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Table of ContentsAbstract:...........................................................................................................................................2
Background:.....................................................................................................................................3
Prostaglandins:............................................................................................................................3
The COX Enzyme.........................................................................................................................3
COX-2 Structure and Funcion:...................................................................................................4
COX Inhibitors:...........................................................................................................................5
Objective:.....................................................................................................................................7
Materials and Methods:...................................................................................................................7
Materials:....................................................................................................................................7
Isolation of the wild-type and Asn580-mutant COX-2 gene:.........................................................8
Transfection of COS-1 cells:.......................................................................................................8
Treatment of the COX-2 expressing cells with COX-2 inhibitors:..............................................8
ELISA for measuring downstream prostaglandin E2 (PGE2) levels:...........................................8
Results:............................................................................................................................................9
Successful transfection of COS-1 cells with the COX-2 gene:....................................................9
Figure 1: COX-2 activity in non-transfected (negative control) and COX-2 transfected COS-1 cells...........................................................................................................................10
Effect of glycosylation at Asn580on the efficacy of COX-2 inhibitors:.......................................10
Figure 2: Effect of COX-2 glycosylation on the efficacy of aspirin.................................11
Figure 3: Effect of COX-2 glycosylation on the efficacy of flurbiprofen........................13
Figure 4: Effect of COX-2 glycosylation on the efficacy of ibuprofen............................15
Figure 5: Effect of COX-2 glycosylation on the efficacy of celecoxib.............................17
Conclusions:..................................................................................................................................18
References:....................................................................................................................................19
1
Abstract:
Cyclooxygenase-2 (COX-2) is an enzyme that catalyzes the rate-limiting step in the
prostanoid synthesis pathway, which plays an important role in a variety of physiological and
pathophysiological processes including inflammation and cancer. COX-2 exists as two major
glycoforms of 72 and 74kDa, the latter resulting from an additional oligosaccharide chain at
amino acid residue Asn580. The purpose of this study is to determine if this additional
glycosylation affects the inhibitory ability of various COX-2 inhibitors. COS-1 cells were
transiently transfected with either the wild-type or Asn580-mutant COX-2 gene. Subsets of both
cell groups were treated with various concentrations of either aspirin, flurbiprofen, ibuprofen, or
celecoxib. After addition of the COX-2 substrate arachidonic acid to inhibitor-treated and
untreated (control) cells, media was collected and subjected to an ELISA which measured levels
of the downstream product prostaglandin E2. Results indicate that, at low concentrations, aspirin
and ibuprofen have a greater inhibitory effect on COX-2 when it is not glycosylated at Asn580.
This indicates that glycosylation of COX-2 at Asn580 influences the efficacy of certain COX-2
inhibitors.
2
Background:
Prostaglandins:
Prostaglandins are 20-carbon chains of unsaturated fatty acids that function as “local
hormones.” All cells, with the exception of the red blood cell, have the capacity to synthesize
prostaglandins [1-3]. Prostaglandins are not stored intracellularly, but rather are synthesized and
released immediately. Prostaglandins interact with G-proteins and mediate their effects through
signal transduction [1-3]. They are involved in several homeostatic and inflammatory processes.
In the cardiovascular system, prostaglandins participate in vasoconstriction and vasodilatation of
arteries, veins and capillaries and in platelet aggregation [1-3]. In the renal system, they are
involved in salt and water excretion [1-3]. They are also extremely important in the reproductive
system where they participate in ejaculation, sperm transport, the induction of labor, and in
ovulation [1-3].
The COX Enzyme
The COX enzyme catalyzes the rate-limiting step in the prostanoid synthesis pathway
that converts arachidonic acid into prostaglandin-G2 (PGG2) via a cyclooxygenase reaction and
then to prostaglandin-H2 (PGH2) via a peroxidase reaction. COX exists in two forms. COX-1 is a
housekeeping enzyme that is constituitively expressed under basal conditions in nearly all human
tissues and is responsible for mediating physiological functions such as platelet aggregation and
cytoprotection of the stomach [4]. COX-2 is expressed by many cell types. For example, COX-2
can be found in cells such as macrophages and monocytes that are involved in inflammatory
responses. The COX enzyme is said to be bifunctional in that it synthesizes both prostaglandins
3
and thromboxanes and also in that the COX isoenzymes are able to produce both physiological
and pathophysiological functions [4][5].
COX-2 Structure and Funcion:
The COX-2 gene is located on human chromosome 1 [6]. The COX-2 gene is about 8 kb
long and encodes for 604 amino acids [7]. The COX-2 protein has five potential glycosylation
sites; three are always glycosylated, one is never glycosylated, and one, Asn580, is glycosylated
50% of the time [4]. The inconsistency in glycosylation of Asn580 leads to the formation of two
different COX-2 glycoforms of 72kDa and 74kDa. These COX-2 glycoforms reside in the
membrane of the endoplasmic reticulum and the nuclear envelope [8].
COX -2 plays a role in many physiological and pathophysiological functions. It plays a
large role specifically in pain perception. During an injury or in the presence of a disease that
causes inflammation, the synthesis of COX-2- dependent prostaglandins is increased [9]. This
sensitizes peripheral nociceptor terminals which produce localized pain and hypersensitivity. For
this reason, COX-2 inhibitors have the capability of producing analgesic effects. One study
carried out by Stewart, et.al. showed that many patients who had taken non-steroidal anti-
inflammatory drugs (NSAIDs) for two or more years significantly reduced their risks for
developing Alzheimer’s [9]. In addition, it has been hypothesized and some studies support that
NSAIDs produce a chemopreventive effect against carcinogenesis, thus suppressing or even
preventing tumors [9].
Although known primarily for its role in pain perception and inflammatory responses,
COX-2 has also been found to have a role in various pathophysiological conditions such as
Alzheimer’s disease, rheumatoid arthritis, and many cancers such as breast, prostate, and
4
colorectal cancer [4]. COX-2 has many other positive physiological functions. It has been shown
to inhibit platelet aggregation thus providing a type of vascular protection [10]. COX-2 may have
a negative impact on the development of atherosclerosis [11]. COX-2 is also imperative to the
female reproductive organs. It is involved in the implantation of the ovum, the angiogenesis
needed for the establishment of the placenta, and in the induction of labor. It has also been
shown to play a positive role in bone metabolism and rennin secretion in the kidneys [12].
One study carried out by Stewart, et.al. showed that many patients who had taken non-
steroidal anti-inflammatory drugs (NSAIDs) for two or more years significantly reduced their
risks for developing Alzheimer’s [9]. In addition, it has been hypothesized and some studies
support that NSAIDs produce a chemopreventive effect against carcinogenesis, thus suppressing
or even preventing tumors [9].
COX Inhibitors:
COX-2 inhibitors can be classified into two distinct categories, selective and non-
selective. Selective COX-2 inhibitors directly target the COX-2 enzyme and were developed
with the goal of providing the anti-inflammatory and analgesic efficacy of traditional NSAIDs
but with a decrease in the GI injury and in the anti-platelet activity associated with traditional
NSAIDs. Non-selective inhibitors simply target cyclooxygenase activity and do not significantly
differentiate between COX-1 and COX-2 [17].
Aspirin is a non-selective COX inhibitor that irreversibly inactivates both COX-1 and
COX-2 by acetylating a serine in the active site and thus interfering with the binding of
arachidonic acid to the COX active site [4]. Aspirin has widely been used for its analgesic
effects. However, more recently, aspirin has been recommended in low doses to patients with
5
atherosclerotic disease due to its role in preventing platelet aggregation and ability to reduce the
risk of myocardial infarctions (heart attacks) in high risk patients [18].
Ibuprofen is a non-selective reversible competitive COX inhibitor that competes with
arachidonic acid for the COX active site. Like aspirin, ibuprofen has also been used extensively
as a pain-reliever and fever reducer. Studies published by Harris et al., however, have recently
shown that individuals who took ibuprofen at a dose of greater than 600 mg three times per week
or more for at least 1 year had a 43% decreased risk of developing breast cancer compared to
controls. This indicates that ibuprofen may be a potential drug of interest in the prevention of
certain cancers [19].
Flurbiprofen is an example of a time-dependant, reversible COX inhibitor. Inhibition is
caused by the formation of a salt bridge between the carboxylate of the drug and arginine120.
Conformational changes then take place. This causes the reversibility of the reaction to be
slowed [4].
Celecoxib is a selective COX-2 inhibitor which makes it different from the classic
NSAID COX-2 inhibitors. The therapeutic benefit of traditional NSAIDs results from the
inhibition of COX-2 at sites of inflammation, whereas many of their adverse effects (GI toxicity
and nephrotoxicity) are primarily due to inhibition of COX-1 [21]. Celecoxib is known
commercially as Celebrex and has a somewhat controversial past. It emerged in the
pharmaceutical market at approximately the same time as refecoxib (Vioxx) as a treatment for
rheumatoid arthritis and osteoarthritis. However, many patients taking these drugs ultimately
were found to have a higher incidence of cardiovascular disease [22]. It was hypothesized that
COX-2 inhibitors, such as Celebrex and Vioxx, may differentially alter the balance between
platelet aggregation and the endothelial-mediated inhibition of aggregation [22]. In 2004, Vioxx
6
was taken off the market, and the Food and Drug Administration (FDA) warned physicians to
limit the number of prescriptions for celecoxib after evidence emerged indicating that celecoxib
causes increased heart risks. It has remained on the market due to its effectiveness in treating
rheumatoid arthritis and several forms of cancer but is still closely watched by the FDA [22].
Objective:
Studies done by Mary Sevigny, et.al, indicate that glycosylation of COX-2 at Asn580 plays
a significant role in regulating COX-2 turnover. This creates a need to better understand COX-2
inhibition in order to prevent negative side effects and to observe the possible connection
between this glycosylation and the effectiveness of COX-2 inhibitors. The purpose of this study
was to determine if additional glycosylation at Asn580 affects the inhibitory ability of various
COX-2 inhibitors.
Materials and Methods:
Materials:
The human COX-2 cDNA in plasmid pcDNA3 was generously provided by Dr. Timothy
Hla from the University of Connecticut, USA. The Asn580 -mutant COX-2 gene was previously
prepared by Dr. Mary B. Sevigny at the Veterans Affairs Medical Center in San Francisco, CA
[5]. The COS-1 cell line was purchased from UCSF Cell Culture Facility. One Shot TOP10
Competent Escherichia coli cells were purchased from Invitrogen (Carlsbad, CA). The QIAPrep
Spin Miniprep Kit and HiSpeed Plasmid Maxi kit were both purchased from QIAGEN
(Valencia, CA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and
penicillin/streptomycin were purchased from Hyclone (Logan, UT). Transfection reagent
TransIT-LT1 Reagent was purchased from Mirus Bio (Madison, WI). The arachidonic acid,
7
ibuprofen, and Prostaglandin E2 EIA-monoclonal kit were all purchased from Cayman Chemical
(Ann Arbor, MI). Aspirin and flurbiprofen were purchased from Sigma Aldrich (St. Louis, MO).
Isolation of the wild-type and Asn580-mutant COX-2 gene:
The wild-type and Asn580-mutant COX-2 genes were propagated in TOP10 Escherichia
coli cells, and pure cultures carrying the plasmids were identified using the QIAprep Spin
Miniprep Kit followed by DNA gel electrophoresis according to manufacturer’s instructions.
QIAGEN’s Hi-Speed Plasmid Maxi Kit was then used to isolate large amounts of the pure wild-
type and Asn580-mutant COX-2 plasmids following the manufacturer’s instructions.
Transfection of COS-1 cells:
COS-1 cells were grown on 6-well plates in DMEM with 5% FBS, 4 mM L-glutamine,
and antibiotics at 37°C, 5% CO2. TransIT-LT1 Reagent was then used to transiently transfect
cells with either the wild-type or Asn580-mutant COX-2 gene according to the manufacturer’s
instructions. Cells were incubated at 37°C, 5% CO2 for ~48 hours in the presence of the
TransIT-LT1/COX-2 DNA complex. Media was then replaced with DMEM, 1% FBS, 4 mM L-
glutamine, and antibiotics, and the cells continued their incubation.
Treatment of the COX-2 expressing cells with COX-2 inhibitors:
Three days after transfection, cells were treated with either aspirin, ibuprofen, celecoxib,
or flurbiprofen. Some cells remained untreated and were controls. Cells were incubated at 37º C.
for one hour. The cells were then treated with the COX-2 substrate arachidonic acid (5mg/ml)
and continued their incubation at 37º C, 5% CO2 for an additional two hours. After the two hour
8
incubation, the media was removed from each sample, frozen, and stored at -70º C for later
analysis of PGE2 levels.
ELISA for measuring downstream prostaglandin E2 (PGE2) levels:
Downstream PGE2 levels were measured from the saved media samples using the
Prostaglandin E2 EIA- Monoclonal kit according to the manufacturer’s instructions.
Results:
Successful transfection of COS-1 cells with the COX-2 gene:
COX-2 activity was determined in COS-1 transfected cells by measuring PGE2 levels in
the media using an ELISA. PGE2, which is much more stable than the COX-2 product PGH2, is a
downstream product of the COX-2 pathway and thus a reliable indicator of COX-2 activity.
Figure 1 indicates that transfection of the COS-1 cells with either the wildtype (72/74
kDa glycoforms) or Asn580-mutant (70/72 kDa glycoforms) COX-2 gene was successful. COS-1
cells do not express endogenous COX-2 and therefore provide a convenient model for studying
the COX-2 gene. These data also show that the 70/72 kDa glycoforms’ activity was almost four
times that of the 72/74 kDa glycoforms. The mutation at Asn580 leads to an increase in total COX-
2 activity; which is consistent with previously published findings [5].
9
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Neg. Control
72/ 74 kDa
70/ 72 kDa
PG
E2
Synt
hesi
s (p
g/m
l)
Figure 1: COX-2 activity in non-transfected (negative control) and COX-2 transfected COS-1 cells. After transfection of COS-1 cells with either the wild-type (72/74 kDa) or mutant (70/72 kDa) COX-2 gene, the cells were treated with arachidonic acid. The media was then analyzed for PGE2 using an ELISA.
Effect of glycosylation at Asn580on the efficacy of COX-2 inhibitors:
Figure 2A represents the overall effect of aspirin over a broad concentration range (1-50
µM) on both the 72/74 kDa and the 70/72 kDa COX-2 glycoforms. Because the effect of the
inhibitor eventually plateaus, the slope of the linear portion of the graph, as seen in Figure 2B, is
used to compare the effect of aspirin between the Asn580-glycosylated and unglycosylated forms.
Figure 2B indicates that aspirin had a two-fold greater effect on the 70/72 kDa glycoform than on
the 72/74 kDa glycoforms.
10
0 10 20 30 40 50 600.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
72/74 kDa70/72 kDa
Concentration of Aspirin (µM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)A
0 5 10 150.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
f(x) = − 262.769094865581 x + 4879.19612111036
f(x) = − 127.614311916395 x + 1829.41013088377
72/74 kDaLinear (72/74 kDa)70/72 kDaLinear (70/72 kDa)
Concentration of Aspirin (µM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)
B
Figure 2: Effect of COX-2 glycosylation on the efficacy of aspirin. (A) Three days after transfection, COS-1 cells transfected with either the wild-type (72/74kDa) or mutant (70/72 kDa) gene were treated with 1,5,10,25, and 50 μM aspirin and then treated with the COX-2 substrate arachidonic acid. The media was analyzed for PGE2 concentration using ELISA. (B) Linear portion of graph (0-10 μM) (n=3). Results are representative of 3 independent experiments.
11
The effect of flurbiprofen (0.02-20 µM) on both the 72/74 kDa and the 70/72 kDa COX-2
glycoforms is shown in Figure 3A. As with the aspirin data, only the linear portion of the graph,
from 0-0.2mMm is used to compare the effect of flurbiprofen between the two different
glycoform groups. Figure 3B indicates that unlike aspirin, flurbiprofen inhibited the COX-2
glycoforms almost equally.
12
0.00 5.00 10.00 15.00 20.00 25.000.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
72/74 kDa70/72 kDa
Concentration of Flurbiprofen (µM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)A
13
0.00 0.05 0.10 0.15 0.20 0.250.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
f(x) = − 15637.1808900467 x + 6827.29533411793
f(x) = − 13313.1576024007 x + 4589.7877445022572/74 kDaLinear (72/74 kDa)70/72 kDaLinear (70/72 kDa)
Concentration of Flurbiprofen (µM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)
B
Figure 3: Effect of COX-2 glycosylation on the efficacy of flurbiprofen. (A) Three days after transfection, COS-1 cells transfected with either the wild-type (72/74kDa) or mutant (70/72 kDa) gene were treated with 0, 0.02,0 .2, 2, 10, and 20 μM flurbiprofen and then treated with the COX-2 substrate arachidonic acid. The media was analyzed for PGE2 concentration using ELISA. (B) Linear portion of the graph (-0.2 μM) (n=3). Results are representative of 3 independent experiments.
Ibuprofen, just as with flurbiprofen, was tested over the concentration range of 0.02-20
µM (Fig. 4A). When comparing the linear portion of the curve (Figure 4B), ibuprofen appears to
be greater than two times as effective on the 70/72 kDa glycoforms as opposed to the 72/74 kDa
glycoforms.
14
0 5 10 15 20 250.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
3500.0
72/74 kDa70/72 kDa
Concentration of Ibuprofen (µM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)A
0 0.5 1 1.5 2 2.50.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
3500.0
f(x) = − 1192.55335802382 x + 3078.24652188349
f(x) = − 547.058853983283 x + 1317.91574863181
72/74 kDaLinear (72/74 kDa)70/72 kDaLinear (70/72 kDa)
Concentration of Ibuprofen (µM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)
B
Figure 4: Effect of COX-2 glycosylation on the efficacy of ibuprofen. (A) Three days after transfection, COS-1 cells transfected with either the wild-type (72/74kDa) or mutant (70/72 kDa) gene were treated with 0.02, 0.2, 2, 10, and 20 μM ibuprofen and then treated with the COX-2 substrate arachidonic acid. The media was analyzed for PGE2 concentration using ELISA. Linear portion of graph (0-2 μM) (n=3). Results are representative of 3 independent experiments.
15
Finally, Figure 5 demonstrates the effect of celecoxib on the 72/74kDa and 70/72kDa
COX-2 glycoforms. As determined by Figure 5A, the linear inhibition range occurs between 0
and 10 µM celecoxib. When this region of the curve is used to compare the effect of celecoxib
between the two different glycoform groups, there doesn’t appear to be a real significant
difference (Fig. 5B). In other words, the presence or absence of glycosylation of COX-2 at Asn580
does not to appear to enhance or interfere with the inhibitory ability of celecoxib.
16
0.00 100.00 200.00 300.00 400.00 500.000.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
3500.0
72/74 kDa70/72 kDa
Concentration of Celecoxib (nM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)A
0.00 2.00 4.00 6.00 8.00 10.00 12.000.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
3500.0
f(x) = − 155.790171131743 x + 2920.90228178673
f(x) = − 112.663694885359 x + 1379.03247371568
72/74 kDaLinear (72/74 kDa)70/72 kDaLinear (70/72 kDa)
Concentration of Celecoxib (nM)
Syn
thes
is o
f P
GE
2 (p
g/m
l)
B
Figure 5: Effect of COX-2 glycosylation on the efficacy of celecoxib. (A) Three days after transfection, COS-1 cells transfected with either the wild-type (72/74 kDa) or mutant (70/72 kDa) gene were treated with 1, 10, 50, 100, and 500 μM celecoxib and then treated with the COX-2 substrate arachidonic acid. The media was analyzed for PGE2 concentration using ELISA. (B) Linear portion of graph (0-10 μM) (n=3). Results are representative of 3 independent experiments.
17
Conclusions:
All of the inhibitors, regardless of whether they were selective, non-selective, reversible,
or irreversible, had a large inhibitory effect on both the 70/72 kDa and the 72/74 kDa glycoform
groups. However, the inhibitory effects of ibuprofen and aspirin, at low concentrations, were
greater on the 70/72 kDa glycoforms. This indicates that the glycosylation at Asn580, which
results in the expression of the 74 kDa glycoform, decreases the effictiveness of ibuprofen and
aspirin.
COX-2 is the center of a lot of research and debate. After the designer drug Vioxx was
taken off the market due to its negative physiological effects such as congestive heart failure
[22], it became extremely obvious that chronic use of drugs that stifle COX-2 activity leads to
serious health problems. Treatments must be devised that will stop the effects of COX-2 over-
expression in COX-2-related diseases (such as arthritis or colon cancer) without interfering with
normal COX-2 functions. This current study reveals a further complexity of COX-2 inhibition,
specifically, that the type of COX-2 glycoform expressed in a particular pathophysiological
condition may determine how effective certain COX-2 inhibitors will be. Future treatments must
therefore be designed with this complexity in mind.
18
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