Tualang Honey Protects Keratinocytes from Ultraviolet Radiation-InducedInflammation and DNA Damage†

Israr Ahmad1, Hugo Jimenez1, Nik Soriani Yaacob3 and Nabiha Yusuf*1,2,3

1Department of Dermatology and Skin Diseases Research Center, Veterans Affairs Medical Center,Birmingham, AL

2Comprehensive Cancer Center, University of Alabama at Birmingham, AL3School of Medical Sciences, Universiti Sains Malaysia, Malaysia

Received 24 October 2011, accepted 17 January 2012, DOI: 10.1111/j.1751-1097.2012.01100.x

ABSTRACT

Malaysian tualang honey possesses strong antioxidant and

anti-inflammatory properties. Here, we evaluated the effect of

tualang honey on early biomarkers of photocarcinogenesis

employing PAM212 mouse keratinocyte cell line. Keratinocytes

were treated with tualang honey (1.0%, v ⁄ v) before a single UVB

(150 mJ cm)2) irradiation. We found that the treatment of

tualang honey inhibited UVB-induced DNA damage, and

enhanced repair of UVB-mediated formation of cyclobutane

pyrimidine dimers and 8-oxo-7,8-dihydro-2¢-deoxyguanosine.Treatment of tualang honey inhibited UVB-induced nuclear

translocation of NF-jB and degradation of IjBa in murine

keratinocyte cell line. The treatment of tualang honey also

inhibited UVB-induced inflammatory cytokines and inducible

nitric oxide synthase protein expression. Furthermore, the treat-

ment of tualang honey inhibited UVB-induced COX-2 expression

and PGE2 production. Taken together, we provide evidence that

the treatment of tualang honey to keratinocytes affords sub-

stantial protection from the adverse effects of UVB radiation via

modulation in early biomarkers of photocarcinogenesis and

provide suggestion for its photochemopreventive potential.

INTRODUCTION

Ultraviolet (UV) B radiation (290–320 nm) induced DNAdamage is one of the earliest molecular events in thedevelopment of skin cancers (1–3). UVB causes DNA damage,

predominantly in the form of cyclobutane pyrimidine dimers(CPDs) and 6-4 photoproducts. Nucleotide excision repair(NER) removes DNA damage by two distinct pathways:

transcription coupled repair and global genome repair of DNA (4).Reactive oxygen species, which are generated endogenously bycellular oxygen metabolism or exogenously by UV, are

environmental mutagens, and produce various types of DNAdamage. 8-oxo-7,8-dihydro-2¢-deoxyguanosine (8-oxo-dG) isone type of oxidative DNA damage that can result in stable

mutations. In mammalian cells, the Ogg1 gene encodes 8-oxo-dG-DNA glycosylase, a repair enzyme, which removes the

oxidized base from DNA. Base excision repair is the mostactive process for correcting these DNA alterations that arise

from the inherent instability of DNA. A growing body ofevidence indicates that components of NER are also involvedin the repair of oxidative damage (5).

UVB irradiation evokes a signaling response through twodifferent pathways: one dependent on and the other indepen-dent of DNA damage. On one hand, DNA damage activates

the tumor suppressor p53, which induces cell-cycle arrest andthe concurrent processes of DNA repair and apoptosis (6,7).On the other hand, UVB irradiation activates the transcription

factor NF-jB (8). Oxidative stress in epidermal cells plays acrucial role in the photodamage pathway, because it contrib-utes to DNA damage (9–11), activation of MAP kinases(12,13), apoptosis (14,15) and secretion of inflammatory

cytokines (16). UVB-induced ROS are responsible for skininflammation, gene mutation and immunosuppression, photo-aging and skin cancer (17,18).

The therapeutic role of honey in the treatment of variousailments has been receiving considerable attention recently,and its therapeutic value has been partly attributed to its

antioxidant properties (19,20). Malaysian tualang honey (TH)is collected from the combs of Asian rock bees (Apis dorsata),which build their hives high up in the tualang tree (Koompassiaexcelsa). TH is used commonly as a medicinal product (21,22)

and as food in Malaysia. Recent data suggest that the elevatedfree-radical scavenging and antioxidant activity observed inTH is due to the increased level of phenolic compounds (23).

In addition to its antibacterial, anticarcinogenic and anti-inflammatory properties, its antioxidant properties make itimportant for human nutrition and health.

The objective of this study was to investigate the effect ofTH on early biomarkers of UVB-induced damage processesemploying murine PAM212 keratinocyte cell line model.

MATERIALS AND METHODS

Cell line and reagents. Murine epidermal keratinocyte cell line PAM212was obtained from Lonza Walkersville, Inc. (Walkersville, MD). THused in this study was supplied by Federal Agricultural MarketingAuthority (FAMA), Malaysia. The primary antibodies for COX-2,iNOS, EP4 and b-actin, and the secondary antibodies, horseradishperoxidase-linked anti-mouse IgG and anti-rabbit IgG, were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for p65and IkBawere procured fromCell Signaling Technology, Inc. (Danvers,

†This paper is part of the Special Issue in Commemoration of the 70th Birthdayof Dr. David R. Bickers.

*Corresponding author email: nabiha@uab.edu (Nabiha Yusuf)� 2012 Wiley Periodicals, Inc.Photochemistry and Photobiology � 2012 The American Society of Photobiology 0031-8655/12

Photochemistry and Photobiology, 20**, **: *–*

1

MA). Anti-8-oxo-dG antibodywas purchased fromMillipore (Billerica,MA) and anti-CPD antibody was purchased from Kamiya BiomedicalCompany (Tukwila, WA). Secondary Alexafluor 488 antibody andAlexafluor 594 antibody were purchased from Invitrogen (Carlsbad,CA). Anti-EP2 antibody and PGE2 ELISA kit were purchased fromCayman Chemical Company (Ann Arbor, MI). ELISA kits for IL-1b,IL-6 and TNF-a were purchased from Invitrogen. Bay 11-7082,celecoxib and aminoguanidine hemisulfate were purchased from Sigma(St. Louis, MO). The DC protein assay kit was obtained from Bio-RadLaboratories (Hercules, CA) and the enhanced chemiluminescenceWestern blotting detection reagents were purchased from AmershamPharmacia Biotech (Piscataway, NJ).

Cell culture and preparation of TH. Murine epidermal keratinocytecell line PAM212 was cultured as monolayer in DMEM supplementedwith 10% heat-inactivated fetal bovine serum, 100 lg mL)1 penicillin-streptomycin (Invitrogen), and maintained in a humidified atmosphereof 95% air and 5% CO2 at 37�C. TH was initially dissolved in serum-free culture medium at a final concentration of 10% (v ⁄ v) and themixture was filter-sterilized using 0.22 lm syringe filter unit (Millipore).The honey mixture was freshly prepared before adding to cell cultures.

UVB irradiation. The source of UVB radiation was a band of fourUVB lamps (Daavlin; UVA ⁄UVB Research Irradiation Unit, Bryan,OH) equipped with an electronic controller to regulate UV dosage at afixed distance of 24 cm from the lamps to the surface of the cell cultureplates. The majority of the resulting wavelengths were in the UVB(290–320 nm; about 80%) and UVA (about 20%) range and the peakemission was recorded at 314 nm.

Treatment of PAM212 keratinocytes. After the determination ofcellular viability by using the MTT assay, we have selected a 1.0%(v ⁄ v) dose of TH for further study. To determine the effects of TH onUVB-induced markers of DNA damage or activation of signalingpathways, the subconfluent (70–80%) cells were treated with 1.0% THfor 24 h before UVB irradiation. In some sets of experiments, we alsoused pharmacological inhibitors like Bay 11-7082 (NF-kB inhibitor,20 lMM), Celecoxib (COX-2 inhibitor, 20 lMM) and aminoguanidinehemisulfate (iNOS inhibitor, 1.0 mMM) in culture to demonstrate theessentiality and specificity of cellular changes mediated by honey.Thereafter, cells were washed three times with PBS to make them TH-free and then cells in fresh 1.0 mL PBS were exposed to 150 mJ cm)2

of UVB. After UVB exposure, PBS was replaced with culture medium,and cells were again incubated. Cells were harvested at desired timepoints, either for preparation of lysates or CPD and 8-oxo-G staining.

MTT assay for cellular viability. The MTT assay is a colorimetricassay for measuring the activity of the cellular reductase enzymes inviable cells that reduce yellow 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma) substrate to purple formazancrystals. The effect of the TH on cell viability was determined by theMTT assay. Briefly, 1 · 104 cells per well in 1.0 mL of complete culturemedium was plated in 96-well culture plates. After overnight incubation,the cells were treated with varying concentrations of TH (0%, 0.1%,0.2%, 0.5%, 1.0%, 2.0%, 3.0% and 5.0%) dissolved in fresh culturemedium supplemented with 2.0% fetal bovine serum and furtherincubated for 24, 48 and 72 h at 37�C in a humidified chamber. At theend of the stipulated period, MTT (50 lL of 50 lg mL)1 stock) wasadded into each well and incubated for 3 h. The 96-well plate consistingof the cells was centrifuged at 280 · g for 5 min at 4�C. The MTTsolution was removed from the wells by aspiration and the resultingformazanwas then dissolved in 150 lLofDMSOand 200 lL transferredinto a 96-well plate. The absorbance of the formazan in each well wasrecorded at 540 nm using a microplate reader (Bio-Rad Laboratories).

Immunocytochemical detection of CPD-positive and 8-oxo-dG-positive cells. UV-induced DNA damage in the form of CPD+ cellswere detected using a protocol described previously with somemodifications (24). Briefly, 1 · 104 cells were plated on round coverslips placed in a 10 mm cell culture dish (Nunc, Denmark). The cellswere allowed to adhere on coverslips overnight and the culture mediumwas replaced with fresh assay medium supplemented with 2.0% fetalbovine serum. The cells were treated with 1.0% of TH for 24 h beforeUVB irradiation. Thereafter, cells were washed three times with PBSand then cells of UVB and UVB + TH group were exposed to the150 mJ cm)2 doses of UVB in 1.0 mL of PBS. After UVB exposure,PBS was replaced with culture medium, and cells were again incubatedin the dark for 30 min to make sure that CPD and 8-oxo-dG weregenerated in UVB-irradiated cells. Thereafter, cells were fixed in 4.0%

formaldehyde for 10 min and washed with PBS and subsequentlypermeabilized with 0.5% Triton-X 100 for 5 min on ice. DNAdenaturation was performed by treating the cells with 2.0 MM HCl atroom temperature for 20 min. Then, cells were washed with PBS andincubated in blocking buffer, 10% anti-goat normal serum, at roomtemperature for 1 h. Cells were then incubated with either CPD-specificor 8-oxo-G-specific mice monoclonal antibody for 1 h at roomtemperature and, after washing, the bound anti-CPD or 8-oxo-Gantibody was detected by incubation with anti-mouse secondary IgGantibodyAlexa Fluor 488 andAlexa Fluor 594, respectively, for 45 min.After washing with PBS, the cells were counterstained with DAPI.CPD+ and 8-oxo-G+ cells were counted under Olympus BX41microscope at five to six different fields and the data were presentedas the mean of the percentage of CPD+ and 8-oxo-dG+ cells ± SDfrom at least three separate experiments.

Preparation of cell lysates and Western blotting. Either whole-celllysates or nuclear lysates were prepared from control, TH, UVB andUVB + TH-treated cells by using cytoplasmic and nuclear extractionkit of Active Motif (Carlsbad, CA). For Western blot analysis, 50–80 lg protein was loaded in each well and resolved on 8%, 10% and12% SDS-polyacrylamide gel and transferred onto nitrocellulosemembranes. Membranes were incubated in blocking buffer for 2 hand then incubated with the primary antibodies in blocking buffer for2 h at room temperature or overnight at 4�C. The membrane was thenwashed with TBS-T and incubated with secondary antibody conju-gated with horseradish peroxidase. Protein bands were visualized usingthe enhanced chemiluminescence detection system (Amersham LifeScience, Inc., Piscataway, NJ). To verify equal protein loading andtransfer of proteins from gel to membrane, the blots were stripped andreprobed for b-actin or histone H3. The band density was analyzedusing IMAGE JIMAGE J software provided by NIH and the values werenormalized to the b-actin band density.

PGE2 immunoassay for quantitation of prostaglandin E2. Theanalysis of PGE2 in cell homogenates was performed using theCayman PGE2 Enzyme Immunoassay Kit (Cayman Chemical Com-pany) following the manufacturer’s instructions. Homogenates werecentrifuged and the supernatants were collected and analyzed forPGE2 concentration according to the manufacturer’s instructions.

Measurement of inflammatory cytokines level. The levels of inflam-matory cytokines were assayed in triplicate by using standardprotocols of enzyme-linked immunosorbent assay (ELISA) (Invitro-gen). The plates were incubated with anti-IL-1b, IL-6 and TNFaantibody (25 lg mL)1; 30 lL per well) at 4�C for overnight and thenwashed with PBS before adding the cell suspensions. Supernatantswere harvested 24 h later after giving UVB radiation. The levels ofcytokines were expressed as picograms per milliliter.

Statistical analysis. Statistical analysis was carried out using ANOVAANOVA

followed by post hoc multiple comparison tests. The chemopreventiveeffect of TH was considered significant if P < 0.05.

RESULTS

TH does not inhibit cell growth of PAM212 murine

keratinocytes

The treatment of PAM212 cells with 0.1–5.0% TH did notresult in significant cell death even after 72 h incubation(Fig. 1). However, a maximum cell death of only about 30%

was observed in 5.0% and 48 h TH-treated cells. Whereasfurther incubation of cells for a longer period, i.e. 72 h resultsin a decrease in cell death. Since up to 1.0% concentration of

TH, more than 90% of the PAM212 cells survived, this is thereason to select this safer dose for the assessment of chemo-preventive potential of TH on PAM212 murine keratinocytes.

TH reduces or repairs DNA damage in UVB-exposed PAM212

keratinocytes

To determine whether TH reduces or repairs UVB-inducedDNA damage in PAM212 keratinocytes, we evaluated the

2 Israr Ahmad et al.

effect of TH on UVB-induced DNA damage in the form of

CPDs and 8-oxo-dG formation. For this purpose, cells wereexposed to UVB (150 mJ cm)2) with or without the treatmentwith TH. Cells were harvested either after 30 min or 24 hafter UVB irradiation and subjected to the analysis of CPD-

positive cells or 8-oxo-G-positive cells following cytostainingusing CPD-specific or 8-oxo-dG-specific monoclonal anti-body. CPD-positive cells were not detectable in both

non–UVB-irradiated cells and TH alone treated cells whereas,in UVB-irradiated cells, it was largely detected. When the cellswere analyzed for CPDs (Fig. 2A) and 8-oxo-dG (Fig. 2B)

immediately after UVB exposure, there was a significantdifference in the cells treated with or without TH in terms ofthe number of CPD-positive cells (Fig. 2A) and 8-oxo-dG-positive cells (Fig. 2B). When the cells were analyzed 24 h

after UVB irradiation, the number and intensity of staining ofCPD-positive cells (Fig. 2A) and 8-oxo-dG-positive cells(Fig. 2B) were markedly decreased in TH-treated cells com-

pared with the cells that were not treated with TH butexposed to UVB, suggesting that TH might accelerate therepair of UVB-induced CPDs and 8-oxo-dG in PAM212

keratinocytes.

Treatment of TH inhibits UVB-induced activation of NF-jB in

PAM212 keratinocytes

Nuclear factor kappa B is present in the cytosol as aheterodimer usually consisting of its p50 and p65 subunitsbound to its inhibitory proteins IjB. One of the critical eventsin NF-jB activation is its dissociation from inhibitory protein

IjB (25). We determined whether the treatment of TH inhibitsUVB-mediated activation of NF-jB. UVB irradiation resultedin degradation of IjBa protein in the cytosol. Our data clearly

demonstrated that the treatment of tualang markedly inhibitedUVB-mediated degradation of IjBa (Fig. 3). We then inves-tigated whether the treatment of TH inhibited UVB-induced

activation and nuclear translocation of NF-jB ⁄ p65 inPAM212 keratinocytes. Employing Western blot analysis, we

found that UVB exposure resulted in the activation andnuclear translocation of NF-jB ⁄ p65, which was markedlyinhibited by TH (Fig. 3) and this is in accordance with theinhibitory potential of the well-known NF-kB inhibitor Bay

11-7082. Histone H3 was used as a loading control for NF-jB.

TH treatment inhibits UVB-induced activation of inflammatory

cytokines

Keratinocytes are the major target of UVR and play a centralrole in the inflammatory and immune-modulatory changesobserved after UV exposure, at least partly via the UV-induced

release of cytokines (IL-1, IL-6, IL-8, IL-10, GM-CSF, TNF-a) (26). Our data show that TH treatment of PAM212keratinocytes resulted in a decrease in proinflammatory

cytokines IL-1b, IL-6 and TNF-a by ELISA (Fig. 4), whichcould be due to the inhibition of NF-kB that resembles thewell-known NF-kB inhibitor Bay 11-7082. Inhibition of UVB-induced inflammatory cytokines signifies the anti-inflamma-

tory potential of TH.

TH inhibits UVB-induced iNOS and COX-2 protein expression

in PAM212 keratinocytes

Exposure to UVB radiation induces the IKKb activity,phosphorylates extracellular signal-regulated kinase (ERK)and p38 MAP kinase, activates NF-jB and elevates the

expression iNOS in mouse skin. Pharmacological inhibition ofUVB-induced IKKb activity results in the inhibition of ERKand p38 MAP kinase phosphorylation, downregulation ofNF-jB activation and suppression of iNOS expression in

hairless mouse skin (27). In order to determine whether THcould downregulate UVB-induced iNOS production, whole-cell extracts were analyzed by Western blotting for iNOS. Our

data show that TH treatment of PAM212 keratinocytesresulted in a decreased expression of inflammatory markeriNOS (Fig. 5) that was confirmed by using a well-known

iNOS inhibitor aminoguanidine hemisulfate. Studies havedemonstrated that the expression of proinflammatory enzymeCOX-2 is induced by UVB exposure (28). Therefore, we

determined the effect of TH on UVB-induced epidermal COX-2 protein expression. Western blot analysis revealed that UVBexposure to PAM212 keratinocytes resulted in a markedincrease in COX-2 protein expression compared with the

control group, whereas prior treatment of TH significantlydecreased the COX-2 protein level (Fig. 4). Celecoxib (COX-2inhibitor) was used to confirm the inhibitory potential of TH

against COX-2.

The inhibitory effect of TH on COX-2 is associated with

inhibition of UVB-induced PGE2 production in PAM212

keratinocytes

As the COX-2 metabolite, PGE2, has been implicated in COX-2-mediated effects, we determined the levels of PGE2 in theTH-treated cells. Our results revealed that treatment with TH

for 24 h resulted in a significant inhibition of PGE2 productionin PAM212 cells (Fig. 6A), suggesting that TH-inducedreduction in PGE2 production is associated with an inhibitory

effect of the TH on COX-2 in these cells, which was furtherconfirmed by using celecoxib.

Figure 1. Effect of tualang honey on the viability of murine PAM212keratinocytes. Dose- and time-dependent effect of tualang honey onthe proliferation of PAM212 keratinocytes was determined using MTTassay as described in the Materials and Methods section. The valuesare represented as the percentage cell death. The data represent themean ± SD of three independent experiments each conducted intriplicate.

Photochemistry and Photobiology 3

TH decreases the levels of UVB-induced PGE2 receptors in

PAM212 keratinocytes

As it has been shown that PGE2 manifests its biological

activity via four known G-protein-coupled receptors (i.e. EP1–EP4) (29), we determined the effect of TH on the UVB-inducedlevels of PGE2 receptors in PAM212 keratinocytes. Westernblot analysis revealed that the treatment of PAM212 kerati-

nocytes with TH for 24 h resulted in a significant attenuationin the levels of EP2 and EP4 (Fig. 6B).

DISCUSSION

The UVB component of solar UV radiation is believed to be

the major cause of the variety of cutaneous disorders includingskin cancers (1–3,30,31). Antioxidants from natural plantproducts consumed by the human population have gained

considerable attention as photoprotective and photochemo-preventive agents against skin cancers (32). Antioxidants,which are abundant in natural honey, are free-radical scav-engers that either reduce the formation of or neutralize free

Figure 2. Inhibitory effect of tualang honey on UVB-induced formation of cyclobutane primidine dimers and 8-oxo-7,8-dihydro-2¢-deoxyguano-sine (8-oxo-dG) in PAM212 keratinocytes. PAM212 were exposed to UVB (150 mJ cm)2) and treated with tualang honey as described in theMaterials and Methods section. Immunocytochemical staining for cyclobutane pyrimidine dimers (CPDs) (A), and 8-oxo-dG (C) was performedusing appropriate antibodies. Representative pictures are shown. The number of CPD-positive cells (B) and 8-oxo-dG-positive cells (D) afterimmunostaining were counted in five different areas of the sections under a microscope. The number of CPD and 8-oxo-dG positive cells arerepresented as percent of CPD- and 8-oxo-dG-positive cells, respectively. The data represent the mean ± SD of cells in triplicate (***P < 0.001and *P < 0.05 vs control; ###P < 0.001 and ##P < 0.01 vs UVB). The representative micrographs are shown from three independentexperiments. Bar = 50 lMM.

4 Israr Ahmad et al.

radicals. It has been reported that the elevated free-radicalscavenging and antioxidant activity observed in TH is due tothe increased level of phenolic compounds. In another study,27 compounds were indentified after methanol extraction of

TH, some of which had antiproliferative properties on keloidfibroblasts. TH that was extracted using methanol containedlipid including fatty acid esters. Phenolic compounds were not

detected in methanolic extraction of TH. Different methodsneed to be used to determine the phenolic content in honey, forexample, using ethyl acetate extract and analyzing with thin

layer chromatography (33). We believe that the medicinalproperties of TH are not based on a single compound but acombination of many different components. More studies need

to be carried out to identify the active compounds in TH.UVB causes a considerable amount of DNA damage,

including oxidative stress. This study was designed to assessthe photochemopreventive effect of TH after single UVB

irradiation to the mouse keratinocyte cell line PAM212. Ourdata clearly demonstrate that the treatment of TH affordssignificant inhibition against UVB-induced inflammation and

DNA damage. UVB radiation to mammalian skin is known toalter cellular function via oxidation of macromolecules, DNAdamage, generation of ROS and alterations in signaling

pathways (9–11).Cyclooxygenase-2 has been implicated in UVB-induced skin

inflammation and photocarcinogenesis, and its overexpressionhas been shown to enhance cell proliferation, induce angio-

genesis, regulate antiapoptotic cellular defenses and augmentimmunological response through the production of PGE2 (32).A considerable body of evidence suggests that the inhibition of

COX-2 expression or activity is important for not onlyalleviating inflammation, but also for the prevention of cancer.Our results demonstrate the inhibitory effect of treatment of

TH against UVB-mediated induction of epidermal COX-2

Figure 3. Inhibitory effect of tualang honey on UVB-induced nucleartranslocation of p65 and degradation of IjBa in PAM212 keratino-cytes. Cells were treated with either tualang honey or Bay 11-7082(20 lMM) and exposed to UVB (150 mJ cm)2) as described in theMaterials and Methods section. Cytosolic and nuclear protein lysateswere prepared and Western blot analysis was performed to determinethe protein expression level of p65 and IkB-a. Equal loading wasconfirmed by stripping the Western blot and reprobing it for b-actin orhistone H3. The representative blots are shown from three independentexperiments (***P < 0.001, **P < 0.01 and *P < 0.05 vs control;##P < 0.01 vs UVB).

Figure 4. Treatment of tualang honey reduces UVB-induced secretion of proinflammatory cytokines by PAM212 keratinocytes. Cells were treatedwith either tualang honey or Bay 11-7082 (20 lMM) and exposed to UVB (150 mJ cm)2) as described in the Materials and Methods section. Cellsupernatant was collected for the estimation of cytokines IL-1b, IL-6 and TNF-a by ELISA. The representative data are shown from threeindependent experiments (***P < 0.001, **P < 0.01 and *P < 0.05 vs control; ##P < 0.01 vs UVB).

Photochemistry and Photobiology 5

protein expression. These results indicate that TH targets

COX-2 expression in exerting its photoprotective effects.UVB is a potent carcinogen known to damage DNA

directly or through the generation of ROS. Both CPDs and 8-

oxo-dG are formed in epidermal DNA after UVB irradiationand are considered as important biomarkers of DNA damage.Our data clearly demonstrated that the treatment of TH to

PAM212 cells resulted in marked reduction in the number ofCPDs and 8-oxo-dG-positive cells and these may be due toenhanced DNA repair. Activated NF-jB is a crucial factor for

the immunoinflammatory responses and is also implicated intumorigenesis (34). Therefore, NF-jB has emerged as one ofthe most promising molecular targets in the prevention ofcancer. NF-jB is sequestered in the cytoplasm as a heterotri-

mer consisting of p50, p65 and IjBa subunits. Upon phos-phorylation and subsequent proteolytic degradation of IjBa,NF-jB activates and translocates to the nucleus where it binds

to DNA and activates the target genes by binding to the DNA

Figure 5. Inhibitory effect of tualang honey on UVB-induced cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)protein expression in PAM212 keratinocytes. Cells were treated witheither tualang honey or with celecoxib (CXB, 20 lMM) and aminogua-nidine (AG, 1.0 mMM) and exposed to UVB (150 mJ cm)2) as describedin the Materials and Methods section. Western blot analysis wasperformed to determine the protein expression using COX-2- andiNOS-specific antibodies. Equal loading was confirmed by strippingthe Western blot and reprobing it for b-actin. The representative blotsare shown from three independent experiments (***P < 0.001,**P < 0.01 and *P < 0.05 vs control; ###P < 0.001 and##P < 0.01 vs UVB).

Figure 6. Treatment of PAM212 keratinocytes with tualang honeydecreases the UVB-induced expression level of PGE2 and PGE2

receptors, EP2 and EP4. (A) Treatment of PAM212 keratinocytes withtualang honey decreases UVB-induced expression level of PGE2 asmeasured by ELISA. (B) Treatment of PAM212 keratinocytes withtualang honey also decreases UVB-induced expression level of PGE2

receptors EP2 and EP4. Cells were treated with either tualang honey orcelecoxib (CXB, 20 lMM) and exposed to UVB (150 mJ cm)2) asdescribed in the Materials and Methods section. Western blot analysiswas performed to determine the levels of EP2 and EP4 receptors usingEP2- and EP4-specific antibodies. Equal loading was confirmed bystripping the Western blot and reprobing it for b-actin. The represen-tative blots are shown from three independent experiments(***P < 0.001 and *P < 0.05 vs control; ##P < 0.01 vs UVB).

6 Israr Ahmad et al.

regulatory element (35). In the present study, we havedemonstrated that the treatment of TH inhibited UVB-induced NF-jB and IKKa activation.

Our data suggest that the treatment of TH to PAM212

keratinocytes affords substantial protection from the adverseeffects of UVB radiation via modulation in early biomarkersof photocarcinogenesis and provide suggestion for its photo-

chemopreventive potential.

Acknowledgement—This work was supported by Pilot & Feasibility

Study to NY from NIH funded UAB Skin Disease Research Center

Grant P30AR050948.

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