Identification of a methylated
tea catechin as an inhibitor of degranulation in human
basophilic KU812 cells. Biosci. Biotechnol. Biochem.
2000; 64: 452–454.
Tachibana H, Koga K, Fujimura Y, Yamada K. A receptor
for green tea polyphenol EGCG. Nat. Struct. Mol. Biol.
2004; 11: 380–381.
Takeda K, Akira S. Toll-like receptors in innate immunity.
Int. Immunol. 2005; 17: 1–14.
Takeuchi O, Akira S. Toll-like receptors: their physiological role and signal transduction system. Int.
Immunopharmacol. 2001; 1: 625–635.
Tanaka M, Narumi K, Isemura M, Abe M, Sato Y, Abe T,
35
Saijo Y, Nukiwa T, Satoh K. Expression of the 37-kDa
laminin binding protein in murine lung tumor cell correlates with tumor angiogenesis. Cancer Lett. 2000; 153:
161–168.
Thepparit C, Smith DR. Serotype-specific entry of dengue
virus into liver cells: identification of the 37-kilodalton/
67-kilodalton high-affinity laminin receptor as a dengue
virus serotype 1 receptor. J. Virol. 2004; 78: 12647–12656.
Thompson HL, Burbelo PD, Segui-Real B, Yamada Y,
Metcalfe DD. Laminin promotes mast cell attachment. J.
Immunol. 1989; 143: 2323–2327.
Tsukamoto S, Hirotsu K, Kumazoe M, Goto Y, Sugihara K,
Suda T, Tsurudome Y, Suzuki T, Yamashita S, Kim Y,
Huang Y, Yamada K, Tachibana H. Green tea polyphenol
EGCG induces lipid raft clustering and apoptotic cell death
by activating protein kinase Cδ and acid sphingomyelinase
through 67-kDa laminin receptor in multiple myeloma
cells. Biochem J. 2012; 443: 525–534.
Turner H, Kinet JP. Signalling through the high-affinity IgE
receptor Fc epsilonRI. Nature 1999; 402: B24–30.
Uckay I, Harbarth S, Pittet D. Management of grampositive bacteraemia. Curr. Opin. Infect. Dis. 2007; 20:
561–567.
Umeda D, Tachibana H, Yamada K. Epigallocatechin-3-Ogallate disrupts stress fibers and the contractile ring by
reducing myosin regulatory light chain phosphorylation
mediated through the target molecule 67 kDa laminin
receptor. Biochem. Biophys. Res. Commun. 2005; 333:
628–635.
Umeda D, Yano S, Yamada K, Tachibana H. Green tea
polyphenol epigallocatechin-3-gallate signaling pathway
through 67-kDa laminin receptor. J. Biol. Chem. 2008a;
283: 3050–3058.
Umeda D, Yano S, Yamada K, Tachibana H. Involvement of
67-kDa laminin receptor-mediated myosin phosphatase
activation in antiproliferative effect of epigallocatechin3-O-gallate at a physiological concentration on Caco-2
colon cancer cells. Biochem. Biophys. Res. Commun.
2008b; 37: 172–176.
Wang KS, Kuhn RJ, Strauss EG, Ou S, Strauss JH. Highaffinity laminin receptor is a receptor for Sindbis virus in
mammalian cells. J. Virol. 1992; 66: 4992–5001.
Xia L, Wurmbach E, Waxman S, Jing Y. Upregulation of
Bfl-1/A1 in leukemia cells undergoing differentiation by
all-trans retinoic acid treatment attenuates chemotherapeutic agent-induced apoptosis. Leukemia 2006; 20: 1009–
1016.
Yang CS, Wang X, Lu G, Picinich SC. Cancer prevention by
tea: Animal studies, molecular mechanisms and human
relevance. Nature Rev. Cancer 2009; 9: 429–439.
Yang G, Shu XO, Li H, Chow WH, Ji BT, Zhang X, Gao YT,
Zheng W. Cancer prospective cohort study of green tea
consumption and colorectal cancer risk in women.
Epidemiol. Biomarkers Prev. 2007; 16: 1219–1223.
Yano S, Fujimura Y, Umeda D, Miyase T, Yamada K,
Tachibana H. Relationship between the biological activities of methylated derivatives of (–)-epigallocatechin-3O-gallate (EGCG) and their cell surface binding activities. J. Agric. Food Chem. 2007; 55: 7144–7148.
Yoon PS, Boxer LA, Mayo LA, Yang AY, Wicha MS. Human neutrophil laminin receptors: activation-dependent
receptor expression. J. Immunol. 1987; 138: 259–265.
doi:10.5047/agbm.2014.00402.0019 © 2014 TERRAPUB, Tokyo. All rights reserved.