Cyclooxygenase-2 inhibition inhibits PI3K/AKT kinase activity in epithelial ovarian cancer

Cyclooxygenase-2 inhibition inhibits PI3K/AKT kinase activityin epithelial ovarian cancer

Shahab Uddin1, Maqbool Ahmed1, Azhar Hussain1, Lina Assad2, Fouad Al-Dayel2, Prashant Bavi1, Khawla S. Al-Kuraya1

and Adnan Munkarah3

1 Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia2 Department of Pathology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia3 Department of Gynecology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia

Cyclooxygenase-2 (COX-2) expression contributes to tumor growth and invasion in epithelial ovarian cancer (EOC). COX-2

inhibitors exhibit important anticarcinogenic potential against EOC, but the molecular mechanisms underlying this effect and

relation with PI3-kinase/AKT signaling remain the subject of intense investigations. Therefore, the role of COX-2 in EOC and

its cross talk with PI3-kinase/AKT pathway were investigated using a large series of EOC tissues in a tissue micro array (TMA)

format followed by in vitro and in vivo studies using EOC cell lines and NUDE mice. Clinically, COX-2 was overexpressed in

60.3% of EOC and was significantly associated with activated AKT (p < 0.0001). Cox-1 expression was seen in 59.9% but did

not associate with AKT. Our in vitro data using EOC cell line showed that inhibition of COX-2 by aspirin, selective inhibitor

NS398 and gene silencing by COX-2 specific siRNA impaired phosphorylation of AKT resulting decreased downstream

signaling leading to cell growth inhibition and induction of apoptosis. Finally, treatment of MDAH2774 cell line xenografts

with aspirin resulted in growth inhibition of tumors in NUDE mice via down-regulation of COX-2 and AKT activity. These data

identify COX-2 as a potential biomarker and therapeutic target in distinct molecular subtypes of ovarian cancer.

More than 85% of human ovarian cancers are epithelial ovar-ian carcinomas (EOCs), which originate from ovarian surfaceepithelial (OSE) cells.1 The high mortality rate associatedwith epithelial ovarian cancer seems to be multifactorial.First, there is a lack of effective screening and early detectionstrategies; therefore, a high percentage of patients are diag-nosed in advanced stage disease.2 In addition, despite goodresponse to primary surgery and chemotherapy treatments,recurrence rates are high and available salvage therapies arenot curative.3–6

Although the exact etiology of EOCs is not determined,an inflammatory process is one factor suggested to participatein tumorigenesis.7 Some studies have reported an associationbetween chronic inflammatory conditions caused by talc orasbestos exposure, endometriosis or other pelvic inflamma-tory diseases and an increased incidence of EOC.8 In addi-tion, repetitive incessant ovulation has been identified as one

of the major risk factors for developing EOC.9 Since severalclassical mediators of inflammation participate in the ovula-tory process, one can speculate that each ovulation event rep-resents a localized inflammatory reaction.10 The importanceof inflammation/ovulation in ovarian tumorigenesis is furthersupported by the observed reduction in the relative risk ofovarian cancer in women with a decrease in the total numberof ovulations during a lifetime, whether due to childbearingor the use of contraceptive pills.11 In addition, evidence fromin vitro and in vivo studies suggests an important role forprostaglandins and their synthesizing enzyme COX in ovar-ian carcinogenesis. Cyclooxygenases (COX-1 and -2) are keyenzymes in the production of prostaglandins (PGs), whichare short-lived lipid signaling molecules involved in a num-ber of biologic functions.12 COX-1 expression is generallyconstitutive, whereas COX-2 is usually induced by stimuliinvolved in inflammatory responses. Prostaglandin E2(PGE2), a primary metabolite of COX-2, has been shown topromote cell survival, proliferation, and angiogenesis andprohibit apoptosis, all processes influencing cancer develop-ment.13 Recently COX-2 overexpression has been shown aslinking factor of chronic inflammation with neoplasticchanges.14 Several reports have shown that either COX-1 orCOX-2 is upregulated in EOC.15–20 Ovarian tumors withincreased content of COX-2 were associated with chemother-apy resistance and poor prognosis while COX-1 was sug-gested to participate in neovascularization.17,21,22 However,how cyclooxygenase (COX2) enhances ovarian cancer pro-gression remains poorly understood.

Key words: Cox-2, AKT, apoptosis, ovarian cancer

Additional Supporting Information may be found in the online

version of this article

The first two authors contributed equally to this work

DOI: 10.1002/ijc.24757

History: Received 21 Dec 2008; Accepted 2 Jul 2009; Online 20

Jul 2009

Correspondence to: Shahab Uddin, Human Cancer Genomic

Research, Research Center, King Faisal Specialist Hospital and

Research Cancer, MBC 98-16, P.O. Box 3354, Riyadh 11211, Saudi

Arabia, Fax: þ966-1-205-5170, E-mail: [email protected]

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Int. J. Cancer: 126, 382–394 (2010) VC 2009 UICC

International Journal of Cancer

IJC

Epidemiologic studies to examine whether nonsteroidolalanti-inflammatory drugs (NSAIDs) prevent or delay the de-velopment of ovarian cancers remain inconclusive. Althoughseveral population- and hospital-based case–control studiespresent evidence for time- and dose-dependent decreases inthe risk of developing ovarian cancers with the consumptionof several NSAIDs,23 other studies failed to find any signifi-cant correlation or found that the reduction in risk was asso-ciated only with the use of particular NSAIDs.24 MostNSAIDs currently in use inhibit both cyclooxygenase (COX-1) and COX-2 at their recommended dosages.25–27

Several investigators have shown that inhibition of COX2activity is cytotoxic to cancer cells in vitro and in vivo.28

Some have reported that these cytotoxic effects are inducedby modulating the activity PI3K/AKT signaling pathway.29,30

It is known that upon activation of PI3K pathway a numberof signaling proteins will be phosphorylated including proteinkinase B (AKT). AKT, once activated exert its oncogenic andantiapoptotic activity by activating several downstream tar-gets such as Bad, Foxo transcription factors and GSK3.

There are conflicting reports regarding role of COX-2 inthe pathogenesis of ovarian cancer. A recent study suggestedthat cellular proliferative activity in ovarian tumors is unre-lated to COX-231; however, there are several other studiesthat indicated a role for COX-2 in ovarian carcinogesis.32,33

This prompted us to analyze the expression of COX-2 pro-tein in a large series of EOC and explore its prognostic sig-nificance. In addition, in vitro and in vivo studies were per-formed to delineate the possible mechanisms by which COX-2 contributes to ovarian cancer pathogenesis.

Material and methodsPatient selection

One hundred fifty-six patients with ovarian carcinoma diag-nosed between 1991 and 2007 were selected from the files ofthe King Faisal Specialist Hospital and Research Centre. Allsamples were analyzed in a tissue microarray (TMA) format.The Institutional Review Board of the King Faisal SpecialistHospital and Research Centre approved the study.

The patients included in this study had their diagnosis,treatment and follow-up care in the departments of Obstet-rics and Gynecology and Oncology at King Faisal SpecialistHospital and Research Centre. The histological subtype ofeach ovarian tumor sample was determined according toestablished criteria.34 The distribution of tumors by histologictype was as follows: 125 serous (80.1%), 22 endometrioid(14.1%), 4 clear cell (2.6%) and 5 undifferentiated/mixed Epi-thelial (3.2%). The median age of the patient population was56 years with a range from 19 to 86 yr. The majority ofpatients underwent primary surgical staging or cytoreduction.In some patients who were not fit for primary surgery, pri-mary neoadjuvant chemotherapy was followed by intervaldebulking surgery. The distribution by FIGO stage at diagno-sis was: Stage I–II in 8 patients (5.1%), Stage III–IV in 137(87.8%), and unknown in 11(6.1%).

Tissue microarray(TMA) construction

Tissue microarrays were constructed from formalin-fixed,paraffin-embedded ovarian cancer specimens as describedpreviously.35,36 In addition, we also constructed a tissuemicroarray comprising of non-neoplastic ovarian surface epi-thelium from FFPE from 13 patients, who had been operatedfor hysterectomy with salpingoopherectomy. Tumor regionswere mapped by a pathologist for coring. The tissue microar-ray was constructed with 0.6-mm diameter cores spaced 0.8mm apart using a modified Tissue Microarrayer (BeecherInstruments, Sun Prairie, WI). The tissue microarray blockwas cut into 5 mm sections, adhered to the slide by an adhe-sive tape-transfer method (Instrumedics Inc., Hackensack,NJ) and UV crosslinked.

Antibodies and immunohistochemistry

Immunohistochemical studies on formalin-fixed, paraffin-em-bedded tissue sections were performed as describer in earlierstudies.35,36 For antigen retrieval, Dako Target Retrieval Solu-tion pH 6.0 (Catalogue number S1700) was used, and theslides were microwaved at 750 W for 5 minutes and then at250 W for 30 minutes. The sections were incubated overnightwith COX-2 (rabbit polycloncal COX-2 Abcam) and COX-1(mouse monoclonal, ABR,12E12) the Dako Envision PlusSystem kit was used as the secondary detection system withDAB as chromogen. IHC for p-AKT was Eperformed bystaining 3–4 lm thick tissue micro array sections with the p-AKT (Ser 473) antibody [Survival Marker: Signal Stain Phos-pho-AKT (Ser 473) IHC detection kit Product No 8100 CellSignaling Technology, Beverly, MA]. The IHC protocolincluded with the kit was followed with no modifications.Incubating the tissue in blocking solution blocked nonspecificbinding. Endogenous peroxidase activity was quenched usingperoxidase quench supplied along with the kit. Endogenousbiotin was blocked and all slides were counterstained withhematoxylin, dehydrated, cleared, and cover slipped with pre-mount. Only fresh cut TMA slides were stained simultane-ously to minimize the influence of slide aging and maximizerepeatability and reproducibility of the experiment. Two typesof negative controls were used. One was the negative controlin the kit in which the primary antibody was omitted. A pre-absorption experiment using p-AKT Ser 473 blocking peptide(Cell Signaling Technology, Beverly, MA, Product No 1140)was used as the second negative control.

Immunohistochemistry assessment

There are no established cut points available for quantitativeCOX-2 expression as there are relatively few articles in ovar-ian carcinomas,37–39 reporting a wide variations in the inci-dence of COX-2 overexpression ranging from 32% to 67.7 %.In this study, COX-2 expression was categorized by doing anH score, which combines intensity of staining in each celland percentage of stained cells. In brief, each TMA spot wasassigned a staining intensity score from 0 to 3(I0, I1–3), and a

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percent of stained tumor cells that was recorded in 5% incre-ments from a range of 0 to 100(P0, P1–3). For each spot ana-lyzed, a score was generated form the product of intensityand percent of tumor cells stained. A final H score (range 0–300) was obtained by adding the sum of individual scoresobtained for each tissue microarray spot (H score ¼ I1 � P1þ I2 � P2 þ I3 � P3). Ovarian tumors were categorized into2 groups based on H score and using the X-tile plat asdescribed below.

X-tile plots are constructed for assessment of biomarkerand optimization of cut off points based on outcome.40,41

The X-Tile plots allow determination of an optimal cut pointwhile correcting for the use of minimum P statistics. Usingthe X-Tile program, an optimal cut point for COX-2 expres-sion was determined at 160, with a Miller-Seigmund p valueof 0.5950 as determined by X-Tile. Tumors with H score<160 were classified as low expressers (n ¼ 58; 39.7%), andthose with H score >160 were classified as high expressers(n ¼ 88; 60.3%; Figs. 1a and 1c).

p-AKT scoring was done as described earlier.42,43 Briefly,phospho-AKT was scored as levels on an intensity scale rang-ing from 0 to 3. Scoring was performed as follows: 0, noappreciable staining in tumor cells; 1, barely detectable stain-ing in tumor cells; 2, appreciable staining of moderate inten-sity, distinctly marking tumor cells and 3, readily appreciablestaining of strong intensity). For purposes of statistical analy-sis, all cases staining at level 0 or 1 were grouped as p-AKTnegative and all cases staining at Level 2 and Level 3 weregrouped as p-AKT positive.

Statistical analysis

The software used for statistical analysis was statview 5.0 (SASInstitute Inc., NC). v2 tests were used to examine relationshipbetween nominal variables. The limit of significance for allanalyses was defined as a p value of 0.05; two-sided tests wereused in all calculations. Survival curves were generated usingthe Kaplan–Meier method and log rank test was used to com-pare survival in patients with COX-2 overexpressing tumorscompared with those with low COX-2 expression.

Cell culture

Epithelial ovarian cancer (EOC) cell lines SKOV3 andMDAH2774 cells were cultured in RPMI 1640 medium sup-plemented with 10% (vol/vol) fetal bovine serum, 100 U/mlPenicillin and 100 U/ml Streptomycin at 37�C in humidifiedatmosphere containing 5% CO2.

Reagents and antibodies

Direct AKT inhibitor and zVAD-fmk inhibitor were pur-chased from Calbiochem (San Diego, CA). MTT and aspirinwere purchased from Sigma (St. Louis, MO). NS398 was pur-chased from Cayman Chemical (Ann Arbor, MI) Antibodiesagainst phospho-AKT, phospho-FKHR, phospho-GSK3,cleaved caspase-3 and BID antibodies were purchased fromCell Signaling Technologies (Beverly, MA). COX-2, cyto-

chrome c, beta-actin, caspase-3, and PARP antibodies werepurchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,CA). Caspase-8 antibody was purchased from R&D (USA).Annexin V kit was purchased from Molecular Probes(Eugene OR). Apoptotic DNA-ladder kit was obtained fromRoche (Penzberg, Germany).

Cell cycle analysis

Cell lines were either treated with and without aspirin for48 hr and resuspended in 500 ll hypotonic staining buffer(sodium citrate 250 mg, Triton X 0.75 ml, propidium iodide25 lg, ribonuclease A 5 lg and 250 ml water) and analyzedby flow cytometry as described previously.44

Annexin V/propidium iodide dual staining

Ovarian cancer cell lines were treated with the indicated con-centrations of aspirin. The cells were harvested and the per-centage of cells undergoing apoptosis was measured by flowcytometry after staining with fluorescein-conjugated Annex-inV/ propidium iodide as previously described.44

Cell lysis and immunoblotting

Cells were treated with aspirin as described in the legends andlysed as previously described.45 Proteins were immunoblottedwith different antibodies and visualized by the enhancedchemiluminescence (Amersham, Piscataway, NJ) method.

Measurement of mitochondrial potential using the JC1 (5,50, 6, 60-teterachloro-1, 10, 3,30- tetraethylbenzimidazolylcar-bocyanine iodide) assay kit.

After treatment of ovarian cancer cell lines with aspirinfor 48 hr, cells were incubated with 10 lM JC1 at 37�C indark for 15 minutes and mitochondrial membrane potential(% of green and red aggregates) was determined by flowcytometry as described previously.46

Assay for cytochrome c release

Release of cytochrome c from mitochondria was assayed asdescribed in a previous publication.47 Briefly, cells weretreated with and without aspirin as described in figure legendand proteins were extracted. Twenty micrograms of proteinsfrom cytosolic and mitochondrial fraction of each sampleswere analyzed by immunoblotting using anti-cytochrome cantibody.

Gene silencing using siRNA

COX-2 siRNA and Scrambled control siRNA were purchasedfrom Qiagen. For transient expression, cell lines were trans-fected by using LipofectAMINE 2000 reagent (Invitrogen)according to the manufacturer’s instructions. After incubatingthe cells for 6 hr, the lipid and siRNA complex was removedand fresh growth medium was added. Cells were lysed 48 hrafter transfection and specific protein levels were determinedby Western Blot analysis with specific antibodies against thetargeted proteins and actin as a loading control.

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384 COX-2 mediated signaling in ovarian cancer


Animals and xenograft study

Six weeks old NUDE mice were obtained from Jackson Labo-ratories (Maine) and maintained in a pathogen free animalfacility at least 1 week before use. All animal studies weredone in accordance with institutional guidelines. For xeno-graft study, mice were inoculated subcutaneously into theright abdominal quadrant with 5 � 106 MDAH2774 cells in200 ll PBS. After 1 week, mice were randomly assigned into3 groups; 2 groups received intraperitoneal aspirin at 1 of 2doses (100 mg/kg or 200 mg/kg); the remaining groupreceived 0.9% saline. The body weight and tumor volume ofeach mouse were monitored weekly. The tumor volume wasmeasured as previously described.48 After 5 weeks treatment,

mice were sacrificed and individual tumors were weighed,then snap-frozen in liquid nitrogen for storage.

ResultsCOX-2 expression and its correlation with p-AKT and

other clinicopathological parameters

Levels of COX-2 were examined by immunohistochemistryin a series of 156 ovarian carcinomas. High levels of COX-2expression were seen in 60.3% (88 of 146) of the ovarian car-cinomas. p-AKT over expression was seen in 52.1 %(75 of144) of the ovarian carcinoma (Fig. 1a).

As shown in Table 1 COX-2 overexpression was signifi-cantly associated with overexpression of p-AKT (p < 0.0001).

Figure 1. Expression of COX-2 and p-AKT in ovarian carcinoma. (a) COX-2 overexpression in ovarian carcinoma, (b) p-AKT overexpression in

ovarian carcinoma, (c) Reduced COX-2 expression in ovarian carcinoma, (d) Reduced p-AKT expression in ovarian carcinoma Original

magnification �200 with the inset showing a higher magnification �400. (immunoperoxidase stain) and (e) Overview of tissue micro array

slides of ovarian carcinoma.

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However, COX-2 overexpression was not associated withpatient’s age, histology type, tumor grade or FIGO stage. COX-2 overexpression was less frequent in clear cell carcinomascompared with the other histologic subtypes. This differencedid not reach statistical significance possibly because of thesmall number of cases in the subgroups. Interestingly expres-sion levels of COX2 was more in EOCs (mean 169.38 6 68.46)as compared with non-neoplastic ovarian surface epithelium(OSE) (128.18 6 57.76; p ¼ 0.0440). Similarly, expression ofphosphorylated AKT was significantly higher in the EOC(mean 175.00 6 71.44) as compared with non-neoplastic ovar-ian surface epithelium (OSE) (136.36 6 50.45; p ¼ 0.0340).Thus our results suggested a progressive increase in COX2 andpAKT expression during ovarian carcinogenesis (Fig. 2).

COX-1 expression and its correlation with p-AKT and

other clinicopathological parameters

Our data showed that COX-1 expression was seen in 59.9%(88 of 147) of the ovarian carcinomas. As shown in Table 2COX-1 over expression is not significantly associated with p-AKT (p ¼ 0.0995) and COX-2 (p ¼ 0.0976). Similarly, COX-1 overexpression was also not associated with patient’s age,

histology type, tumor grade or FIGO stage. Expression ofCox-1 and/or COX-2 was seen in 81% (116 of 142) EOCs.Of the 38.7% ovarian cancers (n ¼ 55) showing lack ofCOX-2 over expression, COX-1 expression was seen in 52.7%(n ¼ 29). Thus, dysregulation in COX-1 or COX-2 expres-sion was seen in majority of the epithelial ovarian cancershighlighting the importance of both these inflammatorymarkers in ovarian carcinogenesis.

Aspirin causes cell growth inhibition and induces

apoptosis ovarian cancer cell lines

We initially sought to determine whether treatment withCOX inhibitors leads to the inhibition of ovarian cancer cellsproliferation. MDAH2774 and SKOV3 cells were cultured inthe presence of 0.5, 1, 5, 10 and 20 mM aspirin for 48 hrand proliferation was assayed using MTT assays. Figure 3ashows that as the dose of aspirin increased from 0.5 to 20mM, cell growth inhibition increased in a dose-dependentfashion in all the ovarian cancer cell lines (p < 0.05). Simi-larly, treatment with NS398, a specific inhibitor for COX-2,resulted in a dose-dependent inhibition of cell growth inboth ovarian cancer cell lines tested (Fig. 3b).

Table 1. Correlation between COX-2 expression status and clinciopathological features in epithelial ovarian carcinoma

Epithelial group N %

Above 160 Below 160

p valueN % N %

Total number of cases 1461 88 60.3 58 39.7

Age (yr)

�50 58 39.7 38 65.5 20 34.5 0.2915

>50 88 60.3 50 56.8 38 43.2

Tumor stage

Stage I–II 8 6.0 3 37.5 5 62.5 0.1527

Stage III–IV 128 94.0 81 63.3 47 36.7

Histopathology

Clear cell 4 2.7 1 25.0 3 75.0 0.4945

Endometriod 22 15.1 13 59.1 9 40.9

Serous 116 79.5 72 62.1 44 37.9

Undifferentiated 4 2.7 2 50.0 2 50.0

FIGO grade

Well differentiated 27 18.5 14 51.8 13 48.2 0.5799

Moderately differentiated 77 52.7 47 61.0 30 39.0

Poorly differentiated 42 28.8 27 64.3 15 35.7

p-AKT (Ser473)

High (2–3) 75 52.8 58 77.3 17 22.7 <0.0001

Low (0–1) 67 47.2 29 43.3 38 56.7

COX-1

Above 25 54 38.0 37 68.5 17 31.5 0.1621

Below 25 88 62.0 50 56.8 38 38.7

1Representative information for COX-2 was available in 146 spots and IHC analysis failure of remaining cases was due to missing spots or fixationartefacts.

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In subsequent experiments, we determined whether theobserved suppressive effects of aspirin and NS398 in MTTassays were due to induction of cell cycle arrest or apoptosis.Ovarian cancer cell lines were treated with 5 and 10 mM ofaspirin for 48 hr or vehicle alone. The cells were stained andcell cycle fractions were determined by flow cytometry. Asshown in Supporting Information Figure 1, the sub-G1 popu-lation of cells increased from 3.77% in control cells to 23.67and 36.85% in MDAH2774 cells treated with 5 or 10 mM as-pirin, respectively. Similarly in SKOV3 cells, the sub-G1 pop-ulation was 4.33% in the control group compared with 18.12and 31.55% in cells treated with 5 or 10 mM aspirin, respec-tively. Annexin V/PI dual staining and DNA laddering detec-tion methods confirmed those apoptotic effects. Ovarian can-cer cells were treated with 5 and 10 mM aspirin for 48 hrand apoptosis was measured by annexinV/PI dual staining.As shown in Figure 3c, treatment of MDAH2774 cells with 5mM and 10 mM aspirin induces apoptosis in 21.10 and39.20% of cells, respectively. Similarly, treatment of SKOV3cells with 5 or 10 mM aspirin induced apoptosis in 21.4 and50%, respectively. Finally, ovarian cancer cells were treated

with 100 lM NS398 or 10 mM aspirin for 48 hr, DNA wasisolated using apoptotic DNA-laddering kit from Roche. Asshown in Figure 3d, inhibition of COX-2 causes fragmenta-tion formation of DNA, a characteristic of apoptotic celldeath. These results confirm that suppression of growth byNS398 and aspirin treatment in ovarian carcinoma cellsoccurs via inducing apoptosis.

Constitutive expression of COX-2 and activation of AKT

signaling pathways in ovarian cancer cell lines

Using 2 ovarian cancer cell lines, we determined the constitu-tive expression of COX-2 and AKT in these cells as well as theeffect of aspirin treatment on that expression. MDAH2774cells were treated for 24 hr with 50 or 100 lM NS398, a spe-cific COX-2 inhibitor. As shown in Figure 4a, both ovariancancer cell lines exhibited constitutive expression of COX-2and p-AKT. Treatment of ovarian cancer cells with NS398(Fig. 4a), and aspirin (Fig. 4b) suppressed COX-2 expressionand induced dephosphorylation of AKT significantly (p <

0.001) (Supporting Information Figs. 2a and 2b). Inhibition ofCOX-2 also suppressed the downstream target of AKT,

Figure 2. Comaprision of COX2 and pAKT expression in non neoplastic ovarian surface epithelium (OSE) and epithelial ovarian carinomas

(EOC). Section of a non-neoplastic ovarian surface epithelium (OSE), (a) heamtoxylin and eosin (H&E) stained (b) COX-2 expression and (c)

p-AKT expression. Original magnification �200 with the inset showing a higher magnification �400 (immunoperoxidase stain). (d and e)

Box plot charts indicate the mean and standard deviation of COX-2 expression in 2 groups—non-neoplastic ovarian surface epithelium(OSE)

and epithelial ovarian carcinomas(EOC).

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FKHR/FOXO1. These data suggest that the activity of AKT isregulated by COX-2. To verify that the effects of aspirin andNS398 are attributed to their COX-2 inhibitory effects, similarexperiments were conducted with siRNA-targeting COX-2.The COX-2-targeting siRNA, down-regulated the expressionof COX-2 protein and decreased the phosphorylation of AKT(Fig. 4c). In addition, downregulation of COX-2 and inhibi-tion of AKT caused activation of caspase-3 On the otherhand, treatment of MDAH2774 cells with specific AKT inhibi-tor or transfection of these cells with AKT-specific siRNAdecreased AKT phosphorylation but did not affect COX-2expression proving that AKT is downstream to COX-2 in thesignal pathway (Fig. 4d and Supporting Information Fig. 2c).

Inhibition of COX-2 with aspirin in ovarian cancer cells

induces apoptosis via mitochondrial pathway and caspase

activation

Next, we sought to determine whether inhibition of COX-2and p-AKT signaling involves the mitochondria in the ovar-ian cancer cells. Activated caspase-8 is capable of cleavingcaspase-3 either directly or by digesting BID to its activeform (tBID) that subsequently leads to loss of mitochondrial

membrane potential and release of cytochrome c from mito-chondria. Aspirin treatment of MDAH2774 and SKOV3 cellsresulted in activation of caspase-8 leading to truncation ofBID in both cell lines tested (Supporting Information Fig.3a). In addition it resulted in loss of mitochondrial mem-brane potential and release of cytochrome c from mitochon-dria into cytosole (Supporting Information Figs. 3b–d). Wethen sought to determine whether aspirin-induced release ofcytochrome c is capable of activation of caspase-3 and PARP.Figure 5a shows that aspirin treatment resulted in the activa-tion of caspase-3 and cleavage of PARP in MDAH2774, andSKOV3 cells. In addition, pretreatment of these cells with 80lM z-VAD-fmk, a universal inhibitor of caspases, followedby aspirin treatment, prevented caspase-3 and PARP activa-tion induced by aspirin and abrogated apoptosis (Figs. 5band 5c), clearly indicating that caspases play a critical role inaspirin induced apoptosis in ovarian cancer cells.

In vivo activity of aspirin against ovarian cancer cells

xenograft

Our observation that ovarian cancer cells exhibit enhancedsensitivity to aspirin-induced apoptosis in vitro suggests a

Table 2. Correlation between COX-1 expression status and clinciopathological features in epithelial ovarian carcinoma

Epithelial group N %

Above 25 Below 25

p valueN % N %

Total number of cases 1471 54 36.7 93 63.3

Age (yr)

�50 57 38.8 23 40.3 34 59.7 0.4701

>50 90 61.2 31 34.4 59 65.6

Tumor stage

Stage I–II 8 5.8 2 25.0 6 75.0 0.5293

Stage III–IV 129 94.2 46 35.7 83 64.3

Histopathology

Clear cell 4 2.7 1 25.0 3 75.0 0.1752

Endometriod 21 14.3 4 19.0 17 81.0

Serous 117 79.6 48 41.0 69 59.0

Undifferentiated 5 3.4 1 20.0 4 80.0

FIGO grade

Well differentiated 27 18.4 10 37.0 17 63.0 0.8553

Moderately differentiated 78 53.1 30 38.5 48 61.5

Poorly differentiated 42 28.6 14 33.3 28 66.7

p-AKT (Ser473)

High (2–3) 73 51.4 30 41.1 43 58.9 0.2541

Low (0–1) 69 48.6 22 31.9 47 68.1

COX-2

Above 160 87 61.3 37 42.5 50 57.5 0.1621

Below 160 55 38.7 17 30.9 38 69.1

1Representative information for COX-1 was available in 147 spots and IHC analysis failure of remaining cases was due to missing spots or fixationartefacts.

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potential therapeutic role in treatment of ovarian cancer. Weexamined the ability of aspirin to inhibit ovarian cancer tu-mor growth in a mouse xenograft model of ovarian cancer.NUDE mice were inoculated subcutaneously in the right ab-dominal quadrant with 5 million MDAH2774 cells. The ani-mals were then treated intraperitoneally with 1 of 2 doses ofaspirin (100 mg/kg/dose and 200 mg/kg/dose) twice perweek,49 or vehicle saline-treated control groups (n ¼ 6). After5 weeks of treatment, mice were sacrificed and tumors werecollected. Aspirin treatment caused a time-dependent regres-sion of MDAH2774 xenograft tumors in mice as comparedwith vehicle-treated mice (6A). The regression reached signif-icance (p < 0.05) at the end of third week of treatment. Asignificant reduction in tumor weight (Fig. 6b) was observedin mice treated with aspirin (p < 0.05). Additionally, images

of tumor before and after necropsy showed that aspirin treat-ment resulted in shrinkage of tumor size (Fig. 6c). The levelof COX-2 and p-AKT proteins were markedly decreased intumors of mice treated with aspirin as compared to vehicle-treated mice as detected by western blotting (Fig. 6d).

DiscussionIn this study, we first investigated the incidence and clinico-pathological associations of both COX-1 and COX-2 expres-sion in a large series of EOC patient samples. COX-2 overex-pression was significantly associated with activated AKT (p-AKT) expression in the clinical samples studied. These datasuggest an interaction between COX-2 enzymatic activity andPI3Kinase/AKT signaling pathway in EOC. COX-1 expres-sion showed a direct trend with expression of p-AKT and

Figure 3. Aspirin (a) and NS398 (b) inhibit the proliferation of ovarian cancer cells. MDAH2774 and SKOV3 cells were incubated with 0.5,

1, 5, 10 and 20 mM aspirin and with 10, 25, 50 and 100 lM NS398 for 48 hr. Cell proliferation assays were performed using MTT as

described in Material and methods. The graph displays the mean 6 standard deviation (SD) of 3 independent experiments with replicates

of 6 wells for all the doses and vehicle control for each experiment *p < 0.05, statistically significant (Student’s t-test). (c) Aspirin-induced

apoptosis detected by Annexin V/PI dual staining. MDAH2774 and SKOV3 cells were treated with 5 and 10 mM for 48 hr and cells were

subsequently stained with flourescein-conjugated annexin-V and propidium iodide (PI). (d) MDAH2774 cells were treated with 10 mM

aspirin and 100 lM NS-398 for 48 hr cells and DNA was extracted and separated by electrophoresis on 1.5% agarose gel. [Color figure can

be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Uddin et al. 389


COX-2. Almost half of the EOCs showing lack of COX2expression showed overexpression of COX-1. Thus, dysregu-lation in COX-1 or COX-2 expression was seen in 81.7%(116 of 142) highlighting the importance of both theseinflammatory markers in ovarian carcinogenesis.

A number of studies have shown that COX-2 inhibitorscan be used for the prevention and/or treatment of a numberof malignancies.50–52 In fact, epidemiological studies andrandomized clinical trials provide convincing evidence thatregular intake of aspirin and other NSAIDs inhibit the

Figure 4. NS398 and aspirin treatment causes downregulation of COX-2 and dephosphorylation of constitutive phosphorylation of AKT and

Foxo1 in ovarian cancer cell lines. (a) MDAH2774 cells were treated with 50 and 100 lM for 24 hr. After cell lysis, equal amounts of

proteins were separated by SDS-PAGE, transferred to immobilon membrane, and immunoblotted with antibodies against COX-2, p-AKT, AKT,

p-Foxo1, Foxo1 and beta actin as indicated. (b) MDAH2774 and SKOV3 cells were treated with 5 and 10 mM for 24 hr. After cell lysis,

equal amounts of proteins were separated by SDS-PAGE, transferred to immobilon membrane, and immunoblotted with antibodies against

COX-2, p-AKT, AKT, p-Foxo1, Foxo1 and beta actin as indicated. (c) MDAH2774 cells were transfected with scrambled siRNA (100 nM) and

COX-2 siRNA (50 and 100 nM) with Lipofectamine 2000. After 48 hr, cells were lysed and proteins were immunoblotted with antibodies

against COX-2, p-AKT-Ser473, AKT, caspase-3 and beta actin. (d) MDAH2774 cells were transfected with scrambled siRNA (100nM) and

AKT1 siRNA (50 and 100 nM) with Lipofectamine 2000. After 48 hr, cells were lysed and proteins were immunoblotted with antibodies

against COX-2, p-AKT-Ser473, AKT, caspase-3 and beta actin. Every experiment is representative of a minimum of 3 independent

experiments. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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development of colon cancer.53 Furthermore, recent random-ized clinical trials of selective COX-2 inhibitors indicate thatcelecoxib suppresses the development of colon adenomas.54,55

The molecular mechanisms underlying this anticancer effectare not fully elucidated. It is known that upon activation ofPI3K pathway a number of signaling proteins are phospho-rylated including protein kinase B (AKT). AKT, once acti-vated exerts oncogenic and antiapoptotic activities by activat-ing several downstream targets such as Foxo transcriptionfactors and GSK3.56,57 In this study, we demonstrated thatinhibition of COX-2 activity using either specific (NS 398) ornonspecific COX-inhibitor (aspirin) resulted in downregula-tion of COX-2, inactivation (dephosphorylation) of AKT aswell as its downstream target Foxo1/FKHRL1.

Gene silencing studies were carried out using COX-2 andAKT siRNAs in order to confirm the relationship betweenAKT and COX-2. COX-2 siRNA induced silencing of COX-2expression, downregulated COX-2 and dephosphorylatedAKT in EOC cells. On the other hand, AKT siRNA failed toaffect the level of COX-2 expression in EOC cell line. Thesefinding suggest that COX-2 AKT activity and modulates isupstream of AKT.

Apoptosis is a multistep process and an increasing num-ber of genes have been identified to be involved in the con-trol or execution of apoptosis.58 Our study demonstrated thatCOX-2 inhibition induced apoptosis via inactivation ofpAKT, resulting in disruption of mitochondrial membranepotential which in turn lead to release cytochrome C into

Figure 5. Activation of caspases-3 and cleavage of PARP induced by aspirin in ovarian cancer cells. (a) MDAH2774 and SKOV3 cells were

treated with 10 mM aspirin for 24 hr. Cells were lysed and 20lg protein were separated by SDS-PAGE, transferred to PVDF membrane, and

immunoblotted with antibodies against pro-caspase-3, cleaved caspase-3, PARP, and beta actin. (b) Effect of z-VAD-fmk on aspirin-induced

activation of caspase-3. MDAH2774 cells were pre-treated with either 80lM z-VAD/fmk for 2 hr and subsequently treated with 10 mM of

aspirin for 24 hr. Cells were lysed and 20 lg protein were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with

antibodies against procaspase-3, cleaved caspase-3, and beta-actin. (c) Effect of z-VAD/fmk on aspirin-induced apoptosis in ovarian cancer

cells. MDAH2774 cells were pretreated with 80 lM z-VAD/fmk for 2 hr and subsequently treated with 10 mM of aspirin for 48 hr and

apoptosis was measured by Annexin/PI dual staining, using flow cytometry. [Color figure can be viewed in the online issue, which is

available at www.interscience.wiley.com.]

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Uddin et al. 391


cytosol. Release of cytochrome C was associated with activa-tion of caspase activity, eventually resulting in apoptosis.

We have used MDAH2774 cell line xenograft tumor inNUDE mice to translate the in vitro results into an in vivo

animal model, Treatment of xenograft tumors with aspirinresulted in a reduction of tumors’ size in a time and dose-de-pendent manner. The body weighs of control and experimen-tal groups of animal did not differ through out the

Figure 6. Aspirin-inhibits growth of MDAH2774 xenograft and down-regulates COX-2 and inactivates AKT in vivo. Nude mice at 6 weeks of

age were injected s.c. with five million MDAH2774 cells. After 1 week, mice were treated with aspirin at 100 mg/kg/dose, 200 mg/kg/dose

or with 5% DMSO in PBS as a vehicle control. (a) Inhibition of MDAH2774 tumor growth by aspirin. The volume of each tumor was

measured every week. The average (n ¼ 6) tumor volume in vehicle-treated control mice and treated with aspirin was plotted, *p < 0.05.

(b) After 5 weeks of treatment, mice were sacrificed and tumor weights were measured *p < 0.05 compared with vehicle-treated mice by

Student’s t-test. (c) Representative tumor images of vehicle and aspirin treated mice. (d) Whole cell lysates from mice treated with vehicle,

aspirin (100 mg/kg) and aspirin (200 mg/kg) were prepared, and 20 lg protein were separated by SDS-PAGE, transferred to PVDF

membrane, and immunoblotted with antibodies against COX-2, p-Akt, Akt and beta-actin.

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experiments suggesting that aspirin treatment significantlyinhibited MDAH2774 xenograft growth without causing anyside effects to mice. Tumor regression was accompanied by adecrease the level of phosphorylated AKT and COX-2 in thetumor cells.

Altogether these data suggest that COX-2 may play rolein tumor growth through regulation of key oncogenic path-ways (PI3K/AKT) on which cancer cells depend for survival.Furthermore, our data also support the hypothesis that COX-2 inhibition may have therapeutic potential in EOC.

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Cyclooxygenase-2 inhibition inhibits PI3K/AKT kinase activity in epithelial ovarian cancer