European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2012) xxx–xxx

G Model

EURO-7720; No. of Pages 6

Induction of apoptosis and inhibition of cell proliferation by the cyclooxgenaseenzyme blocker-nimesulide in the Ishikawa endometrial cancer cell line

S. Sinan Ozalp a, Ceren Yıldız Eren a, Rakibe Beklem Bostancıoglu b, Ays e Tansu Koparal b,*a Eskis ehir Osmangazi University, Faculty of Medicine, Department of Obstetrics and Gynecology, 26470 Eskis ehir, Turkeyb Anadolu University, Faculty of Sciences, Department of Biology, 26470 Eskis ehir, Turkey

A R T I C L E I N F O

Article history:

Received 18 August 2011

Received in revised form 18 February 2012

Accepted 6 May 2012

Keywords:

Nimesulide

COX-2

Endometrial cancer

Cytotoxicity

Apoptosis

A B S T R A C T

Objectives: Endometrial cancer remains a leading cause of death in women and therefore the

development of new therapies is essential. The present study evaluated the effects of nimesulide

alone, cisplatin alone, and combination of cisplatin and nimesulide on an Ishikawa cell line with respect

to cytotoxicity and induction of apoptosis in vitro.

Study design: Ishikawa cells were treated with increasing doses of nimesulide alone, cisplatin alone, and a

combination of cisplatin and nimesulide. Subsequently their effects on cytotoxicity were investigated by

MTT assay, while apoptosis was investigated by DAPI and JC-1 staining and caspase-3 colorimetric assays.

Results: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that nimesulide

alone and combination of cisplatin and nimesulide have growth inhibitory effect on Ishikawa cells.

Nimesulide alone and the combination of cisplatin and nimesulide induced apoptosis. Apoptosis induced

by nimesulide might be related to caspase-3 activation.

Conclusions: These results suggest that nimesulide treatment is as effective as cisplatin treatment in

Ishikawa cells. The combination of cisplatin and nimesulide treatment is more effective than cisplatin

alone in Ishikawa cells.

� 2012 Elsevier Ireland Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

European Journal of Obstetrics & Gynecology andReproductive Biology

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /e jo g rb

1. Introduction

Endometrial cancer is the most common malignant tumor ofthe female genital tract [1]. Although the incidence has remainedstable, the death rate has increased over 100% in the last twodecades [2]. Tumor cells have some features: (1) they are self-sufficient in mitogenic/growth signals; (2) they have intensiveanti-growth signals; (3) they have limitless replication perfor-mance; (4) they undergo sustained angiogenesis and (5) theyachieve tissue invasion and metastasis [3]. These features allowtumor cells to evade apoptosis, a cell suicide program to eliminatedamaged and unwanted cells in order to maintain homeostasis [4].Induction of apoptosis is not only an important defense againstcancer, but also has recently attracted attention as a novel targetfor cancer chemoprevention [5].

Non-steroidal anti-inflammatory drugs (NSAIDs) have analge-sic, antipyretic and anti-inflammatory effects [6]. NSAIDs areparticularly interesting due to their properties of interfering with anumber of cellular functions and a variety of biochemical eventswhich, alone or in combination, may affect cell growth [7]. A

* Corresponding author. Tel.: +90 222 3350580/4710; fax: +90 222 3204910.

E-mail addresses: akoparal@anadolu.edu.tr, beklemb@gmail.com (A.T. Koparal).

Please cite this article in press as: Ozalp SS, et al. Induction of apoptosiblocker-nimesulide in the Ishikawa endometrial cancer cell lij.ejogrb.2012.05.018

0301-2115/$ – see front matter � 2012 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ejogrb.2012.05.018

growing body of experimental and epidemiological evidencesuggests that the use of NSAIDs may decrease the incidence ofmammary cancer tumor burden and tumor volume [8]. The targetsof NSAID are the COX enzyme, which converts arachidonic acid toprostaglandins and other eicosanoids. COX-2, the inducibleisoform, is undetectable in most normal tissues but is activatedin response to pro-inflammatory cytokines, growth factors andtumor promoters [9]. Many studies have reported on COX-2 over-expression in tumors and its association with increased cellproliferation, metastasis and decreased apoptosis [10]. Over-expression of COX-2 is associated with malignant proliferation[11] and seen in endometrial cancer [12,13]. The roles of COX-2 intumorigenesis of endometrium, such as promotion of angiogenesisand inhibition of apoptosis, have been shown in various studies[14,15]. To date, several experimental and clinical studies haveestablished potent anti-cancer activity of NSAID and other COX-2inhibitors, with most investigators reporting on the COX-2 relatedtumor inhibition in head and neck carcinoma [16], non-small celllung cancer cells [17], endometrial carcinoma [18], uterineendometrial carcinoma [19], esophageal squamous cell carcinoma[20]. Moreover, most of them explain this strong effects resultsfrom inhibition the production of prostaglandins [14–20]. COX-2inhibitors reduce the vascularity of in vivo angiogenesis andtumorigenesis [21]. There is evidence that COX-2 inhibitors also

s and inhibition of cell proliferation by the cyclooxgenase enzymene. Eur J Obstet Gynecol (2012), http://dx.doi.org/10.1016/

Fig. 1. Anti-proliferative effect of nimesulide on Ishikawa cell line. The SPSS

software was used for the statistical analyses of assessments of the MTT assay. Data

were evaluated using one-way ANOVA followed by the Tukey test. A value of

p < 0.05 was considered significant.

S.S. Ozalp et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2012) xxx–xxx2

G Model

EURO-7720; No. of Pages 6

inhibit vascular endothelial growth factor-A (VEGF-A)-mediatedangiogenesis, and PGE2-induced VEGF expression in synovialfibroblasts in vitro [22].

Nimesulide (4-nitro-2-phenoxymethane-sulfoanilide) is a sul-foanilide non-steroidal anti-inflammatory drug with preferentialCOX-2 inhibitory activity and has been available in some Asian,African and European countries since 1985 [6,23]. It is chemicallydifferent from other NSAIDs because of the sulfonanilide moiety.For COX-2 inhibition, the sulfonamide group is very critical innimesulide [23].

Thigpen et al. demonstrated that cisplatin is an active agent forthe treatment of endometrial cancer [24]. Cisplatin [CP, cis-diamminedichloroplatinum (II)]-based combination therapy regi-mens are used in the treatment of many types of cancer [25].Clinical resistance to cisplatin, however, is a major problem.

In cancer therapy, it is possible to increase the effect of an agentwith another agent. In this regard, different agents influencing avariety of oncogenic pathways were used in combination to obtaina synergistic cytotoxic effect in the present study. For optimizingindividual therapy, combination therapy is a significant choice. It isdesirable to identify novel agents and novel drug combinationswith activity in endometrial cancer. The aim of the current study,therefore, was to characterize the effects of nimesulide and acombination of cisplatin and nimesulide in the Ishikawa cell line.

2. Materials and methods

2.1. Cell culture

The Ishikawa human cell line was kindly provided by Dr. A. Bilir(Istanbul University, Turkey), and cultured in DMEM:F12 (1:1)(Biological Industries) supplemented with 10% (v/v) heat-inacti-vated fetal calf serum (Sigma) and penicillin–streptomycin(Sigma). The cells were cultured in a humidified atmospherecontaining 5% (v/v) CO2 at 37 8C.

2.2. Cell proliferation

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT; Sigma, St. Louis, MO, USA) was used to measurenumber of viable cells. Briefly, 1 � 104 cells/well were seeded in a96-well microplate (Techno Plastic Products AG) in a final volumeof 100 ml. After 24 h of seeding, cells were treated with differentconcentrations of cisplatin (Sigma) alone, nimesulide (Sigma)alone, and cisplatin plus nimesulide for 24 and 48 h. MTT assay wasperformed as previously described Koparal et al. [26].

2.3. DAPI staining

The morphological changes in the nuclear chromatin of the cellsundergoing apoptosis were detected by staining with the DNA-binding fluorochrome DAPI. The cells were grown on coverslips(Marienfeld, Germany) at a density of 35 � 103 cells per 12 well.After cisplatin alone, nimesulide alone and a combination ofcisplatin and nimesulide treatments (12 h), the assay wasperformed as previously described [27]. Apoptotic nuclei can beidentified by the reduced nucleolar size, condensed chromatingathering at the periphery of the nuclear membrane or a totalfragmented morphology of nuclear bodies.

2.4. Mitochondrial membrane potential

The cells were grown on coverslips (Marienfeld, Germany) at adensity of 35 � 103 cells per 12 well. After cisplatin alone,nimesulide alone and combination of cisplatin and nimesulide

Please cite this article in press as: Ozalp SS, et al. Induction of apoptosiblocker-nimesulide in the Ishikawa endometrial cancer cell lij.ejogrb.2012.05.018

treatments (12 h), the cells were stained by method of Castedoet al. [28,29].

2.5. Caspase 3 activity assay

The cultures were initiated in flasks at a density of 104 cell/cm2.The cells were allowed 24-h attachment and then incubated in theabsence and presence of nimesulide alone, cisplatin alone and thecombination of cisplatin and nimesulide for 24 h. Caspase-3activity in cell lysates was determined using a colorimetric assaykit based on the manufacturer’s protocol (R&D Systems, Minnea-polis, MS, USA). SPSS software was used for the statistical analysesof assessments of the caspase-3 assay. Data were evaluated usingone-way ANOVA followed by the Tukey test. A value of p < 0.05was considered significant.

3. Results

3.1. Inhibition of proliferation by nimesulide in endometrial cancer

cells

Ishikawa cells were treated with increasing doses of nimesulidealone, cisplatin alone and the combination of cisplatin andnimesulide; in addition, cell viability was assessed by MTT assay.The percentage of growth inhibition by nimesulide at various doseson the Ishikawa cells was determined as the percentage of viable-treated cells in comparison with viable cells of untreated controls.Nimesulide treatment displayed a significant dose and time-dependent decrease in cell proliferation (Fig. 1). Two-day exposureof nimesulide at all studied doses caused decreases in the numberof Ishikawa cells. The results of two-day exposure to nimesulidetreatment are more efficient than two-day exposure to cisplatintreatment (Fig. 2). After one day, exposure to 20 mM cisplatintreatment caused a 35% decrease in cell proliferation. On the otherhand, the cisplatin plus nimesulide combination (C20 + N200)caused a 44% decrease in the number of viable cells (Fig. 3). Aftertwo days exposure, the combination of cisplatin and nimesulide(C20 + N200) reduced the number of viable cell (data not shown).

3.2. Detection of apoptosis by DAPI staining

Morphological analysis with DAPI staining showed nuclei withchromatin condensation and the formation of apoptotic bodies inthe cells exposed to nimesulide alone. Fig. 4 shows representativeDAPI-staining fluorescence photomicrographs of cultured Ishi-kawa cells treated with nimesulide alone, cisplatin alone and the

s and inhibition of cell proliferation by the cyclooxgenase enzymene. Eur J Obstet Gynecol (2012), http://dx.doi.org/10.1016/

Fig. 2. Anti-proliferative effects of cisplatin on Ishikawa cell line. The SPSS software

was used for the statistical analyses of assessments of the MTT assay. Data were

evaluated using one-way ANOVA followed by the Tukey test. A value of p < 0.05

was considered significant.

S.S. Ozalp et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2012) xxx–xxx 3

G Model

EURO-7720; No. of Pages 6

combination of cisplatin and nimesulide treatments, respectively.In control cultures (Fig. 4a), the nuclei of Ishikawa cells appearedwith regular contours and were round and large in size. Ishikawacells with smaller nuclei and condensed chromatin were rarelyseen. In contrast, most nuclei of nimesulide-treated Ishikawa cells(Fig. 4c) appeared hypercondensed.

3.3. The effect of nimesulide on mitochondrial membrane potential

The loss of mitochondrial membrane potential is a hallmark ofapoptosis. It is an early event coinciding with caspase activation. Inhealthy cells, red-orange fluorescence is attributable to a potential-dependent aggregation in the mitochondria even though greenfluorescence reflecting the monomeric form JC-1 appeared in thecytosol. In apoptotic cells, green fluorescence dominates aftermitochondrial membrane depolarization. The red aggregates haveabsorption/emission maxima at 585/590 nm while the greenfluorescence has its maxima at 510/527 nm [29]. Fig. 5 demonstratesrepresentative JC-1 stains of apoptotic and non-apoptotic cells.

3.4. The effect of nimesulide on caspase-3 activity

Treatment of tumor cells with DNA-damaging agents such ascisplatin is associated with activation of the intrinsic apoptotic

Fig. 3. Anti-proliferative effect of cisplatin and nimesulide combination on Ishikawa cell li

assay. Data were evaluated using one-way ANOVA followed by the Tukey test. A value

Please cite this article in press as: Ozalp SS, et al. Induction of apoptosiblocker-nimesulide in the Ishikawa endometrial cancer cell lij.ejogrb.2012.05.018

pathway. Caspase-3 is considered to be a specific marker forepithelial apoptosis. Active caspase-3 level was analyzed fornimesulide-treated, cisplatin-treated, and combination of cisplatinand nimesulide-treated cells in comparison with the level inuntreated cells. The Ishikawa cells treated with nimesulide had asignificantly higher activity of caspase-3 than that of no treatment.After 200 mM nimesulide treatment, the maximal activity ofcaspase-3 was increased 82.79% (std. error � 1.92) compared tocontrols. After 20 mM cisplatin treatment, the maximal activity ofcaspase-3 was increased 42.19% (std. error � 4.58). After the 20 mMcisplatin + 200 mM nimesulide combination, the caspase-3 activitywas increased 22.03% (std. error � 2.96), which was more effectivethan the control group. But the combination was not as effective ascisplatin or nimesulide alone. The reason for the lesser effect of thecombination may be related to the evaluation of caspase-3 activityonly after 24 h. Caspase-3 activity of the combination was evaluatedevery 3 h, and the increased level of caspase-3 activity could possiblybe observed. The results clearly show that nimesulide inducesapoptosis in Ishikawa cells. Significant and dose-dependent activa-tion of caspase-3 was observed in response to nimesulide (Fig. 6).

Yu et al. [20] showed that celecoxib antagonizes the cytotoxici-ty of cisplatin by decreasing intracellular cisplatin and DNAplatination. The combination treatment also shows no beneficialeffect compared with cisplatin monotherapy in vivo for esophagealsquamous cell carcinoma. So, the combination’s lower effect mightbe due to that same antagonistic effect and time-dependenttreatment.

4. Comments

A number of studies have demonstrated that COX-2, aninducible isozyme of cyclooxygenase, plays a crucial role incarcinogenesis and cancer progression in both human and animalmodels [30]. COX-2 over-expression protected the cancer cellsagainst several apoptotic stimuli [31]. Several researchers reportedthat COX-2 inhibition prevented carcinogenesis in a variety oftissues including colon, lung, breast, bladder, pancreas and skin[32,33]. COX-2 is a potential pharmacologic target to prevent andtreat a variety of malignancies.

Nimesulide is widely prescribed as a potent anti-inflammatorydrug, and therefore, achievable and safe plasma concentrations areclear from the publications [9]. Studies suggest that nimesulide caninduce apoptosis in liver and lung cancer cells, it also suppressesthe development of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced mammary gland carcinogenesis in rats

ne. The SPSS software was used for the statistical analyses of assessments of the MTT

of p < 0.05 was considered significant.

s and inhibition of cell proliferation by the cyclooxgenase enzymene. Eur J Obstet Gynecol (2012), http://dx.doi.org/10.1016/

Fig. 4. DAPI staining results on Ishikawa cells. A1: control, A2: ethanol, A3: DMSO, B1: cisplatin 10 mM, B2: cisplatin 20 mM, B3: cisplatin 40 mM, C1: nimesulide 25 mM, C2:

nimesulide 100 mM, C3: nimesulide 200 mM, D1: cisplatin 20 mM + nimesulide 100 mM, D2: cisplatin 20 mM + nimesulide 200 mM.

S.S. Ozalp et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2012) xxx–xxx4

G Model

EURO-7720; No. of Pages 6

[24]. Previous studies reveal that nimesulide and its analogs couldinhibit breast cancer cell growth [34].

The expansion ability of tumor cells depends on not only therate of cell proliferation but also the rate of cell death [35].Programmed cell death (apoptosis) is initiated after cells areexposed to cytotoxic stresses including UV, IR irradiation,chemotherapeutic drugs, hypoxia, serum deprivation and TRAIL[36], and it is a major component of this attrition [3]. Itsbiochemical process is activated via two well-studied pathways:the death receptor-mediated pathway and the mitochondria-mediated pathway. Activation of caspase-3 is considered to be thefinal step in many apoptosis pathways [37]. The effects of cisplatin,nimesulide and a combination of cisplatin and nimesulide onapoptosis were examined by DAPI staining, JC-1 staining andcaspase-3 activity in the present study. The result of this studyindicated that nimesulide is highly effective in suppressing thegrowth of Ishikawa cells. The number of viable cells exposed onlyto 20 mM cisplatin was decreased by 35%, while the application ofthe same dose of cisplatin in combination of 200 mM nimesulidedecreased the number of viable Ishikawa cells by 44% (Fig. 1). Inaddition, nimesulide-induced apoptosis-like changes in the cellmorphology were observed in the Ishikawa cells in a time- anddose-dependent manner.

Please cite this article in press as: Ozalp SS, et al. Induction of apoptosiblocker-nimesulide in the Ishikawa endometrial cancer cell lij.ejogrb.2012.05.018

Mitochondria have received considerable attentions as aprincipal target of drug-induced toxicity. Mitochondria carry outa variety of biochemical processes, including oxidative phosphor-ylation, which provide most of the energy for the cell. Mitochon-drial oxidative phosphorylation is dependent upon a protonelectrochemical gradient (DCm) generated by respiration andmaintained by the impermeability of the inner membrane toprotons. If the mitochondrial membrane is rendered permeable toprotons, the membrane potential dissipates. Singh et al. [38]reported that nimesulide significantly depolarized mitochondria at100 mM, but 250 mM and 500 mM concentrations of nimesulidecaused no further decrease in mitochondrial electron flow in theirstudy. Therefore, 100 mM and 200 mM concentrations of nimesu-lide were used in combination experiments of the present study.The Ishikawa cells’ mitochondrial membrane potential changedafter nimesulide treatment, and the percentage of Ishikawa cellswith green fluorescence increased after nimesulide treatment.

Caspase activation is the final common pathway to induction ofapoptosis in many systems. Therefore, the role of caspase-3 onIshikawa cell apoptosis was explored in this study. The resultsdemonstrated that the activity of caspase-3 increased after 24 h ofexposure to nimesulide, which demonstrated similar kinetics tothe occurrence of apoptosis. The results of caspase-3 enzyme

s and inhibition of cell proliferation by the cyclooxgenase enzymene. Eur J Obstet Gynecol (2012), http://dx.doi.org/10.1016/

Fig. 5. A1: control, A2: ethanol, A3: DMSO, B1: cisplatin 10 mM, B2: cisplatin 20 mM, B3: cisplatin 40 mM, C1: nimesulide 25 mM, C2: nimesulide 100 mM, C3: nimesulide

200 mM, D1: cisplatin 20 mM + nimesulide 100 mM, D2: cisplatin 20 mM + nimesulide 200 mM.

S.S. Ozalp et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2012) xxx–xxx 5

G Model

EURO-7720; No. of Pages 6

activity and mitochondrial membrane potential experiments alsorevealed that combinations of these agents induce apoptosisthrough mitochondrial membrane potential and a caspase-3-dependent pathway.

According to our experimental results nimesulide can have abetter killing effect than the well known DNA damaging agent,cisplatin. We can explain nimesulide’s better killing effect due tospecific prostaglandin inhibition. The COX-2 product, prostaglan-din E2 (PGE2), acts through a family of G protein-coupled receptors

Fig. 6. Effects of cisplatin, nimesulide and combination

Please cite this article in press as: Ozalp SS, et al. Induction of apoptosiblocker-nimesulide in the Ishikawa endometrial cancer cell lij.ejogrb.2012.05.018

designated EP1-4 that mediate intracellular signaling by multiplepathways [39]. In this content, EP antagonists may exert stronganti-cancer effects too. Yu et al. [20] reported that four COXinhibitors (nimesulide, celecoxib, 4-[5-(4-chlorophenyl)-3-(tri-fluoromethyl)-1H-pyrazol-1-yl]-benzenesulfonamide (SC-236)and indomethacin), all substantially suppressed prostaglandinE2 production.

There is a lack of literature evaluating nimesulide (a COX-2inhibitor) in endometrial cancer, despite other evidence regarding

of cisplatin and nimesulide on caspase-3 activity.

s and inhibition of cell proliferation by the cyclooxgenase enzymene. Eur J Obstet Gynecol (2012), http://dx.doi.org/10.1016/

S.S. Ozalp et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2012) xxx–xxx6

G Model

EURO-7720; No. of Pages 6

different types of tumors. This study demonstrated that nimesu-lide alone and nimesulide–cisplatin combinations act as potentcytotoxic agents in endometrium cancer by inhibiting cellproliferation and inducing apoptosis, which suggests that nime-sulide and nimesulide–cisplatin combinations can be potentiallydeveloped as anti-cancer agents to combat endometrial cancer.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

References

[1] Li H, Takai N, Yuge A, et al. Novel target genes responsive to the anti-growthactivity of triptolide in endometrial and ovarian cancer cells. Cancer Letters2010;297:198–206.

[2] Sorosky JI. Endometrial cancer. Obstetrics and Gynecology 2008;111:436–47.[3] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.[4] Vaux DL, Korsmeyer SJ. Cell death in development. Cell 1999;96:245–54.[5] Sun SY, Hail JRN, Lotan R. Apoptosis as a novel target for cancer chemopre-

vention. Journal of the National Cancer Institute 2004;96:662–72.[6] Koseoglu BG, Ozturk S, Kocak H, et al. The effects of etodolac, nimesulide and

naproxen sodium on the frequency of sister chromatid exchange afterenclused third molars surgery. Yonsei Medical Journal 2008;49(5):742–7.

[7] Beata RN, Wojciech F, Stanisław S, et al. The influence of anti-inflammatorydrugs on the proliferation of fibroblast derived from nasal polyps. Auris NasusLarynx 2005;32:225–9.

[8] Harris RE, Chlebowski RT, Jackson RD, et al. Breast cancer and nonsteroidalanti-inflammatory drugs: prospective results from the Women’s Health Ini-tiative. Cancer Research 2003;63:6096–101.

[9] Deasy BM, O’Sullivan-Coyne, O’Donovan TR, et al. Cyclooxygenase-2 inhibitorsdemonstrate anti-proliferative effects in oesophageal cancer cells by prosta-glandin E2-independent mechanisms. Cancer Letters 2007;256:246–58.

[10] Dannenberg AJ, Altorki NK, Boyle JO, et al. Cyclo-oxygenase 2: a pharmaco-logical target fort he prevention of cancer. Lancet Oncology 2001;2:544–51.

[11] Gasparini G, Longo R, Sarmiento R, et al. Inhibitors of cyclo-oxygenase 2: a newclass of anticancer agents? Lancet Oncology 2003;4:605–15.

[12] Ferrandina G, Legge F, Ranelletti RO, et al. Cyclooxygenase-2 expression inendometrial carcinoma: correlation with clinicopathological parameters andclinical outcome. Cancer 2002;95:801–7.

[13] Kilic G, Gurates B, Garon J, et al. Expression of cyclooxygenase-2 in endome-trial adenocarcinoma. European Journal of Gynaecological Oncology2005;26:271–4.

[14] St-Germain ME, Gagnon V, Mathieu I, et al. Akt regulates COX-2 mRNA andprotein expression in mutated-PTEN human endometrial cancer cells. Inter-national Journal of Oncology 2004;24:1311–24.

[15] Masferrer JL, Leahy KM, Koki AT, et al. Antiangiogenic and antitumor activitiesof cyclooxygenase-2 inhibitors. Cancer Research 2000;60:1306–11.

[16] Abrahao AC, Castilho RM, Squarize CH, et al. A role for COX2-derived PGE2 andPGE2-receptor subtypes in head and neck squamous carcinoma cell prolifera-tion. Oral Oncology 2010;46:880–7.

[17] Hazra S, Dubinett S. Ciglitazone mediates COX-2 dependent suppression ofPGE2 in human non-small cell lung cancer cells. Prostaglandins Leukotrinesand Fatty Acids 2007;77:51–8.

[18] Genc S, Attar E, Gurdol F, et al. The effect of COX-2 inhibitor, nimesulide, onangiogenic factors in primary endometrial carcinoma cell culture. Clinical andExperimental Medicine 2007;1:6–10.

[19] Hasegawa K, Ohashi Y, Ishikawa K, et al. Expression of cyclooxygenase-2 inuterine endometrial cancer and anti-tumor effects of a selective COX-2inhibitor. International Journal of Oncology 2005;26:1419–28.

Please cite this article in press as: Ozalp SS, et al. Induction of apoptosiblocker-nimesulide in the Ishikawa endometrial cancer cell lij.ejogrb.2012.05.018

[20] Yu L, Chen M, Li Z, et al. Celecoxib antagonizes the cytotoxicity of cisplatin inhuman esophageal squamous cell carcinoma cells by reducing intracellularcisplatin accumulation. Molecular Pharmacology 2011;79:608–17.

[21] Dormond O, Foletti A, Paroz C, Guegg C. NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migrationand angiogenesis. Nature Medicine 2001;7:1041–7.

[22] Ben-Av P, Crofford LJ, Wilder L, Hla T. Induction of vascular endothelial growthfactor expression in synovial fibroblasts by prostaglandin E interleukin-1: apotential mechanism for inflammatory angiogenesis. FEBS Letters1995;72:83–7.

[23] Chen B, Su B, Chen S. A COX-2 inhibitor nimesulide analog selectivelyinduces apoptosis in Her2 overexpressing breast cancer cells via cyto-chrome c dependent mechanisms. Biochemical Pharmacology2009;77:1787–94.

[24] Thigpen T, Blessing J, Homesley H, Creasman WT, Sutton G. Phase II trial ofcisplatin as first-line chemotherapy in patients with advanced or recurrentendometrial carcinoma: a Gynecologic Oncology Group study. Cancer Treat-ment Reports 1989;33:68–70.

[25] Delord JP, Puozzo C, Lefresne F, Bugat R. Combination chemotherapy ofvinorelbine and cisplatin: a phase I pharmacokinetic study in patients withmetastatic solid tumors. Anticancer Research 2009;29:553–60.

[26] Koparal AT, Ulus G, Zeytinoglu M, Tay T, Turk AO. Angiogenesis inhibition by alichen compound olivetoric acid. Phytotherapy Research 2010;24:754–8.

[27] Bostancioglu RB, Is ık K, Genc H, Benkli K, Koparal AT. Studies on the cytotoxic,apoptotic and antitumoral effects of Au(III), Pt(II) complexes of 1,10-phenan-throline on V79 379A and A549 cell lines. Journal of Enzyme Inhibition andMedicinal Chemistry 2012;27(3):458–66. http://dx.doi.org/10.3109/14756366.2011.596835.

[28] Castedo M, Feri K, Roumier T, Metivier D, Zamzami N, Kroemer G. Quantitationof mitochondrial alterations associated with apoptosis. Journal of Immuno-logical Methods 2002;265:39–47.

[29] Sebastian A, Allan E, Allan D, et al. Addition of novel degenerate electricalwaveform stimulation with photodynamic therapy significantly enhances itscytotoxic effect in keloid fibroblasts: first report of a potential combinationtherapy. Journal of Dermatological Science 2011;64(3):174–84.

[30] Pang RP, Zhou JG, Zeng ZRET-AL>. Celecoxib induces apoptosis in COX-2deficient human gastric cancer cells through Akt/GSK3B/NAG-1 pathway.Cancer Letters 2007;251:268–77.

[31] Tiju JW, Liao YH, Lin SJET-AL>. Cyclooxugenase-2 overexpression in humanbasal cell carcinoma cell line increases antiapoptosis, angiogenesis, and tu-morigenesis. Journal of Investigative Dermatology 2006;126:1143–51.

[32] Kashfi K, Rigas B. Non-COX-2 targets and cancer: expanding the moleculartarget repertoire of chemoprevention. Biochemical Pharmacology2005;70:969–86.

[33] Dannenberg AJ, Subbaramaiah K. Targeting cyclooxygenase-2 in human neo-plasia: rationale and promise. Cancer Cell 2003;4:431–6.

[34] Su B, Darby MV, Brueggemeier RW. Synthesis and biological evaluation ofnovel sulfonanilide compounds as antiproliferative agents for breast cancer.Journal of Combinatorial Chemistry 2008;10:475–83.

[35] Wang D, Fung JNT, Tuo Y, Hu L, Chen C. TWEAK/Fn14 promotes apoptosis ofhuman endometrial cancer cells via caspase pathway. Cancer Letters2010;294:91–100.

[36] Kuribayashi K, Mayes PA, El-Deiry WS. What are caspases 3 and 7doing upstream of the mitochondria? Cancer Biology and Therapy2006;5:763–5.

[37] Wang L, Chen T, Qu J, Wei X. Quantitative analysis of caspase-3 activationby fitting fluorescence emission spectra in living cells. Micron2009;40:811–20.

[38] Singh BK, Tripathi M, Pandey PK, Kakar P. Nimesulide aggravates redoximbalance and calcium dependent mitochondrial permeability transitionleading to dysfunction in vitro. Toxicology 2010;275:1–9.

[39] Ma X, Kundu N, Rifat S, et al. Prostaglandin E receptor EP4 antagonism inhibitsbreast cancer metastasis. Cancer Research 2006;66:2923–7.

s and inhibition of cell proliferation by the cyclooxgenase enzymene. Eur J Obstet Gynecol (2012), http://dx.doi.org/10.1016/