Introduction
Epidermal growth factor receptor (EGFR) is a
receptor tyrosine kinase in the cell transmembrane that serves an
essential role in proliferation, metastasis, angiogenesis and
chemo-resistance of oral cancer cells (1,2). More than
80% patients with oral cancer and oral cancer cell lines exhibit
overexpression of EGFR (3–6). High expression of EGFR protein in oral
cancer is associated with poor prognosis, decreased survival time
and increased metastatic potential (7,8).
Inhibition of EGFR and regulation of downstream signaling
represents a novel approach for oral cancer therapy (9,10). Various
strategies have been developed to disrupt EGFR function and to
interfere with downstream signaling (11,12).
Anti-EGFR mAbs and EGFR inhibitors have been investigated the most
extensively (3,5,13).
Cetuximab is an EGFR-targeting mAb and the first
novel targeted agent for oral cancer treatment to obtain Food and
Drug Administration approval in the United States (11,13). In
combination with chemotherapeutic agents, cetuximab has been
demonstrated to increase the overall survival rate of patients with
oral cancer, and to have less toxicity (7,10,14). In oral cancer therapy, combination of
cetuximab with cisplatin, 5-fluorouracil (5-FU), docetaxel
(Taxotere) or paclitaxel (Taxol) has become the new standard
advanced treatment (8,9,15,16).
Curcumin is a traditional Chinese medicine isolated
from the rhizome of Curcuma longa (17–20).
Curcumin has been shown to exert anti-inflammatory, anti-oxidant,
and anticancer effects, it is pharmacologically safe and has
minimal toxicity (17,19,20). The
anticancer activities of curcumin are attributable to its
anti-proliferative, anti-angiogenic, anti-metastatic, pro-apoptotic
and autophagic characteristics (21–25). In
vitro studies reported that curcumin inhibited cell
proliferation in various oral cancer cell lines, including CAL 27,
1483, SCC-1, SCC-9, KB, SAS and SCC15 (26). Curcumin also suppressed EGFR
expression and its downstream signaling molecules (NF-κB, JNK, p38
and ERK) which are vital for oral cancer pathogenesis (27–29).
Furthermore, curcumin enhanced cisplatin cytotoxicity in PE/CA-PJ15
cells in vitro (30). The
combination of 5-FU, doxorubicin or cisplatin with curcumin
exhibited inhibited proliferation and induced apoptotic cell death
of NT8e oral squamous cell carcinoma cells (31). However, the molecular mechanism of the
suppression of cell proliferation and apoptotic induction of
drug-resistant oral cancer cells following co-incubation with
cetuximab and curcumin remains poorly understood. Herein, the
synergistic effects and underlying molecular mechanism of the
effect of combined treatment of cetuximab and curcumin in
cisplatin-resistant oral cancer CAR cells was explored.
Materials and methods
Chemicals and reagents
Erbitux (the active ingredient of cetuximab) was
provided by Hualien Tzu Chi Hospital (Taiwan) and originally
purchased from Merck KGaA (Darmstadt, Germany). Dulbecco's modified
Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine and
penicillin-streptomycin solution were purchased from HyClone (GE
Healthcare, Logan, UT, USA). Caspase-3 and Caspase-9 colorimetric
assay kits were sourced from R&D Systems (Minneapolis, MN,
USA). All primary antibodies and anti-mouse/-rabbit immunoglobulin
G (IgG) horseradish peroxidase (HRP)-conjugated antibodies were
purchased from GeneTex (Hsinchu, Taiwan). Curcumin, Thiazolyl Blue
Tetrazolium Bromide (MTT) and other reagents were of analytical
grade from Sigma-Aldrich (Merck KGaA, Darmstadt Germany), unless
otherwise stated.
Cell culture
The human oral cancer cell line, CAL 27, was
obtained from American Type Culture Collection (ATCC; Manassas, VA,
USA). The cisplatin-resistant subline of CAL 27, CAR, was generated
in our laboratory, as previously described (32–34) and
exposed to increasing concentrations of cisplatin to generate a
stable subline with resistance to ≥80 µM cisplatin. CAR cells were
maintained in an environment of 5% CO2 at 37°C in DMEM
supplemented with 10% FBS, 2 mM L-glutamine, 100 µg/ml
streptomycin, 100 Units/ml penicillin and 80 µM cisplatin.
Cetuximab was diluted with cultured medium (DMEM with
supplementation as described above), and curcumin was dissolved in
dimethyl sulfoxide (DMSO).
Cytotoxicity assay
Cell viability was estimated by MTT assay. In brief,
CAR cells (1×104 cells/well) were plated in 96-well
tissue culture plates and treated with curcumin (10, 20, 40 or 50
µM), cetuximab (10, 20, 40 or 50 µg/ml) or 20 µg/ml cetuximab and
10, 20 or 40 µM curcumin for 24 h. Following exposure and removal
of the medium, the cells were cultured with 0.5 mg/ml MTT for an
additional 2 h. The blue formazan product was dissolved in 100 µl
DMSO and spectrophotometrically measured at a wavelength of 570 nm
using an ELISA plate reader (Anthos Labtec Instruments GmbH,
Salzburg, Austria), as previously described (35). The percentage of living cells was
calculated, and the ratio of optical density of the experimental
wells and control wells was calculated as % of control. Combination
index (CI) was determined using the Chou-Talalay method, as
previously described (36). A value
<1.0 indicated a synergistic effect.
Morphological determination
CAR cells (1×105 cells per well) were
seeded into a 24-well plate and treated with 20 µg/ml cetuximab and
10, 20 or 40 µM curcumin for 24 h. The cells were visualized using
a phase-contrast microscope to check for apoptotic characteristics
and photographed, as previously described (37).
Caspase-3 and −9 activity
measurement
CAR cells were seeded at a density of
5×106 cells per 75T flask and incubated with 20 µg/ml
cetuximab, 40 µM curcumin, or 20 µg/ml cetuximab and 40 µM curcumin
for 24 h. The cell lysate was collected, and the cell fraction was
analyzed for caspase-3/-9 activity using Caspase-3 and Caspase-9
Colorimetric Assay kits (R&D Systems, Inc., Minneapolis, MN,
USA), according to the manufacturer's protocol.
Western blot analysis
CAR cells (5×106 cells per 75T flask)
were treated with either 20 µg/ml cetuximab, 40 µM curcumin or both
for 24 h. Then, the cells were harvested and lysed with PRO-PREP
Protein Extraction Solution (iNtRON Biotechnology, Seongnam-si,
Gyeonggi-do, Korea). The protein concentration was determined using
the Pierce BCA protein assay kit (Thermo Fisher Scientific, Inc.,
Waltham, MA, USA), and 40 µg protein was loaded per lane of 8–10%
SDS-PAGE gels. The protein was thereafter transferred into
Immobilon-P Transfer Membranes (Merck Millipore, Billerica, MA,
USA). Each membrane was blocked in 5% non-fat dry milk in
phosphate-buffered saline with Tween-20 (PBST; 8 mM
Na2HPO4, 0.15 M NaCl, 2 mM
KH2PO4, 3 mM KCl, 0.05% Tween-20, pH 7.4) for
1 h. The membraned were then incubated at 4°C overnight with
primary antibodies against p-EGFR (cat. no. GTX61353), EGFR (cat.
no. GTX100448), p-ERK (cat. no. GTX59568), ERK (cat. no. GTX59618),
p-JNK (cat. no. GTX52326), JNK (cat. no. GTX52360), p-p38 (cat. no.
GTX48614), p38 (cat. no. GTX110720) (all 1:1,000 dilution), and
β-actin (cat. no. GTX109639) (1:5,000 dilution) (GeneTex).
Following washing with PBST, the membrane was incubated with
appropriate anti-mouse (cat. No GTX213111-01)/-rabbit (cat. no.
GTX213110-01) HRP-conjugated secondary antibodies (1:10,000
dilution) for 1 h at room temperature. Proteins were visualized by
enhanced chemiluminescence (Immobilon Western Chemiluminescent HRP
Substrate; Merck Millipore) and using the LAS-4000 imaging system
(Fuji, Tokyo, Japan), as previously described (38–40). The
density of the immunoblots was analyzed using ImageJ (version 1.47;
National Institutes of Health, Bethesda, MA, USA).
Statistical analysis
The values are presented as the mean ± standard
deviation of 3 independent experiments. Comparisons between the
drug-treated and -untreated groups were made using one-way analysis
of variance followed by Dunnett's test using SPSS software (version
16.0; SPSS, Inc., Chicago, IL, USA). P<0.001 was considered to
indicate a statistically significant difference.
Results
Effects of curcumin, cetuximab and
combination treatment on the viability of cisplatin-resistant oral
cancer CAR cells
The cytotoxicity of curcumin and cetuximab on CAR
cells. The cells were cultured with various concentrations of
curcumin (10, 20, 40 or 50 µM), or cetuximab (10, 20, 40 or 50
µg/ml), or 20 µg/ml cetuximab combined with 10, 20 or 40 µM
curcumin, for 24 h. Cell viability was evaluated by MTT assay. The
results demonstrated that curcumin markedly decreased the viability
of CAR cells in a concentration-dependent manner, and the viability
at 20, 40 and 50 µM was 87.1, 48.5 and 12.3%, respectively
(Fig. 1A). It was also revealed that
20, 40 and 50 µg/ml cetuximab reduced CAR-cell viability to 85.8,
72.8 and 67.4%, respectively, another concentration-dependent
effect (Fig. 1B). However, 10 µg/ml
cetuximab demonstrated no significant inhibition. Thus, CAR cells
were more sensitive to curcumin than that to cetuximab. The cells
were treated with a combination of 20 µg/ml cetuximab and 0, 10, 20
and 40 µM curcumin for 24 h, and significant potentiation of
cytotoxicity and synergy of CAR cells was demonstrated by
viabilities of 83.7, 75.7, 70.3 and 38.6%, respectively (Fig. 1C). The combination index (CI) was 0.9,
0.8 and 0.6 at treatments of 20 µg/ml cetuximab and 10, 20 and 40
µM of curcumin, respectively, indicating the synergistic effects of
cetuximab and curcumin. The results imply that a significant
increase in cytotoxic effects was achieved with simultaneous
administration of cetuximab and curcumin to CAR cells.
Effects of curcumin and cetuximab,
alone or combined, on the morphology of CAR cells
Photomicrographs demonstrated that combined
treatment resulted in cell shrinkage, cytoplasmic membrane blebbing
and cell death, compared with exposure to 20 µg/ml cetuximab alone
and untreated control (Fig. 2).
Furthermore, combined treatment resulted in increased inhibition in
viability of CAR cells compared with single-drug (cetuximab)
treatment and untreated control (Fig.
2). These data indicated that concurrent exposure to cetuximab
and curcumin synergistically induced apoptosis and reduced
proliferation of CAR cells.
Effects of curcumin and cetuximab,
alone or in combination, on caspase-3/-9-dependent apoptosis of CAR
cells
To further examine whether the observed suppression
of cell viability involved in apoptotic machinery, the cells were
treated with 20 µg/ml cetuximab, or 40 µM curcumin, or both, for 24
h prior to determination of caspase-3 and caspase-9 activities.
Individual treatment with cetuximab and curcumin induced 1.6- and
4.9-fold increases in caspase-3 activity compared with control,
whereas combination treatment stimulated a 6.8-fold increase in the
activity of caspase-3 of CAR cells (Fig.
3A). Similarly, caspase-9 activity was synergistically
increased in CAR cells when treated with cetuximab and curcumin
(6.0-fold increase; Fig. 3B).
However, either curcumin or cetuximab alone stimulated more minor
effects on caspase-9 activity, causing 1.5- and 3.6-fold increases
in CAR cells. These results indicate the synergistic cytotoxicity
of curcumin and cetuximab, and that the apoptotic mechanism was
caspase-3/-9-dependent in CAR cells.
Effect of curcumin and/or cetuximab
treatment on EGFR and MAPKs-regulated molecular signaling in CAR
cells
Cetuximab was reported to inhibit tumor growth,
invasion, angiogenesis and metastasis by binding to the
extracellular domain of EGFR by regulating the MAPKs pathway
(12,13). Furthermore, it has been documented
that colorectal cancer-cell resistance to cisplatin chemotherapy
may involve MAPK signaling (41). The
present study further analyzed the expression levels of EGFR and
the downstream MAPK pathway in CAR cells prior to cetuximab or
curcumin treatment, alone or in combination, using western blot
analysis. It was demonstrated that treatment combination treatment
effectively inhibited the phosphorylation of EGFR, but no effect on
total EGFR protein level was observed in CAR cells (Fig. 4). The levels of phosphorylated MAPKs
(ERK, JNK and p38) were also synergistically decreased compared
with cetuximab or curcumin treatment alone. However, there was no
effect on total ERK, JNK or p38 protein expression levels in any
treatment group (Fig. 4). Taken
together, these results suggest that combined use of cetuximab and
curcumin triggered a dramatic increase in CAR-cell apoptosis by
suppressing EGFR and MAPKs signaling (Fig. 5), suggesting that the synergic effects
resulted in enhanced oral anticancer activity compared with
single-drug treatment in resistant oral cancer cells.
 | Figure 4.Effects of curcumin and cetuximab on
EGFR and MAPK-regulated signaling in CAR cells. Protein expression
of p-EGFR, EFGR, p-ERK, ERK, p-JNK, JNK, p-p38 and p38 were
determined, and a β-actin control was applied to ensure equal
loading. Representative images were taken from 3 independent
experiments. EGFR, epidermal growth factor receptor; MAPK, mitogen
activated protein kinase; p-, phosphorylated; ERK, extracellular
signal-regulated kinase; JNK, c-Jun N-terminal kinase. |
Discussion
Surgery and brachytherapy are the major therapies
for oral cancer in the T1, T2 and artificial T3 groups
(Tumor-Node-Metastasis classification) in clinical practice
guidelines for head and neck cancer in Japan (42). Platinum-based chemotherapy (cisplatin
or carboplatin) is used for advanced stage cancer (42,43). In
1978, cisplatin was approved by the Food and Drug Administration
(FDA) for oral cancer treatment (44). Cisplatin is used for oral cancer
chemotherapy and functions via direct reaction with cellular
nucleophiles to achieve inter- and intra-stand DNA cross-links and
protein cross-links with DNA and RNA (45). However, oral cancer cells have gained
resistance to chemotherapeutic agents (46,47).
Several molecular mechanisms are involved in cisplatin-resistance:
i) Increased activity of transporter protein function (MDR1 or
p-glycoprotein); ii) activated drug metabolism activity by enzymes;
iii) decreased drug binding to DNA; iv) promoted ROS production; v)
stimulated DNA repair; vi) increased tolerance to DNA damage; vii)
altered transcription of target genes; viii) changes in cell
cycle-associated events and ix) inhibition of cell death (46–48).
Recently, EGFR has been demonstrated as an important therapeutic
target in oral cancer, and it is expressed more highly in oral
cancer tissue than in normal tissues (7,10). In
addition, a correlation between high EGFR expression and
radio-resistance was demonstrated in patients with oral cancer
(49). Kuroda et al (50) demonstrated that cisplatin-resistance
is associated with EGFR-mediated signaling in lung cancer A549
cells. Chemo-sensitivity to cisplatin was restored by an
EGFR-selective tyrosine kinase inhibitor (AG1478) in A549 cells,
suggesting that the EGFR inhibitor may be a therapy for
cisplatin-resistance (50). To the
best of our knowledge, this is the first study to report the
synergistic inhibitory effect of cetuximab (an EGFR inhibitor) and
curcumin in cisplatin-resistant human oral cancer cells.
Evidence indicates that the molecular mechanisms of
cetuximab anticancer activity take 2 forms (51,52).
Firstly, EGF binding to the EGFR extracellular domain to inhibit
subsequent receptor dimerization/activation and to induce EGFR
degradation is inhibited. Secondly, antibody-dependent cellular
cytotoxicity or complement-dependent cell-mediated cytotoxicity
(51,52). Curcumin has been historically used in
traditional Chinese medicine, and its anticancer effects on various
types of solid cancers, such as colon cancer, multiple myeloma and
pancreatic cancer have reached phase II and III clinical trials
(53,54). Curcumin is also a potential
therapeutic agent for the treatment of diabetic patients (55,56). It
has been reported that curcumin is able to directly but partially
inhibit the enzymatic activity of the EGFR intracellular domain.
Inhibition of EGFR phosphorylation and induction of EGFR
ubiquitination then block the EGFR signaling pathway in cancer
cells (57). However, it has not been
reported whether combined cetuximab with curcumin synergistically
inhibit drug-resistant oral cancer-cell proliferation. The present
results demonstrated that a combined treatment of cetuximab and
curcumin synergistically inhibited cell viability (Figs. 1B and 2), induced cell death (Fig. 2), and stimulated caspase-3 and
caspase-9 activities (Fig. 3).
Furthermore, curcumin dramatically enhanced cetuximab-suppressed
phosphorylation of EGFR, ERK, JNK, and p38 levels in CAR cells
(Fig. 4). A previous study by Son
et al (58) demonstrated the
effect of cetuximab combined with cisplatin on colon cancer growth.
Cetuximab significantly decreased phosphorylation of EGFR and
phosphorylated p38/p38 ratio at 30 µg/ml, but there was no effect
on the phosphorylated ERK/ERK ratio in colon cancer HCT116 cells.
The present study also demonstrated that cetuximab treatment
decreased the protein level of phosphorylated EGFR and
phosphorylated p38. In addition, Li et al (59) demonstrated that the EGFR monoclonal
antibody, cetuximab, mildly evoked apoptosis of human vulvar
squamous carcinoma A431 cells. This is consistent with the present
finding that cetuximab triggered a non-significant increase in
caspase-3 and caspase-9 activities in CAR cells. However, the
activities of caspase-3 and caspase-9 were dramatically enhanced in
CAR cells prior to treatment with cetuximab in combination with
curcumin; or with exposure only to curcumin. The present results
are also consistent with previous studies (26,31)
showing that curcumin is effective against various types of cancer
via intrinsic apoptotic function. Although curcumin possesses
powerful biological activities, it does not reach the criteria of a
good drug candidate because it lacks adequate water solubility and
high bioavailability, and undergoes rapid in vivo metabolism
(60). To overcome these limitations,
novel forms of curcumin targeting, including nanoparticles,
liposomes, cyclodextrin encapsulation, micelles and phospholipid
complexes, have been synthesized and tested in recent years
(61,62).
In conclusion, combined cetuximab and curcumin
treatment is a novel therapeutic option for oral cancer treatment,
exhibiting synergistic anti-proliferative activity. The mechanism
results in a decreased activated EGFR level in cisplatin-resistant
oral cancer cells. With the results presented in the present study,
curcumin could be used as an adjuvant drug, and the combination of
cetuximab and curcumin may be a strategy to pursue in clinical
trials.
Acknowledgements
The authors would like to thank Dr Hong-Yi Chiu of
Department of Pharmacy, Hualien Tzu Chi Hospital (Taiwan) for
providing cetuximab (Erbitux) as a gift.
Funding
The present study was supported by China Medical
University Hospital (grant no. DMR-107-1230).
Availability of data and materials
The datasets used and/or analyzed during the current
study are available from the corresponding author on reasonable
request.
Authors' contributions
CFC, HJH and TDW conceived and designed the
experiments; CFC, CCL, JHC, HYC, JSY, and CYL performed the
experiments. CFC, CCL, JHC, and JSY analysed the data; CFC, HJH and
TDW wrote and modified the manuscript. All authors read and
approved the final manuscript.
Ethics approval and consent to
participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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