Jie Xu1, Xia Shen2, Daozhong Sun1, Yanjie Zhu3. 1. Department of General Surgery, Zhejiang Greentown Cardiovascular Hospital, Hangzhou, Zhejiang, China. 2. Department of Emergency, Zhejiang Greentown Cardiovascular Hospital, Hangzhou, Zhejiang, China. 3. Department of Dermatology, The Second People's Hospital of Yuhang District, Hangzhou, Zhejiang, China. Email: 1048950853@qq.com.
Colon cancer is one of the most common malignancies
(1). Surgical resection, chemotherapy, and molecular
targeted therapy are the main treatment strategies for
colon cancer (2). Nonetheless, approximately 50% of
colon cancer patients develop chemoresistance (3).
Therefore, the search for possible therapeutic agents
is important to improve the prognosis of colon cancer
patients.Natural products represent a rich source for the discovery and development of anti-cancer
drugs (4- 6). Cordycepin, also known as 3’-deoxyadenosine, is a kind of nucleoside analog,
which is extracted from Cordyceps militaris (7, 8). Cordycepin has various
pharmacological effects, including anti-tumor, antibacterial, and anti-aging activity,
immunomodulation, scavenging of free radicals and anti-ischemia/ reperfusion injury activity
(8-10). Reportedly, cordycepin can inhibit the growth of colon cancer cells, suggesting that
cordycepin is promising in the treatment of colon cancer (11). However, the detailed
mechanism by which cordycepin exerts its tumorsuppressive functions is not clear.Kinases play a critical role in cellular signal
transduction, and many of them are associated
with tumorigenesis and cancer progression (12).
Glycogen synthase kinase 3 (GSK3), a serine/
threonine kinase, which has two isoforms, GSK3α and
GSK3β (13). GSK3β has been found to regulate the
survival and proliferation of colon cancer cells (14).
Interestingly, GSK3β is reported to be involved in the
phosphorylation of β-catenin, thereby activating the
E3 ubiquitin ligase subunit β-Trcp and inducing the
proteasome degradation of β-catenin (15). In addition,
elevated levels of nuclear β-catenin is considered to be
a hallmark of aggressive colon cancer, which activates
Wnt-related targets including c-myc, cyclin D1, MMP2
and MMP9, thereby promoting cell proliferation,
invasion and migratory potential (16-19). The present
study aimed to investigate the role and mechanism of
GSK3β/β-catenin/cyclin D1 pathway in colon cancer.
Materials and Methods
Cell lines and cell culture
In this experimental study, Cordycepin (3′-deoxyadenosine, CAS: 73-03-0) was obtained
from Sigma-Aldrich (St. Louis, MO, USA). Human immortalized colon mucosa cell line FHC,
and human colon cancer cell lines (HT-29 and LoVo) were provided by the Shanghai Cell Bank
of the Chinese Academy of Sciences (Shanghai, China), and the cells were cultured in
Roswell Park Memorial Institute-1640 (RPMI1640) medium (Gibco, Waltham, Waltham, MA, USA)
supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA), 0.1 mg/mL
streptomycin (Gibco, Waltham, MA, USA) and 100 U/mL penicillin (Gibco, Waltham, MA, USA)
at 37˚C in a humidified environment with 5% CO2 . The medium was changed every
1-2 days, and when the confluence reached 80%, 0.25% trypsin was used for trypsinization,
and the cells were passaged.
The cells were trypsinized, resuspended and then inoculated in 96-well plates at
5×103 cells/well, with 3 replicate wells for each group of cells. HT-29 and
LoVo cells were treated with different concentrations of cordycepin (20, 40 and 80 μM) for
24 hours, respectively. Cells with no treatment served as the control group. After adding
10 μL of MTT (SigmaAldrich, St. Louis, MO, USA) per well, the cells were further incubated
for 2 hours. Then dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) was added
into the wells, and the crystals were dissolved, and the absorbance values were measured
by a spectrophotometer at 490 nm.
Wound healing experiment
HT-29 and LoVo cells were inoculated into 6-well culture plates (5×105
cells/well) and cultured. When the confluency of the cells reached 90%, a scratch was made
in the middle of the monolayer cells with a sterile 200 μL pipette tip. Then, the cells
were gently washed 3 times with phosphate buffered saline (PBS), the width of the scratch
was detected, and then the cells were cultured with serum-free medium. After 24 hours, the
scratch was detected under the microscope again. Scratch healing rate (%)=(0 hours scratch
width−24 hours scratch width)/0 h scratch width×100%.
Transwell assay
Cell invasion assays were performed with Transwell chambers (Corning, NY, USA). Matrigel
(1:10; BD Biosciences, Franklin Lakes, NJ, USA) was used to cover the filter of Transwell
chambers. The density of HT-29 cells was modulated to 1×106 cells/mL with
serum-free medium, and 100 μL of cell suspension and 600 μL of the complete medium were
supplemented to the upper compartment and lower compartment, respectively. After 2 days,
the cells on the upper surface of the filter were removed, and the cells on the below
surface of the filter were fixed with 4% paraformaldehyde, and subsequently stained with
0.1% crystal violet. The number of cells that passed through the filter were counted in
five random fields of view, and the average was calculated to indicate the invasion
ability of the cells.
Flow cytometry
The cells were trypsinized with EDTA-free trypsin,
and the cells were collected by centrifugation.
According to the manufacturer’s instructions for
Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen
Biotech Co., Ltd., Shanghai, China), the colon cells
were washed twice with PBS and resuspended with
1× binding buffer and accordingly incubated with 5
μL of Annexin V-FITC staining solution and 10 μL of
propidium iodide (PI) staining solution for 15 min at
ambient temperature in the dark. After the cells were
washed by PBS, a flow cytometer (BD Biosciences,
San Jose, CA, USA) was used to quantify apoptosis
within 1 hours.
Lung metastasis assay
All animal experiments were approved by the animal Ethical Committee of Zhejiang
Greentown Cardiovascular Hospital (2017A044). 12 nude mice (6-week-old, male) weighing
12-15 g, purchased from the Experimental Animal Center of Zhejiang University, were
randomly grouped into 2 groups (n=6 in each group). The mice in each group were injected
with HT-29 cells (1×107 cells per mouse) via the caudal vein, and then treated
with or without cordycepin (20 mg/kg). After 2 weeks, the mice were euthanized, and the
fresh lung tissues were harvested for histopathology analysis. The number of metastatic
nodules in the lung of each mouse was counted with naked eyes. Next, formalin-fixed and
paraffin-embedded lung tissues were prepared, and 4 μm of thick sections were stained with
hematoxylin and eosin and then observed under the microscope (NikonEclipseE600; Nikon,
Thornwood, NY, USA).
Western blot
The colon cancer cells were lysed in RIPA lysis
solution (Beyotime Biotechnology, Shanghai, China),
and the BCA protein assay kit (Beyotime Institute
of Biotechnology, Haimen, China) was utilized to
quantify the protein concentration. After adding protein
loading buffer and boiling to denaturation, sodium
dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was performed. The protein samples
were transferred onto the polyvinylidene fluoride (PVDF) membranes, and then were blocked at ambient
temperature for 1 hours using 5% skimmed milk. Then
the membranes were incubated with the following
primary antibodies: anti-GSK3β (1:2000, ab32391,
Abcam, Shanghai, China), anti-β-catenin (1:2000,
ab16051, Abcam, Shanghai, China) and anti-cyclin D1
(1:2000, ab16663, Abcam, Shanghai, China) overnight
at 4˚C. The following day, the secondary antibody,
Goat Anti-Rabbit IgG H&L (1:5000, ab6721, Abcam,
Shanghai, China), was added, and the membrane was
incubated for 1 hour at 37˚C. After the membranes
were washed by tris buffered saline tween (TBST), the
ECL chemiluminescence kit (Beyotime Biotechnology,
Shanghai, China) was used for luminescence
development, and an Odyssey imaging system was
utilized to analyze the grayscale value of each band.
Statistical analysis
Statistical product and service solutions (SPSS)
software (version 23.0, SPSS, Chicago, IL, USA) was
adopted to process the data represented as “mean ±
standard deviation”. To make the comparison between
two groups, a One-Sample Kolmogorov-Smirnov test
was used to detect the normality of the data. If the data
were normally distributed, an independent sample t
test was utilized; paired sample Wilcoxon signed rank
test was adopted to compare the data with skewed
distribution. One-way ANOVA test was performed to
make the comparison among three or more groups.
If there was a significant difference, Newman-Keuls
analysis was performed to make the comparison
between 2 groups. The differences were considered
statistically significant at P<0.05.
Results
Cordycepin suppresses the malignant phenotypes of
colon cancer cells
The molecular structure of cordycepin is shown in Figure 1A. To probe the biological
effect of cordycepin on the phenotypes of FHC and colon cancer cells, the proliferation of
HT-29, LoVo and FHC cells was detected by the MTT method after treatment with different
concentrations of cordycepin (20 μM, 40 μM and 80 μM). As shown, the viability of colon
cancer cells was decreased by cordycepin treatment in a dose-dependent manner
(P<0.05, Fig .1B); notably, only high doses of cordycepin (80 μM) could
significantly suppress the viability of FHC cells (Fig .S1A, See Supplementary Online
Information at www.celljournal. org), which suggested that cordycepin selectively kills
cancer cells. The results of the scratch healing assay and Transwell assay showed that
cordycepin treatment reduced the migration and invasion of HT-29 and LoVo cells compared
with the control group (P<0.01, Fig .1C, D). In addition, flow cytometry revealed
that cordycepin treatment promoted the apoptosis of HT29 and LoVo cells compared with the
control group (P<0.05, Fig .1E). Additionally, a lung metastasis model in nude mice
was used to evaluate the metastasis of HT-29 cells in vivo, and it showed
that cordycepin treatment inhibited the pulmonary metastasis of HT29 cells in
vivo (Fig .S1B, See Supplementary Online Information at
www.celljournal.org).
Fig.1
Effects of cordycepin on the biological behaviors of colon cancer cells. A. The
molecular structure of cordycepin. B. Cell viability of HT-29 and LoVo
cells was detected by MTT assay after treatment with different concentrations of
cordycepin (control, 20 µM, 40 µM and 80 µM). C. Cell migration (scale
bars: 50 μm) of HT-29 and LoVo cells was detected by scratch healing assay after
treatment with different concentrations of cordycepin (control, 20 µM, 40 µM and 80
µM). D. Cell invasion (scale bars: 50 μm) of HT-29 and LoVo cells was
detected by Transwell assay after treatment with different concentrations of
cordycepin (control, 20 µM, 40 µM and 80 µM). E. Cell apoptosis of HT-29
and LoVo cells was detected by flow cytometry after treatment with different
concentrations of cordycepin (control, 20 µM, 40 µM and 80 µM). All experiments were
repeated at least 3 times and were performed in triplicates, and data were presented
as mean ± SD. *; P<0.05, **; P<0.01, and ***; P<0.001.
Effects of cordycepin on the biological behaviors of colon cancer cells. A. The
molecular structure of cordycepin. B. Cell viability of HT-29 and LoVo
cells was detected by MTT assay after treatment with different concentrations of
cordycepin (control, 20 µM, 40 µM and 80 µM). C. Cell migration (scale
bars: 50 μm) of HT-29 and LoVo cells was detected by scratch healing assay after
treatment with different concentrations of cordycepin (control, 20 µM, 40 µM and 80
µM). D. Cell invasion (scale bars: 50 μm) of HT-29 and LoVo cells was
detected by Transwell assay after treatment with different concentrations of
cordycepin (control, 20 µM, 40 µM and 80 µM). E. Cell apoptosis of HT-29
and LoVo cells was detected by flow cytometry after treatment with different
concentrations of cordycepin (control, 20 µM, 40 µM and 80 µM). All experiments were
repeated at least 3 times and were performed in triplicates, and data were presented
as mean ± SD. *; P<0.05, **; P<0.01, and ***; P<0.001.
Effects of cordycepin and GSK-3β inhibitor
(CHIR99021) on GSK3β protein expression
To investigate the mechanism by which cordycepin suppresses the malignancy of colon
cancer cells, Western blot was used to detect the expressions of GSK3β protein in colon
cancer cells. Cordycepin treatment was found to promote GSK3β protein expression in HT-29
and LoVo cells in a dose-dependent manner (P<0.001, Fig .2A). After the cotreatment
with CHIR99021, the promoting effect of cordycpin on GSK-3β protein expression was
reversed (P<0.001, Fig .2B).
Fig.2
Effects of cordycepin and CHIR99021 on GSK3β expression. A. GSK3β protein
expression in HT-29 and LoVo cells was detected by Western blot after treatment with
different concentrations of cordycepin (control, 20 µM, 40 µM and 80 µM).
B. GSK3β protein expression in HT-29 and LoVo cells was detected by
Western blot after co-treatment with 80 μM cordycepin and 10 μM CHIR99021. All
experiments were repeated at least 3 times and were performed in triplicates, and data
were presented as mean ± SD. ***; P<0.001.
Effects of cordycepin and CHIR99021 on GSK3β expression. A. GSK3β protein
expression in HT-29 and LoVo cells was detected by Western blot after treatment with
different concentrations of cordycepin (control, 20 µM, 40 µM and 80 µM).
B. GSK3β protein expression in HT-29 and LoVo cells was detected by
Western blot after co-treatment with 80 μM cordycepin and 10 μM CHIR99021. All
experiments were repeated at least 3 times and were performed in triplicates, and data
were presented as mean ± SD. ***; P<0.001.
Effects of cordycepin and CHIR99021 on the biological
behaviors of colon cancer cells
To investigate that whether the tumor-suppressive
effects of cordycepin on colon cancer cells are
dependent on GSK3β, after the HT-29 and LoVo cells
were treated with cordycepin and CHIR99021, MTT
assay, scratch healing assay, Transwell assay and flow
cytometry were used to detect the viability, migration,
invasion and apoptosis of the colon cells, repectively.
It was found that, cordycepin treatment inhibited
the viability, migration and invasion, and promoted
apoptosis of HT-29 and LoVo cells; remarkably, cotreatment with CHIR99021 reversed the above effects
of cordycepin (P<0.001, Fig .3A-D).
Fig.3
Effects of cordycepin and CHIR99021 on the viability, migration and apoptosis of colon cancer
cells. A. Viability of HT-29 and LoVo cells was detected by MTT assay
after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours.
B. Migration of HT-29 and LoVo cells was detected by scratch healing
assay after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours.
C. Invasion of HT-29 and LoVo cells was detected by Transwell assay
after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours.
D. Apoptosis of HT-29 and LoVo cells was detected by flow cytometry
after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours. All
experiments were repeated at least 3 times and were performed in triplicates, and data
were presented as mean ± SD. ***; P<0.001.
Effects of cordycepin and CHIR99021 on the viability, migration and apoptosis of colon cancer
cells. A. Viability of HT-29 and LoVo cells was detected by MTT assay
after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours.
B. Migration of HT-29 and LoVo cells was detected by scratch healing
assay after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours.
C. Invasion of HT-29 and LoVo cells was detected by Transwell assay
after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours.
D. Apoptosis of HT-29 and LoVo cells was detected by flow cytometry
after treatment with 80 μM cordycepin and 10 μM CHIR99021 for 24 hours. All
experiments were repeated at least 3 times and were performed in triplicates, and data
were presented as mean ± SD. ***; P<0.001.
Effects of cordycepin and CHIR99021 on cyclin D1
and β-catenin protein expressions
Next, we investigated the regulatory effects of
cordycepin and GSK3β on cyclin D1 and β-catenin,
the expression levels of cyclin D1 and β-catenin
proteins were detected by Western blot. The results
showed that the expression levels of cyclin D1 and
β-catenin protein were reduced upon cordycepin
treatment; however, co-treatment with CHIR99021
inhibited the effects of cordycepin on cyclin D1 and
β-catenin protein expression (P<0.001, Fig .4A, B).
These data suggested that cordycepin could regulate
the expression level of cyclin D1 and β-catenin in
colon cancer cells via activating GSK3β.
Fig.4
Effects of cordycepin and CHIR99021 on cyclin D1 and β-catenin protein expressions. A, B.
Cyclin D1 and β-catenin protein expressions of HT-29 and LoVo cells were
detected by Western blot after co-treatment with 80 μM cordycepin and 10 μM CHIR99021.
All experiments were repeated at least 3 times and were performed in triplicates, and
data were presented as mean ± SD. ***; P<0.001.
Effects of cordycepin and CHIR99021 on cyclin D1 and β-catenin protein expressions. A, B.
Cyclin D1 and β-catenin protein expressions of HT-29 and LoVo cells were
detected by Western blot after co-treatment with 80 μM cordycepin and 10 μM CHIR99021.
All experiments were repeated at least 3 times and were performed in triplicates, and
data were presented as mean ± SD. ***; P<0.001.
Discussion
Colon cancer patients with distant metastasis or recurrence after surgery have poor
prognosis (20). In recent years, several studies have implied that cordycepin can induce
apoptosis of cancer cells and promote DNA damage to inhibit the proliferation and metastasis
of cancer cells, and it may also increase the chemosensitivity of cancer cells (21, 22). For
example, cordycepin inhibits the proliferation of tongue cancer cells in a dose-dependent
manner (23). Cordycepin can down-regulate the expression of C-X-C chemokine receptor type 4
in hepatocellular carcinoma cells in a dose-dependent manner, significantly inhibiting the
migration and invasion of hepatocellular carcinoma cells (24). Another study shows that
cordycepin can regulate the expressions of cyclin-dependent kinase 1 and cyclin B1, leading
to cell cycle arrest of esophageal cancer cells (25). In addition, multiple previous studies
report that cordycepin has the potential to block the progression of colon cancer (26- 28).
Specifically, cordycepin can inhibit the migration and invasion of HCT116 cells by
regulating the expression of Prostaglandin E2 receptor 4 and the transduction of AMPK-CREB
signaling pathway (27). Additionally, cordycepin can induce the activation of JNK1, leading
to cell cycle arrest of HCT116 cells (28). Consistently, in the present study, cordycepin
was found to significantly inhibit the proliferation, migration and invasion of colon cancer
cells and promote their apoptosis.GSK3β is a serine/threonine kinase partaking in
modulating cell proliferation, DNA repair, cell cycle
progression, signal transduction and metabolic pathways
(29). Importantly, the dysfunction/dysregulation of GSK3β is involved in tumorigenesis, and it may exert cancerpromoting function or tumor-suppressive function in
different cancers (30-32). GSK3β promotes epithelialmesenchymal transition (EMT) of triple-negative breast
cancer cells, and GSK3β inhibitors selectively kill cancer
cells with mesenchymal properties and are considered
as potential therapeutic targets for triple-negative breast
cancer (30). Resveratrol can inhibit the EMT of colon
cancer through the AKT/GSK‑3β/Snail signaling pathway
(31). The occurrence, progression and drug resistance of
pancreatic ductal carcinoma are thought to be related to
the expression of GSK3β, and the inhibition of GSK3β
induces apoptosis and slows the growth of tumors and
metastases (32). β-catenin is a crucial component in Wnt
signaling pathway; the abnormal activation of the Wnt/βcatenin signaling pathway facilitates the accumulation
of β-catenin in the nucleus and the transcription of
many oncogenes such as c-Myc and Cyclin D1, thereby
contributing to the occurrence and development of
a variety of cancers including colon cancer (33, 34).
Importantly, GSK3β can phosphorylate β-catenin,
leading to the ubiquitination and proteasomal-dependent
degradation of β-catenin (15, 35). The dysregulation of the
GSK3β/β-catenin pathway is involved in the regulation
of the malignant biological behaviors of cancer cells (36-
38). For instance, upregulated gene 4 can promote the
proliferation of osteosarcoma cells through the GSK3β/
β-catenin/cyclin D1 pathway (37). PIK3CD induces
malignant phenotypes of colorectal cancer by activating
AKT/GSK-3β/β-catenin signaling (38). Herein, we found
that cordycepin could increase GSK3β expression and
inhibit the expression levels of β-catenin and cyclin D1,
which partly explains the mechanism of cordycepin’s
tumor-suppressive effects in colon cancer.
Conclusion
This study not only confirms the tumor-suppressive role of cordycepin in colon cancer, but
also reports that the biological function of cordycepin on colon cancer cells is mediated by
GSK3β/β-catenin/cyclin D1 pathway. It’s worth noting that, even though
cordycepin/Cordyceps militaris is widely used in traditional Chinese
medicine, clinical trials are still needed to further validate its safety and efficacy in
treating colon cancer.
Authors: Geraldine Vidhya Vijay; Na Zhao; Petra Den Hollander; Mike J Toneff; Robiya Joseph; Mika Pietila; Joseph H Taube; Tapasree R Sarkar; Esmeralda Ramirez-Pena; Steven J Werden; Maryam Shariati; Ruli Gao; Mary Sobieski; Clifford C Stephan; Nathalie Sphyris; Noayuki Miura; Peter Davies; Jeffrey T Chang; Rama Soundararajan; Jeffrey M Rosen; Sendurai A Mani Journal: Breast Cancer Res Date: 2019-03-07 Impact factor: 6.466
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