Nutrients 2018, 10, 1936; doi:10.3390/nu10121936 www.mdpi.com/journal/nutrients
Molecular Targets of Epigallocatechin—Gallate
(EGCG): A Special Focus on Signal Transduction and
, Valeria Naponelli
, Federica Rizzi
and Saverio Bettuzzi
Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126 Parma, Italy;
email@example.com (A.N.); firstname.lastname@example.org (F.R.); email@example.com (S.B.)
National Institute of Biostructure and Biosystems (INBB), Viale Medaglie d’Oro 305, 00136 Rome, Italy
Centre for Molecular and Translational Oncology (COMT), University of Parma,
Parco Area delle Scienze 11/a, 43124 Parma, Italy
* Correspondence: firstname.lastname@example.org; Tel.: +39-0521-033790
† These authors contributed equally to this work.
Received: 7 November 2018; Accepted: 4 December 2018; Published: 6 December 2018
Abstract: Green tea is a beverage that is widely consumed worldwide and is believed to exert
effects on different diseases, including cancer. The major components of green tea are catechins, a
family of polyphenols. Among them, epigallocatechin-gallate (EGCG) is the most abundant and
biologically active. EGCG is widely studied for its anti-cancer properties. However, the cellular and
molecular mechanisms explaining its action have not been completely understood, yet. EGCG is
effective in vivo at micromolar concentrations, suggesting that its action is mediated by interaction
with specific targets that are involved in the regulation of crucial steps of cell proliferation,
survival, and metastatic spread. Recently, several proteins have been identified as EGCG direct
interactors. Among them, the trans-membrane receptor 67LR has been identified as a high affinity
EGCG receptor. 67LR is a master regulator of many pathways affecting cell proliferation or
apoptosis, also regulating cancer stem cells (CSCs) activity. EGCG was also found to be interacting
directly with Pin1, TGFR-II, and metalloproteinases (MMPs) (mainly MMP2 and MMP9), which
respectively regulate EGCG-dependent inhibition of NF-kB, epithelial-mesenchimal transaction
(EMT) and cellular invasion. EGCG interacts with DNA methyltransferases (DNMTs) and histone
deacetylases (HDACs), which modulates epigenetic changes. The bulk of this novel knowledge
provides information about the mechanisms of action of EGCG and may explain its
onco-suppressive function. The identification of crucial signalling pathways that are related to
cancer onset and progression whose master regulators interacts with EGCG may disclose
intriguing pharmacological targets, and eventually lead to novel combined treatments in which
EGCG acts synergistically with known drugs.
Keywords: green tea catechins; epigallocatechin-gallate (EGCG); 67LR; cancer apoptosis; cell death;
chemoprevention; gene expression
Green tea is produced from Camellia sinensis and it represents the second most consumed
beverage in the world after water, being used primarily in Asia and in the Middle East .
Several observational and intervention studies have demonstrated that green tea consumption
has beneficial effects on many human diseases, including obesity, metabolic syndrome,
neurodegenerative disorders inflammatory diseases, and cancer [2–6]. The major polyphenolic
component of dried green tea extracts is epigallocatechin-gallate (EGCG) EGCG is the most
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abundant and biologically active catechin from green tea, accounting for at least 50% of the total
catechin content in green tea leaves . The biological effects of green tea were initially ascribed to
pro- or anti-oxidative properties of catechins. Most of the studies have been conducted
administrating green tea extracts or pure EGCG. A typical weakness of many studies is related to
data collected in vitro and cell culture systems, following the administration of doses of green tea
extracts (or EGCG) much higher than those that were reached in human plasma after green tea
consumption. In vivo, administration of the equivalent of two or three cups of green tea leads to a
peak in the plasma levels of tea catechins in the sub-micromolar range in humans [8,9].
Several in vitro, in vivo, and clinical studies have shown multiple EGCG anticancer actions.
Among them there are anti-proliferative, pro-apoptotic, anti-angiogenic, and anti-invasive functions
. Furthermore, EGCG has been observed to impair other processes that are involved in
carcinogenesis as inflammation, oxidative stress and hypoxia and to target tumor microenviroment
components (e.g., cancer stem cells, fibroblasts, macrophages, and microvasculature) . In several
in vitro and in vivo cancer types, EGCG has been shown to act synergistically with other natural
compounds (e.g., curcumin, ascorbic acid, quercetin, genestein, caffeine) [10,12] and it has also been
testing in combination with currently used chemotherapeutic drugs (e.g., doxorubicin, cisplatin,
sunitinib) [13–16]. Furthermore, in order to improve EGCG bioavailability and stability, novel
formulations of the catechin encapsulated in nanoparticles have been developed [17–20].
Even if the anti-tumoural effect of green tea catechins (and specifically EGCG) has been
extensively demonstrated in vitro, their molecular and cellular mechanisms are not yet completely
The anti-cancer effect of EGCG and green tea extracts is mediated through several mechanisms,
including stimulation of anti-oxidant activity and activation of detoxification system [23,24],
alteration of the cell cycle , suppression of mitogen-activated protein kinase (MAPK) and
receptor protein kinase (RTKs) pathways [26,27], inhibition of clonal expansion of the
tumour-initiating stem cell population , and production of epigenetic changes in gene expression
. These mechanisms (reviewed recently in [30–32]) are not completely understood yet. Green tea
catechins are thought to function both as powerful radical scavengers, in particular, under increased
oxidative stress conditions , and as ROS generators leading to the inhibition of cancer cell growth
through the induction of apoptotis [24,34]. Moreover, they have been shown to induce apoptosis in
several ways, such as modulating pro- and anti-apoptotic protein (Bax, Bcl-2, Bcl-XL) and cell cycle
regulator proteins (cyclins, CDKs) . Green tea catechins are also able to target genes and proteins
that are associated with cell proliferation and apoptosis, including RTKs (receptor tyrosine kinases).
Several studies described the inhibitory effect of green tea catechins on these receptors and on
Ras/extracellular signal-regulated kinase (ERK)/MAPK and phosphatidylinositol 3-kinase
(PI3K)/Akt, which are RTKs-related downstream pathways that are often constitutively activated in
tumor cells. EGCG negatively modulates the expression of various transcription factors, including
Sp1, AP-1, and NF-kB preventing cancer formation [36,37]. Another mechanism that can explain the
pleiotropic effects exerted by green tea catechins is represented by the epigenetic changes in gene
expression and chromatin organization. The major epigenetic mechanisms are DNA methylation,
histone modifications, and expression of noncoding regulatory micro RNA (miRNAs). Green tea
catechins can induce an epigenetic reactivation of genes silenced during carcinogenesis or an
epigenetic downregulation of oncogenes through the inhibition of DNA methyltransferases
(DNMTs) or histone deacetylases (HDACs) activity and the reduction of their expression [38,39].
Micromolar concentrations of EGCG have been shown to exert a wide array of different effects
in a cancer cell. The current understanding is that catechins may either interact with a single critical
regulator affecting the activity of key enzymes that are involved in important pathways, or by
hitting multiple targets in parallel, thereby modulating different pathways simultaneously [40,41].
First it is necessary to identify proteins that bind to catechins with high affinity, which may
represent the master regulators controlling one or multiple pathways. Using in vitro models, several
research teams have identified proteins that are targeted by EGCG. Among these are vimentin, Fyn,
ZAP70, insulin-like growth factor 1 receptor, and glucose regulated protein 78 kDa [42–46].
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However, the functional effect of green tea catechins on the target protein activity has been
demonstrated only at much higher concentrations of EGCG than Kd values, probably because of the
non-specific binding of EGCG to other proteins competing for the target . In Table 1 are listed the
principal EGCG molecular targets that were identified in cancer cells.
We think that EGCG-protein binding can be important for the beneficial effect of green tea
catechins. Green tea catechins bind to a plethora of proteins and the process of the interaction is
highly dependent on the folding status and on the conformational properties of the target protein. In
this review, we decided to take into consideration only few proteins that, after direct binding to
EGCG, alter and affect their downstream pathways promoting anti-cancer effects. These data could
be used for a rational drug design of green tea catechins derivatives exploitable for more specific and
effective anti-cancer therapies. We will focus particularly on the onset and progression of cancer,
describing and discussing the possible molecular mechanisms through which catechins exert their
Table 1. Epigallocatechin-gallate (EGCG) molecular targets that are involved in cancer onset and
MMP-2 * 
DNMT1 * 
p18  Bad  MMP-9 *  IL-8  DNMT3A
 MRLC 
p21  Bak  MMP-14  IGF-1R * 
 MYPT1 
p27  Bcl-2 *  uPA  VEGF  HDAC1 *  eEF1a 
Cyclin D 
Cyclin E  Bcl-xs  E-cadherine
 CCL-2  HAT  RAR-β 
Cyclin A 
Cyclin B 
HSP90 * 
CDK4  Caspase9  Vimentin *  eNOS [74–78] GRP78 * 
CDK6  Apaf-1  Twist  PECAM-1
CDK2  Puma  N-cadherine
 miR-16 
CDK1  XIAP  HIF-1α 
Erk1/2  Cytochrome
C  β-catenin  miR-210 
Pin *  p53  Wnt  miR34a 
PPA2  Survivin  TIMP-3  miR145 
ZAP70 * 
AR  PARP  TRAF-6 * 
67LR *  Oct4 
EGFR  Notch1 
HGFR  Nanog 
TGFR-II * 
cGMP  
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* EGCG direct interactors.
2. 67-kDa Laminin Receptor Signalling Pathways
One of the most interesting targets of EGCG action is the 67-kDa laminin receptor (67LR), a
non-integrin cell surface receptor whose expression has been shown to be increased in several
cancers, such as blood, prostate, breast, gastric, and colon [101–106]. The receptor expression is
usually correlated with drug resistance, and it contributes positively to cancer cells viability, tumour
progression, metastatic diffusion, and neo-angiogenesis [101,102,107,108].
In 2004, Tachibana et al. identified for the first time the 67LR as a specific EGCG membrane
receptor using surface plasmon resonance. The study revealed that 67LR was able to bind EGCG
with a Kd value of 39.9 nM. This interaction enabled EGCG to reduce the growth of the lung cancer
cell line A549 , thus exerting anticancer activity. Other green tea components, such as caffeine,
quercetin, epicatechin (EC), and epigallocatechin (EGC) were tested for binding to 67LR, but none
were specific ligands of the receptor or showed tumour suppressive effects . Therefore, EGCG
appears to be the only catechin able to bind 67LR. Subsequently, a putative EGCG binding site
corresponding to the region between the residues 161 and 170 of the receptor has been identified
. The direct binding between EGCG and 67LR has been confirmed in prostate cancer cells by Yu
et al. . Using MVD (Molegro Virtual Docker, an integrated platform for predicting protein
ligand interactions), these authors identified a binding site for EGCG with the same sequence of the
laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933
sequence of β1 chain of 67LR .
Many studies have explored the signalling cascades that are triggered by EGCG-67LR
interaction, some of which will be discussed in this review. In many cases, the tumour suppression
pathway affected ordered microdomains of the cell membrane known as lipid rafts, where the 67LR
has been located . Lipid rafts differ from the surrounding membrane, because their composition
is enriched in specific lipids (sphingomyelins and glycosphingolipids) and cholesterol, which are
tightly packed to form liquid ordered assemblies . Lipid rafts are dynamic, heterogeneous
structures whose composition is extremely variable, not only in relation to the lipid and sterols
content, but also because of the several proteins that can be recruited (e.g., BCR, FcεRI) or harboured
(e.g., Scr tyrosin kinases) [112–115]. Lipid rafts are rich in tyrosine kinase receptors (RTKs), such as
EGFR [116–118], IGF1R , and HER2 . These receptors have been found to be inhibited by
EGCG in several in vitro and in vivo cancer models (e.g., colon, lung, liver and breast cancers)
[92,121–123]. The functional proteins recruited by lipid rafts allow these structures to play complex
roles. Lipid rafts can float within the plasma membrane or can cluster in larger and stabilized
platforms in response to different stimuli. In most cases, the lipid rafts clustering allows for the
activation of the proteins . Furthermore, modifications in lipid rafts/protein interaction can lead
to alterations in lipid and sterol content, which can, in turn, influence lipid raft functions. Thanks to
their capability to interact with several cellular and molecular factors as caveolae , viruses ,
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bacteria, inflammatory molecules [126,127], and growth factors [128–130], these microdomains are
involved in a plethora of biological functions, like cell polarization, membrane trafficking ,
pathogen internalization , and regulation of a wide spectrum of signal transduction pathways
. Because most of these pathways can control cancer development, progression, rate of cell
proliferation , migration, invasion [132,133], and apoptosis , lipid rafts composition and
functions have received much attention. In addition, many anti-cancer agents (e.g., edelfosine, avicin
D, resveratrol) exert their anti-tumour activity, at least in part, by altering or disrupting the structure
of lipid rafts [135,136].
EGCG has been found to bind to the plasma membrane by interacting with the lipid rafts. The
first evidence of this association was shown in the basophilic cell line KU812, where the suppressive
action of EGCG on the expression of the high-affinity immunoglobulin E receptor (FcεRI) was
triggered by direct binding to lipid rafts. This was mediated by the inhibition of Erk1/2 kinases
phosphorylation and activation . Shortly after, the same research team observed that the
down-regulation of FcεRI was driven by EGCG through binding to 67LR, a receptor associated with
lipid rafts . Others reported that the EGCG inhibitory effect on EGFR in colon cancer cell line
HT29 , and on HGFR in prostate cancer cell line DU145  was mediated by the alteration of
lipid rafts. The collection of signalling pathways affected by lipid rafts structure/function via
EGCG/67LR quickly increased in number, as reported in several tumour models, such as multiple
myeloma, mammary and epidermiod carcinoma, and chronic myeloid leukemia [52,137–139].
2.1. Lipid Rafts-Mediated Apoptosis
2.1.1. EGCG/67LR/Akt/eNOS/NO/cGMP/PKCδ/aSMase Pathway
Together with the inhibition of cell proliferation, migration, and angiogenesis, induction of
apoptosis is one of the main mechanisms through which EGCG exerts its anti-tumour activity
[140,141]. Several studies reported that EGCG is able to affect the expression and function of
anti-apoptotic factors (e.g., Bcl-2, Bcl-xl) and to up-regulate pro-apoptotic molecules (e.g., Bax,
caspase-3) in several cancer models [58,142–144]. However, the mechanisms through which EGCG
modulates key cell death regulators are not completely understood. Some studies reported that 67LR
plays a relevant role in triggering apoptosis after binding its ligand, EGCG, in haematological
malignancies, such as acute myeloid leukemia and multiple myeloma [145,146]. More recently, a
signalling pathway inducing EGCG/67LR-dependent apoptosis through the activation of protein
kinase Cδ (PKCδ), acid sphingomyelinase (aSMase), and lipid rafts clustering has been described in
multiple myeloma models  (Figure 1). The enzyme aSMase is responsible for the catabolism of
sphingomyelin (SM) and is known to be part of the signalling cascades that mediates lipid
raft-dependent apoptosis . It can be activated in response to external pro-apoptotic stimuli as
physical agents (e.g., radiation, UVA light) [148,149], anti-cancer drugs (e.g., cisplantin, doxorubicin)
[150,151], and pro-apoptotic receptors (e.g., Fas, TNF-R) [147,152]. One of the best described
mechanisms of aSMase activation is triggered by Fas receptor. The binding between death receptor
Fas, harboured in lipid rafts , and its ligand FasL lead to the recruitment of adaptor
Fas-associated protein with death domain FADD, which in turn recruits and activates pro-caspase 8.
The final death-inducing signalling complex (DISC) then activates aSMase, which migrates from the
cytoplasmic compartment to lipid rafts, where it generates the sphingolipid ceramide from SM [154–
156]. In response to ceramide generation, cholesterol is displaced from lipid rafts, thus leading to an
increase of membrane fluidity . Ceramide plays a role as second messenger in the signal
transduction, inducing lipid raft clustering and the stabilization of DISC complex, amplification of
Fas/FasL signalling, finally leading to apoptosis [155,158,159]. A similar mechanism has been
hypothesized in the case of cervical, prostate and colon cancer, where EGCG administration induces
cell apoptosis through aSMase activation and ceramide increase [160–162].
Studies on multiple myeloma cell lines in vitro and in vivo, in patients or murine models,
showed that the activation of 67LR through EGCG binding induces the activation of PKCδ after
phosphorylation of Ser664. Activation of PKCδ leads, in turn, to aSMase activation, and finally to cell
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apoptosis . These authors pointed out that treatment with 5 µM EGCG led to an increase of
nitric oxide (NO) . NO is an inorganic signalling messenger triggering a wide range of cellular
pathways. The increase in NO levels is due to the activation of endothelial nitric oxide synthase
(eNOS), after phosphorylation in the residue Ser1177 by Akt kinase . Production of NO causes an
increase of cGMP, produced by NO-dependent soluble guanylate cyclase (sGC) activation, and then
the phosphorylation of PKCδ  (Figure 1). Furthermore, more recently, it has been observed that
the anticancer agent coptisine induces apoptosis in hepatocellular carcinoma (HCC) cells via the
67LR/cGMP pathway . Conversely, several studies reported that EGCG negatively regulates
eNOS/NO production in different cancer types [75–78] and also sGC/cGMP amount .
However, the fact that administration of 5 µM EGCG was sufficient to enhance NO production,
but not a significant increase of cGMP levels to induce cell apoptosis, gives rise to the question of
whether other factors might interfere with cGMP-mediated aSMase activation (Figure 1). Enzyme
phosphodiesterase 5 (PDE5), one of the major cGMP negative regulators, was found to be highly
expressed in multiple myeloma patients as compared to healthy donors, suggesting that PDE5 could
be a target for a possible combinatorial therapy with 5 µM EGCG . This experimental approach
has been implemented. The combined treatment of PDE5 inhibitor Vardenafil and 5 µM EGCG
caused a strong reduction of cell viability not only in multiple myeloma, but also in other models as
prostate, gastric, pancreatic, breast cancer, and in acute myeloid and chronic lymphocytic leukemia
cell lines [74,164,165]. Vardenafil and EGCG synergistic action has been found to cause a significant
reduction of IC
of EGCG [74,164,165]. The tumour suppressive effects of the combinatorial therapy
have also been confirmed in vivo in xenograft murine models of multiple myeloma, treatment that
resulted in the reduction of tumour volume and increased survival without hepatotoxicity, a
possible side effect of high EGCG administration . In this model, controls (namely cell lines and
primary cultures, as well as healthy animal models), were not affected by EGCG alone or in
combination with Vardenafil.
Figure 1. EGCG modulates cell division and apoptosis via 67LR. EGCG binding to 67-kDa laminin
receptor (67LR) activates apoptosis program through enhanced nitric oxide (NO) and cGMP
production, acid sphingomyelinase (αSMase) activation and ceramide generation. Ceramide
metabolization in sphingosine-1-phosphate (S1P) reduces cell apoptosis. EGCG binding to 67LR
inhibits via eukaryotic translation elongation factor 1a (eEF1A) cell cytokinesis inducing myosin
phosphatase target subunit (MYPT1) dephosphorylation and activation and myosin II regulatory
light chain (MRLC) dephosphorylation and inactivation.
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2.1.2. EGCG/67LR/Ceramide/SphK1/S1P Pathway
The formation of larger platforms of cholesterol-enriched lipid rafts in cancer cells is often
associated with aberrant activation of RTKs, resulting in increased proliferation, survival, and
metastatic spread . Instead, ceramide causes cholesterol displacement from lipid rafts,
formation of ceramide-enriched lipid rafts, and induction of cell apoptosis . Therefore, ceramide
catabolism/degradation may produce anti-apoptotic effects. Ceramide can be deacetylazed and
converted to sphingosine, which can be phosphorylated to sphingosine-1-phosphate (S1P) by the
sphingosine kinase 1 (SphK1), an enzyme that is highly expressed in several cancers. The S1P can
activate protein G-coupled receptors that can in turn activate pro-survival and anti-apoptotic
signalling. In prostate cancer models, treatment with high doses of EGCG (75 µM) suppressed
tumour growth in vitro and in vivo through the inhibition of SphK1/S1P signalling  (Figure 1).
The lesson from these data is that, in a particular cell system, a correct balance between
ceramide synthesis and catabolism is fundamental [168,169]. Treatment with 1 µM and 5 µM EGCG
in the multiple myeloma cell line U266 caused the induction of aSMase activity . However,
ceramide accumulation has been observed only after giving high concentrations of EGCG (10 µM
and 20 µM EGCG) . Treatment with these high doses of EGCG leads to the disruption of
cholesterol-enriched lipid rafts and the inhibition of phosphorylation and the activation of several
RTKs (e.g., EGFR, ErbB2, ErbB3, HGFR, IGF1R, Mer, and Flt3). IGFR inhibition has been
demonstrated to be dependent on 67LR and aSMase expression . Because the amount of SphK1
has been found to be increased in multiple myeloma cell lines and specimens from patients, the
combinatorial treatment of 5 µM EGCG and SphK1 inhibitor Safingol was tested. The data
demonstrate that the combination of the two drugs caused an increase in ceramide content, the
disruption of cholesterol-enriched lipid rafts, inhibition of RTKs phosphorylation, and finally an
increase of cell apoptosis . Furthermore, the combination treatment also affected another cell
death mediator that was activated by ceramide, the death-associated protein kinase 1 (DAPK1),
causing the de-phosphorylation of DAPK1 inhibitory residue Ser308 and leading to its activation
Thus, like the inhibition of PDE5 with Vardenafil, the simultaneous action on two related
pathways employing two agents in combination, produced a synergistic effect that strongly reduced
of EGCG . The onco-suppressive action of the double treatment (EGCG plus Safingol)
has been found effective in vitro in acute myeloid leukemia, chronic myeloid leukemia, and in
chronic lymphocytic leukemia models . Absence of toxicity of the combined therapy has also
been shown in vivo .
2.2. Cancer Cell Growth Inhibition
2.2.1. EGCG/67LR/eEF1a/MYPT1/MRLC Pathway
EGCG can exert anticancer functions inducing cell cycle arrest. Several studies reported the
blockade of the cell division cycle by EGCG administration in G0, G1, S, and G2 phases. EGCG may
act through the indirect downregulation of pro-proliferative factors, such as cyclin D1, cyclin E,
cyclin A, cyclin B, CDK4, CDK6, CDK2, and CDK1, as well as by the upregulation of
anti-proliferative effectors, such as CDK inhibitors p27, p21, p16, and p18 [35,48,56,66,171,172]. In
addition, EGCG has been found to act on cytokinesis, a critic step of cell division, by interacting with
67LR receptor [55,61,173].
Cytokinesis is the final step of cell division, leading a mother cell to be divided into two
daughter cells. Early events of the process require the formation of an actomyosin ring, also known
as contractile ring, that allows the formation of the cleavage furrow at the equator of mitotic cells
[174,175]. Generation of the furrow enables the equal division of genetic material between the two
forming cells and their subsequent separation. The interaction between actin filaments (F-actin) and
myosin motors is controlled by different processes among which is the
phosphorylation/dephosphorylation of the myosin II regulatory light chain (MRLC). Myosin II is
one of the main motors involved in cytokinesis, activated through MRLC phosphorylation at
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Ser19/Thr18 by kinases, such as MLCK, ROCK, and Citron kinase . Ser19 phosphorylation
favours the interaction with F-actin, the contractile ring formation, and filaments assembly. A
di-phosphorylation seems to be involved in the assembly of filaments, but the role of
phosphorylation in Thr18 alone is less clear [177–179]. Conversely, MRLC dephosphorylation in
Ser19 or Ser19/Thr18 by the myosin phosphatase leads to myosin inactivation. The MRLC activity is
also indirectly regulated through the phosphorylation of myosin phosphatase itself. When the
largest region of myosin phosphatase, called myosin phosphatase target subunit (MYPT1), is
phosphorylated in at least one of the inhibitory sites (e.g., Thr696, Thr853), its activity is inhibited,
and, as a consequence, MRLC remains active, thus providing a positive signal triggering cytokinesis
EGCG has been found to be able to interfere with the cytokinesis of HeLa cells through its
action on MRLC phosphorylation status, thereby affecting the cellular growth . EGCG activates
the signalling cascade that is responsible for the impaired MRLC phosphorylation through binding
to its membrane receptor 67LR  (Figure 1). At first, it was reported that the treatment of HeLa
cells with 10, 20, and 50 µM EGCG resulted in the disruption of stress fibers, reduction of the
contractile ring formation, increment of cells blocked in G2/M phases, and inhibition of cell growth
. Further analyses revealed that EGCG treatment also caused, via 67LR, a reduction in single
Ser19 and in double Ser19/Thr18 MRLC phosphorylation, which effects on MRLC phosphorylation
might reasonably trigger the effects shown on cell division and growth . Under similar
conditions, EGCG was also found to decrease the phosphorylation of MYTP1 at inhibitory site
Thr696 both in vitro and in vivo, thus preventing myosin phosphatase inactivation. According to the
literature, the ability of EGCG to interfere with MRLC phosphorylation could be the indirect
consequence of MYPT1 loss of inhibition . Recently, another factor has been added to the
members of the EGCG signalling pathway, believed to be responsible for impaired cancer cell
cytokinesis: the eukaryotic translation elongation factor 1a (eEF1a), which has been found to be
necessary to enable EGCG to alter MYPT1 phosphorylation status . eEF1a is mainly known as a
component of the eukaryotic translation machinery, but it also takes part in other cellular processes,
such as senescence, oncogenic transformation, and cell proliferation [181–183]. eEF1a is able to bind
to MYPT1 and F-actin . In vitro and in vivo experiments demonstrated that no significant
reduction in MYPT1 and MRLC phosphorylation, actin disassembly and cell proliferation was
observed after EGCG administration in eEF1a knockout models . This evidence has been further
corroborated by the observation that when eEF1a levels are restored and 67LR is absent, the effects
that are described above disappear as well. Thereby, eEF1a is thought to be downstream of 67LR and
upstream of MYPT1 in the signalling pathway that is triggered by EGCG  (Figure 1).
2.2.2. EGCG/67LR/cAMP/PKA/PP2A Pathway
67LR surface receptor is involved in the selective anti-tumour activity exerted by EGCG in
melanomas. Tsukamoto et al. identified protein phosphatase 2A (PP2A) as a downstream target of
67LR in melanoma cells . PP2A is a Ser/Thr phosphatase that is involved in important cellular
processes, such as proliferation, signal transduction, and apoptosis, and it is considered to be a
tumor suppressor that is functionally inactivated in cancer [185,186].
By performing functional genetic screening, Tsukamoto and colleagues showed that EGCG
binding to 67LR receptor induces PP2A activation mediated by the cAMP/PKA pathway , which
led to the suppression of melanoma tumor cell growth. Even though the direct interaction between
EGCG and PP2A was demonstrated using very high EGCG concentrations [84,187,188], 1 µM EGCG
was sufficient to activate 67LR/PP2A pathway. PP2A directly interacts with p70S6k and
down-regulates mTOR signaling , which is usually aberrantly activated in melanomas.
Therefore, it represents an important contribution to chemotherapeutic resistance of commonly used
BRAF inhibitor treatment. The EGCG-activating 67LR/PP2A pathway exerts a strong synergistic
effect with PLX4720, a BRAF inhibitor, in drug-resistant melanomas.
Another effect that is mediated by the 67LR/PP2A signaling is the activation of Merlin, a tumor
suppressor protein that is encoded by the NF2 gene at physiological concentrations of EGCG, as low
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as 1 µM . Merlin activity seems to target cell surface RTKs and adhesion/extracellular matrix
receptors, regulating cell proliferation, survival and motility . PKA, p21-activated kinase 1 and
2 (PAK 1/2), or MYPT can activate Merlin by dephosphorylation at Ser-518. In the study by
Tsukamoto et al. , EGCG was demonstrated to be an activator of Merlin via 67LR/PP2A pathway.
In prostate cancer cell lines the absence or inactivation of Merlin contributes to tumor development
and progression toward a highly invasive and chemo-resistant state [191–193].
Recently published data show that 10 µM EGCG up-regulates let-7b miRNA expression not
only in melanoma cell lines, but also in metastatic melanoma tumours in vivo . miRNAs are
non-coding RNAs transcripts that are able to regulate fundamental biological activities related to
mRNA degradation or translational inhibition . Yamada et al. demonstrated that 67LR is
involved in the EGCG-elicited let-7b increase, which leads to the inhibition of melanoma tumor
progression . Let-7b recognizes multiple target genes that are related to tumor progression, such
as the high mobility group A2 (HMGA2), decreased in EGCG-treated melanoma cells , or Ras
Furthermore, the data indicated that PP2A inactivation caused the induction of let-7b, which is
generally down-regulated in cancer (including melanoma and prostate cancer) [81,197], even if it is
not clear whether let-7b transcription or let-7b processing is modulated by EGCG-induced PP2A
Zhou et al. confirmed that EGCG induced miRNAs profile changes in a mouse model of lung
tumor. They highlighted that the miRNAs affected by EGCG and target genes are different from
those that were previously identified by in vivo studies .
2.3. Modulation of Cancer Stem Cells Properties
EGCG was shown to affect the survival of cancer stem cells (CSCs). EGCG inhibits CSCs growth
and stemness in several malignancies, such as breast , lung [54,200], colorectal cancer ,
osteosarcoma , and neuroblastoma .
Kumazoe M. et al.  describe the effects of EGCG on the features of pancreatic CSCs (i.e., the
capability to form colonies and spheroids) through the activation of the EGCG/67LR/cGMP axis. The
same research team had observed that spheroid formation in pancreatic CSCs colonies was inhibited
by cGMP targeting of the Forkhead box O3 (FOXO3)/CD44 axes . Transcriptional factor FOXO3
is known to be a cancer suppressor, but it also induces the high expression of CD44, a master
regulator (and also a marker) of CSCs . FOXO3 has been shown to be a direct target of EGCG in
tumours, like pancreatic and breast cancer. In pancreatic cancer treatment with EGCG suppressed
tumour growth, accompanied by FOXO3 downregulation . By contrast, in breast cancer, a
positive regulation of FOXO3 exerted by the EGCG has been described [70,204,205]. Although the
reported modulations seem to be opposite, the action of EGCG on FOXO3 seems to lead to cancer
suppression altogether. Recently, the role of EGCG in inhibiting cancer stem cells (CSC) growth and
altering their features is emerging [54,95,199]. According to this literature, EGCG seems to act by
downregulating CD44 expression in tumours, like non-small cell lung cancer and pancreatic cancer
[200,202]. In pancreatic cancer cell lines expressing CD44, the isoform 3A of the enzyme
phosphodiesterase (PDE3A) is highly expressed . Like other members of the same family,
PDE3A is a negative regulator of cGMP . In pancreatic cancer cells, low EGCG administration
did not lead to a significant increase in cGMP amount, or to the reduction of colony and spheroid
formation . Further experiments were conducted using low doses of EGCG combined with the
administration of a PDE3A inhibitor, Trequinsin. The combination therapy decreased the protein
levels of FOXO3 and CD44, caused an increase of cGMP, and a strong reduction in the CSCs
capability to form both colonies and spheroids. The combination of EGCG and Trequinsin is
synergistic and it reduces the IC
of EGCG, thus allowing for its use at physiological concentration.
These observations have also been confirmed in vivo . Surprisingly, as for the other signalling
pathway that is discussed above, the effects of EGCG alone, or in combination with other agents, are
always specific for cancer cells, and they do not affect normal cells. This highly specific effect of
EGCG is still waiting for an explanation.
Nutrients 2018, 10, 1936 10 of 24
3. Other EGCG-Interacting Proteins
Another interesting protein that was shown to interact directly with green tea catechins is the
human peptidyl prolyl cis/trans isomerase (Pin1). Pin is a protein with two domains: an N-terminal
WW-domain and a C-terminal PPIase domain; both are necessary for its function. Although many
PPIases have been identified some with an established role in cancer, only Pin1 acts distinctively and
specifically on phosphorylated proteins. Pin1 catalyzes the cis/trans isomerization of the peptidyl
proline bond of proteins. This activity causes major changes in the conformation of the target
protein, with a consequent alteration of its function or stability. In this way, Pin1 affects and
modulatse different pathways involving kinase-dependent signaling, such as NF-kB,
activator-protein 1 (AP-1), nuclear factor of activated T cells (NFAT), or b-catenin . Pin1 has
been demonstrated to have a major role in oncogenic signaling [208,209] and is highly expressed in
several cancers [210,211], including prostate cancer .
Urusova et al. used crystallographic and biochemical data to show that EGCG interacts directly
with both the PPase and WW domains of Pin1, which inhibits its tumour-promoting activity.
Therefore, Pin1 represent a possible target for anti-cancer therapies [83,213]. The dissociation
constant of EGCG and Pin1 has been calculated as 21 µM, both by protease-coupled and isothermal
titration calorimetric assays: this value is similar to the concentration of EGCG that was found to
exert anti-cancer effects in experimental cancer models . Since the Kd value that resulted was
quite high, the interaction between EGCG and Pin1 was described as “not strong”. Urusova and
colleagues crystallized the Pin1-EGCG complex, resolving its structure at 1.9 Å resolution by X-ray
diffraction. The crystal structure has revealed that a molecule of EGCG was bound to Pin1 WW
domain (aminoacids 1–31), which is responsible for the interaction with the substrate, while another
molecule of EGCG was bound to the Pin1 PPIase domain, necessary for the isomerization reaction. A
recent study demonstrated that galloyl group in EGCG is required for Pin1 inhibition . Binding
between EGCG and Pin1 in solution has been studied recently by combining fluorescence spectrum,
far-UV circular dichroism spectrum with molecular dynamics simulations. The analysis of the
binding energy confirmed the strong inhibitory effect that is exerted by EGCG on Pin1 activity .
To analyze the functional consequence of Pin1-EGCG binding, Urusova and colleagues used
mouse embryonal fibroblasts (MEF) collected from PIN1 KO and WT mice, and showed that Pin1
expression is required for EGCG (10–40 µM) inhibitory effect on MEFs growth. Furthermore, the
formation of the EGCG-Pin1 complex prevented the binding of the Pin1 substrate c-Jun. Finally,
EGCG effect on transcriptional regulation of AP-1 and NF-kB has been shown to be mediated by
Green tea catechins are mainly believed to prevent cancer. However, several epidemiological
studies suggest that their activity also works against cancer progression; the interaction of EGCG
with proteins that are involved in cancer progression and metastatic spread has been considered.
One of the effects exerted by EGCG is the inhibition of TGF-β signaling transduction. TGF-β is a
multifunctional cytokine that induces epithelial-mesenchymal transition (EMT) of cancer cells, and it
is also responsible for the maintenance of EMT, a critical event during early metastatic growth. The
mechanism by which EGCG modulates TGF-β pathway has not been completely elucidated. It has
been shown that the binding between TGF-β and its receptor, TGFR-II, activates two different
pathways leading to EMT: the canonical Smad-dependent pathway and the mitogen-activated
protein kinase (MAPK) pathway. Tabuchi et al. used immunoprecipitation and affinity
chromatography assays to demonstrate binding between EGCG and TGFR-II protein. This
interaction may be responsible for the inhibitory effect of EGCG on the expression of alpha-SMA
(considered a marker of the EMT) via the TGF-beta Smad2/3 pathway in human lung fibroblast cells
EGCG has also been shown to bind to metalloproteinases (MMPs). MMPs are matrix degrading
enzymes that are involved in tumor invasion and metastasis  whose expression is regulated by
several growth factors, including TGF-β1 [216–219]. Sazuka et al. have demonstrated that EGCG
inhibits the collagenase activity of MMP-2 and MMP-9 produced by lung carcinoma cells. The
authors suggest that the mechanism of inhibition relies on direct binding between EGCG and MMP
Nutrients 2018, 10, 1936 11 of 24
proteins, as proved by affinity gel chromatography experiments . In 2017, Chowdhury et al.
performed a preliminary in silico analysis and then showed a strong interaction of pro-/active
MMP2 with the galloyl group of EGCG and ECG in pulmonary artery smooth muscle cell culture
supernatant. They showed that EGCG and ECG were better inhibitors of proMMP2 when compared
to MMP2, and they demonstrated that a strong interaction with MT1/MMP is involved in the
conversion of proMMP2 to active MMP2 . Further, investigating the interactions of pro-/active
MMP-9 with green tea catechins by computational methods, they showed strong interactions
between pro-/active MMP9 and EGCG/ECG .
4. EGCG Epigenetic Regulation
Another mechanism that can explain the pleiotropic effects exerted by green tea catechins in
tumor cells is the epigenetic change in gene expression and chromatin organization. Mutations in
oncogenes and tumor suppressor genes are often the cause of cancer development and alterations of
gene expression count for cancer progression.
Many biologically active compounds, including EGCG, have been demonstrated to modulate
DNA methylation and histone acetylation status .
DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) are enzymes that are
involved in transcriptional gene silencing and histone acetyl transferases (HATs) positively regulate
gene expression regulation [223,224]. Several studies reported EGCG contribution in epigenetic
control acting on DNMTs, HDACs and HATs expression and activity in different tumours. We will
briefly mention different genes whose expression is enhanced or reduced by EGCG-dependent
Fang et al. demonstrated that EGCG binds to DNMT and competitively inhibits the enzymatic
activity (Ki of 6.89 µM), yielding the reactivation of methylation-silenced genes in prostate cancer
PC3 cells . Molecular modeling and docking studies supported the binding of EGCG to
DNMT3B and HDAC1 .
In HeLa cell line, it has been observed that EGCG can direct bind to and inhibit DNMT1,
DNMT3B, and HDAC1 activity, causing a reduction in DNA hypermethylation and restoring the
expression of repressed genes as retinoic acid receptor β (RARβ), CDH1 (e-cadherine gene), and
DAPK1 [29,39]. Furthermore, in the same the same cell line, EGCG combination with
eugenol-amrogentin (active compounds of clove and Swertia Chirata, respectively) reduces DNMT1
expression with the consequent hypomethylation of the cell cycle inhibitors p16 and LimD1
promoters . In acute promyelocytic leukemia cells, EGCG down-regulates DNMT1, HDAC1,
HDAC2, G9a, and Polycomb repressive complex 2 (PRC2) core components expression and favours
the binding of hyperacetylated H4 and acetylated H3K14 histones to promoter regions of p27, CAF,
C/EBPα, and C/EBPε genes . In the lung cell line PC-9, EGCG combination with Am80 (a
synthetic retinoid used for acute promyelocytic leukemia therapy) causes a decrease of HDAC4,
HDAC5, and HDAC6 protein levels and reduction of HDAC activity, leading to increased p53 and
α-tubulin acetylation . In in vitro and in vivo models of lung cancer, EGCG has been found to
resensitize tumor cells to Cisplatin (DDP)-based combination chemotherapy through DNMT and
HDAC activity inhibition, and the subsequent re-expression of GAS1, TIMP4, ICAM1, and WISP2
genes . In in vivo model of lung cancer, EGCG epigenetic action in down-regulating DNMT1 is
accompanied by phospho-histone H2AX (γ-H2AX) and p-AKT reduction . In skin cancer cells,
it has demonstrated EGCG capability in reducing DNMT1, DNMT3A, and DNMT3B activity and
expression, and also in increasing histones H3 and H4 acetylation. As a consequence of the described
epigenetic changes, a restored expression of the cell cycle inhibitors p16 and p21 has been observed
. In breast cancer cells, EGCG-dependent reduction of HDAC1 and zeste homolog 2 (EZH2)
protein levels leads to tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) gene transcriptional
activation . In prostate cancer cell lines, it has been observed that the EGCG-dependent reduction
of the acetylated androgen receptor (AR) gene might be induced by EGCG reduction in HAT activity
. EGCG also acts on teleomerase, reducing its activity in different tumor types as esophageal
carcinoma , glioma , cervical cancer , breast cancer , nasopharyngeal carcinoma
Nutrients 2018, 10, 1936 12 of 24
, ovarian cancer , laryngeal squamous cell carcinoma , and lung cancer . It has also
been shown that EGCG can translocate from the cytoplasm to the nucleus where it can bind to DNA,
suggesting a possible role in gene expression regulation also through the direct binding to nucleic
acid [236,237]. However, the effects of EGCG/DNA direct interaction need to be clarified.
Because of their anti-proliferative, pro-apoptotic, and anti-oxidative properties, green tea
catechins and especially EGCG are receiving much attention in cancer biology. Several in vitro, in
vivo, and clinical studies, have demonstrated that EGCG exerts anti-cancer effects in different
models through the activation/inhibition of several signalling pathways, most of which are triggered
by the direct interaction between EGCG and specific protein targets. The array of EGCG interactors
is wide and growing, and it includes intracellular molecules, membranes receptors, membrane
microdomains, and the plasma membrane itself. One of the first EGCG direct target identified was
67LR, but in recent years, others interactors, such as Pin1 or TGFR-II, have been recognized.
Appropriate identification and study of EGCG direct targets will allow a better understanding of its
mechanisms of action and a better exploitation of its anti-cancer properties. From 2004, when the
67LR was first identified as direct target of EGCG by Tachibana et al., several research teams have
investigated the pathways modulated by EGCG-67LR interaction. Today, we know that the
anti-proliferative action of EGCG is mediated by the binding to 67LR, whose expression is increased
in tumour cells. Convincing experimental data also showed that membrane composition is involved
in the inhibitory activity of EGCG in some cancer cells lines. Since 67LR is generally located in lipid
rafts, EGCG-mediated microdomains composition and the alteration of their functions triggers the
downstream signalling cascades. In addition, new experimental data have brought to light novel
EGCG signalling cascades leading to cell apoptosis, cell cycle arrest, reduction in CSC colony and
spheroid formation, as well as regulation of miRNAs expression. EGCG binding to membrane
receptors, such as TGFR-II, intracellular molecules, such as Pin1 and secreted enzymes, such as
MMPs, provided noteworthy information about the mechanisms of EGCG-mediated tumour
suppression. Another mechanism to explain the pleiotropic anti-cancer effects that are exerted by
EGCG and green tea catechins that is gaining the attention of the researchers is the modulation of
epigenetic processes. Long-term administration of green tea catechins leads to the re-activation of
tumour suppressor genes that are silenced during carcinogenesis and downregulation of oncogenes
through the inhibition of enzymes, such as DNMTs and HDACs involved in DNA methylation and
chromatin remodelling. Further studies on the interaction of EGCG with protein targets will provide
new insights enabling the development of more pharmacological treatments targeting
EGCG-activated master regulators of key pathways.
Author Contributions: A.N., V.N., F.R. and S.B. critically reviewed the literature and wrote the manuscript.
Funding: This research received no external funding.
Acknowledgments: V.N. was supported by a fellowship by Fondazione Umberto Veronesi. We thank Paul
Wegener for English editing support.
Conflicts of Interest: The authors declare no conflict of interest.
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