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MINI REVIEW P 01-17
Natural products targeting STAT3 signaling
pathways in cancer cells
Yena Jin1,2, Younghwan Kim1, Yu-Jin Lee1, Dong Cho Han1,2 and Byoung-Mog Kwon1,2,*
1
Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, 2University of Science and
Technology in Korea, 125 Gwahakro Yoosunggu, Daejeon 34141, Republic of Korea.
*Correspondence: [email protected]
Cancer is a leading cause of death worldwide. The roles and significance of Signal Transducer and Activator of
Transcription 3 (STAT3) in human cancers have been extensively studied. STAT3 is a promising therapeutic target for cancer
drug discovery. Herbal medicine-derived secondary metabolites efficiently impact all cancer hallmarks; therefore, natural
products are a major source of new cancer drug development. Many natural products can inhibit STAT signaling pathways
in cancer cells by targeting different pathways. In this review, we describe the structures of STAT3 inhibitors isolated from
herbal medicines and discuss their targeting molecules and modes of action.
INTRODUCTION
Despite scientific and technological advancements that have
led to tremendous progress in treating and preventing cancer,
cancer is a leading cause of death in countries of all income
levels. In 2012, an estimated 14.1 million new cancer cases
and 8.2 million cancer deaths occurred worldwide (Torre et al.,
2016). Dramatically improving our understanding of the genomic
aberrations of tumors has allowed for the development of new
drugs directed at specific targets; one example is the tyrosine
kinase inhibitor imatinib used to treat blood cancers. One
strategy for developing cancer drugs is to target drivers of cancer
cell proliferation, metastasis, and survival, as well as cancer stem
cells (Gonda and Ramsay, 2015; Medema, 2013).
As shown Figure 1, Signal Transducer and Activator of
Transcription 3 (STAT3) proteins play crucial roles in cellular
signaling pathways that regulate diverse biological processes,
including cell proliferation, differentiation, survival, inflammatory
response, immunity, cancer stem cells, and angiogenesis (Yu et
al., 2009). Seven members of the STAT family (STAT 1, 2, 3, 4,
5a, 5b, and 6) have been identified in the mammalian system;
these members are activated by signals from cytokines and
growth factor receptors and are translocated to the nucleus
to regulate target gene transcription (Bromberg et al., 2000).
STAT3 has received particular attention owing to the constitutive
activation of STAT3 in a variety of tumors, including melanoma,
and pancreatic, lung, colorectal, ovarian, endometrial, cervical,
breast, brain, renal, prostate cancers, as well as head and
neck squamous cell carcinoma (HNSCC), glioma, lymphoma,
and leukemia (Yuan et al., 2015). Many studies have shown
that aberrant STAT3 activation contributes to cell proliferation,
differentiation, migration, and survival, as well as cancer stem
support (Xiong et al., 2014). This unforeseen STAT3 activation
could partially account for the drug resistance that often follows
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the primary targeted therapies and chemotherapies (Spitzner et
al., 2014).
Cragg et al reported that nature is a valuable source for potential
chemotherapeutic agents (Cragg et al., 2014). Herbal medicine
has played a vital role in disease treatment since prehistoric
times (Assefa et al., 2010). It has been reported that 48.6% of
antitumor medicines are derived, either directly or indirectly,
from natural products (Cragg et al., 2009). Nutraceuticals can
modulate inflammatory pathways and thus affect tumor survival,
proliferation, invasion, angiogenesis, and metastasis (Gupta et
al., 2010).
This review focuses on STAT3 inhibitors isolated from herbal
medicines and their target molecules and modes of action for the
modulation of STAT3 activity.
STAT3 IN CANCER
Cancer is a class of diseases characterized by deregulated
cell proliferation, cell invasion, and cell capacity to expand to
secondary sites of the body. Cancer progression is a multistep
and complex process that begins with abnormal cells with
malignant potential or neoplastic characteristics and continues
with tumor growth, stromal invasion, and metastasis. These
events reply not only on intrinsic tumor characteristics but also
on tumor microenvironment, which includes the surrounding and
supportive stroma, humoral factors, different effectors of the
immune system, and the vasculature (Harris and McCormick,
2010).
STAT3 is crucial in tumor cell proliferation, invasion, and
migration, and is capable of inducing the epithelial-mesenchymal
transition (EMT), regulating the tumor microenvironment and
promoting cancer stem cell (CSC) self-renewal and differentiation
which all benefit the progression of cancer (Siveen et al., 2014).
Therefore, constitutively activated STAT3 may be a therapeutic
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Natural products targeting STAT3 signaling pathways in cancer cells
FIGURE 1 I JAK/STAT3 signaling pathways. JAK/STAT3 pathways are activated through
the binding of recruited cytokines or growth factors. Activated STAT3 forms a homo- or
heterodimer and translocates from the cytoplasm to the nucleus where it binds DNA and
coactivators and induces gene transcription.
target for the treatment of human cancers. Table 1
outlines pre-clinical studies of pSTAT3 expression
in various human tumors (Wang et al., 2012).
There was a significant correlation between higher
pSTAT3 and worse tumor outcomes, such as breast
cancers exhibiting invasiveness, and head and
neck as well as gastric cancers exhibiting nodal
metastasis and/or existing in late clinical stages. As
shown in Figure 1, STAT3 is activated by cytokines
or growth factors (Debnath et al., 2012). STAT3
molecules form homodimers after phosphorylation
of the tyrosine-705 residue that translocates
into the nucleus. These activated STAT3 dimers
promote proliferation primarily by stimulating the
transcription of key cancer genes linked to tumor
cell proliferation, such as cyclin D1, cyclin B and
cdc2, which are involved in the regulation of the cell
cycle. Activated STAT3 contributes to malignancy
by preventing apoptosis pathways, which involves
the increased expression of several anti-apoptotic
proteins, such as survivin and members of the
B-cell lymphoma (Bcl) family (Bcl-xL, Bcl-2 and
TABLE 1 I Constitutive STAT3 activation and oncogenesis
2
Cancer type
Constitutively
activated STAT3
Mediators
(or regulators)
Breast cancer
Human cell lines
EGF/EGFR, Src, or JAK
Endometrial and
cervical cancer
Human cell lines
Down-stream regulation
and target genes
Cell cycle progression
Anti-apoptosis by caspase-3
Genes: Bcl-XL, survivin and Mcl-1
Lung cancer
Human cell lines
EGF/EGFR, IL-6 and
HGF, followed by Src,
or mutant EGFR or
CXCL12/CXCR4-JAK2
Multiple myeloma
Human cell lines
IL-6/JAK
Ovarian cancer
Human cell lines
Genes: Bcl-XL, cyclin D1
Sarcomas
Human cell lines
Proliferation and anti-apoptosis
by caspases
Head and neck
cancer
Human HNSCC lines
primary cultures of oral
keratinocytes
IL-6/gp130, TGF-α
Or EGFR
Proliferation, tumor growth
Genes: cyclin D1, Bcl-2 and Bcl-XL
Prostate cancer
Human cell lines
In vivo mouse models
JAK1 or JAK2
Proliferation, tumor growth, anti-apoptosis by
caspase-3
Melanoma
Human cell lines
In vivo mouse models
Src, but not EGFR or JAK
Proliferation and anti-apoptosis
Genes: Bcl-XL, Mcl-1
Hepatocellular
carcinoma
Human cell lines
In vivo mouse models
TGF-α, or dysfunctional
TGF-β signaling
Proliferation, Genes: cyclin A,
c-jun, c-fos, and c-myc
Pancreatic cancer
Human cell lines
In vivo mouse models
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Anti-apoptosis proliferation
Anti-apoptosis , Gene: Bcl-XL
cyclin D1 and p-STAT3:
mutually exclusive events
Tumor growth, angiogenesis and metastasis
Gene: VEGF
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Yena Jin, Younghwan Kim, Yu-Jin Lee, Dong Cho Han and Byoung-Mog Kwon
Mcl-1), which are also known as direct target genes of STAT3
(Yu and Jove, 2004). STAT3 has also been found to be involved
in colorectal cancer cell invasion and migration through the
regulation of gene expression such as E-cadherin, VEGF, and
matrix metalloproteinases (MMPs) (Wendt et al., 2014). CSCs,
have been identified in a range of different types of cancer, and
CSCs have a high potential for metastasis; in addition, owing to
their resistance to cancer therapy, they often cause relapse after
treatment (Visvader and Lindeman, 2012). It has been reported
that STAT3 plays essential roles in maintaining cancer stemness
and tumorigenic potential (Fouse and Costello, 2013).
INHIBITION OF STAT3 ACTIVITY
signaling (SOCS).
Therefore, STAT activity could be modulated by a variety of
methods including blocking ligand-receptor binding, inhibiting
upstream kinases, and activating or inducing the expression of
negative modulators, as well as indirect modulations such as
reactive oxygen species (ROS). Many STAT3 inhibitors have been
identified using a variety of techniques, including screens of large
compound libraries (Schust and Berg, 2004), computer assisted
virtual screens using the STAT3 crystal structure (Siddiquee
et al., 2007), STAT3 inhibitors isolated from herbal medicines
(Arumuggam et al., 2015), fragment-based design for a novel
STAT3 inhibitor (Yu et al., 2013), and STAT3-binding peptides
(Dhanik et al., 2012). The various targets for the modulation of
STAT3 activity are summarized in Figure 2.
Herbal medicines have been used for the treatment of cancer
and vinca alkaloids such as vinblastine and vincristine were the
first plant-derived anti-cancer drugs (Cragg et al., 2009). Many
of the recently reported natural compounds exhibiting anti-tumor
activities inhibit the activity of tumor-promoting proteins such as
phosphatidylinositol-3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2),
and STAT3 (Chinembiri et al., 2014). Many natural products can
inhibit STAT3 activity, and several natural or dietary compounds
have been shown to possess in vitro and/or in vivo inhibitory
effects against STAT3 (Arumuggam et al., 2015). In the present
review, we focus on the STAT3 inhibitors isolated from herbal
medicines, and discuss the targets and mode of actions of
the STAT3 inhibitors. As previously mentioned, small molecule
inhibitors targeting various members of the STAT3 signaling
Since the first reported as an oncogene in 1995, STAT3 has
been observed to be activated in many types of cancer (Watson
and Miller, 1995). STAT3 plays major roles in tumor progression
and metastasis by modulating the tumor microenvironment.
Furthermore, JAK2/STAT3 signaling is important in tumorassociated immune cells and cancer stem-like cells (Yu et al.,
2014). Therefore, STAT3 is a promising therapeutic target for
cancer drug discovery (Yuan et al., 2015).
As shown in Figure 1, STAT3 is activated through the binding of
cytokines or growth factors to cell surface receptors. Cytokines,
such as the interleukins IL-6, IL-10, and IL-11, as well as
growth factors such as EGF, fibroblast growth factor (FGF),
and vascular endothelial growth factor (VEGF), can activate the
tyrosine phosphorylation cascade. Once ligands bind to their
corresponding receptors, the receptors form a dimer complex.
The ligand receptors recruit janus kinases
(JAKs). JAKs lead to their activation
via phosphorylation which in turn
phosphorylates the cytoplasmic tyrosine
residues on the receptors that serve as
a dock for the SH2 domain of STAT3.
STAT3 becomes activated (pSTAT3)
through the phosphorylation of its Tyr705
residue located within its Src Homology
2 (SH2) domain. The activation of
STAT3 triggers pSTAT3 to form a dimer
via the interaction of the pTyr705 of
one monomer and the SH2 domain of
another. The activated dimer dissociates
from the receptor and subsequently
translocates from the cytoplasm to the
nucleus where it binds specific DNA
sequences and induces transcription. In
addition to JAKs, STAT3 can be activated
by nonreceptor tyrosine kinases such
as Src and ABL. Activated STAT3 is
negatively regulated by phosphatase,
FIGURE 2 I Targets for STAT3 modulation. There are different approaches to inhibiting STAT3
such as Src homology region 2 domainsignaling, including by the direct interactions of small molecules with the proteins and the inhibition
containing phosphatase-1 (SHP-1) and
of upstream kinases that activate STAT3. STAT3 activity can also be modulated through negative
regulators, such as phosphatases.
SHP-2, and suppressors of cytokine
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Natural products targeting STAT3 signaling pathways in cancer cells
pathways have been employed to disrupt STAT3 signaling
and activity. We briefly discuss selected inhibitors that target
the STAT3-SH2 domain, the upstream kinase of STAT3, and
phosphatase, which induces dephosphorylation of STAT3. STAT3
inhibitors modulating STAT3 activity via indirect pathways such
as the induction of ROS are also discussed.
DIRECTLY TARGETING STAT3 PROTEIN
STAT3 exists in the cytoplasm as a monomer. STAT3 molecules
form homodimers after phosphorylation of the Try-705 residue
that translocates into the nucleus. These activated STAT3 dimers
regulate the expression of cell cycle-regulatory proteins and
genes associated with cell survival. Therefore, the best approach
may be to inhibit STAT3 protein directly. The three domains of
STAT3 (Levy and Darnell Jr, 2002), including the NH2-terminal,
DNA-binding and SH2 domains, were identified as selective targets for the development of STAT3 inhibitors based on
the understanding of the structure and function of STAT3. The
most popular target is the SH2 domain because it is necessary for the recruitment of STAT3 to any activated receptor. In
addition, inhibition of this target can block STAT3 dimerization,
and consequently inhibit nuclear translocation that leads to the
regulation of the expression of STAT3 target genes. Structurebased drug design and computational docking have been
used to discover small-molecule inhibitors and many synthetic
inhibitors of the STAT3 SH2 domain have been reported; some
SH2 domain inhibitors are under clinical study (Zhao et al., 2016).
Many natural products have been introduced as STAT3
inhibitors via blocking SH2 binding, inhibiting upst-ream kinases,
or expressing phosphatase (Yang et al., 2013). In this section,
we describe the STAT3 SH2 inhibi tors isolated from herbal
medicines. Multiple novel small molecule inhibitors targeting the
STAT3 SH2 domain have been identified from natural products
(Figure 3).
Cryptotanshinone (CTS), which is the one of the major
representative components isolated from the root of Salvia
FIGURE 3 I Structure of STAT3-SH2 domain inhibitors.
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miltiorrhiza Bunge (Danshen), has been reported to have
various pharmacological effects. An antitumor activity of CTS
has also been reported in several studies (Chen et al., 2013).
Cryptotanshinone and tanshinone IIA are well-known active
components of Danshen and they have anti-tumor activities
(Akaberi et al., 2015). It was also reported that tanshinone IIA
induced apoptosis in human hepatocellular carcinoma cells. CTS
was identified as a potent STAT3 inhibitor. CTS inhibited STAT3
Tyr705 phosphorylation in DU145 prostate cancer cells as well
as the growth of the cells. CTS down-regulates the expression
of STAT3 downstream target proteins such as cyclin D1, survivin,
and Bcl-xL in DU145 cells. CTS only inhibited STAT3 Tyr705
phosphorylation and the activity of STAT3 upstream kinases was
not affected (Shin et al., 2009).
Computational modeling showed that CTS can bind to the
STAT3 SH2 domain. Modeling also showed that cryptotanshinone
bound to the SH2 domain via a number of hydrogen bonds with
nearby residues, including Arg609 and Ile634 (Shin et al., 2009).
The computationally proposed mechanism of CTS was confirmed
by localization experiments conducted with confocal microscopy,
suggesting that CTS bound to STAT3 molecules directly. When
the cells were treated with CTS, most STAT3 was localized in the
cytoplasm, as was CTS. This co-localization of CTS with STAT3
implied that CTS binds directly to STAT3. Because STAT3 Tyr705
phosphorylation is related to STAT3 dimerization, whether CTS
inhibited STAT3 dimerization was assessed using native PAGE.
CTS inhibited STAT3 dimerization in a time-dependent manner.
Six hours after CTS treatment, dimerized STAT3 was significantly
decreased. It was further confirmed that CTS decreased the
amount of STAT3 dimers using an electrophoretic mobility shift
assay (EMSA). The results of the experiment indicated that the
DNA-binding activity of STAT3 decreased after a 3-hour treatment
and was almost completely lost after a 6-hour treatment. These
data suggest that CTS binds to STAT3 monomers, thereby
blocking dimerization, and inhibiting STAT3 transcriptional
regulatory activity. Therefore, the antitumor effects of CTS might
be mediated by blocking STAT dimerization through the inhibition
of the STAT3 SH2 domain and then inhibiting the expression of
STAT3-target genes such as Bcl-xL, survivin, and cyclin D1. The
down-regulation of these proteins inhibited cell cycle progression
and led to the inhibition of cell growth. The down-regulation of
cyclin D1 expression by CTS was induced by the arrest in the
G1 phase of the cells. Additionally, the suppression of Bcl-xL
expression by CTS appeared to induce cell death. Based on
these data, CTS is a promising molecule for the development of
STAT3 SH2 domain inhibitors.
Alantolactone, a major component of Inula helenium, has been
reported to possess various pharmacological activities, such as
anti-inflammation and antitumor activities (Rasul et al., 2013).
Alantolactone inhibited STAT3 phosphorylation at tyrosine 705 in
a dose- and time dependent manner; however, this compound
did not inhibit STAT3 phosphorylation at serine 727 in MDA-
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Yena Jin, Younghwan Kim, Yu-Jin Lee, Dong Cho Han and Byoung-Mog Kwon
MB-231 cells. The total STAT3 protein levels were not affected
by alantolactone. Because STAT3 phosphorylation at tyrosine
705 leads to STAT3 dimerization and then nuclear translocation,
consistent with the inhibition of STAT3 phosphorylation,
alantolactone inhibited STAT3 translocation to the nucleus and
also blocked the DNA-binding activity of STAT3. The STAT3
inhibition activity of alantolactone was tested in several breast
cancer cell lines, including MDA-MB-231, MCF-10A, and MCF7. The results demonstrated that alantolactone significantly
inhibited STAT3 phosphorylation in MDA-MB-231 cells, which
are STAT3 activated tumor cells. As previously mentioned, the
STAT family includes STAT1, STAT3, STAT5, and STAT6, and
alantolactone did not inhibit the phosphorylation of STAT1
and only slightly inhibited STAT5 and STAT6 phosphorylation;
however, STAT3 phosphorylation was almost completely inhibited
in alantolactone-treated breast cancer cells, suggesting that
alantolactone selectively inhibits STAT3 activation.
Upstream kinases, such as JAK1/2, AKT, and EGFR, were
not inhibited by alantolactone. Therefore, a structure-based
molecular-docking study was performed and it was found that
alantolactone could interact with the STAT3 SH2 domain. (Chun
et al., 2015). The result suggests that alantolactone suppressed
the inducible and constitutively activated STAT3 and blocked the
nuclear translocation and the DNA-binding of STAT3 in MDAMB-231 cells by the inhibition of the STAT3 SH2 domain. It was
reported that alantolactone also inhibited migration, invasion,
adhesion, and colony formation in MDA-MB-231 cells and the
compound significantly suppressed the growth of human breast
cancer cell xenograft tumors. These results support the potential
of alantolactone as a STAT3 inhibitor as well as a potential agent
against triple-negative breast cancers.
Scoparone (6,7-dimethoxycoumarin) is a coumarin-type
natural product. More than 300 coumarins have been isolated
from plants (Hoult and Paya, 1996). Scoparone is isolated from
the Chinese herb Artemisia capillaris (yin chin) and has been
used for the treatment of neonatal jaundice in Asia. Scoparone
possesses several other biological properties, including anticoagulant, hypolipidemic, vasorelaxant, anti-oxidant, and antiinflammatory effects (Hung and Kuo, 2013). The inhibition of the
transcriptional activity of the nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) appears to be responsible
for its anti-inflammatory activity. It was recently reported that
scoparone inhibited the growth of DU145 prostate-cancer cells
by the inhibition of STAT3 activity (Kim et al., 2013). Scoparone
repressed both constitutively and IL-6-induced activity of STAT3.
Consistent with this finding, scoparone decreased the nuclear
accumulation of STAT3, but did not reduce the phosphorylation of
JAK2 or Src, the major upstream kinases responsible for STAT3
activation. Therefore, scoparone suppressed the transcription
of STAT3 target genes such as cyclin D1, c-Myc, survivin,
Bcl-2, and Socs3, which was confirmed by Western blot and
quantitative real-time PCR analyses. Because scoparone down-
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regulates the STAT3-target genes, scoparone inhibits the growth
of DU145 cells and induces apoptosis. Furthermore, scoparone
treatment suppressed anchorage-independent growth in soft
agar as well as the tumor growth of DU145 xenografts in nude
mice, concomitant with a reduction in STAT3 phosphorylation.
Because scoparone inhibits STAT3 activity and does not affect
the activity of JAK2 or Src, it was postulated that scoparone
might regulate STAT3 activity by directly binding to STAT3
through the SH2 domain, which was confirmed by a structurebased molecular modeling and docking study. The refined model
suggested that two oxygen atoms within the lactone ring of
scoparone might form hydrogen bonds with amino acid residues
within the SH2 domain of STAT3 (Kim et al., 2013). These results
suggest that scoparone inhibits the phosphorylation of STAT3
by binding to the SH2 domain of STAT3. Furthermore scoparone
suppressed the growth of STAT3-actavted DU145 cells more
potently than that of STAT3-negative PC-3 cells. Therefore,
these results suggest that STAT3 is a novel molecular target
for scoparone and that scoparone represents an anticancer
drug candidate for the treatment of cancers in which the STAT3
signaling pathways are constitutively active tumor.
A variety of STAT3 inhibitors block STAT phosphorylation,
dimerization, and translocation to the nucleus, and inhibit
STAT3 target gene expression. In this section, we discuss a few
SH2 domain binders isolated from herbal medicines. To date,
many natural products that target the SH2 domain and inhibit
STAT3 dimerization have been reported, including cardamonin
(2,4-dihydroxy-6-methoxychalcone), a class of chalcone
compounds isolated from the spicy plant Alpinia conchigera Griff
(Zingiberaceae); garcinol and polyisoprenylated benzophenone,
isolated from the dried rind of the fruit Garcinia indica, plumbagin
(5-hydroxy-2-methyl-1,4-naphthoquinone) isolated from the root
of the plant Plumbago zeylanica; withaferin A originally isolated
from the plant Withania somnifera; and tectochrysin (flavonoid
compound) isolated from A. oxyphylla Miquel (Kamran et al.,
2013). These compounds selectively inhibited STAT3 activity
and suppressed the expression of STAT3 target genes such as
cyclin D1, survivin, Bcl2, VEGF, and Bcl-xL, which led to inhibited
proliferation and angiogenesis, and induced apoptosis in a
variety of tumor cells.
MODULATION OF STAT3 ACTIVITY
BY KINASE INHIBITION
The phosphorylation of JAK via the activation of cytokine
receptors activates the cytoplasmic STAT proteins via
phosphorylation. Additionally, the activation of STATs is also
modulated by several growth factors such as EGF, HGF, and
PDGF. The activated STATs form a dimer, which shifts to
the nucleus, binds to specific DNA response elements, and
regulates the genes related to cell growth, differentiation, and
angiogenesis. The activation of the JAK2/STAT3 signaling
pathway in human tumor tissues was recently confirmed, which
therefore may provide a novel therapeutic strategy for human
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Natural products targeting STAT3 signaling pathways in cancer cells
cancer. In this section, we discuss natural products that inhibit
STAT3 activity by the inhibition of JAK2 kinase (Yuan et al, 2015).
Most JAK2/STAT3 pathway inhibitors isolated from herbal
medicines inhibit STAT3 phosphorylation by the modulation of
multiple kinases. In this review, we discuss JAK2/STAT3 pathway
inhibitors that target JAK2 such as brevilin A and thymoquinone,
as well as JAK2/STAT3 pathway inhibitors, such as cucurbitacin
analogues and 4-methoxydalbergione (Figure 4).
Brevilin A is a pseudoguaiane sesquiterpene isolated from Litsea
glutinosa. Brevilin A was identified as a STAT3 pathway inhibitor
through the cell-based luciferase assay against 1,440 natural
products. The human prostate carcinoma DU145 and breast
cancer MDA-MB-468 cell lines are well known tumor cells with
constitutively activated STAT3 (Chen et al., 2013). Brevilin A
inhibited STAT3 activity in a dose- and time-dependent manner in
both DU145 and MDA-MB-468. Brevilin A also blocks JAK-JH1
tyrosine kinase activity in vitro. To test JAK-specific inhibition,
the phosphorylation levels of p65, AKT and glycogen synthase
kinase 3 beta (GSK-3β) were analyzed in brevilin A-treated tumor
cells. Interestingly, brevilin A did not inhibit the phosphorylation
of these proteins, indicating that brevilin A is a specific JAK
kinas inhibitor. Brevilin A repressed both constitutive and IL6-induced phosphorylation of STAT3 at Try705 in a time- and
dose-dependent manner. The inhibition of STAT3 activity blocks
the translocation of STAT3 into the nucleus and then the downregulated expression of STAT3 target genes, e.g., c-Myc and
cyclin D1. After the DU145 and MDA-MB-468 cells were treated
with brevilin A for 24 and 48 hours, both c-Myc and cyclin D1
expression levels were reduced. Increased cleaved PARP was
also observed, indicating that brevilin A induced DU145 and
MDA-MB-468 apoptosis after 24 hours of treatment (Chen et al.,
2013). These results are consistent with reports indicating that
blocking STAT3 activity led to cell growth inhibition in DU145 (Shin
et al., 2009) and MD-AMB-468 cells (Siddiquee et al., 2007).
Furthermore brevilin A has exhibited preferential cell growth
inhibition of DU145 and MDA-MB-468 cells, the growth of which
is dependent on STAT3 signaling, over that of human nontransformed telomerase-immortalized fibroblasts BJ cells.
JAKs family members contain tyrosine Janus homology
domain 1 (JH1 domain), which is the tyrosine kinase domain
and usually exhibits constitutive enzymatic activity. Because
brevilin A inhibits JAK2 phosphorylation in tumor cells, to find
the molecular target of brevilin A, JAK2-JH1 was over-expressed
in HEK239T cells, and it was observed that STAT3 Tyr705
phosphorylation was increased. Brevilin A exhibited a significant
inhibition of pSTAT3, induced by the overexpression of the JAK2JH1 domain. Interestingly, the compound did not inhibit pSTAT3
at Tyr705 in c-Src-overexpressed HEK293T cells compared with
a known Src inhibitor, PD-18097. In addition, brevilin A did not
inhibit phosphorylation in HEK293T cells with overexpressed
JAK1-JH1 or JAK3-JH1 domains. Based on these results, brevilin
A modulates the JAK2/STAT3 signal pathway via the inhibition of
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FIGURE 4 I Structure of JAK2-STAT3 inhibitors.
JAK2 kinase activity.
Thus, these findings provide evidence that brevilin A is a
selective JAK/STAT inhibitor and may act as a potential drug for
targeting diseases caused by JAK/STAT abnormalities.
Cucurbitacin B is one of several naturally occurring cucurbitacins that constitute a group of oxygenated triterpenes, which
are characterized by the tetracyclic cucurbitacin nucleus skeleton
and are present in many plants, such as β-glucosides (Chen et
al., 2005). Cucurbitacins are essential herbs for a large number
of traditional Chinese medicines and the bitter principles of
Cucurbitaceae. Cucurbitacin B, a member of the cucurbitacins, is
isolated from Trichosanthes kirilowii Maximowicz (Cucurbitaceae
family). This active ingredient has antiproliferative effects on
several types of malignancies and is known to be a dual inhibitor
of the activation of both JAK2 and STAT3 in some malignancies
(Thoennissen et al., 2009).
JSI-124, a cucurbitacin analogue, blocked the activation of
STAT3 in several human cancer cell lines that contain high levels
of constitutively activated tyrosine-phosphorylated STAT3 such
as A549, MDA-MB-468, and MDA-MB-231 cells. Subsequently
the compound inhibited STAT3 DNA-binding activity and STAT3dependent gene expression. JSI-124 also decreased the levels
of tyrosine-phosphorylated JAK2, but it did not decrease those
of Src. JSI-124 was a highly selective JAK/STAT3 inhibitor
because it did not inhibit other tumor survival pathways, such
as those mediated by AKT, pERK1/2, or JNK (Blaskovich et
al., 2003). Because JSI-124 suppresses the cellular levels
of pSTAT3 and pJAK2 but not those of pERK1/2, pJNK, and
pAKT, it is suggested that a STAT3 tyrosine kinase is a possible
molecular target for JSI-124. Consistent with a direct inhibition
of the enzymatic activity of a tyrosine kinase is the fact that the
suppression of the STAT3 phosphotyrosine levels was rapid (i.e.,
was observed as early as 30 min and was complete after only
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Yena Jin, Younghwan Kim, Yu-Jin Lee, Dong Cho Han and Byoung-Mog Kwon
2 hours of treatment). There are two well-characterized STAT3
tyrosine kinases, JAK and Src kinase. JSI-124 did not inhibit
the kinase activities of JAK1, JAK2, and Src in an in vitro kinase
assay as confirmed by in vitro kinase assays in which JAK2 and
JAK1 enzymatic activities were inhibited by AG490, a known
JAK inhibitor, but were not inhibited by JSI-124. Similarly, Src
kinase activity was inhibited in vitro by the known Src kinase
inhibitor PD180970 but was not inhibited by JSI-124, indicating
that Src kinase is not a target. Although STAT3 is not known as a
direct target of JSI-124, the ability of JSI-124 to increase mouse
survival, to inhibit the growth of human and murine tumors and
oncogene- transformed NIH 3T3 tumors in mice with high levels
of constitutively activated STAT3 and to not inhibit the growth of
those tumors with low levels of activated STAT3 further validates
the interference of STAT3 signaling as a sound approach to
cancer chemotherapy; therefore, JSI-124 is a good candidate
for developing tumor therapeutics targeting JAK/STAT-activated
tumors.
4-Methoxydalbergione (4-MD) is isolated from the dried
heartwoods of Dalbergia odorifera and Dalbergia odorifera is
mainly distributed in China; its heartwood is used for treating
blood disorders, ischemia, swelling, necrosis, and rheumatic pain
in China and Korea (Beldjoudi et al., 2003). In addition, it has
been reported that 4-methoxydalbergione has anti-proliferative
and apoptotic effects in human osteosarcoma cells. The
inhibitory effects of 4-MD were compared between aggressively
growing osteosarcoma (MG63) and mildly growing osteosarcoma
(U-2-OS) cells (Park et al., 2016). 4-MD exhibited significant
inhibition effects in a concentration-dependent manner in both
MG63 and U-2-OS cells. However, 4-MD inhibited cell growth
more strongly in MG63 cells than in U-2- OS cells. To determine
whether the 4-MD-induced growth inhibition of osteosarcoma
cells was associated with the induction of apoptosis, the cells
were treated with 4-MD and were assessed using two apoptosis
assays, the Annexin V-FITC and TUNEL assays. 4-MD inhibited
the constitutive phosphorylation of JAK2 and STAT3 in a doseand time-dependent manner, and 30 μM MD completely inhibited
pSTAT3 and pJAK2. In a time-course study, it was found that the
inhibition of JAK2 phosphorylation occurred within 1 hour after
the treatment, whereas the inhibition of STAT3 phosphorylation
occurred at 3 hours after the treatment. These results suggest
that the inhibition of STAT3 Tyr705 phosphorylation by 4-MD
is caused by inhibited JAK2 activity. Because the nuclear
translocation of STAT3 is mandatory for its oncogenic functions,
whether 4-MD could suppress the nuclear translocation of STAT3
was determined. 4-MD inhibited the translocation of STAT3 to
the nucleus in osteosarcoma cells. 4-MD also down-regulated
the expression of STAT3 target proteins such as Bcl-2, Bcl- xL,
and survivin. These results indicate that JAK2/STAT3 signaling is
involved in the anti-osteosarcoma effect of 4-MD and that 4-MD
induced apoptosis by the down-regulation of anti-apoptotic
proteins. The in vivo antitumor activity of 4-MD was assessed
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in a xenograft mouse model; 4-MD significantly decreased
tumor weight by 22.25±11.46% compared with that of the
control. To determine whether growth inhibition and apoptosis
were responsible for the observed antitumor activity of 4-MD,
immunohistochemistry for proliferation, STAT3 signaling, and
anti-apoptotic proteins were used to assess xenograft tissue. The
results showed that 4-MD effectively suppressed the expression
of anti-apoptotic molecule (e.g., survivin) and therapeutic target
molecules (e.g., pSTAT3) in tumor tissues.
Many natural products, including curcumin, inhibit the JAK/
STAT pathways by modulating the activity of multiple-kinases.
Mitogen-activated protein kinases (MAPK) signaling cascades,
including ERK1/2, c-Jun N-terminal kinase (JNK) and p38 MAPK
as well as cAMP response element-binding protein (CREB),
are also important for the activation of STAT3 (Steelman, et al.,
2004). Thus, the inhibitory activity of 4-MD against a variety of
kinases was examined. In 4-MD-treated tumor cells, the activity
of ERK1/2, JNK and p38 MAPK, as well as that of CREB was
significantly reduced in a dose-dependent manner. To determine
the downstream consequences of these effects on MAPK
and CREB, the effects of 4-MD on the expression of PTEN
(phosphatase and tensin homolog deleted on chromosome
ten) were examined. The results showed a concentrationdependent increase in PTEN in osteosarcoma cells treated with
4-MD compared with that in the untreated cells. These results
suggested that 4-MD can regulate the activity of CREB and
MAPK, consequently increasing the expression of PTEN and
inhibiting JAK2, leading to STAT3 inactivation and osteosarcoma
cell growth.
Thymoquinone (TQ), a compound isolated from black seed oil
(Nigella sativa), has been reported to possess anti-inflammatory
and anticancer activities (Amin and Hosseinzadeh, 2016). TQ
inhibited the constitutive phosphorylation, nuclear localization
and reporter gene activity of STAT3 (Kundu et al., 2014). TQ
down-regulated the expression of STAT3 target gene products,
such as survivin, c-Myc, cyclin-D1, and cyclin-D2, and enhanced
the expression of cell cycle inhibitory proteins p27 and p21.
Several upstream kinases, such as EGFR tyrosine kinase, JAK2,
and Src, are known to regulate STAT3 activation. The effect of
TQ on the constitutive activation of these kinases in HCT116
cells was examined. Treatment with TQ markedly diminished the
phosphorylation of EGFR at the tyrosine-1173 residue, as well as
the phosphorylation of JAK2, and Src kinase. Although JAK2 can
directly phosphorylate STAT3, it has been reported that JAK2
and Src function as upstream kinases to EGFR, which transmits
activating signals to STAT3. The pretreatment of cells with
AG490, a JAK2 inhibitor, attenuated the phosphorylation of EGFR
and STAT3. Moreover, when tumor cells were treated with PP2,
a Src kinase inhibitor, the tyrosine phosphorylation of EGFR and
STAT3 was completely inhibited in HCT116 cells. The treatment
of cells with gefitinib, an EGFR antagonist, also blocked STAT3
phosphorylation without affecting the levels of phosphorylated
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Natural products targeting STAT3 signaling pathways in cancer cells
JAK2 and Src. Collectively, these results revealed that TQ
induced apoptosis in HCT116 cells by blocking STAT3 signaling
via the inhibition of JAK2- and Src-mediated phosphorylation of
EGFR tyrosine kinase. It was also found that TQ inhibited both
constitutive and IL-6-inducible STAT3 phosphorylation which
correlated with the inhibition of c-Src and JAK2 activation in
multiple myeloma cells. TQ-treated multiple myeloma cells also
exhibited down-regulated expression of STAT3-regulated genes,
such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and VEGF.
Therefore, TQ induced the accumulation of cells in the sub-G1
phase, inhibited proliferation and induced apoptosis, as indicated
by PARP cleavage. TQ is a good example of a STAT3 inhibitor
acting through the regulation of upstream kinases activity.
INHIBITION OF STAT3 ACTIVITY
BY PHOSPHATASES
STAT3 phosphorylation is also tightly regulated by dephosphorylation, which is medicated by the STAT3 protein tyrosine
phosphatases (PTPs) SHP-1, SHP-2, TC-PTP, and PTPRT (Table 2,
Böhmer and Friedrich, 2014). MEG2 also dephosphorylates STAT3
at Tyr705 via a direct interaction (Tremblay, 2013). Most natural
products targeting phosphatases regulate the expression of SHP1 and/or SHP-2. In this section, we discuss the modes of action of
natural products regulating the expression of SHP-1 and/or SHP-2
(Figure 5).
SHP-1 was first identified in hematopoietic cells and is
predominantly expressed in hematopoietic and epithelial
cells. SHP-1 belongs to a family of non-receptor protein
TABLE 2 I PTP family members with relevance for regulation of JAK-STAT signaling
Systematic name
(also gene name)
Common
synonyms
Subfamily
PTPN1
PTP1B
Nontransmembrane
PTP family 1
435 aa
PTP for JAK2 and TYK2, STAT6, and several RTKs; can
activate SRC-family kinases; knockout mice are resistant
to high-fat diet, and exhibit increased insulin and leptin
sensitivity; promotes tumorigenesis in mouse models of
ERB2/neu-driven breast cancer
PTPN2
TCPTP
NT1
415 aa (48 kDa,
TCPTP48); 387
(45 kDa,
TCPTP45)
PTP for several STATs, JAK1, and JAK3, and several RTKs;
knockout mice die between 3-5 week of age from anemia
and systemic inflammation; inactivating mutations found in
ALL patients
Hematopoietic
(595 aa),
epithelial (597 aa),
long form
(SHP-1L, 624 aa)
2 SH2 domains, negative regulation by SH2-domaincatalytic-domain interaction,
C-terminus with regulatory function (lipid binding,
pTyr residues) contains NLS; cytoplasmic to nuclear
translocation
observed; negative regulator of cytokine, growth factors
and immunoreceptor signaling (e.g., Epo, CSF-1, BCR);
substrates receptors, adaptor proteins, JAKs
593 aa
2 SH2 domains, negative regulation by SH2-domaincatalytic-domain interaction,
C-terminus with regulatory function
(pTyr residues); largely cytoplasmic; knockout
embryonically lethal; positive
regulator of Ras signaling downstream of several RTKs
and cytokines (e.g., PFGFRβ,
IL-6) by dephosphorylation of inhibitory molecules;
activating mutations in Noonan Syndrome and JMML
1304 aa
Transmembrane molecule with two intracellular PTP
domains,
membrane-proximal domain carries most of PTP activity;
activates SRC-family kinases (e.g., LCK, FYN) by
dephosphorylating their C-terminal
inhibitory phosphosite; also reported as PTP for JAK1;
knockout mice exhibit severe combined immunodeficiency;
inactivating mutations in a subset of
T-ALL patients
PTPN6
PTPN11
PTPRC
8
SHP-1,
SH-PTP1,
HCP, PTP1C
SHP-2,
SH-PTP2,
PTP1D
CD45, LCA
NT2
Protein, alternate
gene products
NT2
Receptorlike PTP
family
1/6
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Regulation, specific biochemical features, and functions
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Yena Jin, Younghwan Kim, Yu-Jin Lee, Dong Cho Han and Byoung-Mog Kwon
FIGURE 5 I Structure of STAT3 inhibitors through SHP-1 and/or SHP-2 expression.
tyrosine phosphatases (PTPs); it is involved in hematopoietic
signaling processes and has also been reported to function as
a tumor suppressor during tumor progression. In contrast to
the predominant expression of SHP-1 in hematopoietic cells,
SHP-2 is a ubiquitously expressed enzyme that appears to be
involved in multiple signaling pathways downstream of a variety
of growth factors and cytokines. SHP-1 contains two SH2
domains, a catalytic PTP domain and a C-terminal tail. Notably,
its phosphatase activity is highly dependent on its structural
variability. For example, the closed-form chemical structure
of SHP-1 is assembled by the N-SH2 domain and protrudes
into the catalytic domain to directly block the entrance into the
active site, and the highly mobile C-SH2 domain is believed to
act as an antenna to search for the phosphopeptide activator.
As previously mentioned, SHP-1 and SHP-2 have very similar
structures and regulate the function of the JAK family of tyrosine
kinases via their SH2 domains. It has been demonstrated that
the hyperphosphorylation of JAK kinases in cells is frequently
associated with a lack of functional SHP-1 or SHP-2, indicating
that SHP-1 and SHP-2 are critical negative regulators of the
JAK2/STAT3 signaling pathway (Bohmer and Friedrich, 2014).
10-Acetoxychavicol acetate (ACA) is isolated from the
rhizomes of the commonly used ethno-medicinal plant Languas
galangal (Zingiberaceae). ACA exhibits antioxidant and antiinflammatory activities via the suppression of xanthine oxidase,
superoxide anion generation, and inducible nitric oxide synthase
expression (Murakami and Ohigashi, 2007). ACA has also been
reported to exhibit antitumor activity against multiple myeloma
by the inhibition of nuclear factor kappa B (Ito et al., 2005). ACA
induces tumor apoptosis by enhancing caspase-3 activity and
inhibits the proliferation and angiogenesis of prostate tumor
growth.
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ACA has been shown to suppress the phosphorylation of
STAT3 (at tyrosine 705 and serine 727 sites) in a dose- and
time-dependent manner, and the maximum inhibition was
obtained at 5–10 μM in MDA-MB-231 breast cancer cells that
expressed constitutively active STAT3. ACA also suppressed the
phosphorylation of JAK2 and Src kinase in MDA-MB-231 cells in
a dose-dependent manner. ACA suppressed IL-6-induced STAT3
phosphorylation in a concentration dependent manner. These
results indicate that ACA has the ability to block constitutive and
induced STAT3 activation in cancer cells. After being activated
by IL-6, activated STAT3 forms dimers via a phosphotyrosineSH2 domain interaction, and the dimer then translocates into
the nucleus. IL-6 induced the accumulation of STAT3 in the
nucleus, whereas pretreatment with ACA markedly decreased
the accumulation of STAT3 in the nucleus. The translocation
of STAT3 dimers to the nucleus results in specific DNA binding
to the promoters of target genes and thereby induces the
expression of target genes. There are various target genes
downstream of STAT3 that participate in the induction of antiapoptotic programs (e.g., survivin, Bcl-xL, and Mcl-1), protooncogene stabilization (e.g., c-Myc and cyclin D1), angiogenesis
(e.g., VEGF and hypoxia inducible factor), and invasiveness (e.g.,
MMP-2 and MMP-9). The analysis of the target gene products
of STAT3 revealed that ACA preferentially down-regulated the
expression of matrix metalloproteinase such as MMP-2 and
MMP-9. In contrast, the expression levels of anti-apoptotic
and proliferative genes, such as cyclin D1, survivin, and Bcl2, were only slightly affected. As confirmed by zymography, the
enzymatic activity of MMP-9 was also decreased by ACA; in
addition, ChIP assay results indicated that the direct interaction
between STAT3 and MMP-2/MMP-9 was significantly reduced
by ACA. Together, these results suggest that ACA specifically
represses the activities of the STAT3 downstream targets MMP-
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Natural products targeting STAT3 signaling pathways in cancer cells
2 and MMP-9 and inhibits cancer cell migration and metastasis
(Wang et al., 2014).
Because ACA inhibited the phosphorylation of STAT3 (at
tyrosine 705 and serine 727 sites), phosphorylation of JAK2 (at
tyrosine 1,007 and 1,008 sites), and Src kinase (at tyrosine 416
site) in MDA-MB-231 cells, phosphatases are the best target
molecule candidate of ACA for the inhibition of JAK2/STAT3
pathway. When sodium vanadate, a broad-acting tyrosine
phosphatase inhibitor, was treated with ACA in breast cancer
cells, the ACA-induced inhibition of STAT3 was rescued
without affecting the basal level of STAT3 phosphorylation. The
involvement of tyrosine phosphatases in the process of ACAmediated STAT3 inhibition has also been suggested. Many PTPs
have been implicated in STAT3 regulation, including SHP-1, SHP2, TC-PTP, PTEN, PTP-1D, CD45, and PTP-e. Among these, ACA
induced the expression of SHP-1 at the transcriptional level in a
concentration-dependent manner. Immunoblot analysis further
confirmed that ACA markedly up-regulated SHP-1 expression
(Wang et al., 2014). The silencing of SHP-1 using siRNA reverses
the inhibition of tumor cell migration by ACA and does not
affect cancer cell viability. Therefore, SHP-1 is a target molecule
for the anti-metastasis effect of ACA. The results showed that
ACA regulated STAT3 activity by the expression of SHP-1 and
then suppressed tumor metastasis without affecting cancer cell
viability. Therefore, ACA is a potential compound for inhibition of
tumor metastasis through the modulation of the STAT3 signaling
pathway.
Guggulsterone (GS), isolated from Commiphora mukul and
used to treat obesity, diabetes, hyperlipidemia, atherosclerosis,
and osteoarthritis, has recently been shown to antagonize the
farnesoid X receptor and decrease the expression of bile acid–
activated genes. GS also inhibits the proliferation and induces
the apoptosis of a wide variety of human tumor cell types,
including leukemia, head and neck carcinoma, multiple myeloma
and melanoma, and breast, lung prostate and ovarian carcinoma
(Almazari and Surh, 2013). GS suppressed STAT3 activation in
U266 cells, and the inhibition was time-dependent with maximum
inhibition occurring at approximately 4 hours and without effects
on the expression of STAT3 protein. GS also inhibited IL-6induced pSTAT3. GS induced the dephosphorylation of STAT3,
which was gradually reversed by washing GS out of the cells. The
reversal was complete within 16 hours, and STAT3 protein levels
did not change. As a known STAT3 inhibitor, GS also abrogates
the translocation of STAT3 to the nucleus, the DNA-binding
ability of STAT3, and the STAT3-dependent expression of genes
such as cyclin D1, Bcl-2, Mcl-1, and VEGF. Because JAK2 is one
of the main kinases involved, GS-treated whole-cell lysates were
immunoprecipitated with anti-JAK2 antibodies and were then
subjected to an immunocomplex kinase assay using GST-JAK2
as a substrate. It was found that GS suppressed the constitutive
phosphorylation of JAK2 in a time-dependent manner. GS also
suppressed the constitutive phosphorylation of c-Src kinase (Ahn
10
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et al., 2008). However, the inhibition of STAT3 Tyr705 was not due
to an inhibition of JAK2 activity because the inhibition of JAK2
phosphorylation occurred after the dephosphorylation of STAT3
(Ahn et al., 2008). These results suggest that the inhibition of
STAT3 Tyr705 phosphorylation by GS is not caused by upstream
kinases.
Treatment of U266 cells with the broad tyrosine phosphatase
inhibitor sodium pervanadate reversed the GS-induced inhibition
of STAT3 phosphorylation. Tyrosine phosphatases might be
involved in the GS-induced inhibition of STAT3 activation
because protein tyrosine phosphatases have been involved in
the modulation of STAT3 activity, and a PTP inhibitor rescued
the GS-induced inhibition of STAT3 phosphorylation. Therefore,
whether GS can modulate the expression of SHP-1 in U266 cells
were examined. GS induced the expression of SHP-1 protein in
a time-dependent manner, with maximum expression occurring
after 120 to 240 minutes. In conclusion, GS induced the
apoptosis of human MM cells by the inhibition of STAT3, which
occurred by the expression of SHP-1.
5-Hydroxy-2-methyl-1,4-naphthoquinone, also known as
plumbagin, an analogue of vitamin K3, is isolated from the
Plumbaginaceae, Droseraceae, Ancestrocladaceae, and
Dioncophyllaceae families. The root of chitrak (Plumbago
zeylanica), a major source of plumbagin, has been used in Indian
medicine as an antiatherogenic, cardiotonic, hepatoprotective,
and neuroprotective agent. The active principle, plumbagin,
is also isolated along with a series of structurally related
naphthoquinones from the roots, leaves, bark, and wood of
Juglans regia (also known as the English walnut, Persian walnut,
and California walnut), Juglans cinerea (butternut and white
walnut), and Juglans nigra (Padbye et al., 2012). Plumbagin has
been shown to exert anticancer effects against a wide variety
of tumor cells, including breast cancer, lung cancer, ovarian
cancer, acute promyelocytic leukemia, melanoma, and prostate
cancer (Padbye et al., 2012). Plumbagin inhibited the constitutive
phosphorylation of STAT3 in U266 cells, with maximum inhibition
occurring at 5 μM (Sandur et al., 2010). The inhibition was
time dependent, with maximum inhibition occurring at 4 hours.
Whether plumbagin modulates the phosphorylation of STAT3
at the serine 727 residue was also examined. Interestingly,
plumbagin also inhibited the serine phosphorylation of STAT3
in a dose-dependent manner. Plumbagin had no effect on the
expression of STAT3 protein under these conditions. EMSA
assays of plumbagin-treated U266 cells showed that the
treatment decreased STAT3 DNA-binding activity in a doseand time-dependent manner. These results suggests that
this compound blocks the dimerization of STAT3 and inhibits
its translocation to the nucleus. Therefore, plumbagin downregulated the expression of STAT3 target genes such as antiapoptotic proteins Bcl-xL, cell cycle regulator protein, cyclin D1,
and VEGF.
Plumbagin suppressed the constitutive phosphorylation of
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Yena Jin, Younghwan Kim, Yu-Jin Lee, Dong Cho Han and Byoung-Mog Kwon
JAK1 at tyrosine 1022/1023 and JAK2 at tyrosine 1007/1008.
The expression levels of JAK1 and JAK2 remained unchanged
cell types, including oral squamous carcinoma, colon carcinoma,
lung carcinoma, pancreatic adenocarcinoma, breast carcinoma,
under the same conditions. The relative activity of plumbagin
compared with that of AG490 (JAK2 inhibitor) in the suppression
of STAT3 phosphorylation was examined. At 5 μM, plumbagin
was found to be more effective than AG490 was at 100 μM. By
immune complex kinase assays, plumbagin was found to affect
JAK2 activity in U266 cells, which indicated that plumbagin also
suppressed the activity of JAK2.
To monitor the effect of PTP on STAT3 tyrosine phosphorylation,
sodium pervanadate (a broad-acting PTP inhibitor) and
plumbagin were treated in U266 cells. The PTP inhibitor
prevented the plumbagin-induced inhibition of STAT3 activation,
suggesting that PTPs are involved in the plumbagin-induced
inhibition of STAT3 activation. SHP-1 is an important negative
regulator of JAK/STAT signaling in leukemias and lymphomas.
Whether plumbagin can modulate the expression of SHP-1 in
U266 cells was examined. Cells were incubated with different
concentrations of plumbagin for 4 hours; whole-cell extracts
were prepared and examined for SHP-1 protein using Western
blot analysis. Plumbagin induced the expression of SHP-1
protein in U266 cells. The suppression of SHP-1 expression by
small interfering RNA (siRNA) would abrogate the inhibitory effect
of plumbagin on STAT3 activation; plumbagin failed to suppress
STAT-3 activation in cells treated with SHP-1 siRNA. These
siRNA results corroborate the previous evidence of the critical
role of SHP-1 in the suppression of STAT-3 phosphorylation by
plumbagin (Sandur et al., 2010).
Because plumbagin down-regulated the expression of cyclin
D1, the gene critical for cell proliferation and the cell cycle, it
inhibited cell proliferation of of a dose-dependent manner and
caused a significant accumulation of the cell population in the
sub-G1 phase. The treatment of U266 cells with plumbagin
induced a caspase-3-dependent cleavage of a 118-kDa PARP
protein into an 87-kDa fragment. In addition, the overexpression
of constitutively active STAT3 can rescue plumbagin-induced
apoptosis. Antitumor activity of vitamin K3 analogues has been
reported; juglone and 1,4-naphthoquinone can modulate NF-κB
and leukemia, by inducing apoptosis and exhibiting significant
chemopreventive effects (Joo and Jetten, 2010).
Lee et al reported that FOH suppressed both constitutive and
inducible STAT3 activation in MM. FOH specifically blocked
STAT3 phosphorylation at Tyr705 but had no effect on STAT3
phosphorylation at Ser-727 (Lee et al., 2015). It is well known that
JAK1/2 and c-Src have also been implicated in STAT3 activation.
FOH inhibited the activation of constitutively active JAK1/2 and
c-Src activation in MM cells. It was reported that pervanadatetreatment in U266 cells reversed STAT3 dephosphorylation
induced by FOH, indicating that the PTP plays a major role in the
dephosphorylation of STAT3. Interestingly, FOH induced SHP2 protein expression that correlated with a down-regulation in
STAT3 phosphorylation in MM cells, and transfection with SHP2 siRNA reversed the observed STAT3 inhibitory effect of FOH.
The expression of several STAT3-target gene products such as
cyclin D1, Bcl-2, Bcl-2, and survivin was suppressed by FOH.
The inhibition of cyclin D1 expression by FOH correlated with
suppressed proliferation and the accumulation of cells in the
subG1 phase of the cell cycle. In addition, down-regulation of
the expression of Bcl-2 and Bcl-xl could contribute to the ability
of FOH to induce apoptosis in MM cells. It was observed that
FOH induced PARP cleavage by the activation of caspase-8, -9,
and -3. FOH significantly suppressed MM growth in nude mice,
down-regulated the expression of pJAK1/2, p-Src, p-STAT3,
and various STAT3-regulated gene products in tumor tissues,
and increased the levels of caspase-3 in the FOH treatment
groups. Overall, these results demonstrate that FOH inhibits both
inducible and constitutive STAT3 activation through the induction
of tyrosine phosphatase, which may account for its antitumor effects observed in vivo. FOH is promising for potential
applications in the treatment of cancer and other diseases.
activation, and unlike plumbagin, shikonin, which is an analogue
of vitamin K, is known to inhibit protein tyrosine phosphates.
Thus, this finding suggests that not all analogues of vitamin
K exhibit anti-tumor activities by the modulation of different
targets. In conclusion, plumbagin is a selective inhibitor of the
STAT3 pathways and is a useful vitamin K analogue for STAT3
modulation and for developing tumor therapeutics targeting JAKSTAT activated tumors with fewer side effects.
Farnesol (FOH) is an isoprenoid, and isoprenoids are one
class of phytochemicals that inhibit tumor cell proliferation
and differentiation and induce apoptosis (Burke et al., 1997).
FOH is used to treat obesity, diabetes, hyperlipidemia, and
atherosclerosis. Additionally, FOH exhibits anticancer potential
by suppressing the proliferation of a wide variety of human tumor
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Capillarisin (CPS) is isolated from Artemisia capillaries Thunb
(Compositae), which has been used to treat various human
diseases including liver cirrhosis, liver cancer, jaundice and
cholecystitis in Asian countries, such as Korea, China, and Japan
(Han et al., 2013). CPS has been reported to be a potent inhibitor
of the NF-κB activation pathway and to have suppressed the
phosphorylation of STAT3 at Tyr705, but not Ser727, without an
effect on the expression of STAT3 protein in U266 cells (Lee et
al., 2014). CPS substantially reduced STAT5 activation in MM
cells, without affecting total STAT5 levels, as demonstrated
by Western blot analysis. CPS also blocked the IL-6 induced
phosphorylation of STAT3 and subsequently suppressed the DNA
binding activity of STAT3. EMSA analysis of U266 cells showed
that CPS decreased STAT3 DNA-binding activity in a dose- and
time-dependent manner. Therefore, CPS suppresses the nuclear
translocation of STAT3. CPS inhibits the activation of upstream
kinases involved in the STAT3 signaling cascade, such as JAK1/2
and Src kinase, in a dose-dependent manner in MM cells.
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Natural products targeting STAT3 signaling pathways in cancer cells
To determine which target molecules are involved in CPSmediated STAT3 inactivation, a broad-acting tyrosine
phosphatase inhibitor (sodium pervanadate) was used to treat
U266 cells, and it was observed that the CPS-induced inhibition
of STAT3 and JAK1/2 phosphorylation was rescued. These data
suggest that tyrosine phosphatases are involved in the CPSinduced inhibition of JAK1/2 and STAT3 activation in MM cells.
Thus, it was examined whether CPS affects the expression
of SHP-1, SHP-2, and PTEN which are nontransmembrane
PTPs expressed abundantly in hematopoietic cells. Treatment
with CPS led to an increased expression of SHP-1 and SHP2, but not PTEN at the protein level. CPS also substantially
enhanced mRNA levels of both SHP-1 and SHP-2 in U266 cells.
Transfection with SHP-1/2 siRNAs reversed the inhibition of
STAT3 and JAK1 activation by CPS. It was also found that CPS
failed to suppress JAK1 and STAT3 activation in cells treated
with SHP-1/2 siRNAs, thereby implicating the pivotal role of
these two phosphatases in the STAT3/JAK1 inhibitory effects of
CPS. The expression of several STAT3-target gene products was
suppressed by CPS. These products included proliferative (cyclin
D1) and anti-apoptotic (Bcl-2, Bcl-xl, survivin, and IAP-1) gene
products. Thus, the down-regulation of the expression of Bclxl could contribute to the ability of CPS to induce apoptosis in
MM cells. The down-regulation of cyclin D1 expression by CPS
correlated with its significant anti-proliferative effects observed
in MM cells. CPS was the first compound found to regulate the
expression of SHP-1/2; therefore, there is sufficient rationale for
investigating the therapeutic efficacy of STAT3 inhibitors isolated
from herbal medicines and CPS is a potential lead molecule
for the development of JAK/STAT3 pathway blockers via the
expression of SHP-1/2 (Lee et al., 2014).
Nimbolide (NL), is a terpenoid limonoid isolated from Azadirachta
indica leaves and it has been found to exhibit diverse
pharmacological activities such as anti-feedant, anti-malarial,
anti-HIV, antimicrobial, and significant anti-cancer responses.
The diverse anticancer effects of NL have been reported in
multiple tumor types including those of breast, colorectal,
brain, and liver cancer (Elumalai and Arunakaran, 2014). Both
constitutive pSTAT3 in DU145 and IL-6-stimulated pSTAT3 levels
in LNCaP were substantially reduced upon NL treatment. In
DU145 cells, both JAK1 and JAK2 phosphorylation levels were
reduced by NL, whereas Src phosphorylation was not affected.
NL also reduced the phosphorylation of both JAK1 and JAK2
stimulated by IL-6 in LNCaP cells. These data indicate that the
STAT3 inhibition caused by NL treatment may be caused by the
down-regulation of upstream activation through JAK1 and JAK2
kinases. In addition, NL blocked STAT3 from binding to DNA and
thereby reduced the transcription of STAT3 target genes.
ROS are maintained at low levels by cellular anti-oxidative
systems under normal physiological conditions and this low
level of ROS can promote both cell survival and proliferation.
However, when ROS accumulate at elevated, non-physiological
concentrations, apoptotic cell death can be caused through
MODULATION OF STAT3 ACTIVITY
THROUGH ROS
Reactive oxygen species (ROS) are critical for the metabolic
and signal transduction pathways associated with cell
growth and apoptosis. Cancer cells exhibit increased ROS
levels compared with those of normal cells because of their
accelerated metabolism. The high ROS levels in cancer cells
renders these cells more susceptible to oxidative stress–induced
cell death, which can be exploited for selective cancer therapy
(Gorrini et al., 2013). Many known natural compounds, such as
hydroxycinnamaldehyde (Han et al., 2004), curcumin, EGCG,
piperlogumin (Raj et al., 2011), and resveratrol, generate ROS to
destroy cancer cells (Sulliva and Chandel, 2014). It was reported
that JAK/STAT-dependent signaling is regulated by redox-related
pathways (Duhe, 2013).
Recent reports suggest that ROS may inhibit JAK2 phosphorylation via posttranslational modification of its critical
cysteine residues as well as STAT3 through PTP activation. In
this section, we discuss natural products (Figure 6) that inhibit
the JAK2/STAT3 pathway through ROS generation in tumor cells.
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FIGURE 6 I Structure of STAT3 inhibitors that act through ROS
modulation.
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Yena Jin, Younghwan Kim, Yu-Jin Lee, Dong Cho Han and Byoung-Mog Kwon
damage of DNA or proteins. N-acetyl-L-cysteine (NAC) and
glutathione (GSH) are two well-known thiol-related antioxidants.
Interestingly, pre-treatment with these two antioxidants
substantially rescued STAT3 inhibition caused by NL treatment,
thereby indicating that ROS may be involved in the inhibitory
effect of NL on STAT3 in PCa cells. Furthermore, NAC/GSH pretreatment was also found to significantly reduce the cellular
apoptosis induced by NL, as observed by flow cytometry and
Western blot analysis, further demonstrating that oxidative stress
may also contribute to the observed pro-apoptotic effects of NL.
It was observed that ROS levels were increased by NL-treatment
in DU145 cells. The GSH/GSSG system is one of the major
intracellular antioxidant systems. The ratio of GSH to oxidized
glutathione (GSSG) is an indicator of cellular oxidative stress.
NL significantly decreased GSH and increased the GSSG/GSH
ratio in DU145 cells, thereby indicating that the exposure of the
cells to NL resulted in an imbalance of the GSH/GSSG system.
To further investigate the role of GSH/GSSG imbalance in
mediating NL-induced oxidative stress, both a GSH synthesis
blocker, buthionine sulfoximine (BSO), and a GSH prodrug,
NAC, were employed. It was found that pretreatment with NAC
significantly prevented NL-induced ROS production, whereas
BSO enhanced ROS production. Therefore, the GSH/GSSG
imbalance in mediating NL-induced oxidative stress was caused
by the down regulation of GSH. Furthermore, NL increased
the level of H2O2 in a time-dependent manner; increased H2O2
production was also prevented by NAC and was enhanced by
BSO. These results demonstrate that H2O2 is the major ROS
induced by NL in PCa cells. In addition, BSO enhanced NLinduced cellular apoptosis when applied in combination with NL,
and the increased apoptosis was also attenuated by NAC pretreatment in PCa cells. Glutathione reductase (GR), an enzyme
involved in the reduction of GSSG to GSH, was inhibited by NL
in a time-dependent manner in NL-treated cells. These results
demonstrate that NL directly suppresses GR and GSH synthesis
by inhibiting their activity and increasing ROS in tumor cell.
It has also been reported that ROS can directly oxidize
the cysteine residues of JAK2 in the catalytic domain, and
are therefore able to suppress its kinase activity as well as
downstream STAT3 activation (Duhe, 2013). NL-induced oxidative
stress inhibited the phosphorylation of JAK2 and STAT3 and also
significantly reduced PCa cell viability, supporting the notion that
the oxidization of JAK2 may contribute to ROS-mediated STAT3
inhibition. NL also inhibited tumor progression and metastasis
in a mouse prostate model. These findings suggest that NL
could be a potent anticancer agent against various malignancies
related to STAT3 activation.
6-Shogaol (6SG), an active compound in ginger (Zingiber
officinale), exhibit anti-proliferative, antimetastatic, and proapoptotic activities. Ginger (Zingiber officinale Roscoe,
Zingiberaceae) is one of the most commonly used dietary
seasonings in the world (Marx et al., 2015). Ginger has long been
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used for the treatment of human diseases, such as the common
cold, nausea, arthritis, migraines, and hypertension (Semwal et
al., 2015). Ginger contains approximately 1.0-3.0% volatile oils
and a number of pungent compounds. Gingerols are the most
abundant compounds in fresh roots. Shogaols, the dehydrated
form of gingerols, are found in only small quantities in the fresh
root (Semwal et al., 2015).
Previous animal studies have conclusively shown that ginger
and its active phenolic constituents such as 6G/6SG can inhibit
skin tumor formation and abrogate the initiation as well as
progression of colon, liver, and breast cancer. Among various
compounds in ginger, 6SG has been intensively studied for their
anti-tumor potential in several tumor cell lines (Wang et al., 2014).
6SG inhibited constitutive STAT3 activation in a variety of
STAT3-activated human cancer cell lines, such as MDA-MB-231,
DU145, SCC4, A549, and HepG2 cells. 6SG clearly inhibited
STAT3 activation in both MDA-MB-231 and DU145 cells with
30 μM 6SG compared with different types of tumor cells. 6SG
also suppressed the phosphorylation and nuclear translocation
of STAT3 in MDA-MB-231 and DU145 cells (Kim et al., 2015).
The expression of STAT3 remained unchanged under the same
conditions. 6SG also suppressed the phosphorylation of JAK2
in both MDA-MB-231 and DU145 cells. Bcl-2, Bcl-xl, IAP1, and survivin have been implicated in cell survival, and act
downstream of the JAK/STAT signaling cascade; 6SG downregulates the expression of anti-apoptotic gene products in a
time-dependent manner.
Previous studies have reported that ROS induces the MAPK
and AKT cascades. Interestingly, 6SG induces the activation
of MAPK cascades including JNK, p38 MAPK, and ERK,
in MDA-MB-231 cells. 6SG substantially up-regulated the
phosphorylation of all three JNK, p38, and ERK MAP kinases
in a time-dependent manner. Therefore, the potential role of
ROS in 6SG mediated ERK, JNK, and p38 MAPK activation in
MDA-MB-231 cells was explored. Whole-cell lysates of MDAMB-231 cells treated with 6SG for 8 hours with or without NAC
pretreatment were analyzed for pJNK, p-p38, and pERK. NAC
pretreatment drastically prevented the cleavage of PARP in
MDA-MB-231 cells treated with 6SG for 24 hours. These results
indicate that increasing ROS levels may mediate the effects of
6SG on PARP cleavage and apoptosis in MDA-MB-231 cells. To
confirm the generation of ROS by 6SG, MDAMB- 231 cells were
treated with 6SG in the presence or absence of the antioxidant
NAC and stained with DCFHDA. Treatment with 6SG resulted
in a large increase in DCF fluorescence intensity compared that
observed for untreated control cells. NAC pretreatment clearly
abrogated the observed effect of 6SG on ROS production.
Although it is unclear how 6SG generates ROS in cancer
cells, collectively, these results indicate that 6SG can inhibit the
constitutive STAT3 signaling cascade and induce ROS-mediated
JNK, p38 MAPK, and ERK activation. Moreover, 6SG induces
apoptosis through the down-modulation of gene products that
mediate tumor cell survival and proliferation in human breast cancer.
Biodesign l Vol.4 l No.1 l Mar 30, 2016 © 2016 Biodesign
13
Natural products targeting STAT3 signaling pathways in cancer cells
Sugiol is an abietane-type diterpene compound and is isolated
from the roots of Salvia prionitisHance (Labiatae), Salvia
miltiorrhiza Bunge, and Salvia viridis (Liu et al., 1995). Many
studies have investigated the biological activities of sugiol,
including its modest anti-tumor, anti-inflammatory, antimicrobial,
and aldose reductase inhibitory activities (Saijo et al., 2015).
Sugiol has also been reported to exhibit very weak antitumor
activity against SW620, MDA-MB-231, and NCI-H23 cells (Son
et al., 2005). However, sugiol selectively inhibited the growth of
DU145 cells, which exhibit constitutively active STAT3, compared
with other, STAT3-independent other cells such as LNCap, PC3,
and non-tumorigenic MCF10A cells (Jung et al., 2015). These
data suggest that sugiol primarily suppresses the proliferation
of STAT3-dependent cancer cells, where STAT3-dependent
cancer cells are those in which STAT3 is activated and the
proliferation rate of the cancer cells is decreased by knockdown
of STAT3. Sugiol dramatically decreased STAT3 phosphorylation
at Try705, whereas the total amount of STAT3 protein remained
nearly unchanged under these conditions in DU145 cells. The
phosphorylation of STAT1 and STAT5 was not inhibited by sugiol
under these conditions. Sugiol inhibited the expression of STAT3
target genes and the cell cycle regulators cyclin D1, cyclin A,
survivin, VEGF, Mcl-1, and Bcl-xL, in DU145 cells. Because
sugiol down-regulated cyclin D1 and A, cell cycle arrest at the
G1/S stage was induced by the compound in DU145 cells.
JAK and Src family proteins are the most well-known upstream
kinases that phosphorylate STAT3 tyrosine residues; however,
treatment with up to 50 µM sugiol did not affect JAK family
proteins, such as JAK2, JAK3, and TYK2. An in vitro kinase
assay was also performed for several upstream kinases of
STAT3, including EGFR, FGFR, JAK2, JAK3, Lyn, and Src, to
examine the effect of sugiol on these tyrosine kinases. At 10 µM,
sugiol did not inhibit the activity of these kinases in an in vitro
assay, which suggests that the inhibition of STAT3 activation is
independent of upstream kinases.
Transketolase (TKT) was identified as a target molecule of sugiol
by affinity chromatography. TKT activity was decreased by sugiol
in a dose-dependent manner in vitro. STAT3 phosphorylation
was reduced in DU145 cells transfected with TKT siRNA. TKT
siRNA knockdown increases ROS levels because TKT downregulates intracellular GSH levels. Sugiol induced a significant
time-dependent decrease in GSH levels, and NAC and GSH
treatment prevented sugiol-induced GSH depletion. Therefore,
sugiol inhibits TKT activity and then reduces GSH levels, which
leads to the generation of ROS in tumor cells. These data
suggest that the interaction of sugiol with transketolase induced
ROS generation and STAT3 dephosphorylation, which inhibited
cell proliferation (Jung et al., 2015).
As previously mentioned, ROS induce the MAPK and AKT
cascades. When DU145 cells were treated with sugiol, the
phosphorylation of ERK and AKT, but not JNK or p38, were
increased and then gradually decreased in a time-dependent
14
Biodesign l Vol.4 l No.1 l Mar 30, 2016 © 2016 Biodesign
manner. The treatment of cells with ROS scavengers NAC or
GSH abrogated sugiol-induced STAT3 dephosphorylation and
ERK phosphorylation, but not AKT phosphorylation. This result
indicates that sugiol-induced ROS inhibit STAT3 activity and
promote ERK activity.
It was reported that ERK phosphorylates STAT3 on Ser727
and recruits tyrosine phosphatase (PTPase) or MAP kinase
phosphatase (MKP) proteins, which dephosphorylate Tyr705 in
cardiomyocytes (Booz et al., 2003). Notably, sugiol increased
Ser727 phosphorylation but decreased Tyr705 phosphorylation
in DU145 cells, as previously reported and the events were
abrogated by pretreatment with the ERK inhibitor U0126. These
results suggest that STAT3 was inactivated because of sugiolinduced ERK activation, and this process was induced by
the direct interaction between STAT3 and p-ERK. Booz et al.
proposed that ERK serves as a scaffolding protein to recruit a
phosphatase to STAT3. Pretreatment with the broad-spectrum
tyrosine phosphatase inhibitor pervanadate prevented the sugiolinduced inhibition of STAT3 activation, and it was found that
the silencing of only MEG2 expression by siRNA abrogated
the inhibitory effects of sugiol on STAT3 activation. The MEG2
overexpression inhibited STAT3 phosphorylation, which suggests
that MEG2 is a critical phosphatase involved in the suppression
of STAT3 phosphorylation by sugiol. Sugiol inhibits TKT activity
and induces the dephosphorylation of STAT3 at Tyr-705 through
ROS-mediated ERK activation, and it is responsible for the
antitumor effect of sugiol in DU145 prostate cells (Jung et al.,
2015). Sugiol exhibits a novel mechanism in inhibiting STAT3
activity via ROS and phosphatase. Therefore, sugiol is a good
probe with which to elucidate the STAT3 signal pathways.
CONCLUDING REMARKS
Evidence indicating that STAT3 is an oncogene and an important
target for therapy continues to accumulate; therefore, these
important findings re-establish STAT3 as a desirable therapeutic
target for human cancer treatment. However, despite intensive
efforts, STAT3 has remained frustratingly elusive as a target for
cancer therapy.
Several natural or dietary compounds have been shown to
possess in vitro and/or in vivo inhibitory effects against STAT3.
However, non-specific effects in targeting STAT3, off-target
effects, variable bioavailability and moderate efficacy constitute
disadvantages of natural products as effective chemopreventive
agents. In this review, we described specific or selective STAT3
inhibitors isolated from herbal medicines including STAT3SH2 domain inhibitors, up-stream kinase inhibitors of STAT3,
STAT3 inhibitors by the regulation of PTP, and STAT3 modulators
through the increase of ROS levels in cancer cells.
A large number of STAT3 inhibitors are currently undergoing
clinical trials, which demonstrates that with technological
advances and the continuous improvement of all approaches,
progress is being made. Most inhibitors described in this review
will require further research and development if they are to
bdjn.org
Yena Jin, Younghwan Kim, Yu-Jin Lee, Dong Cho Han and Byoung-Mog Kwon
become the next generation of cancer therapies. Therefore, more
STAT3 inhibitors may be isolated from herbal medicines for the
development of cancer treatments, with less toxicity and greater
activity.
ACKNOWLEDGEMENTS
This work was supported by the KRIBB Research Initiative Program, the
Bio-Synergy Research Project (2012M3A9C404877), the Bio & Medical
Technology Development Program (2015M3A9B5030311), the Foreign
Plant Extract Library Program (2011-00497), funded by the Ministry of
Science, ICT& Future Planning.
AUTHOR INFORMATION The Authors declare no potential conflicts of interest.
Original Submission: Feb 20, 2016
Revised Version Received: Mar 18, 2016
Accepted: Mar 19, 2016
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