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Design, synthesis and evaluation oflantadene A congener with hydroxylfunctionality in ring A as an antitumouragentManu Sharma a , Anurag Rakhi b , Nandita Dalal b & NeetikaSharma ca Department of Biotechnology and Bioinformatics , JaypeeUniversity of Information Technology , Waknaghat, Solan 173215 ,Himachal Pradesh , Indiab Department of Pharmaceutical Chemistry , Rayat Institute ofPharmacy , Railmajra, Nawanshahr 144533 , Punjab , Indiac Department of Chemistry , Maharaja Singh College , Saharanpur247001 , Uttar Pradesh , IndiaPublished online: 09 Jul 2010.

To cite this article: Manu Sharma , Anurag Rakhi , Nandita Dalal & Neetika Sharma (2011) Design,synthesis and evaluation of lantadene A congener with hydroxyl functionality in ring A as anantitumour agent, Natural Product Research: Formerly Natural Product Letters, 25:4, 387-396, DOI:10.1080/14786411003792173

To link to this article: http://dx.doi.org/10.1080/14786411003792173

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Natural Product ResearchVol. 25, No. 4, February 2011, 387–396

Design, synthesis and evaluation of lantadene A congener with

hydroxyl functionality in ring A as an antitumour agent

Manu Sharmaa*, Anurag Rakhib, Nandita Dalalb and Neetika Sharmac

aDepartment of Biotechnology and Bioinformatics, Jaypee University of InformationTechnology, Waknaghat, Solan 173215, Himachal Pradesh, India; bDepartment ofPharmaceutical Chemistry, Rayat Institute of Pharmacy, Railmajra, Nawanshahr 144533,Punjab, India; cDepartment of Chemistry, Maharaja Singh College, Saharanpur 247001,Uttar Pradesh, India

(Received 4 November 2009; final version received 19 March 2010)

Pentacyclic triterpenoid lantadene A congener with hydroxyl functionalityin ring A was designed and synthesised on the basis of enhancement ofpolarity and bioactivity. The new synthesised compound 22�-angeloyloxy-methyl-2-hydroxy-3-oxoolean-1,12-dien-28-oate (6) was screened for cyto-toxicity against human cancer cell lines (HL-60, HeLa, Colon 502713 andA-549) and showed a better cytotoxicity than the parent compound(p5 0.05). Further, compound 6 was screened for in vivo antitumouractivity in a two-stage squamous cell carcinogenesis model, using femaleSwiss albino mice. Compound 6 showed a better tumour inhibition profilethan the parent compound. Compound 6 also exhibited a marked decreasein protein expression of activator protein-1 (c-jun), nuclear factor-kappa B(p65) and p55. The results inferred that an increase in polarity of the leadmolecule not only increased the antitumour activity but also reduced thedose, which may be linked to the deregulation of the abovementionedmolecular targets, and warrants further optimisation of the structure tomake it a drug-like candidate.

Keywords: lantadene A; antitumour; AP-1 (c-jun); NF-iB (p65) and p55

1. Introduction

Lantana camara L. is one of the most noxious weeds in the world, which grows wildin tropical and subtropical regions (O. Sharma, Vaid, & P. Sharma, 1991). Its wildgrowth provides a huge amount of biomass and currently there is immense interest toexploit its natural products for drug research (Ghisalberti, 2000). The methanolicleaf extract of Lantana showed chomopreventive effects on 7,12-dimethylbenz[a]anthracene (DMBA)-induced squamous cell carcinogenesis inSwiss albino mice (M. Sharma, P. Sharma, & Bansal, 2007a). The triterpenoids ofL. camara are named lantadenes (1–4; Figure 1) and have been found to exhibit awide spectrum of pharmacological activities, including antitumour effects (Inadaet al., 1995, 1997; M. Sharma, P. Sharma & Bansal, 2007b). These compounds differ

*Corresponding author. Email: lantadene@hotmail.com

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2011 Taylor & Francis

DOI: 10.1080/14786411003792173

http://www.informaworld.com

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in the structure of the side chain attached at the C-22 position through ester linkage,and there are indications that these structural variations may play an importantrole in their pharmacological activity (M. Sharma, P. Sharma, & Bansal, 2008;M. Sharma, P. Sharma, Bansal, & Singh, 2007c). Lantadene A (LA) as well as theircongeners (synthesised by us) are highly nonpolar compounds and are required inhigher doses to attain bioactivity (Kaur, M. Sharma, P. Sharma, & Bansal, 2008).Therefore, it was decided to increase the polarity of the LA methyl ester (LAM) andto evaluate its effect on antitumour activity. In this article, we report the synthesis of22�-angeloyloxy-methyl-2-hydroxy-3-oxoolean-1,12-dien-28-oate (6). This com-pound was screened for its cytotoxicity against four human cancer cell lines, andfurther studied for its in vivo tumour inhibitory potential on two-stage squamous cellcarcinogenesis in Swiss albino mice, induced by DMBA and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). The effect of this compound was alsoevaluated against the deregulation of AP-1 (c-jun), NF-iB (p65) and p55 by ELISA.

2. Results and discussion

In continuation of our research to optimise pharmacophore of the pentacyclictriterpenoid LA, we report the synthesis of a novel pentacyclic triterpenoid congener

1; R= Lantadene A (LA)

2; R = Lantadene B (LB)

3; R = Lantadene C (LC)

4; R = Lantadene D (LD)

O

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23 24

5

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910

1112

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Figure 1. Structures of lantadenes (1–4) found in lantana.

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with hydroxyl functionality in ring A, which was designed on the basis of anenhancement of polarity and bioactivity. 22�-Angeloyloxy-methyl-2-hydroxy-3-oxoolean-1,12-dien-28-oate (6) was synthesised as shown in Figure 2. LA (1) wasisolated and transformed to LAM (5) as described previously (M. Sharma &P. Sharma, 2006; Sharma et al., 2007c). Compound 5 was stirred with ButOK inButOH at room temperature with provision of efficient access of air to the reactionmixture to obtain 22�-angeloyloxyl-methyl-2-hydroxy-3-oxoolean-1,12-dien-28-oate(6) in 58.5% yield. All structures were confirmed by using spectroscopic techniquesand elemental analysis.

The LA (1) and its congeners (5 and 6) were studied for their cytotoxicity againsthuman cancer cell lines (HL-60, HeLa, Colon 502713 and A-549), and the results aresummarised in Table 1. Compound 6 further showed a better cytotocity profile than5 (p5 0.01). Carcinogenesis is a multistep process involving the sequential phases ofinitiation, promotion and progression (Sporn & Suh, 2000). Chemoprevention is onedesirable strategy to reverse, arrest or inhibit carcinogenesis. The mouse skincarcinogenesis model is often used to study the genetic and biological changesassociated with chemical initiation of lesions and their subsequent transition tosquamous cell carcinoma. DMBA is a polycyclic aromatic hydrocarbon capable ofinducing skin lesions when applied repeatedly on mouse skin (Shukla, Baqar, &Mehrotra, 1998). The primary bioactivation of DMBA occurs via the formation ofDMBA-3,4-dihydrodiol. DMBA-diol-epoxide has been suggested to be the ultimatecarcinogen responsible for inducing chronic inflammation, reactive oxygen species(ROS) production and oxidative damage of DNA, resulting in the transformation ofnormal cells to tumour cells (Slaga, Gleason, Digiovanni, Sukumaran, & Harvey,1979). When mouse skin is repeatedly exposed to DMBA, the Langerhans cells willbe depleted, and local immunosuppression takes place (Qu, Muller, & Woods, 1979).This leads to the induction of tumours on the skin of the mice. TPA is a phorbolester, which was isolated from croton oil. This is an active constituent of croton oil,responsible for the promotion of tumours. Squamous cell carcinogenesis was inducedin Swiss albino mice (LACCA/female) by DMBA and promoted by TPA. The onset

O

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Lantana leaf powder

1) Extraction with methanol

2) Chloroform partitioning

Etheral CH2N2

O2 -ButOk-ButOH

(1)

(5) (6)

Figure 2. Sequence of steps involved in the isolation and transformation of LA (1) to LAM(5) and 22�-angeloyloxy-methyl-2-hydroxy-3-oxoolean-1,12-dien-28-oate (6).

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of papillomas was observed in the 4th week in the DMBA/TPA-treated mice andfound to be 36.3%. There was a gradual rise in the incidence of tumours that reached100% during the 8th week. The incidence of DMBA/TPA-induced papillomas wasdelayed for 3 weeks by 1 and 5 and for 4 weeks by 6. The animals treated with 6

showed a significant decrease in the incidence of cancer (17.2% vs. 100%) at the endof 20 weeks as compared with the DMBA/TPA-treated group (p5 0.001). Theoverall papilloma incidence in those treated with 5 and 1 was 19.6% (p5 0.001,p5 0.01) and 17.9% (p5 0.001, p5 0.05), respectively, in comparison withthe DMBA/TPA-treated group. The survival rate decreased significantly inDMBA/TPA-treated mice as compared with the vehicle-treated group (30% vs.80%). Survival rates with compound-treated animals were significantly higher incomparison to the DMBA/TPA-treated group (p5 0.01, p5 0.05). The 1, 5 and6-treated groups showed similar survival rates (80%). The average body weight ofDMBA/TPA-treated mice decreased significantly. However, there was a slightincrease in the average body weight of 1, 5 and 6-treated mice at the termination ofthe study (p5 0.01, p5 0.05). Histopathological examination of the depilated backof mice revealed normal skin and the presence of subcutaneous tissue in acetone-treated mice. Twice weekly application of DMBA for 8 weeks on depilated miceinduced well-differentiated squamous cell carcinoma with the formation of keratinpearls. There was marked infiltration of cancer cells in the underlying dermis. Theskin section of mice treated with synthesised compounds showed hyperplasticpapillomatous lesions with no evidence of infiltration or cytological atypia. Thepretreatment of animals with derivatives could be associated with their ability tointerfere with the initiation of carcinogenesis, which is a relatively rapid process, andthe continuous treatment after TPA with the promotion, which is a slow process.These agents also increased the survival time of the animals. Slight weight gain in 1, 5and 6-treated mice could be a result of recovery from the effect of DMBA/TPA or,alternatively, with better papilloma control.

Several transcriptional factors including activator protein-1 (AP-1), nuclearfactor-kappa B (NF-�B), and signal transducers and activators of transcription(STATs) have been linked to the tumour promotion (Angel, Szabowski, & Schorpp-Kistner, 2001; Siebenlist, Franzoso, & Brown, 1994). AP-1 is an important nucleartranscription factor involved in many cellular functions such as cell proliferation,death, survival and differentiation (Shaulian & Karin, 2002). In our previous study,we observed marked decrease in the protein expression of AP-1 (c-jun), NF-�B (p65)

Table 1. Cytotoxicity of LA (1) and its congeners (5 and 6) against human cancer cell lines(IC50 mmol/mL�1).

Compound HL-60 HeLa Colon 502713 A-549

1 0.035� 0.001a 0.042� 0.008b 0.038� 0.005b 0.039� 0.001a

5 0.034� 0.014a 0.037�0.005a 0.037� 0.008b 0.033� 0.005a

6 0.026� 0.006b 0.031� 0.005b 0.032� 0.001a 0.028� 0.004b

Paclitaxelc 50.0023 50.0023 50.0023 50.0023

Notes: Data is represented in mean � SD; n¼ 3. aSignificant difference relative to control50.05. bSignificant difference relative to control 50.01. cPositive control.

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and p53 by compounds 1 and 5 in comparison to DMBA/TPA-treated group.Therefore, it was decided to observe the effect of compound 6 on the proteinexpression of AP-1 (c-jun), NF-�B (p65) and p53. Transcription factor AP-1comprises homodimers and heterodimers composed of a basic region leucine zipperprotein that belongs to the Jun (c-jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, Fra-2),Jun dimerisation partners (JDPs, JDP2) and the closely related activationtranscription factors. AP-1 proteins have target genes regulating cell proliferationand death. The Jun group controls cell life and death through their ability to regulatethe expression and function of cell cycle modulators. A functional role for AP-1components in the epidermis of the skin has been suggested for differentiation,carcinogenesis, UV-response, photoageing and wound repair (Angel et al., 2001).This might be due to regulation of AP-1 which can be achieved at different levels bythe changes in transcription of genes encoding AP-1 subunits, by controlling thestability of the mRNAs, by post-translational processing, turnover and modificationby phosphorylation of AP-1 proteins, and by specific interaction between AP-1proteins and other transcription factors and cofactors (Hess, Angel, & Schorpp-Kistner, 2004). There was a significant increase (p5 0.001) in the level of c-junprotein as analysed by ELISA in DMBA/TPA-treated tumours of group II (Table 2)as compared to control mice skin in group I, while there was a significant decrease(p5 0.01) observed in the level of c-jun in DMBA/TPAþ 5 and 6 treatment tumoursas compared to DMBA/TPA-treated tumours. Also, a significant decrease(p5 0.05) in the levels of c-jun was observed in DMBA/TPAþ 1 treatment tumoursas compared to DMBA/TPA-treated mice tumours.

NF-iB has been implicated in carcinogenesis as it plays a critical role in cellsurvival, cell adhesion, inflammation, differentiation and cell growth. Cancer is ahyperproliferative disorder that results from tumour initiation and promotion andultimately produces tumour metastasis. Several genes involved in cellular transfor-mation, proliferation, invasion and angiogenesis are regulated by NF-iB (Aggarwal,Sethi, Nair, & Ichikawa, 2006). Oxidant stress can result in the degradation ofcytoplasmic NF-iB inhibitor, IiB and its translocation to the nucleus (Asehnoune,Strassheim, Mitra, Yeol-Kim, & Abraham, 2004). In this study, the proteinexpression of p65 increased significantly in the DMBA/TPA-treated group. Thisindicates activation of NF-iB, which is probably due to inhibition of the IiB proteinthat resides in the cytoplasm and hence the increased level of p65 expression inthe nuclear fraction. The constitutive activation of NF-iB also appears to have arole in cell proliferation (Asakura et al., 2002). A significant decrease (p5 0.01) wasobserved in the level of p65 in DMBA/TPAþ 5 and 6-treated tumours as compared

Table 2. Quantification of protein expression in various treatment groups by ELISA.

Groups Control DMBA/TPA DMBA/TPAþ 6 DMBA/TPAþ 5 DMBA/TPAþ 1

AP-1 (c-jun) 0.226� 0.08 0.568� 0.01a 0.221� 0.01c 0.327� 0.02b 0.421� 0.04a

NF-�B (p65) 0.150�0.02 0.345� 0.07b 0.167� 0.04a 0.177� 0.06c 0.206� 0.08c

p53 0.226� 0.05 0.732� 0.04a 0.311� 0.02a 0.456� 0.02c 0.554� 0.02b

Notes: The values are mean � SD of six independent observations. ap5 0.05; bp5 0.01;cp5 0.001 with respect to control mice and DMBA/TPA-treated mice.

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to DMBA/TPA-treated tumours. Also, a significant decrease (p5 0.05) in the

levels of p65 was observed in DMBA/TPAþ 1 treatment tumours as compared to

DMBA/TPA-treated mice tumours. This may be due to suppression of NF-iB by

these compounds. Several chemopreventive agents are inhibitors of NF-iB activa-

tion. These inhibitors can block any one or more steps in the NF-iB signalling

cascade, the translocation of NF-iB into the nucleus, DNA binding of the dimers

and interactions with the basal transcription activation.The tumour suppressor gene, p53, is regarded as a key factor in maintaining the

balance between cell growth and death (Agarwal, Taylor, Chernov, Chernova, &

Stark, 1998; Mowat, 1998). Due to its role in the regulation of the cell cycle,

alterations in p53 are critical events in carcinogenesis (Arora, Siddiqui, & Shuklka,

2004). In this study, a decrease in the protein expression of p53 was seen in the

control (group I) and a significant increase in the p53 expression was observed in

DMBA/TPA (group II)-treated mice tumours, while there was a significant decrease

(p5 0.001) observed in the level of p53 in DMBA/TPAþ 5 and 6 treatment tumours

as compared to DMBA/TPA-treated tumours. Also, a significant decrease

(p5 0.001) in the levels of p53 was observed in DMBA/TPAþ 1 treatment tumours

as compared to DMBA/TPA-treated mice tumours. The results inferred that an

increase in polarity of the lead molecule (5) not only decreased the dose, but also led

to an increase in the antitumour activity.

3. Experimental

3.1. General experimental procedures

Melting points were determined on a Buchi melting point apparatus and are

uncorrected. For thin layer chromatography (TLC), glass plates coated with silica gel

G (E. Merck) were used. The TLC plates were activated at 110�C for 30 min and

visualised by the exposure to iodine vapours. Glass columns of appropriate sizes

were used. Silica gel (60–120mesh) was used as an adsorbent. IR spectra were

recorded on a Perkin Elmer 882 spectrometer using potassium bromide pellets.1H-NMR and 13C-NMR spectra were recorded with a Bruker AC 300F 300MHz

spectrometer using CDCl3 as solvent, and with a Micromass 70-VSE mass

spectrometer at 70 eV using electron ionisation (EI). Elemental analysis of

compounds was within (0.04% of the theoretical values). All solvents were freshly

distilled and dried prior to use, according to the standard procedures. All chemicals

were purchased from SD Fine Chemicals, Qualigen and Loba Chemicals. DMBA

and TPA were purchased from Sigma Chemicals Ltd.

3.2. Plant material

Leaves of L. camara were collected in the month of September from Palampur (HP),

India. The leaves were shade-dried and powdered. A voucher specimen (LC; 097

UIPS) was deposited in the Herbarium at the University Institute of Pharmaceutical

Sciences, Panjab University, Chandigarh, India.

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3.3. Extraction and isolation of LA (1)

LA was isolated as described in our previous studies. The spectral data of thecompound was found to be identical to the previously reported data (M. Sharma &P. Sharma, 2006).

3.4. Synthesis LAM (5)

LAM was synthesised as described in our previous studies (Sharma et al., 2007c).

3.5. Synthesis of 22b-angeloyloxy-methyl-2-hydroxy-3-oxoolean-1,12-dien-28-oate (6)

A solution of 0.2 g of LAM (0.35mmol) in ButOH (6mL) and ButOK in excess wasvigorously stirred at 20–25�C for 4 h with provision of sufficient access of air to thereaction mixture. The reaction mixture was diluted with MeOBut (25mL) andneutralised with 1mol HCl (15mL) on cooling to 0�C. The organic layer wasseparated, washed successively with water (2� 20mL) and a saturated solutionof NaCl (10mL), dried with anhydrous sodium sulphate and concentrated in vacuo.The residue was recrystallised with a methanol–water mixture to obtain a colourlessglassy product. (0.12 g; 58.5%), m.p. 132–133�C. IR (KBr): 2910 (aliphatic C–Hstretch), 1730 (C¼O, C-22 ester), 1710 (C¼O, C-17 ester), 1650 (C¼O, 3- keto), 3569(O–H stretch) cm�1. 1H-NMR (CDCl3) �: 0.83, 0.93, 1.00, 1.02, 1.03, 1.05, 1.19(s, 7�CH3),1.72 (3H, d, J¼ 1.3Hz, 50 CH3), 1.94 (3H, s, 40CH3), 3.01 (1H, dd,J¼ 14.2; 4.0Hz, C-18-H),3.53 (3H, s, COOCH3), 4.52 (1H, t, J¼ 3Hz, C-22-H), 5.28(1H, t, J¼ 3.5Hz, C-12-H), 5.93 (1H, q, J¼ 1.3Hz, 30 H), 6.32 (1H, s, C-1-H), 10.2(1H, broad s, O–H). 13C-NMR (CDCl3) �: 132.6 (C-1), 144.4 (C-2), 211.7 (C-3), 47.2(C-4), 55.1 (C-5), 19.3 (C-6), 32.7 (C-7), 39.3 (C-8), 47.2 (C-9), 36.4 (C-10), 23.2(C-11), 122.1 (C-12), 143.7 (C-13), 42.1 (C-14), 27.2 (C-15), 24.3 (C-16), 50.2 (C-17),38.3 (C-18), 46.3 (C-19), 30.7 (C-20), 37.4 (C-21), 75.3 (C-22), 26.1 (C-23), 21.2(C-24), 15.3 (C-25), 16.2 (C-26), 25.6 (C-27), 177.2 (C-28), 33.3 (C-29), 26.7 (C-30),166.4 (C-10), 127.3 (C-20), 138.4 (C-30), 20.3 (C-40), 15.3 (C-50), 51.6 (COOCH3). MSm/z: 580.3 [Mþ, 100%], 500.3, 499.3, 483.3, 424.3, 279.3, 264.3, 223.3. Anal. Calcd:C, 74.45; H, 9.02. Found: C, 74.47; H, 9.00.

3.6. Animals

Female Swiss albino mice (6-weeks-old) weighing 18–22 g were obtained from theCentral Animal House of Panjab University. Animals were kept in the DepartmentalAnimal House with a controlled temperature of 23� 5�C, 60� 5% humidity, and a12 h light/dark cycle. They were fed a basal diet and water. The mice wereacclimatised for one week before experimentation.

3.7. Cell culture and MTT assay

Four cell lines (HL-60, HeLa, colon 502713 and A-549) were obtained from NCCS,Pune, India, and maintained in RPMI medium supplemented with 10% foetal bovine

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serum, 100 mgmL�1 streptomycin, and 100 IUmL�1 penicillin. Cells were incubatedat 37�C in a humidified atmosphere of 5% CO2 for 48 h for the comparison ofcytotoxicity between compounds. The cell lines (2� 10�4 per 0.1mL well) weretreated with serial dilutions of compounds in 96-well culture plates (Costar) for 48 h.During the last 4 h, cells were reacted with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) at 37�C for a colorimetric MTT-based cytotoxicityassay. The reaction product, formazan, was extracted with DMSO, and theabsorbance was read at 540 nm (Mosmann, 1993). Data represents the mean valuesand SD of triplicate assays in at least one experiment.

3.8. In vivo antitumour activity

Squamous cell carcinogenesis was induced in Swiss albino mice (LACCA/female)according to the established method (Azuine & Bhide, 1992). Animal care andhandling was conducted according to the guidelines set by the World HealthOrganisation (WHO), Geneva, Switzerland, and the Indian National ScienceAcademy (INSA), New Delhi, India. Depilatory cream was used to remove hairfrom the back of mice. The animals were left for two days and divided into fourgroups. Animals in group I (10 animals) were treated with 100 mLof vehicle(acetone). The acetone was topically applied on the depilated back of each mousetwice weekly for 20 weeks. The animals of group II (10 animals) were topicallytreated with DMBA (100 nmol per 100 mLof acetone) on the depilated back of eachmouse for two weeks and promoted with twice weekly applications of TPA (1.7 nmolper 100 mLof acetone) for the next 18 weeks. The animals of groups III–V (10animals) were orally treated with compounds 1, 5 and 6, respectively, suspended inwater and carboxymethyl cellulose. Treatments started one week before DMBAapplication and continued for 20 weeks thereafter.

3.9. Histology

The incidence of skin lesions, tumours per mouse, body weight of mice and numberof mice that survived the 20 week period were recorded. The body weight, number ofdeaths and papillomas appearing on depilated skin were recorded at weeklyintervals. Only those papillomas that persisted for two weeks or more wereconsidered for final evaluation of the data. The skin nodules were excised and fixedin Zenker’s fixative, routinely processed, and embedded in paraffin. Sections(7 mmthick) were stained with haematoxylin and eosin and examined under a lightmicroscope to carry out histopathology.

3.10. Nuclear extract preparation and protein estimation by ELISA

The nuclear extract was prepared by homogenising the tumour and control skin(10%) in 2mL of buffer A (50mmol NaCl, 10mmol Hepes pH: 8, 500mmol sucrose,1mmol EDTA). After homogenising, 0.2% Triton-X 100 and 1mmol phenyl-methylsulphonyl fluoride (PMSF) were added. This mixture was then centrifuged at5000 rpm for 2min under cold conditions. Then, the supernatant, which was knownas cytoplasmic extract, was removed. The pellet was re-suspended in 500 mLof buffer

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B (50mmol NaCl, 10mmol Hepes pH: 8, 25% glycerol, 1mmol EDTA, 1mmolPMSF). This mixture was then centrifuged at 5000 rpm for 3 min under coldconditions. Then, the supernatant was discarded and the pellet was re-suspended in50 mL of buffer C (350mmol NaCl, 10mmol Hepes pH: 8, 25% glycerol, 1mmolEDTA and 1mmol PMSF). This was then incubated on ice for 30min with constantshaking and then centrifuged at 10,000 rpm for 10min under cold conditions. Thesupernatant, which was known as nuclear extract, was removed. The proteinestimation in nuclear extract was carried out by the method of Lowry (Sharma et al.,2007a). ELISA was carried out in the nuclear extract to quantitate the c-jun, p65,and p53 in the control skin and tumours. The assay was standardised by titrating thedifferent concentrations of antigens and antibodies. Wells were coated with 5 mg forc-jun, p65 and p53 proteins in 100 mL of 0.05mol carbonate buffer (pH 9.6) and keptovernight at 4�C, in a moist chamber. The plate was flicked to remove the unboundantigen solution and the wells were blocked with 1% BSA in 0.1mol phosphatebuffered saline (pH 7.2) for 1 h at 37�C. The wells were flicked and washed thricewith 200 mL of PBS containing 0.05% (v/v) Tween 20. Wells were then incubatedwith anti-c-jun (1 : 250), anti-p65 (1 : 1000) and anti-p53 (1 : 300) and primaryantibody, respectively, diluted in PBS (containing 0.05% Tween 20 and 1% BSA)and kept for 2 h at 37�C. Plate was again washed and incubated with anti-rabbitsecondary antibody (peroxidase labelled) (Sigma–Aldrich 1 : 1000) for c-jun and p65while for p53 (Sigma–Aldrich 1 : 300), wells were incubated with anti-sheep IgG(Sigma–Aldrich 1 : 1000) for 2 h at 37�C. Wells were further washed thrice asdescribed above and colouring developed by addition of 2,20-azino-di-(3-ethyl)-benzothiozolinsulphonic acid reagent; absorbance at 405 nm was measured byELISA reader.

3.11. Statistical analysis

Statistical analysis of the data was performed by analysis of variance (ANOVA) andStudent’s t-test. The value of p5 0.05 was considered significant.

Acknowledgement

The authors are thankful to the Rayat Educational and Research Trust for providing thefunds and facilities to carry out this work.

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