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Journal of the Chinese Medical Association 76 (2013) 146e152
www.jcma-online.com
Original Article
Reappraisal of the anticancer efficacy of quercetin in oral cancer cells
Su-Feng Chen a, Shin Nien b, Chien-Hua Wu c, Chia-Lin Liu c, Yun-Ching Chang c,
Yaoh-Shiang Lin d,e,f,*
a
Department of Dental Hygiene, China Medical University, Taichung, Taiwan, ROC
Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC
c
Graduate Institute of Pathology and Parasitology National Defense Medical Center, Taipei, Taiwan, ROC
d
Department of OtolaryngologydHead and Neck Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ROC
e
Department of Nutritional Science, Toko University, Chia-Yi County, Taiwan, ROC
f
Department of OtolaryngologydHead and Neck Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC
b
Received May 21, 2012; accepted August 8, 2012
Abstract
Background: Despite increased experience in therapy, the overall outcome of oral squamous cell carcinoma (OSCC) has not improved because of
the relative resistance to chemotherapeutic drugs in addition to local invasion and frequent regional lymph node metastases. Quercetin (Qu) is
a principal flavonoid compound and an excellent free-radical-scavenging antioxidant that promotes apoptosis. Limited reports regarding the
molecular or cellular role of Qu in anticancer properties on OSCC have been presented. This study was conducted to clarify the efficacy of Qu on
OSCC in vitro and further to evaluate the possible mechanism(s).
Methods: Cultured OSCC cells (SCC-25) and human gingival fibroblasts (HGFs) were treated with different concentrations of Qu. Cell viability
and cell colony-forming potential were detected with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and colony
growth assays. Cell-cycle analysis and apoptosis were measured by flow cytometry. Cell migration and invasion were tested using the micropore
chamber assay.
Results: Cell viability and colony-forming potential were decreased in a dose-dependent manner following Qu treatment. Qu also dosedependently inhibited the proliferation of SCC-25 cells via both G1 phase cell cycle arrest and mitochondria-mediated apoptosis. In addition, Qu also decreased the abilities of migration and invasion of SCC-25 cells in a dose-dependent manner.
Conclusion: Qu effectively inhibits cell growth and invasion/migration of SCC-25 cells in vitro. The cellular and molecular mechanisms are via
cell cycle arrest accompanied by mitochondria-mediated apoptosis. Our findings suggest that Qu may have potential as a new chemopreventive
agent or serve as a therapeutic adjuvant for OSCC.
Copyright Ó 2012 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved.
Keywords: apoptosis; invasion; migration; oral squamous cell carcinoma; quercetin
1. Introduction
Oral squamous cell carcinoma (OSCC) is one of the most
common and lethal head and neck malignancies in Taiwan.
OSCC is now a global health problem with increasing
* Corresponding author. Dr. Yaoh-Shiang Lin, Department of OtolaryngologydHead and Neck Surgery, Kaohsiung Veterans General Hospital,
386 Ta-Chung 1st Road, Kaohsiung 813, Taiwan, ROC.
E-mail address: [email protected] (Y.-S. Lin).
incidence and mortality rates. The major environmental risk
factors responsible for the development of OSCC include betel
nut chewing, cigarette smoking, alcohol consumption, and
exposure to high-risk human papillomavirus.1 OSCC is a very
difficult disease to treat because of multidisciplinary and
diverse treatment strategies and the varied natural behavior of
the cancer. Local invasion and frequent regional lymph node
metastases together with relative resistance to chemotherapeutic drugs lead to an unpredictable prognosis. Despite
increased experience in therapy, including improvements in
1726-4901/$ - see front matter Copyright Ó 2012 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved.
http://dx.doi.org/10.1016/j.jcma.2012.11.008
S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
147
surgical technology and adjuvant treatments, the overall outcome of OSCC has not improved, indicating an urgent need
for a new strategy for OSCC treatment.
Accumulating evidence suggests that natural dietary factors, among which complementary and alternative medicines
are consistently represented, may inhibit the process of carcinogenesis and may also effectively reduce the risk of many
human cancers.2e4 Quercetin (Qu) is the principal flavonoid
compound (3,30,40,5,7-penta hydroxyl flavanone) commonly
extracted from cranberries, blueberries, apples, and onions. It
possesses a wide spectrum of biopharmacological properties
and may offer promising new options for the development of
more effective chemopreventive and chemotherapeutic strategies because of its powerful antioxidant and free-radicalscavenging properties.5,6 A review of the literature shows
that Qu treatment has been associated with selective antiproliferative effects and induction of cell death, probably
through an apoptotic mechanism, in breast or other cancer cell
lines but not in normal cells.7,8 The characteristics of the
extensive anticarcinogenic and antiproliferative effects of Qu
have attracted a great deal of attention and have led to it being
considered a potential candidate for cancer prevention and
treatment of oral cancer.
Few studies of the anticancer properties of Qu in oral
cancer cells have been reported. Therefore, the current study
was undertaken to test the exact antiproliferative effects of Qu
on OSCC in vitro. The study also aimed to evaluate the possible mechanism(s) of Qu, with special emphasis on cellular
and molecular changes in the growth and invasion potential of
OSCC cells.
mL in 24-well plates, 100 mL/well, and treated with Qu
(Sigma-Aldrich, St. Louis, MO, USA) at different dosages for
24 hours. After incubation, the medium was replaced with
50 mL of MTT reagent (2 mg/mL) and incubated in 5% CO2 at
37 C for 1 hour.
2. Methods
2.5. Apoptosis analysis
2.1. Cell lines and culture conditions
Cells that were treated for 24 hours were trypsinized and
washed twice with phosphate-buffered saline, then suspended
in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and
2.5 mM CaCl2). Cells were stained with 5 mL Annexin V
conjugated with fluorescein isothiocyanate (FITC) at room
temperature in the dark for 30 minutes. They were then analyzed by flow cytometry to measure the fluorescence intensity
using the FL-1H channel to detect FITC. Untreated cells
served as the negative control.
An OSCC cell line, SCC-25, taken from squamous cell
carcinoma of the tongue, was kindly provided by Dr Jenn-Han
Chen (from National Defense Medical Center, Taipei, Taiwan). The cell line was cultured in RPMI 1640 medium
(Gibco, Grand Island, NY, USA). Human gingival fibroblasts
(HGFs) were obtained from surgical specimens of patients
with head and neck squamous cell carcinomas. The fibroblasts
were maintained in Dulbecco’s Modified Eagle Medium and
Ham’s Nutrient Mixture-F12 culture medium at a ratio of 3:1.
Both SCC-25 and HGFs were kept in a medium supplemented
with 10% FBS and 1% penicillin/streptomycin (Biological
Industries, Beit Haemek, Israel) at 37 C and 5% CO2. The use
of HGFs was approved by the institutional review board of the
Tri-Service General Hospital, Taipei, Taiwan (TSGHIRB No.:
1-101-05-052).
2.2. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide assay
Cell viability were determined using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay
(Sigma). Cells were suspended at a density of 2 104 cells/
2.3. Anchorage-independent colony growth assay
Tumor cells were seeded at a density of 3 103 cells/well
in a six-well plate and treated with Qu at different dosages
then incubated in 5% CO2 at 37 C for 14 days. On day 14, the
colonies were stained with 2 mg/mL MTT. Colonies of >50
cells were counted and the colony-forming potential of Qutreated OSCC was expressed as the percentage of colonies
of the untreated cells.
2.4. Cell cycle analysis
The cells were harvested by Qu treatment for 24 hours and
fixed in 70% ethanol at 20 C for 1 hour; next, the cells
were collected by centrifugation and reconstituted in
phosphate-buffered saline (10 mM HEPES, pH 7.4, 140 mM
NaCl, and 2.5 mM CaCl2). Cells were then stained with
propidium iodide solution (20 mg/ml propidium iodide and
10 mg/ml RNase A) at 37 C in the dark for 30 minutes. The
stained cells were examined by flow cytometry using FL-2A
to score the DNA content of cells. The percentages of cells in
the G1, S, and G2/M cell-cycle phases were determined using
the Modfit software (Verity Software House, Inc., Topsham,
ME, USA).
2.6. Migration analysis
The migration ability was determined using a micropore
chamber assay. Cells (1 104) were seeded onto the top
chamber of a 24-well micropore polycarbonate membrane
filter with 8-mm pores (Becton Dickinson Labware, Lincoln
Park, NJ, USA), and the bottom chamber was filled with RPMI
1640 containing 10% fetal bovine serum as a chemoattractant.
After 24 hours of incubation in 5% CO2 at 37 C, cells on the
upper surface were carefully removed with a cotton swab, and
cells that had migrated to the lower surface of the filter were
stained by hematoxylin. Cell migration was quantified by
counting the average migrated cells in five random highpowered fields per filter.
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S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
2.7. Invasion analysis
Invasion assay was performed in modified Boyden chambers
with 8-mm pore size filter inserts for 24-well plates. Filters were
coated with 10e12 mL of ice-cold basement membrane
extracellular matrix in 8e12 mg of protein/mL. Cells (1 104)
were added to the upper chamber in 200 mL of RPMI1640
lacking serum. The lower chamber was filled with 500 mL of
RPMI1640 medium supplemented with 10% fetal bovine
serum. After 24 hours of incubation, the cells were fixed with
methanol and stained by hematoxylin. Cells that remained in
the basement membrane or attached to the upper side of the
filter were removed with paper towels. Cells on the lower side of
the filter were examined using light microscopy and counted.
2.8. Western blotting
Cells that had been treated for 24 hours were washed twice
with ice-cold phosphate-buffered saline and lysed in 0.5 mL of
homogenization buffer (10 mM TriseHCl at pH 7.4, 2 mM
EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 50 mM
NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1:100/v:v proteinase inhibitor cocktail) at 4 C for 30
minutes. Cell lysates were then ultracentrifuged at 100,000 g
for 30 minutes at 4 C, and the supernatants were used as the cell
extracts. The protein concentration of the cell extract was
determined by BCA protein assay, and the extracts were adjusted
to 2 mg/mL with homogenization buffer. For immunoblot
analysis, the cell extract was subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis, and the resolved bands were
electrotransferred to PVDF membranes using semidry blot
apparatus (Bio-Rad) at 3 mA per cm2 of the gel in transfer buffer
(25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol) at
room temperature for 30 minutes. The free protein binding sites
on the PVDF membrane were blocked by incubation with 5%
nonfat milk in TTBS buffer (20 mM Tris at pH 7.4, 0.15 M NaCl,
and 0.2% Tween 20) at 25 C for 2 hours. The membrane was
then immunoblotted with 0.1 mg/mL primary antibody. Mouse
monoclonal anticaspase 3, B cell lymphoma 2 (Bcl-2), cyclin A,
cyclin B1, cyclin D1 and cyclin E antibodies, rabbit polyclonal
anti-p27 Kip1, goat polyclonal anti-p21Cip1, poly[ADP-ribose]
polymerase (PARP), Bcl-2 associated protein X (Bax), heat
shock protein 27 (Hsp27) (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA), p38MAP kinase (p38MAPK), phosphorylated
p38MAP kinase (pp38MAPK) (Cell Signaling), phosphorylated
heat shock protein 27 (pHsp27) (R&D Systems, Inc., Minneapolis, MN, USA) in Tris-Buffered Saline Tween-20 buffer
containing 3% nonfat milk at 4 C overnight and subsequently
with secondary antibody conjugated with peroxidase (1:1000) at
25 C for 1 hour. The immunoblots were developed using an
enhanced chemiluminescence system, and the luminescence
was visualized on X-ray film.
2.9. Statistical analysis
All data are expressed as means SD unless stated otherwise. The differences between groups were calculated using
Student’s unpaired t-test. Dose-dependent effects were calculated using simple linear regression. A p value <0.05 was
regarded as statistically significant. All statistical analyses
were performed using SPSS version 12.0 (SPSS, Inc., Chicago, IL, USA).
3. Results
3.1. Qu exerted an antiproliferative effect on SCC-25
cells
The growth inhibitory effect of Qu on SCC-25 cells was
evaluated by cell viability and anchorage-independent colony
growth assays. HGF (control) was used to test if Qu has a negative effect on normal cells and was evaluated by cell viability.
The survival rate of SCC-25 cells showed a significant dosedependent decrease following treatments with Qu (Fig. 1A).
Testing with a dose range of 10e100 mM Qu showed that,
expressing cell viability as the percentage of that in untreated
cells, a 50% cell survival rate of SCC-25 cells was obtained at
a dose of 50 mM. Three doses (25, 50 and 75 mM) were chosen as
test concentrations for the remainder of the experiments
(Fig. 1B). An anchorage-independent colony growth assay
further demonstrated a significant inhibitory effect of Qu, with
the number of colonies of SCC-25 cell lines being markedly
decreased by Qu treatment in a dose-dependent manner
(Fig. 1C). The result was also confirmed by quantitative and
statistical analyses (Fig. 1D). The data reported from the above
two assays were from three independent experiments expressed
as means standard deviation (SD).
3.2. Qu affected the cell cycle of SCC-25 cells
The distribution of Qu-treated cells in the cell cycle was
assessed by flow cytometry, and the expression of cell cycle
proteins was measured by immunoblotting. Following treatment with different doses of Qu (25e75 mM), the proportion
of G1 phase cells was increased with a concomitant decrease
in S phase cells compared with untreated controls and
a gradual increase in the proportion of G1 phase cells with
a concurrent increase of sub-G1 phase cells, indicating the
induction of apoptosis (Fig. 2A). Quantitative and statistical
analyses showed an obvious increase in G1 phase cells at a Qu
dose of 50 mM (Fig. 2B). Analysis of cell cycle proteins
showed a dose-dependent decrease in cyclin D1 and increase
in p21Cip1 (Fig. 2C).
3.3. Qu induced apoptosis in SCC-25 cells
Qu-treated SCC-25 cells were stained with Annexin
VeFITC and then analyzed by flow cytometry to assess the
proportion of apoptotic cells. When compared with the control
groups, there was a significant dose-dependent increase in the
proportion of Annexin V-positive OSCC cells following Qu
treatment (Fig. 3A), and this increase was also confirmed by
quantitative and statistical analyses (Fig. 3B). Immunoblotting
analysis showed that the level of the antiapoptotic oncoprotein
S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
A
Cell viability (% of control)
B
HGF
SCC-25
120
149
100
80
60
40
20
0
0
10
20
30
40
50
60
70
80
90
100
Quercetin concentration (µM)
D
120
Colony ( % of control )
C
100
80
60
40
20
0
0
25
50
75
Quercetin concentration (µM)
Fig. 1. Growth inhibitory effects of quercetin (Qu) on SCC-25 cells. (A) The growth inhibitory effect of Qu on SCC-25 cells was initially evaluated in a cell viability
assay. Qu did not affect human gingival fibroblasts (HGF). (B) Viable cells were viewed directly under a microscope. (C) Anchorage-independent growth of SCC25
cell lines in soft agar under 100 magnification after 2 weeks in culture. (D) The colony growth assay showed a significant inhibitory effect of Qu on SCC-25 cells.
B
80
Perecentsge (%)
A
Quercetin 0µM
Quercetin 25µM
Quercetin 50µM
Quercetin 75µM
60
40
20
0
G1
C
Cont.
S
Cell cycle phase
25µM
50µM
G2
75µM
Cyclin D1
Cyclin E
Cip1/P21
Kip1/P27
α-tubulin
Fig. 2. Effects of quercetin (Qu) on the cell cycle of SCC-25 cells. Flow cytometry demonstrated that the percentage of cells in G1 phase was increased and that in
S phase, SCC-25 cells treated with Qu were concomitantly decreased compared with controls. (B) Quantitative and statistical analyses of results shown in panel A.
(C) Western blot analysis of cell cycle proteins following Qu treatments showed a significant dose-dependent decrease in the expression of cyclin D1, and
a significant increase in p21Cip1.
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S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
B
45
40
35
30
25
20
15
10
5
0
% Annexin V - positive
A
0
C
25
50
75
Quercetin concentration (µM)
Cont.
25µM
50µM
75µM
Bax
Bcl-2
CI-Caspase3
CI-PARP
α-tublin
Fig. 3. Apoptotic effect of quercetin (Qu) on SCC-25 cells. (A) Apoptosis analysis with Annexin VeFITC staining showed that the proportion of fluorescent cells
was significantly enhanced after Qu treatment. (B) Quantitative and statistical analyses of results shown in panel A. (C) Immunoblotting of proteins involved in
apoptosis showed that following Qu treatment, the level of antiapoptotic oncoprotein Bcl-2 was dramatically decreased, and the level of the proapoptotic protein
Bax was increased in SCC-25 cells. The expression of the active form of caspase 3 and PARP were increased.
Bcl-2 decreased dramatically, whereas the level of the proapoptotic protein Bax was increased. Both of the expressions of
the active form of caspase 3 (cleaved caspase 3) and the poly
ADP-ribose polymerase (cleaved PARP) were increased.
3.4. Qu effectively inhibited the migration and invasion
of SCC-25 cells
A transwell Boyden chamber migration assay showed that
Qu treatment significantly decreased the migration ability of
SCC-25 cells in a dose-dependent manner (Fig. 4A). Quantitative and statistical analyses also demonstrated a significant
decrease in migration (Fig. 4B). An invasion assay demonstrated that Qu treatment also significantly reduced this invasive ability in a dose-dependent manner (Fig. 4C); this was
confirmed by quantitative and statistical analyses (Fig. 4D).
4. Discussion
Growing evidence from epidemiological studies and
experimental data from animal models indicate that lifestyle
modifications, particularly a vegetable- and fruit-rich diet,
may substantially reduce the incidence of certain types of
cancers.9 Qu, as described earlier, is one of the most abundant
flavonoids in vegetables and fruits. This pure compound is
a powerful antioxidant and free-radical scavenger. Several
studies in vitro and in vivo have further revealed that Qu can
inhibit malignant growth and metastasis in a variety of cancer
cells by exhibiting a strong ability to induce apoptosis,
potentially making it an additional option for cancer management.5,10 To the best of our knowledge, little is known
about the cellular and molecular changes mediating the
anticancer properties of Qu or the mechanisms of its effects
on the proliferation, apoptosis, invasion, and migration of oral
cancer cells.
In this study, we used representative SCC-25 cells to clearly
demonstrate the effective dose-dependent inhibition of growth
and invasion following Qu treatment. The findings represent
the first evidence of the cellular and molecular mechanisms of
Qu inhibition of SCC-25 cells growth and invasion. As
described above, our study showed that Qu treatment of SCC25 cells exerted a marked and dose-dependent inhibitory effect
on cell growth. The viability test showed a similar responsiveness of SCC-25 cells to Qu treatment. An anchorageindependent colony growth assay further validated the clear
dose-dependent inhibitory effect of Qu on SCC-25 cells. These
findings indicate that Qu may have cytotoxic effects on SCC25 cells, similar to those reported in a previous study of oral
cancer.11,12 Many oncoproteins and tumor suppressors, such as
cyclin A, B, D1, and E, Bcl-2, Bax, p21Cip and p27Kip1, are
associated with the cancer cell cycle and tumorigenesis.13
Immunoblotting analysis of the cell cycle proteins in SCC25 cells showed dose-dependent increases of p21Cip1 following Qu treatment, which might facilitate the arrest of SCC-25
in late G1 phase. Using appropriate doses of 50 mM Qu, the
increased proportion of G1 phase cells gradually declined
S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
B
120
Migration (% of control)
A
151
100
80
*
60
*
40
*
20
0
0
D
120
Invasion (% of control)
C
25
50
75
Quercetin concentration (μM)
100
80
*
60
40
*
*
20
0
0
25
50
75
Quercetin concentration (μM)
Fig. 4. Inhibitory effects of migration and invasion of quercetin (Qu) on SCC-25 cells. (A) Boyden chamber migration assay showed that Qu significantly decreased
the migration ability of SCC-25 cells in a dose-dependent manner. (B) Quantitative and statistical analyses of results shown in panel A. (C) Boyden chamber
migration assay demonstrated that Qu significantly decreased the invasion ability of SCC-25 cells in a dose-dependent manner. (D) Quantitative and statistical
analyses of the results shown in panel C.
because of the accumulation of sub-G1 phase cells resulting
from the early stages of the induction of apoptosis.
Induction of cell cycle arrest and apoptosis is a wellestablished method for treating some forms of cancer.14
Based on our data, we propose that cell cycle arrest without
repair accompanied by cellular apoptosis may be the main
cellular and/or molecular mechanism(s) occurring in SCC-25
cells following Qu treatment. The mitochondrial apoptosis
pathway is mediated by the Bcl-2 family and Bax proteins and
the expression of Bcl-2. The ratio of Bcl-2 to Bax proteins
contributes to the susceptibility of cells to induction of
apoptotic cell death. Our immunoblotting analysis revealed
that the level of Bcl-2 decreased dramatically in Qu-treated
SCC-25 cells, whereas the level of the proapoptotic protein
Bax was increased in SCC-25 cells. PARP activity was altered
by the upstream apoptosis, the active form of caspase 3, which
then induced the downstream apoptosis. Thus, the response of
the SCC-25 cells to induction of apoptosis by Qu treatment
involved a sustained decrease in expression of the antiapoptotic oncoprotein Bcl-2 along with an elevated level of
proapoptotic protein Bax, resulting in an increased Bax/Bcl-2
ratio. These results highlight the importance of the Bax/Bcl-2
ratio in the life and death of oral cancer cells.13,15 Together,
these results demonstrate that the cellular and molecular
mechanisms of Qu effects on SCC-25 oral cancer cells mainly
depend on cell cycle arrest accompanied by mitochondriamediated apoptosis.
Research has revealed that Qu and polyphenol can inhibit
the activity of matrix metalloproteinases (MMPs), especially
MMP-2 and MMP-9.16 These two key regulators associated
with invasion and metastasis can be activated by E2F transcription factor, which plays a pivotal role in cell proliferation
by regulating the expression of genes involved in G1eS
transition and DNA synthesis.17 Our study also demonstrated
that Qu significantly and dose-dependently decreased the invasion and migration abilities of SCC-25 cells.
In conclusion, our study demonstrates that Qu treatment
inhibits cell growth and invasion/migration on SCC-25 cells
in vitro. The cellular and molecular mechanisms of the biopharmacological effects of Qu were mainly via cell cycle arrest accompanied by mitochondria-mediated apoptosis. Our
findings clearly suggest that Qu may have potential as a new
chemopreventive or therapeutic agent for OSCC.
Acknowledgments
This study was partly supported by the Tri-Service General
Hospital, Grant No. TSGH-C100-007-009-10-S02, TSGHC100161, TSGH-C100-155, ND-I-29 and the Department of
Dental Hygiene, China Medical University, Grant No.
152
S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
CMU99-N1-04-1, and by the National Science Council, ROC.
and Grants No. NSC 99-2320-B-039-028-MY3 and NSC 1002320-B-016-009.
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