Simultaneously Targeting Epidermal Growth Factor Receptor
Tyrosine Kinase and Cyclooxygenase-2, an Efficient
Approach to Inhibition of Squamous Cell
Carcinoma of the Head and Neck
Zhuo (Georgia) Chen,1Xin Zhang,1
Mengfeng Li,4Zhiqiang Wang,2
H. Samuel Wieand,3Jennifer R. Grandis,5and
Dong M. Shin1
1Department of Hematology/Oncology, Winship Cancer Institute,
Emory University, Atlanta, Georgia;2Division of Hematology-
Oncology,3Biostatistics Facility, and4Department of Pathology,
University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania;
and5Department of Otolaryngology, University of Pittsburgh School
of Medicine, Pittsburgh, Pennsylvania
Purpose: Epidermal growth factor receptor (EGFR)
and cyclooxygenase-2 (Cox-2) contribute to development of
squamous cell carcinoma of the head and neck (SCCHN).
Simultaneously blocking both EGFR and Cox-2–mediated
pathways may be an efficient means of inhibiting cancer cell
growth in SCCHN.
Experimental Design: A combination of EGFR-selective
tyrosine kinase inhibitors (TKIs) AG1478 or ZD1839 (Iressa
or gefitinib) with a Cox-2 inhibitor (Cox-2I) celecoxib (Ce-
lebrex) was studied for its effects on cell growth, cell cycle
progression, and apoptosis in SCCHN cell lines by cell
growth assay, clonogenic assay, flow cytometric analysis,
and terminal deoxynucleotidyl transferase-mediated nick
end labeling assay. A potential effect of EGFR TKIs and
Cox-2I on angiogenesis was examined by endothelial capil-
lary tube formation assay. Primary and secondary targets of
EGFR TKIs and Cox-2I were also examined using immuno-
blotting and immunoprecipitation after the combined treat-
Results: The combination of AG1478 or ZD1839 with
celecoxib either additively or synergistically inhibited
growth of the five SCCHN cell lines examined, significantly
induced G1arrest and apoptosis, and suppressed capillary
formation of endothelium. Furthermore, the combination
showed strong reductions of p-EGFR, p-extracellular signal-
regulated kinase 1/2, and p-Akt in SCCHN cells as compared
with the single agents. Both AG1478 and ZD1839 inhibited
expression of Cox-2 protein, whereas celecoxib mainly
blocked the production of prostaglandin E2.
Conclusions: These results suggest that cell growth in-
hibition induced by a combination of EGFR TKIs and
Cox-2I is mediated through simultaneously blocking EGFR
and Cox-2 pathways. This combination holds a great poten-
tial for the treatment and/or prevention of SCCHN.
Approximately 40,000 new cases of squamous cell carci-
noma of the head and neck (SCCHN) occur in the United States
each year, with a death rate of ?12,000 patients annually (1).
The survival rate for the patients with SCCHN remains poor
despite advances in diagnosis and treatment (2). Head and neck
cancers usually develop in area of the carcinogen-exposed epi-
thelium and likely result from the accumulation of cellular and
genetic alterations, leading to aberrant expression of many pro-
teins involved in cell growth regulation (3–5). Blockade or
modification of the function of one or several of these proteins
may impede or delay the development of cancer. An effective
preventive approach that could be implemented before the de-
velopment of invasive cancer to reduce the incidence of SCCHN
would be highly desirable. Among the potential chemopreven-
tive agents, retinoids have been investigated extensively as
treatment for premalignant lesions of the upper aerodigestive
tract (6, 7). However, toxicity is significant, and the duration of
responses has been limited. Thus, a new strategy is needed for
chemoprevention of upper aerodigestive tract malignancies.
As a potential investigational approach for prevention and
treatment of SCCHN, we studied a combination of two types of
molecular targeted agents; epidermal grow factor receptor
(EGFR)-selective tyrosine kinase inhibitors (TKIs) AG1478 and
ZD1839 (Iressa or gefitinib; AstraZeneca Pharmaceuticals,
Cheshire, United Kingdom), and the cyclooxygenase-2 inhibitor
(Cox-2I) celecoxib (Celebrex; Pharmacia Corporation/Pfizer,
Inc., G. D. Searle & Co., Chicago, IL). These two types of
agents act on different biological targets: tyrosine phosphoryl-
ated EGFR (p-EGFR) and Cox-2, respectively. Both targets
have been shown to contribute to SCCHN carcinogenesis.
EGFR is a 170-kDa transmembrane protein with intrinsic tyro-
sine kinase activity that regulates cell growth in response to
binding of its ligands such as EGF and transforming growth
factor ?. EGFR expression has been documented extensively in
a wide variety of malignant tumors, including SCCHN. Over-
expression of EGFR and its ligand transforming growth factor ?
Received 12/3/03; revised 5/12/04; accepted 5/19/04.
Grant support: National Cancer Institute Grant U01 CA101244
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Dong M. Shin, Winship Cancer Institute, Emory
University. 1365-C Clifton Road, Suite C3090, Atlanta, GA 30322. Phone:
(404) 778-5990;Fax:(404) 778-5520;E-mail:dong_shin@emoryhealthcare.
©2004 American Association for Cancer Research.
5930 Vol. 10, 5930–5939, September 1, 2004
Clinical Cancer Research
was observed in 80 to 100% of SCCHN specimens (8–11). Use
of EGFR-selective TKIs has been one of the approaches to
block EGFR activity in both preclinical and clinical studies (12).
Cox catalyzes the synthesis of prostaglandins (PGs) from
arachidonic acid. Two members of the Cox family have been
identified. Cox-1 is constitutively expressed in most tissues and
responsible for the synthesis of PGs that mediate normal phys-
iologic functions (13). In contrast, Cox-2 expression is not
detected in most normal tissues. It is induced by inflammatory
or mitogenic stimuli such as cytokines, growth factors, tumor
promoters, and viral infection, resulting in increased synthesis of
PGs in inflamed or neoplastic tissues (14). Cox-2 is overexpressed
expression is found to be up-regulated at both mRNA and protein
levels (16, 17). Treatment using Cox-2Is in cancer chemopreven-
tive trials reduced the risk of developing familial adenomatous
polyposis, some of which will inevitably progress to full-fledged
colon cancer (18). Currently, there are ?20 ongoing cancer che-
mopreventive trials using Cox-2Is, including celecoxib (19).
In general, combination therapies have proven to be more
effective than single agents in the prevention and treatment of
cancer. They not only enhance clinical response but also dimin-
ish the probability of developing drug resistance. There have
been some promising results with combination chemoprevention
strategies for cancer (20, 21). In the current study, we evaluated in
TKIs (i.e., AG1478 or ZD1839) and a Cox-2I (i.e., celecoxib) on
SCCHN cells. We also examined protein levels of primary targets
of EGFR TKI and Cox-2I, p-EGFR and Cox-2, respectively, and
downstream signaling molecules of EGFR and Cox-2–mediated
pathways after the combined treatment.
MATERIALS AND METHODS
Cell Lines and Reagents.
were used in this study. Cell line Tu177 was established from
the larynx. Tu212 and 212LN were established from a primary
hypopharyngeal tumor and a lymph node metastasis, respec-
tively, from a single patient. Cell lines 686LN and 886LN were
established from lymph node metastases of SCC of the tongue
and larynx, respectively. These cell lines were obtained from Dr.
Gary L. Clayman (University of Texas M. D. Anderson Cancer
Center, Houston, TX) and Dr. Peter G. Sacks (New York
University College of Dentistry, New York, NY; refs. 22, 23).
They were grown in DMEM:Ham’s F-12 (1:1) with supple-
mented 10% fetal bovine serum.
Of the EGFR-selective TKIs, AG1478 was purchased from
Calbiochem (San Diego, CA), and ZD1839 (Iressa or gefitinib)
was provided by AstraZeneca Pharmaceuticals. Both AG1478
and ZD1839 are competitive inhibitors for ATP binding in the
TK domain of EGFR. The Cox-2 inhibitor celecoxib was pro-
vided by Pharmacia Corporation/Pfizer, Inc., G. D. Searle & Co.
All three drugs can be dissolved in DMSO in appropriate
concentrations and stored at ?20°C until use.
Cell Growth Assay.
SCCHN cell lines were plated at a
concentration of 5 ? 103cells/well into 96-well plates in quad-
ruplicate. Twenty-four hours later, the drugs were added in a
range of concentrations as single agents [AG1478 (0–30 ?mol/
L); ZD1839 (0–10 ?mol/L); and celecoxib (0–100 ?mol/L)]. In
Several SCCHN cell lines
another experiment, cells were treated with two drugs in fixed
concentrations (AG1478 at 10 ?mol/L or ZD1839 at 0.5 ?mol/L
plus celecoxib at 25 ?mol/L). Cell growth inhibition was meas-
ured by determining cell density with sulforhodamine B assay
(24) at 72 hours after addition of the drugs. Percentage of
inhibition was determined by comparison of cell density in the
drug-treated cells with that in the untreated cell controls in the
same incubation period (percentage of inhibition ? 1 ? cell
density of a treated group per cell density of the control group).
All experiments were repeated three times.
To study effect of the EGFR-selec-
tive TKIs and the Cox-2I on clonogenicity of SCCHN cells,
exponentially growing cells from two cell lines, Tu177 and
686LN, were seeded into 6-well plates at concentration of 2 ?
103/well in triplicates. After 24 hours, ZD1839 and celecoxib
were added as either single or double agents in concentrations of
0.2 and 20 ?mol/L, respectively. An equivalent amount of
DMSO used to dissolve both agents was added to the control
cells. The cells were incubated in the presence of the drugs for
10 to 14 days to form colonies. The cell colonies were stained
in crystal violet (0.5%). They were counted under a microscope
using the standard definition of a colony that should contain at
least 50 cells. The experiment was repeated twice.
Soft agar clonogenic assay was also performed. SCCHN
cells from two cell lines, Tu177 and 686LN, were plated into
6-well plates at concentration of 10 ? 103/well in 0.5% agarose
with 1% agarose underlay in triplicates. ZD1839 and celecoxib
were mixed with the top agarose as either single or double
agents at final concentration of 0.1 and 10 ?mol/L, respectively,
before plating of the cells. The equivalent amount of DMSO was
also mixed with the top agar and SCCHN cells as the control.
Cells were incubated for 21 days, and colonies ? 0.02 mm were
counted. The total numbers of the colonies were recorded as
average of three counts with a SD as indicated in Table 1. The
experiment was repeated twice.
Flow Cytometric Analysis.
selective TKIs and Cox-2I were analyzed for both cell cycle and
apoptosis using flow cytometry. Tu177 cells were incubated
with AG1478 (5 ?mol/L), ZD1839 (0.5 ?mol/L), and celecoxib
(25 ?mol/L), alone, as single agents, or with combinations of
either AG1478/celecoxib or ZD1839/celecoxib for 72 and 96
hours. The cells were stained with propidium iodide (200 ?g/
mL) and RNase A (10 ?g/mL) and then analyzed by flow
cytometry (Epics XL-MCL; Beckman Coulter, Miami, FL). Cell
The effects of the EGFR-
Numbers of colonies in clonogenic assay
ZD ? celecoxib
ZD ? celecoxib
110 ? 27 (100)
37 ? 6.6 (34)
8 ? 2.6 (7)
0 ? 0 (0)
115 ? 8.5 (100)
55 ? 12 (47)
10 ? 5.2 (9)
5 ? 2.0 (4)
217 ? 19 (100)
163 ? 22 (75)
29 ? 0.6 (13)
0.7 ? 0.6 (0.3)
104 ? 14 (100)
56 ? 4.9 (54)
21 ? 7.0 (20)
9.3 ? 3.7 (0.9)
* Average ? SD with percent control.
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