Cordycepin Suppresses Integrin/FAK Signaling and Epithelial-Mesenchymal Transition in Hepatocellular
Carcinoma
Wen-Ling Yao1,2, Bor-Sheng Ko1,3,Tzu-An Liu1, Shu-Man Liang1, Chia-Chia Liu1, Yi-Jhu Lu1, Shean-Shong Tzean2,
Tang-Long Shen2*, and Jun-Yang Liou1,4*
1
Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Miaoli County 350,
Taiwan
2
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan
3
Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan
4
Graduate Institute of Basic Medical Science, China Medical University, Taichung 404, Taiwan
Running Title: Cordycepin suppresses Integrin/FAK signaling
Yao WL and Ko BS contributed equally to this work.
*Address correspondence to Jun-Yang Liou at the Institute of Cellular and System Medicine, National Health
Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan. Tel: 886-37-246166 ext. 38309; Fax:
886-37-587408; E-mail: jliou@nhri.org.tw; or Tang-Long Shen at the Department of Plant Pathology and
Microbiology, National Taiwan University, Taipei 106, Taiwan E-mail: shentl@ntu.edu.tw.
1
ABSTRACT
Cordycepin, also known as 3-deoxyadenosine, is an analogue of adenosine extracted from the traditional
Chinese medicine “Dong Chong Xia Cao”. Cordycepin is an active small molecular weight compound and is
implicated in modulating multiple physiological functions including immune activation, anti-aging and anti-tumor
effects. Several studies have indicated that cordycepin suppresses tumor progression. However, the signaling
pathways involved in cordycepin regulating cancer cell motility, invasiveness and epithelial-mesenchymal transition
(EMT) remain unclear. In this study, we found that cordycepin inhibits hepatocellular carcinoma (HCC) cell
proliferation and migration/invasion. Treatment of cordycepin results in the increasing expression of epithelial
marker, E-cadherin while no significant effect was found on N-cadherin, α-catenin and β-catenin. Furthermore,
although the expression of focal adhesion kinase (FAK) was slightly reduced, the level of phosphorylated FAK was
significantly reduced by the treatment of cordycepin. In addition, cordycepin significantly suppresses the expression
of integrin α3, integrin α6 and integrin β1 which are crucial interacting partners of FAK in regulating the focal
adhesion complex. These results suggest cordycepin may contribute to EMT, anti-migration/invasion and growth
inhibitory effects of HCC by suppressing E-cadherin and integrin/FAK signaling. Thus, cordycepin is a potential
therapeutic or supplementary agent for preventing HCC tumor progression.
Keywords: cordycepin, E-cadherin, focal adhesion kinase, hepatocellular carcinoma, integrin
2
INTRODUCTION
Dong Chong Xia Cao, a popular traditional Chinese medicine, has been extensively used as a herbal
supplement in Far-East Asia [1,2]. It is a complex comprised of the fungus Cordyceps sinensis and the infected
larvae of Hepialus armoricanus [1,2]. Extracts from Dong Chong Xia Cao are considered highly potent in treating
various diseases and have been found to be immune activators, anti-aging and anti-tumor effectors [1,2]. Cordycepin,
an analogue of adenosine (Fig. (1)), is a major active ingredient found in Dong Chong Xia Cao’s extract. Several
reports have shown that cordycepin induces apoptosis and has anti-tumor properties in various types of cancer cells
[3-12]. Furthermore, cordycepin was reported to suppress hematogenic metastasis of mouse melanoma cells [13,14]
and lung carcinoma cells [15]. Thus, cordycepin is a potential natural effective therapeutic agent for the suppression
of tumor progression.
Multiple physiological processes occur during the epithelial-mesenchymal transition (EMT) which increases
the invasiveness and metastatic potential of cancer cells. Therefore, EMT is implicated as a critical stage in the
conversion of early stage tumors into aggressive malignancies [16-18]. EMT is typically characterized by the loss of
E-cadherin, a gain of N-cadherin and vimentin, and the translocation of -catenin from the membrane and cytosol to
the nuclear compartment [19,20]. Among these EMT markers, E-cadherin expression is often inversely correlated
with tumor malignancy and patient survival [19,20]. For instance, gene silencing and reduced expression of
E-cadherin was reported in the malignant progression of hepatocellular carcinoma (HCC) [21,22] and E-cadherin
expression has been associated with greater survival of HCC patients [23].
Focal adhesion kinase (FAK) is a non-receptor cytoplasmic protein tyrosine kinase and the expression of FAK
is increased in distinct types of tumors [24-34]. FAK is overexpressed in HCC [31-34] and is associated with
invasiveness and aggressiveness to promote tumor progression [34,35]. Furthermore, expressions of both FAK and
Src are upregulated in HCC and the expression of Src, and the active form of Src are highly correlated with FAK
expression [36]. Overexpression of FAK was found to be associated with hepatitis B virus (HBV) infections, which
is a high risk factor for HCC [37]. FAK responds to a wide range of stimuli including the extracellular cellular
matrix (ECM), growth factors and integrins. Stimuli that activate integrin signaling induce FAK
autophosphorylation at tyrosine 397 (Tyr-397), thus increasing the subsequent phosphorylation of FAK and the
binding affinity for Src as well as to other complex partners. An increase of FAK activation by autophosphorylation
at Tyr-397 is highly correlated with advanced tumor stages, vascular invasion and intrahepatic metastasis in HCC
[38]. Therefore, FAK expression and activation play important roles in HCC tumor progression. Targeting FAK
expression or activation should be regarded as a potential approach for anti-HCC therapy.
Integrins are heterodimeric glycoproteins which locate on the plasma membrane and interact with the ECM
and with intracellular focal adhesion complexes to modulate multiple cellular functions, such as survival, migration
and proliferation [39,40]. The intracellular domain of certain integrins interacts with FAK thereby activating
downstream signaling pathways, particularly those that regulate tumor progression. In HCC, previous studies have
shown that an upregulation of several integrins is significantly associated with cancer cell migration and tumor
metastasis [40,41]. Among these tumor-associated integrins, integrin β1, integrin α3 and integrin α6 are the most
3
crucial integrins overexpressed in HCC [42-54]. Several studies indicated that integrin β1 forms heterodimers with
integrin α3 and integrin α6 to promote HCC tumor growth, EMT, migration/invasiveness and metastasis. For
example, integrin α6β1 was reported to be an essential factor for the matrix-dependent activation of FAK and
mitogen-activated protein (MAP) kinase to regulate HCC cell migration [48]. Moreover, both integrin α6β1 and
CD151 are overexpressed and synergize with phosphatidyl-inositol-3-kinase (PI3-K)/protein kinase B (PKB)
signaling to promote EMT of HCC cells [49]. In addition, integrin β1 also associates with integrin α3 and
contributes to cell migration and invasion of HCC cells. It was shown that transforming growth factor-β1 triggers
HCC invasiveness through an integrin α3β1 dependent manner [51] and the enhanced expression of integrin α3β1 is
directly involved in the malignant phenotype of HCC via increasing cell invasion and metastasis [52]. The
overexpression of integrin α3 was shown to promote HCC tumor growth and the knockdown of integrin α3 caused
the loss of the proliferative activity in HCC cells [47]. These results indicate that increased expression of integrins,
particularly integrin α6β1 and α3β1 contribute to HCC tumor progression. Targeting tumor-associated integrins is
therefore considered a potential strategy for HCC therapy [55].
We have sought to investigate the molecular mechanisms involved in cordycepin’s regulation of HCC cell
migration/invasion and EMT. We show for the first time that cordycepin inhibits integrin α6β1 and α3β1 expression,
thereby abrogating the activation of FAK. Our data also provide evidence that the expression of E-cadherin is
enhanced by cordycepin in HCC. Cordycepin is thus a potential therapeutic small molecular compound extracted
from a natural herbal supplement that can halt HCC progression.
4
MATERIALS AND METHODS
Cell Culture
SK-Hep-1 human hepatoma cells were maintained in DMEM (Gibco, Gaithersburg, MD) supplemented with
10% fetal bovine serum (FBS; Hyclone Thermo Fisher Scientific, Waltham, MA), 100 units/ml penicillin, and 100
units/ml streptomycin, in a humidified incubator with 5% CO 2 at 37°C.
Cell Proliferation and Migration/Invasion Assay
Proliferation of SK-Hep1 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay described previously [56]. Briefly, cells were plated for 24 to 72 hrs then treated with
cordycepin and the absorbance was measured at 570 nm. The cell migration/invasion assay was performed in a
Boyden chamber with bio-coat cell migration chambers (Becton Dickinson, Pont-de-Claix, France) as described
previously [56,57]. Cordycepin or vehicle treated SK-Hep1 cells were trypsinized, re-suspended with 0.1% bovine
serum albumin (BSA)–DMEM and added to the upper wells (2 × 104 cells). Cells then migrated to the bottom wells
containing medium with 100 μg/mL fibronectin (Becton Dickinson, Pont-de-Claix, France), epidermal growth factor
(20 ng/mL), and 10% BSA. The cells that remained on the upper side were removed and the migrating cells on the
bottom side were stained and fixed with 0.1% crystal violet containing 20% ethanol and 1% formaldehyde for 20
minutes. Efficiency of cell migration was quantified by counting the total number of migrating cells.
Western Blot Analysis
Western blotting analysis was performed as previously described [56,57]. In brief, cordycepin treated cells
were harvested and lysed in ice-cold RIPA buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid,
10% NP-40, 10 mM EDTA; Millipore, Temecula, CA) containing cocktail protease inhibitors (Roche, Indianapolis,
IN). Cell lysates were clarified by centrifugation at 15,000 rpm for 20 minutes at 4°C. Protein concentrations were
determined by a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). 20 μg protein of each sample was
applied to a gradient SDS-PAGE gel and immunoblotted onto PVDF membranes. The membranes were blocked for
1 hour in PBST (0.1% Tween 20, 2.67 mM KCl, 1.47 mM KH 2PO4, 137.93 mM NaCl, 8.1 mM Na2HPO4, pH 7.4)
containing 5% nonfat dry milk. Membranes were incubated with primary antibodies overnight and washed 3 times
with PBST for 5 minutes. The membranes were immersed in PBST containing horseradish peroxidase-conjugated
secondary antibody for 1 hour and protein levels were determined by use of enhanced chemiluminescence reagents.
Antibodies against E-cadherin, N-cadherin, α-catenin and β-catenin were purchased from BD Biosciences, San Jose,
CA; phosphorylated-FAK (p-FAK) at tyrosine 397 was purchased from Millipore, Temecula, CA; FAK, integrin α3,
integrin α6, integrin β1 and integrin β4 were purchased from Santa Cruz Biotechnologies, Heidelberg, Germany; and
actin was purchased from Sigma-Aldrich, St. Louis, MO.
Statistical Analysis
The Student’s t test was used to analyze differences among 2 groups. A p value less than 0.05 was considered
5
statistically significant.
6
RESULTS
Cordycepin inhibits cell proliferation, migration and invasion of HCC
Previous studies indicated that cordycepin suppresses the cell growth of various tumors. In this study, we
examined whether cordycepin inhibits HCC cell proliferation. SK-Hep1 cells, a poorly differentiated HCC cell line
with a high capacity for cell migration and invasion, were incubated with different concentrations of cordycepin for
24-72 hr. The rate of cell proliferation was determined by an MTT assay. Cordycepin significantly reduced SK-Hep1
cell proliferation in a concentration- and time course-dependent manner (Fig. (2)). To further examine the effect of
cordycepin on cell motility and invasiveness of HCC, SK-Hep1 cells were treated with 200 μM cordycepin followed
by two-chamber migratory and invasive analysis. Cordycepin significantly suppressed SK-Hep1 cell migration (Fig.
(3A)) and invasion (Fig. (3B)).
Cordycepin induces E-cadherin expression in HCC cells
To explore whether cordycepin plays role on modulating the EMT of cancer cells, we examined the
expression of EMT markers and related tight junction proteins, including E-cadherin, N-cadherin, α-catenin and
β-catenin in cordycepin-treated SK-Hep1 cells. At the basal level, expression of E-cadherin was barely detected in
SK-Hep1 cells (Fig. (4)) indicating that SK-Hep1 cells are typically a mesenchymal type of cancer cells. Our results
show that there was a dose dependent increase of E-cadherin expression after treatment with cordycepin, which is
the hallmark of EMT for suppressing tumor metastasis (Fig. (4)). In contrast, treatment of SK-Hep1 cells with
cordycepin had no effect on expression of N-cadherin, α-catenin and β-catenin (Fig. (4)).
Regulation of Integrins/FAK signaling by cordycepin
Since treating cells with cordycepin inhibits HCC cell migration and invasion, we assumed that cordycepin
influences the signaling factors involved in regulating the focal adhesion complex. To investigate the effect of
cordycepin on the expression of focal adhesion related factors, we first examined the expression and
phosphorylation level of FAK. SK-Hep1 cells were treated with 200 μM cordycepin for 1 to 72 hrs and the
expression of FAK and phosphorylated–FAK (p-FAK) were determined by Western blot analysis. Cells treated with
cordycepin for 48 to 72 hrs only slightly reduced FAK expression (Fig. (5), upper panel). However, p-FAK was
significantly suppressed by cordycepin (Fig. (5), middle panel). These results suggest that cordycepin does not
directly inhibit FAK expression but instead, impairs the upstream signaling of FAK activation. Certain integrins are
known to interact with FAK and play a pivotal role in the activation of FAK. We next examined the expression of
these integrins in cordycepin-treated HCC cells. SK-Hep1 cells were treated with indicated concentrations of
cordycepin for 24-72 hrs and the expression of integrin β1, integrin β4, integrin α6 and integrin α3 were determined
by Western blot analysis. Cordycepin slightly reduced integrin α6 expression but significantly reduced integrin β1
and integrin α3 expression (Fig. (6)). These results suggest that cordycepin suppresses certain integrins expression
and consequently, abolishing the activation of FAK.
7
DISCUSSION
In the past few decades there has been a rising interest in the use of complementary or alternative medicines as
supplementary to western medicine. The herbal supplement Dong Chong Xia Cao has been used for generations as a
valuable herbal medicine in China and in the Far-East because of its effective properties. Cordycepin is one of the
active small molecular compounds extracted from Dong Chong Xia Cao and has been shown to have potential
anti-tumor properties in various tumors. However, the molecular mechanisms of how cordycepin suppresses tumor
progression have not been elucidated. In this study, we found that cordycepin significantly inhibits HCC cell
proliferation (Fig. (2)), motility and invasiveness (Fig. (3A) and (3B)). In addition, our data show for the first time,
that cordycepin suppresses integrin α3, integrin α6 and integrin β1 expression (Fig. (6)), thereby reducing FAK
activation (Fig. (5)). In addition, cordycepin increased E-cadherin expression (Fig. (4)), suggesting that cordycepin
may prevent EMT and metastasis of HCC (Fig. (7)). Since cordycepin has been isolated from an alternative
medicine that has been used for many years there are fewer issues with the safety profile of this drug. Cordycepin
can be considered as a herbal supplementary or an alternative compound for preventing HCC metastasis.
Tumor-associated integrins are implicated in contributing to tumor migration/invasion, EMT and eventually
metastasis. Specific integrins interact with FAK, inducing FAK autophosphorylation at Tyr-397 followed by the
activation of downstream signaling. Our results demonstrate that although cordycepin has no significant effect on
FAK levels, it suppresses integrin β1 and integrin α3, integrin α6 expression in HCC cells. These results suggest that
cordycepin affects HCC tumor metastasis. The detailed mechanisms of how cordycepin regulates integrin expression
are currently under investigation. In addition, our unpublished findings have revealed that cordycepin suppression of
cell proliferation and tumor progression is mediated through different signaling and mechanism in distinct
malignancies. Therefore, the signaling pathways affected by cordycepin are tissue and tumor type specific. Further
studies are currently ongoing to validate this hypothesis.
Our previous study indicated that bortezomib, a small molecular weight proteasome inhibitor, suppresses FAK
expression via inactivation of NFκB [58]. Suppressed FAK expression results in induced cancer cell apoptosis and
reduced cell migration [58]. Since the activation and overexpression of FAK is implicated in the progression of
malignant tumors, we postulated that targeting of FAK by bortezomib is a potential strategy for preventing cancer
metastasis [59]. From the results of our current study, we propose that studies into the efficacy of administering
cordycepin in combination with other anti-cancer agents, such as bortezomib, for the treatment and prevention of
advance HCC progression should be considered.
8
LIST OF ABBREVIATIONS
EMT: epithelial-mesenchymal transition
FAK: focal adhesion kinase
HCC: hepatocellular carcinoma
ECM: extracellular cellular matrix
NFκB: nuclear factor kappa B
9
ACKNOWLEDGEMENTS
This work was supported by the National Health Research Institutes (01A1-CSPP07-014), the National
Science
Council
(98-2320-B-400-008-MY3,
100-2314-B-002-062)
(DOH100-TD-C-111-001) of Taiwan.
10
and
the
Department
of
Health
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FIGURE LEGENDS
Fig. 1. Structure of adenosine (C10H13N5O4) and cordycepin (C10H13N5O3).
Fig. 2. Cordycepin inhibits cell proliferation.
SK-Hep1 cells were treated with 0-200 μM cordycepin for 24 to 72 hrs. Cell proliferation was determined by an
MTT assay.
Fig. 3. Cordycepin suppresses cell migration and invasion.
(A) Cell migration and (B) invasion efficiency of SK-Hep1 cells treated with or without cordycepin were analyzed
by a two chamber assay.
Fig. 4. Cordycepin induces E-cadherin expression.
SK-Hep1 cells were treated with indicated concentrations of cordycepin for 72 hrs and the expression of E-cadherin,
N-cadherin, α-catenin and β-catenin was analyzed by Western blot analysis. Actin was used as a loading control.
Fig. 5. Cordycepin reduces FAK phosphorylation.
SK-Hep1 cells were treated with 200 μM cordycepin for 72 hrs. Expression levels of FAK and phosphorylated FAK
(p-FAK) were determined by Western blot analysis. Actin was used as a loading control.
Fig. 6. Effect of cordycepin on integrins expression.
SK-Hep1 cells were treated with indicated concentrations of cordycepin for 24-72 hrs. Integrin β1, integrin β4,
integrin α6 and integrin α3 levels were determined by Western blot analysis. Actin was used as a loading control.
Fig. 7. A proposed mechanistic model of cordycepin in regulating Integrin/FAK signaling of HCC.
16