Experimental and Molecular Pathology 88 (2010) 394–400

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Experimental and Molecular Pathology

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Induction of pulmonary carcinogenesis in Wistar rats by a single dose of 9, 10Dimethylbenz(a)anthracene (DMBA) and the chemopreventive role of Diclofenac

Poonam Thakur, S.N. Sanyal ⁎Department of Biophysics, Panjab University, Chandigarh 160014, India

Abbreviations: CMC, carboxy-methyl cellulose; PBS,⁎ Corresponding author.

E-mail addresses: poonamthkur@gmail.com (P. Thaksanyal@pu.ac.in (S.N. Sanyal).

0014-4800/$ – see front matter © 2010 Elsevier Inc. Adoi:10.1016/j.yexmp.2010.03.005

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 17 November 2009and in revised form 4 March 2010Available online 15 March 2010

Keywords:DiclofenacCOX-2ApoptosisInflammation

Objectives: To evaluate the chemopreventive efficacy of Diclofenac, a preferential cyclooxygenase-2 (COX-2)inhibiting non steroidal anti-inflammatory drug (NSAID) in the 9, 10 Dimethylbenz(a)anthracene (DMBA)induced experimental lung carcinogenesis.Methods: Animals were divided into 4 groups. Control group received normal saline intratratracheally. DMBAgroup was given DMBA (20 mg/kg of body weight) in the similar manner. DMBA+Diclofenac group wasgiven daily oral dose of Diclofenac (8 mg/kg of body weight) in addition to DMBA while the last groupreceived Diclofenac only. Animals were sacrificed after 24 weeks. COX-2 expression was studied byimmunohistochemistry (IHC) and Western immunoblotting. For apoptosis study DNA fragmentation onagarose gel and florescent staining of alveolar macrophages were done.

Results: The incidence and burden of tumor were reduced by the Diclofenac treatment. Diclofenac caused thereduction in the COX-2 levels which were increased in the DMBA treated group. It also caused the inductionof apoptosis as seen by both techniques.Conclusion: From all these results it can be concluded that Diclofenac might have a chemopreventive role forlung carcinogenesis which is mediated by suppression of COX-2 enzyme and induction of apoptosis.

© 2010 Elsevier Inc. All rights reserved.

Introduction

Cyclooxygenase (COX) enzyme catalyzes the formation of proin-flammatory prostaglandins such as PGE2 from the arachidonic acid.These compounds have various carcinogenesis accelerating roles(Castelao et al., 2003). COX has two major isoforms; COX-1 isconstitutively expressed in many tissues and involved in maintainingvascular homeostasis while COX-2 is an inducible early response geneproductwhose expression is regulated by inflammatorymediators suchas cytokines, growth hormones, endotoxins etc. (Hilario et al., 2006).COX-2 is upregulated in inflammation, premalignant lesions, adeno-carcinomas and various other disorders (Asgari et al., 2004). Its role inlung cancer progression and development has been well investigatedand it is one of the major molecules targeted for chemopreventivestrategies (Lee et al., 2008; Subbaramaiah and Dannenberg, 2003). It isprogressively upregulated (Bauers et al., 2000) as the phenotype ofcancer changes from premalignant lesion to the full grown adenomas.Also, its expression is associated with poor prognosis in lung cancerpatients (Riedl et al., 2004). Many reports suggest that COX-2 due to itsperoxidase activity can bio-activate the environmental carcinogens likepolycyclic aromatic hydrocarbons (PAHs) (Wiese et al., 2001). It is also

phosphate buffer saline.

ur), sanyalpu@gmail.com,

ll rights reserved.

associated with an increase in invasiveness (Gridelli et al., 2002);increasing angiogenesis by releasing proangiogenic factors like iNOS,interleukin-6, vascular endothelial growth factor etc. (Gately, 2000) andconferring apoptosis resistance (Lin et al., 2001) in tumorous tissue.

Non steroidal anti-inflammatory drugs (NSAIDs) are commonlyused drugs for management of pain, fever, inflammation, arthritis etc.(Hilario et al., 2006). They act mainly through inhibition of COXenzyme. Numerous epidemiological studies have shown theirantineoplastic effects (Asgari et al., 2004). NSAIDs are classified intotwo classes based on their COX inhibiting activity — nonspecificNSAIDs like aspirin which inhibit both isoforms of the enzyme andCOX-2 selective molecules like celecoxib, etoricoxib and NS.398.Despite showing promise, their use as chemopreventives has how-ever, been hindered due to the unwanted side effects associatedwith both classes (Hilario et al., 2006; Grosch et al., 2006). In thisscenario, Diclofenac holds its unique position as it has a preferentialselectivity for COX-2 (Giuliano and Warner, 1999) therefore it mighthave lesser side effects compared to those associated with othertraditional NSAIDs. In addition to COX-2 inhibiting effects it alsopossesses Lipooxygenase (LOX) inhibitory effects thereby producingthe lower level of leukotrienes (Cryer and Feldman, 1998). Thisdual inhibition can provide a beneficial effect in the delay of cancerprogression. It has a half-life of 2 h in normal tissue and around 6 h ininflamed tissue in rat body (Schweitzer et al., 2009).

In the present study, we studied the chemopreventive action ofDiclofenac in Dimethylbenz(a)anthracene (DMBA) induced lung

Fig. 1. Body weight profile of animals during the 24 week study.

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cancer in female Wistar rats. DMBA is an environmental pollutantpresent in cigarette smoke, automobile exhaust, grilled food, woodsmoke etc. (Granberg et al., 2000). It is a highly potent carcinogen andimmunosuppressive agent (Buters et al., 2003;Davilla et al., 1996). It is aprocarcinogen which is metabolized by cytochrome P450 enzymesinto the ultimate carcinogenic form 7,12-DMBA-3,4-dihydrodiol-1,2-epoxide (Kleiner et al., 2002). It forms adducts with DNA and producesmutations (Moody et al., 2003). These adducts can persist in the bodywith a half-life of around 7 days (Świercz et al., 2006). It was used toproduce lung cancer in rats by single intratracheal instillation. In thismodel the antineoplastic efficacy of Diclofenac was studied in terms ofCOX-2 inhibition and induction of apoptosis, which is the end effect ofmost of the chemopreventives.

Materials and methods

Experimental design

Female Wistar rats were obtained from the Central Animal Houseof Panjab University, Chandigarh. Animals were housed in polypro-pylene cages on rice husk (changed regularly), with a maximum of 4rats in one cage. They were kept under standard laboratory conditionswith ambient temperature and 12 hour day/night photoperiod. Theywere fed standard pellet diet (obtained from Ashirwad industries,

Fig. 2. Gross anatomical changes in the rat lungs after 24 weeks of DMBA

Kharar, Punjab, India) and water ad libitum. Their body weight wasrecorded every week in a single pan balance throughout the durationof study. After acclimatization for 1 week, animals were randomlydivided into 4 groups having 10 animals each. The first group servedas control and was given a single intratracheal instillation of 0.5 mlsaline, which was the vehicle of DMBA. A single intratracheal dose ofDMBA (20 mg/kg body weight) was given to the second group usingthe dose and protocol as established in our lab earlier (Saini andSanyal, 2008). Briefly, animals were anesthetized with ketaminehydrochloride (130 mg/kg body weight) given intraperitoneally. Anincision was made on the neck and the trachea was exposed. DMBA,suspended in the normal saline at the concentration of 100 mg/ml,was instilled with the syringe by slowly injecting it into the trachea.The wound was sutured with a silk thread and a coat of antibiotic wasapplied. The third group was the co-treatment group, which receiveda single dose of DMBA as mentioned above. After a recovery periodof 1 week, Diclofenac (8 mg/kg body weight) dissolved in 0.2%CMC (carboxy-methyl cellulose, sodium salt) at the concentration of3 mg/ml, was given daily by gavage for 24 weeks. The last group wasadministered Diclofenac alone in a similar manner throughout thestudy period. The dose chosen for Diclofenac (NSAID) is within thetherapeutic anti-inflammatory range as based on the reported ED50

value for rats. Also, this dose was based on the anti-inflammatoryeffects on carrageenan induced hind paw edema test in rats in the

administration. Tumor nodules and angiogenic lesions can be seen.

Table 1The effect of Diclofenac on the DMBA induced lung tumorigenesis, showing the tumorand lesion incidence, burden and multiplicity at 24 weeks post treatment (n=10).

Groups Tumorincidence

Tumorburden

Tumormultiplicity

Lesionincidence

Lesionburden

Lesionmultiplicity

Control Nil Nil Nil Nil Nil NilDMBA 100% 19 19 100% 4.33 4.33DMBA+Df Nil Nil Nil 100% 1 1Diclofenac Nil Nil Nil Nil Nil Nil

Df — Diclofenac; DMBA — 9, 10-Dimethylbenz(a)anthracene.Incidence: percentage of animals bearing tumor or lesion.Multiplicity: total no. of tumors or lesions/no. of tumor or lesion bearing rats.Burden: total no. of tumors or lesions/total no. of rats.

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present lab (Kaur and Sanyal, 2009). All the procedures wereperformed according to the conditions laid down by the ethicscommittee on the use of experimental animals of Panjab University.DMBA was obtained from Sigma-Aldrich (St. Louis, MO, USA) whileDiclofenac was a generous gift from Ranbaxy (Gurgaon, Haryana,India).

Lung tumor analysis

After 24 weeks rats in all the groups were sacrificed by an overanesthesia of diethyl ether. Lungswere cleared of blood by perfusionwithchilled phosphate buffer saline (PBS). They were removed and all fivelobeswere separated and examined thoroughly using a handheld lens forthe presence of any tumors and any tumors or lesions found werecounted.

Histology

Tissue portions from two different lobes were cut and fixed in 10%neutral buffered formalin for at least 48 h. Paraffin wax (58 °C–60 °C)

Fig. 3. The effect of various treatments on the histoarchitecture of the rat lungs after 24 wee(20×), D — DMBA (20×) and E — DMBA (40×).

was used to embed the tissue according to the standard protocol(Pearse, 1968) and cut into thin sections (5 µM) by a hand operatedmicrotome. Sections were dewaxed, passed through a graded series ofalcohol and stained with hematoxylin and eosin.

Immunohistochemistry

After dewaxing in xylene and hydrating through a graded series ofalcohol, lung sections were incubated with 2% BSA in PBS for 30 min ina moist chamber to block the non specific staining. Sections wererinsed in PBS and incubated with primary antibody against COX-2(1:1000 dilution; Santa Cruz Biotechnology Inc., CA, USA) with 1% BSAin PBS for 2 h in a moist chamber at 37 °C. After washing twice withPBS-tween 20 (50 µl/100 ml), slides were incubated with thesecondary antibody (1:10,000 dilution; Genei, Bangalore, India) in1% BSA for 2 h at 37 °C. Washing with tween 20 was repeated andcolor developed using NBT/BCIP solution (Genei, Bangalore, India).Counter staining was done with methyl green.

DNA fragmentation

DNA was isolated from the lung tissue using a phenol chloroformextraction method (Sambrook et al., 1998). It was quantitated at260 nm; electrophoresed on 1.5% agarose gel in TAE buffer andvisualized by Ethidium bromide under UV light. Photograph wastaken using a GelDoc machine (Upland, CA, USA).

Fluorescence microscopy for apoptosis study in alveolar macrophages

For macrophage isolation, animals were sacrificed and perfusionwas done with chilled PBS. Lungs were removed, and after washing inPBS, chopped finely. Incubation with protease was done for 1 h. Thenit was filtered through amuslin cloth and centrifugation of filtrate wasdone at 700 rpm for 5–7 min. The pellet obtained was suspended in

ks of treatment. A — control (20×), B — DMBA+Diclofenac (20×), C — Diclofenac alone

Fig. 4. The effect of DMBA, DMBA+Diclofenac and Diclofenac only treatment on the expression of COX-2 in the rat lungs. A — control (20×), B — DMBA+Diclofenac (20×),C — Diclofenac alone (20×) and D — DMBA (20×).

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PBS. For viability assay methyl green staining was done. Giemsastainingwas used to assay the purity of preparationwhich was alwaysfound to be more than 97%. 10 µl of isolated cells was mixed withequal volume of both Acridine orange and Ethidium bromide orPropidium iodide and Hoechst-33342 (Schwartz and Osborne, 1995).After putting the cover slip these were observed under a florescencemicroscope (Nikon, Japan). For Acridine orange–Ethidium bromidelive cells appear green, apoptotic cell yellow while necrotic cell red,while for Propidium iodide–Hoechst-33342, live cells appeared blue,apoptotic cells pink and necrotic cells red. For quantitation a total of100 cells from 4 different slides were counted and percent apoptoticcells were calculated for each group.

Western blotting

Western blotting for COX-2 was done (Sambrook et al., 1998) tostudy the protein expression for the enzyme. 0.2 g of lung tissue wastaken and a 20% homogenate was prepared in the lysis buffer(100 mM tris–Cl, 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100).Homogenate was centrifuged for 30 min at 10,000 rpm. Supernatantwas collected and estimated for the protein content. For proteinestimation Bradford method (Bradford, 1976) was followed. Afterestimation it was run on SDS-PAGE and transferred onto thenitrocellulose membrane. The quantitative transfer was checked bystaining with Ponceau S. Primary antibody for COX-2 was used for

Fig. 5. Study of the effect of DMBA, DMBA+Diclofenac and Diclofenac only treatmenton the expression of COX-2 in the rat lungs by Western immunoblot.

blotting following the method of Towbin et al. (1979). NBT/BCIPsolution was used for developing the blots after reacting with thealkaline phosphatase conjugated secondary antibody.

Fig. 6. Apoptosis as seen by DNA fragmentation by DMBA, DMBA+Diclofenac andDiclofenac only treatment.

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Statistical analysis

Statistical significance of the data was performed by the analysis ofvariance (one way ANOVA) following a post-hoc test using the leastsignificance difference (LSD) method with the help of SPSSv10computer software.

Results

The present study was designed to establish the chemopreven-tive efficacy of Diclofenac in the 9, 10-Dimethylbenz(a)anthracene(DMBA) induced lung cancer in female Wistar rats. Body weightof the animals was recorded every week. It was observed that ani-mals in control, DMBA+Diclofenac and Diclofenac only groupshowed linear growth in body weight with time but DMBA treatedanimals did not gain any appreciable weight (Fig. 1). After 24 weeksanimals were sacrificed and lungs were observed for the presenceof tumors. In the DMBA treated animals full blown tumor noduleswere visible along with the angiogenic lesions (Fig. 2) which werepresent in all the five lobes of the lungs. These lesions were alsoobserved in the DMBA+Diclofenac treated animals. Table 1 showsthe incidence of tumors and angiogenic lesions in the differenttreatment groups. 100% tumor incidence was observed in DMBAtreated animals. It was seen that with the Diclofenac co-treatmentthe tumor incidence was found to be nil, although lesions were

Fig. 7. Macrophages visualized under fluorescence microscope (10×) for the 24 week treatmHoechst-33342 co-staining. (C) Histogram showing the percent apoptotic cells visualized by**p≤0.01 w.r.t. control group,*p≤0.05, +++p≤0.001 w.r.t. DMBA group and +p≤0.05 w

present but the burden had decreased significantly compared to theDMBA treated group.

Changes in the histological structure were also observed. Distinctchanges were present in DMBA treated animals (Figs. 3D and E).Thetumor nodules presented themselves as adenomas with no alveolarstructure, hyperplasia and dysplasia. The histoarchitecture waslargely preserved in DMBA+Diclofenac group although alveoli wereconstricted compared to the control.

The NSAIDs exert their beneficial chemopreventive effects mainlyby the inhibition of COX-2. Immunohistochemical analysis of COX-2showed the highest expression in DMBA group where intense bluestaining pertaining to its expression can be seen at the tumor region(Fig. 4D), while comparatively lesser expression was seen inDiclofenac co-treatment group. In Fig. 5Western blotting also showeda very high expression of COX-2 in DMBA group and the lowering of itin DMBA+Diclofenac group.

Fig. 6 shows the DNA fragmentation in various treatment groups.In DMBA treated group the intensity of the bands of fragmentedDNA which is indicative of the apoptosis process, is the lowest. WithDiclofenac treatment however, the intensity has increased indicatingthe restoration of apoptosis process.

Apoptosis was also studied in the alveolar macrophages. Stainingwith fluorescent dyes, Ethidium bromide/Acridine orange (Fig. 7A)and Propidium iodide/Hoechst (Fig. 7B) revealed that apoptosis wasdiminished in DMBA treated group significantly (p≤0.001) compared

ent group. (A) Ethidium bromide/Acridine orange co-staining. (B) Propidium iodide/fluorescence microscopy in various treatment groups (***p≤0.001 w.r.t. control group,.r.t. DMBA group).

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to the control group (Fig. 7C). With Diclofenac co-treatment thenumber of apoptotic macrophages was increased significantly(p≤0.001) when compared to DMBA group, thus showing the abilityof Diclofenac to restore apoptosis, although the levels were still lowerthan control (p≤0.05 for EtBr/AO and p≤0.001 for PI/Hoechst). Even,in the Diclofenac alone group the apoptosis was somehow diminishedcompared to the control.

Discussion

In this study a single intratracheal dose of DMBA at 20 mg/kgbody weight was used to induce carcinogenesis in rat lungs. DMBAis an environmental carcinogen which is commonly used to producecancer in a variety of organs which include the skin, mammarygland etc. (Li et al., 2003; Suzuki et al., 2003). However, it requiresmultiple applications to the site of interest. In the current studywe used a model which utilizes the deposition of a single dose ofDMBA intratracheally. It allows the carcinogen to be relativelysafely handled as the instillation was needed only once and themethod requires a minimum of specialized equipment with mini-mum fatality. The present observations confirm that this dose wassufficient to produce tumors after 24 week as the tumor as well aslesion incidence was 100% in the DMBA treated group. Diclofenaccauses a delay in the cancer progression as evidenced by the absenceof any tumor nodules in DMBA+Diclofenac co-administrationgroup.

COX-2 has diverse roles in lung cancer progression. It increasesangiogenesis and metastasis (Zhu et al., 2008). COX-2 inhibition alsoresults in the blockage of endothelial cell migration (Ruegg et al.,2003). There is also evidence that it is associated with the molecularmechanism of proliferation and diffusion of cancerous cells. Numer-ous studies have provided the evidence that COX-2 inhibition has achemopreventive action. Diclofenac is a NSAID which has thepreferential COX-2 activity (Giuliano and Warner, 1999) conferringit a better side effect profile compared to other COX-2 specific ortraditional NSAIDs. In addition, NSAIDs like Diclofenac cause degra-dation of IκBα, which causes NF-kB translocation into the nucleus.This induces transcription of many genes which produce the proteinsinvolved both in inflammation and apoptosis (Cho et al., 2005). Theresults of IHC andWestern immunoblot show that the levels of COX-2were higher in the DMBA treated group which was however loweredby Diclofenac treatment. Hence, it can be speculated that the ability ofthe Diclofenac to delay cancer progression may be due to theinhibition of COX-2 protein.

Programmed cell death or apoptosis is central to the pathogen-esis of many disease processes including cancer (Piazza et al., 1997).Reduction in cancer incidence by NSAIDs like Diclofenac is asso-ciated with the increased incidence of apoptosis (Hofer et al., 2002),while carcinogenic changes by DMBA involve diminution ofapoptosis which helps in rapid proliferation and spread of cancerouscells. Results from DNA fragmentation experiment clearly show thediminished apoptosis in DMBA treatment group, which helps in thegrowth of the cancerous cells. Diclofenac offers a chemopreventiveeffect here by inducing apoptosis, the mechanism for which isbelieved to be mediated by the activation of caspase-3 (Chang andWeng, 2001).

Macrophages have a very important role in inflammation andcarcinogenesis in the lungs (Chen et al., 2005). Growing tumorssecrete a variety of factors which in turn activate macrophages tosecrete proinflammatory, angiogenic factors, cytokines etc. thatenhance metastasis and growth of tumor (Balkwill and Mantovani,2001). Florescence staining was used to study the apoptosis inalveolar macrophages. The apoptosis was drastically reduced incancerous lungs in DMBA treated animals confirming their importantrole in tumor progression. Diclofenac co-treatment increased the

number of apoptotic macrophages which helps to prevent the spreadof tumor.

In conclusion, the results of the present study clearly show that asingle treatment of DMBA is able to induce lung cancer whichprogresses with time. Diclofenac, a NSAID exerts its chemopreventiveaction on this model by being able to restore apoptosis as seen byvarious physicochemical methods which acts to kill the growingcancer cells. It also inhibits the expression of COX-2 enzyme which isassociated with the development of lung cancer and poor prognosis.From all these parameters it can be concluded that Diclofenac has achemopreventive action in DMBA induced experimental lung carci-nogenesis. Its mechanism of action at the molecular level however,needs to be further explored.

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