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Carcinogenesis vol.32 no.3 pp.368–380, 2011doi:10.1093/carcin/bgq278Advance Access publication December 22, 2010

Guggulsterone (GS) inhibits smokeless tobacco and nicotine-induced NF-kB and STAT3pathways in head and neck cancer cells

Muzafar A.Macha, Ajay Matta1, S.S.Chauhan,K.W.Michael Siu1 and Ranju Ralhan1,2,3,4,�

Department of Biochemistry, All India Institute of Medical Sciences, NewDelhi-110029, India, 1Department of Chemistry and Center for Research inMass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario,Canada M3J 1P3, 2Alex and Simona Shnaider Laboratory of MolecularOncology and Department of Pathology and Laboratory Medicine, MountSinai Hospital, Joseph and Wolf Lebovic Health Complex, 600 UniversityAvenue, Room 6-500 Toronto, ON, Canada M5G 1X5, 3Joseph and MildredSonshine Family Center for Head and Neck Diseases and Department ofOtolaryngology—Head and Neck Surgery, Mount Sinai Hospital, Toronto,ON, Canada M5G 1X5 and 4Department of Otolaryngology—Head and NeckSurgery, University of Toronto, ON, Canada M5G 2N2

�To whom correspondence should be addressed. Alex and Simona ShnaiderLaboratory of Molecular Oncology, Department of Pathology and LaboratoryMedicine, Mount Sinai Hospital, Joseph and Wolf Lebovic Health Complex.600 University Avenue, Room 6-500, Toronto, ON, Canada M5G 1X5.Tel: þ1 416 586 4800 6426. Fax: þ1 416 586 8628;Email: [email protected]

Understanding the molecular pathways perturbed in smokelesstobacco- (ST) associated head and neck squamous cell carcinoma(HNSCC) is critical for identifying novel complementary agentsfor effective disease management. Activation of nuclear factor-kappaB (NF-kB) and cyclooxygenase-2 (COX-2) was reportedin ST-associated HNSCC by us [Sawhney,M. et al. (2007) Expres-sion of NF-kappaB parallels COX-2 expression in oral precancerand cancer: association with smokeless tobacco. Int. J. Cancer,120, 2545–2556]. In search of novel agents for treatment ofHNSCC, we investigated the potential of guggulsterone (GS),(4,17(20)-pregnadiene-3,16-dione), a biosafe nutraceutical, in in-hibiting ST- and nicotine-induced activation of NF-kB and signaltransducer and activator of transcription (STAT) 3 pathways inHNSCC cells. GS inhibited the activation of NF-kB and STAT3proteins in head and neck cancer cells. This inhibition of NF-kBby GS resulted from decreased phosphorylation and degradationof nuclear factor of kappa light polypeptide gene enhancer inB-cells inhibitor, alpha the inhibitory subunit of NF-kB. Impor-tantly, treatment of HNSCC cells with GS abrogated both ST- andnicotine-induced nuclear activation of NF-kB and pSTAT3 pro-teins and their downstream targets COX-2 and vascular endothe-lial growth factor. Furthermore, GS treatment decreased thelevels of ST- and nicotine-induced secreted interleukin-6 in cul-ture media of HNSCC cells. In conclusion, our findings demon-strated that GS treatment abrogates the effects of ST and nicotineon activation of NF-kB and STAT3 pathways in HNSCC cells thatcontribute to inflammatory and angiogenic responses as well as itsprogression and metastasis. These findings provide a biologicrationale for further clinical investigation of GS as an effectivecomplementary agent for inhibiting ST-induced head and neckcancer.

Introduction

Tobacco is used by .1.3 billion people worldwide (1); it kills .5million people annually and the World Health Organization estimatesthis number to increase to 8 million by 2030 (2). Tobacco use is thesingle largest cause of cancer globally (3,4). The number of adultsmokers has declined in the USA (3,4), but the use of smokelesstobacco (ST) products (e.g. chewing tobacco, khaini and snuff) isincreasing worldwide, and these products can be a gateway for life-long addiction (1,5). ST consumption is emerging as a major riskfactor for head and neck squamous cell carcinoma (HNSCC) (6,7),the most common cancer in men in many Asian countries andthe sixth most common cancer in USA (8). The causal associationbetween smoking and HNSCC has been unequivocally established(5,9–12). The evidence that ST causes oral cancer was confirmedby the International Agency for Research on Cancer and ST wasclassified as a human carcinogen, with nitrosoamines being one of themajor carcinogenic constituents (13). In a recent study, 23 polycyclicaromatic hydrocarbons have been identified in ST (14). Nicotine,a major component of ST, besides causing addiction has been shownto regulate cell proliferation, angiogenesis and inhibit apoptosis in-duced by anticancer drugs (15). An indepth understanding of themolecular mechanism(s) underlying ST-related head and neck carci-nogenesis will lead to the development of effective strategies to pre-vent and treat ST-related HNSCC.

Studies carried out in our laboratory and others have shown aberrantexpression of genes involved in ST-associated HNSCC (16–20). ST hasbeen shown to increase oxidative stress (21) plays a major role in acti-vation of nuclear factor-kappaB (NF-jB) and pSTAT3 pathways,involved in inflammation, survival and proliferation of cancer cells(21–24). Our laboratory also reported exposure of oral premalignantcultures and cancer cells (AMOS III) to ST resulted in increased cellproliferation and activation of NF-jB; association of ST consumptionwith overexpression of NF-jB and cyclooxygenase-2 (COX-2) wasobserved in clinical oral squamous cell carcinoma (SCC) tissue samplesalso (23–25). The persistent activation of NF-jB can lead to elevatedexpression and secretion of interleukin (IL)-6. Interestingly, blockingNF-jB diminished the accumulation of active signal transducer andactivator of transcription (STAT) 3 in HNSCC cells, suggesting existenceof cross talks between NF-jB and STAT3 pathways (25). Recently ourgroup observed nuclear pSTAT3 accumulation in clinical oral squamouscell carcinoma tissue samples was significantly associated with ST con-sumption habits (26), indicating a plausible link between STAT3 and ST.Therefore, it is important to identify agents that can abrogate these effectsof ST in head and neck cancer cells for developing therapies to preventand treat ST-related head and neck cancer.

Guggulsterone (GS), (4,17(20)-pregnadiene- 3,16-dione), derivedfrom the plant Commiphora mukul, is widely used to treat obesity,diabetes, hyperlipidemia, atherosclerosis and osteoarthritis (27). GSsuppresses inflammation by inhibiting inducible nitric oxide synthe-tase (28) and NF-jB induced by various carcinogens and tumor pro-moters (29). Our group and several other reports have shown thattreatment with GS induces apoptosis and suppress proliferation ofwide variety of human tumor cell types (28–33). GS inhibits invasion,angiogenesis and metastasis of tumor cells and reverses chemoresist-ance (31–33). GS has also been shown to inhibit both constitutive andinducible STAT3 pathways in head and neck cancer cell lines (34,35).Recently, Leeman-Neill et al. (35) showed antiproliferative effects ofGS are partially dependent on STAT3 inactivation. They showedknocking down expression of STAT3 using small interfering RNAin head and neck cancer cells reduced GS-induced cell death in com-parison with the no transfection controls. In addition, our group incollaboration with Dr Bharat Aggarwal’s laboratory showed that GSinhibits inducible and constitutive STAT3 activation through the

Abbreviations: CLSM, confocal laser scan microscopy; COX-2, cyclooxyg-enase-2; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycol-bis(aminoethylether)-tetraacetic acid; HEPES, N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid; HNSCC, head and neck squamous cell carcinoma; GS,guggulsterone; IL, interleukin; IkBa, nuclear factor of kappa light polypeptidegene enhancer in B-cells inhibitor, alpha; NF-jB, nuclear factor-kappaB; PBS,phosphate-buffered saline; SCC, squamous cell carcinoma; SDS, sodium dodecylsulfate; STAT, signal transducer and activator of transcription; ST, smokelesstobacco; VEGF, vascular endothelial growth factor.

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induction of tyrosine phosphatase, which makes it a potentially effec-tive suppressor of tumor cell survival and proliferation (34). We furthershowed that activation of STAT3 by IL-6 is completely abrogated bypretreatment with GS in melphalan sensitive MM.1S cells. Similarresults of GS-induced inhibition of STAT3 activation have also beenreported in colon cancer cells (36). Together, these reports clearlysuggest inhibition of STAT3 activation is an important mechanism ofGS-induced cell death. Hence, it will be interesting to determine theeffect of GS on ST- and nicotine-induced head and neck carcinogenesis.

In the current study, we determined the effect of ST and/or nicotinetreatment on NF-jB and STAT3 signaling in head and neck cancercells. Furthermore, we challenged ST- and/or nicotine-induced activa-tion of NF-jB and STAT3 signaling by pretreatment of head and neckcancer cells (SCC4 and HSC2) with GS, to evaluate its therapeuticpotential. This may help us in designing novel therapies based on suchbiosafe nutraceutical agents that target ST-induced inflammation, sur-vival and proliferation in head and neck cancer cells.

Materials and methods

Antibodies and reagents

Z-GS, nicotine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideand propidium iodide were purchased from Sigma–Aldrich (St Louis, MO).Z-GS was dissolved in dimethyl sulfoxide as a 100 mM stock solution andstored at �20�C; Penicillin, streptomycin, Dulbecco’s modified eagle medium/F12 medium, fetal bovine serum, L-glutamine, sodium pyruvate, vitamins andminimum essential medium were obtained from Invitrogen (Grand Island, NY).Rabbit polyclonal antibodies against NF-jB (p65 subunit) (sc-109), NF-jB (pp65subunit) (sc-101749, Ser-276), nuclear factor of kappa light polypeptide geneenhancer in B-cells inhibitor, alpha (IkBa) (sc-371), pIkBa (sc-101713, Ser-32), Jak1 (sc-295), pJak1 (sc-16773, Tyr 1022) and vascular endothelialgrowth factor (VEGF) (sc-152); goat polyclonal antibodies against COX-2(sc-1746) and pSTAT3 (sc-7993, Tyr 705) and mouse monoclonal antibodiesagainst STAT3 (sc-8019) and a-tubulin (sc-5386) were obtained from SantaCruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit, anti-goat, antimouse-horseradish peroxidase conjugates were purchased from DAKO Cy-tomations (Glostrup, Denmark), and goat anti-rabbit Alexa 594 was pur-chased from Molecular Probes (Eugene, OR). Bacteria-derivedrecombinant human IL-6 was obtained from Biosource International (Cama-rillo, CA). ST extract was prepared as described before by us (37).

Cell culture

Head and neck cancer cells (SCC4 and HSC2) were grown in monolayercultures in Dulbecco’s modified eagle medium (Sigma, St Louis, MO) supple-mented with 10% fetal bovine serum (Sigma), 1 mM L-glutamine, 1 mMminimum essential medium, 100 lg/ml streptomycin and 100 U/ml penicillinin a humidified incubator (5% carbon dioxide, 95% air) at 37�C as describedearlier (38).

Preparation of nuclear extracts

Nuclear extracts were prepared from control, untreated and treated head andneck cancer cells (SCC4 and HSC2) as described earlier (39). Briefly head andneck cancer cells (SCC4 and HSC2) (2 � 107) cells were treated, harvested,washed with cold phosphate-buffered saline (PBS, pH 5 7.2) and incubatedon ice for 15 min in hypotonic buffer A [10 mM N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid (HEPES) (pH 5 7.9), 10 mM KCl, 0.1 mMethylenediaminetetraacetic acid (EDTA), 0.1 mM ethyleneglycol-bis(ami-noethylether)-tetraacetic acid (EGTA), 1 mM dithiothreitol, 0.5 mM phenyl-methylsulfonyl fluoride and 0.6% NP-40]. Cells were vortexed gently forlysis, and nuclei were separated from the cytosol by centrifugation at12 000g for 1 min. Nuclei were resuspended in buffer C [20 mM HEPES(pH 5 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMdithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride] and shaken for30 min at 4�C. Nuclear extracts were obtained by centrifugation at12 000g for 10 min and stored at �80�C till use.

Western blotting

Western blotting was carried out as described previously by us (38). Whole-celllysates were prepared from treated and untreated control cells SCC4 and HSC2cells and protein concentration was determined using the Bradford reagent(Sigma–Aldrich) and equal amounts of proteins (80 lg/lane) were resolvedon 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel. The proteins werethen electrotransferred onto polyvinylidenedifluoride membrane. After block-ing with 5% non-fat milk in Tris-buffered saline (0.1 M, pH 5 7.4), blots were

incubated with specific antibodies as per manufacturer’s recommended pro-tocol at 4�) served as a control for protein loading in each lane. Membraneswere incubated with horseradish peroxidase-conjugated secondary antibodies,(DAKO Cytomation), diluted at an appropriate dilution in 1% bovine serumalbumin, for 2 h at room temperature. After each step, blots were washed threetimes with Tween (0.1%)-Tris-buffer saline. Protein bands were detected bythe enhanced chemiluminescence method (Santa Cruz Biotechnology) on XO-MAT film. The band intensities were measured as integrated density valuesusing AlphaEase FC Software (version 3.1) with ChemiImager IS-4400 (AlphaInnotech Corporation, San Leandro, CA).

Confocal laser scan microscopy

For confocal laser scan microscopy (CLSM), 5 � 103 SCC4 cells were platedon coverslips and grown for 24 h. Expression and localization of p65 proteinwas observed in treated and untreated, control SCC4 cells using CLSM asdescribed by us (38). Briefly, treated and control SCC4 cells were, fixed inmethanol for 20 min at �20�C, rinsed with PBS (pH 5 7.2) and incubatedwith p65 antibody overnight at 4�C. After rinsing in PBS, the coverslips wereincubated with biotinylated secondary antibody (LSAB plus Kit; DAKOCytomations) for 45 min at 37�C followed by incubation with streptavidin-conjugated fluorochrome, fluorescein isothiocyanate (DAKO Cytomations).Thereafter, the coverslips were counterstained with propidium iodide(10 mg/ml; Sigma) for 30 s. Coverslips were rinsed and mounted in mountingmedium. Slides were examined using a CLSM-LSM510 scanning module (CarlZeiss Microscopy; Jena GmbH, Germany).

IL-6 assay

Cell-free supernatants were collected from GS (50 lM), ST extract (20 lg/ml),nicotine (10 lM) and IL-6-treated SCC4 cells and filtered through a 0.22 lmlow protein-binding polyethylsulfonate membrane filter. Aliquots of 100 llwere removed, and IL-6 contents were determined by enzyme-linked immu-nosorbent assay kit (Biosource International, Camarillo, CA).

Chromatin immunoprecipitation assay

For chromatin immunoprecipitation assay, treated and untreated control SCC4cells were cross-linked with 1% formaldehyde at 37�C for 10 min. The cross-linking reaction was quenched by washing the cells several times with coldPBS, buffer I (0.25% Triton X 100, 10 mM EDTA, 0.5 mM EGTA, 10 mMHEPES, pH 5 6.5) and buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA,10 mM HEPES, pH 5 6.5). Cells were centrifuged, resuspended incold immunoprecipitation buffer (1% SDS, 10 mM EDTA, 50 mM Tris,pH 5 8.1 and protease inhibitors) and sonicated to an average fragment sizeof 300–600 bp. Solubilized chromatin was clarified by centrifugation at21 000g, and the supernatant was preincubated for 2 h with protein-A/G aga-rose beads. Precleared chromatin was incubated overnight at 4�C with 2 lganti-STAT3 antibody. Preimmune rabbit serum was used as a negative control.Immune complexes were bound to protein-A agarose beads at 4�C for anadditional 2–3 h. Beads were washed sequentially for 10 min each in bufferI (20 mM Tris, pH 8.1, containing 0.1% SDS, 1% Triton X 100, 2 mM EDTAand 150 mM NaCl), buffer II (20 mM Tris, pH 5 8.1, containing 500 mMNaCl, 0.1% SDS, 1% Triton X 100 and 2 mM EDTA), buffer III (0.25 M LiCl,1% NP-40, 1% deoxycholate 1 mM EDTA, 10 mM Tris, pH 5 8.1) and 10 mMTris, pH 8.1 and 2 mM EDTA (TE buffer). Protein–DNA complexes wereeluted from the protein-A agarose beads with 1% SDS and 0.1M NaHCO3 atroom temperature for 10 min with gentle agitation. The supernatant was trans-ferred to a fresh tube, and cross-links were reversed overnight at 65�C. Sampleswere treated with Proteinase K (Sigma Chemical Co., St Louis, MA) at 37�Cfor 2 h and were extracted once with phenol, and the DNA was precipitatedwith 2.5 vol ethanol plus 20 lg glycogen as carrier. Precipitated DNA waspelleted, washed once with 70% ethanol, dried and resuspended in water. Thepurified DNA was used as a template for polymerase chain reaction amplifica-tion using primers (forward, 5#-TTGGTGCCAAATTCTTCTCC-3#; reverse, 5#-CACACGTCCTCACTCTC GAA-3#) flanking the putative STAT-binding siteslocated at �848 and �630 in human VEGF promoter region. The polymerasechain reaction products were resolved on 1.2% agarose and stained with ethi-dium bromide, and the band intensities were quantified using Chemimager 4400(Alpha Innotech Corporation).

Results

Effect of GS on ST- and nicotine-induced phosphorylation andlocalization of p65 subunit of NF-jBPhosphorylation of p65 subunit of NF-jB complex is required for itsnuclear translocation and transcriptional activation. Therefore, we in-vestigated the effect of GS on ST- and nicotine-induced phosphoryla-tion of p65 in head and neck cancer cells. SCC4 and HSC2 cells were

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treated with GS (50 lM)/ST (20 lg/ml)/nicotine (10 lM) for differenttime intervals, and protein extracts obtained from cytosolic fractionwere checked for phosphorylated p65 protein. As shown in Figure1a, GS completely inhibited the phosphorylation of p65, whereas eitherST- or nicotine-induced the p65 phosphorylation in a time-dependentmanner with significant increase of phosphorylated p65 (pp65) ob-served after 2 h of treatment in SCC4 cells (Figure 1b and c). Interest-

ingly, we observed that treatment with GS (50 lM) for 4 h completelyabrogated the ST- or nicotine-induced phosphorylation of p65, respec-tively, in both the cell lines (Figure 1d and e). Thus, GS inhibited ST- ornicotine-induced NF-jB activation by inhibiting p65 phosphorylation.

GS treatment decreased nuclear accumulation of p65 as comparedwith the untreated control (Figure 2a), whereas ST or nicotine in-creased nuclear accumulation of p65 as shown by western blotting

Fig. 1. Effect of GS on ST- and nicotine-induced phosphorylation p65. SCC4 cells were treated with (a) 50 lM GS for 0 and 30 min and 1, 2, 4 and 6 h; (b) ST(20 lg/ml) for 0,1, 2, 4, 6 and 9 h; or (c) 10 lM nicotine for 0, 30 min, 1, 2, 3 and 4 h. SCC4/HSC2 cells were pretreated with 50 lM GS for 0, 1, 2, 4, 6 and9 h, followed by ST for 4 h (d) or with nicotine for 1h (e). Cytoplasmic extracts were prepared, separated on 10% SDS–PAGE. Proteins were then electrotransferredon polyvinylidenedifluoride membrane followed by blocking with 5% non-fat milk overnight. Blots were incubated with specific antibody against pp65.Protein expression was determined using enhanced chemiluminescence method. The same blots were stripped and reprobed with a-tubulin antibody to show equalprotein loading.

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Fig. 2. (a–e) Effect of GS on ST- and nicotine-induced nuclear translocation of p65. SCC4 cells were treated with (a) 50 lM GS for 0, 30 min, 1, 2, 4 and 6 h;(b) ST (20 lg/ml) for 0, 1, 2, 4, 6 and 9 h; or (c) 10 lM nicotine for 0 and 30 min and 1, 2, 3 and 4 h. SCC4/HSC2 cells were pretreated with 50 lM GS for 0,30 min, 1, 2, 4, and 6 h, followed by ST for 4 h (d) or with nicotine for 2 h (e). Cytoplasmic and nuclear extracts were prepared according to materials and methodsand separated on 10% SDS–PAGE. Proteins were then electrotransferred on polyvinylidenedifluoride membrane followed by blocking with 5% non-fat milkovernight. Blots were incubated with specific antibody against p65. Protein expression was determined using enhanced chemiluminescence method. The sameblots were stripped and reprobed with a-tubulin or laminin B antibody to show equal protein loading in cytoplasmic and nuclear fraction respectively. (f) Effect ofGS on ST- and nicotine-induced nuclear translocation of p65 using immunofluorescence. SCC4 cells grown on coverslips, were kept untreated (i–iii) ortreated with GS (50 lM) for 4 h (iv–vi); ST (20 lg/ml) for 6 h (vii–ix); with nicotine (10 lM) for 2 h (x–xii); pretreated with 50 lM GS for 4 h and stimulated with ST(20 lg/ml) for 6 h (xiii–xv) or with nicotine (10 lM) for 2 h (xvi–xviii).Then immunolabelled with anti-p65 antibody followed by streptavidin-conjugated fluorescein


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using cytoplasmic and nuclear fractions obtained from treated andcontrol SCC4 cells (Figure 2b and c). However, treatment with GS(50 lM) for 4 h completed abrogated the ST- or nicotine-inducednuclear translocation, with concomitant increase in cytoplasmic ac-cumulation of p65 in both SCC4 and HSC2 cells (Figure 2d and e).These findings were further confirmed by CLSM of p65 subunit usingcontrol and GS-treated SCC4 cells (Figure 2f).

Effect of GS on ST- and nicotine-induced IjBa phosphorylation

The degradation of IjBa and subsequent release of NF-jB (p65 andp50) requires prior phosphorylation at Ser-32 and Ser-36 residues.Therefore, to investigate whether the inhibitory effect of GS is medi-ated through the alteration of IkBa phosphorylation, SCC4 cells weretreated with GS (50 lM)/ST (20 lg/ml)/nicotine (10 lM) for differenttime intervals and western blotting was performed for phospho-IjBa

Fig. 2. Continued.

isothiocyanate (FITC)-labeled tertiary antibody (green fluorescence) and nuclei were counterstained with propidium iodide (PI) (red fluorescence). Panels(i), (iv), (vii), (x), (xiii) and (xvi) represents nuclei showing red fluorescence of PI; panels (ii), (v), (viii), (xi), (xiv) and (xvii) shows Green fluorescence forp65 protein in cytoplasm and nuclei; panels (iii), (vi), (ix), (xii), (xv) and (xviii) shows increased Green fluorescence for p65 protein in cytoplasm of SCC4cells. (original magnification �100 for all photomicrographs).

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Fig. 3. Effect of GS on ST- and nicotine-induced IjBa phosphorylation and IjBa degradation. SCC4/HSC2 cells were treated with GS (50 lM) for 0, 30 min, 1, 2,4 and 6 h; ST (20 lg/ml) for 0, 1, 2, 4, 6 and 9 h; or 10 lM nicotine for 0, 30 min, 1, 2, 3 and 4 h. SCC4 cells were pretreated with 50 lM GS for 0, 1, 2, 4 and 6 h,followed by ST for 4 h or with nicotine for 2 h. Whole-cell extracts were prepared, separated on 10% SDS–PAGE. Proteins were then electrotransferred onpolyvinylidenedifluoride membrane followed by blocking with 5% non-fat milk overnight. Blots were incubated with specific antibody against pIjBa (a–e) andIjBa (f–j). Protein expression was determined using enhanced chemiluminescence method. The same blots were stripped and reprobed with a-tubulin antibody toshow equal protein loading.


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(Ser�32/36). As shown in Figure 3a, GS treatment decreased consti-tutively expressed phosphorylated IjBa (Ser-32/36) as revealed bywestern blotting. However, either ST or nicotine increased the expres-sion of pIjBa with a significant increase in expression observed after4 and 1 h on ST and nicotine treatment, respectively (Figure 3b and c).Interestingly, treatment with GS (50 lM) completely abrogated ST/nicotine-induced phosphorylation of IjBa in SCC4 and HSC2 cells(Figure 3d and e).

We determined whether the GS inhibition by either ST- or nicotine-induced NF-jB activation was due to inhibition of IjBa degradation. GS

induced the expression of IjBa in a time-dependent manner (Figure 3f),with maximum expression observed at 4 h; however, ST- or nicotine-induced IjBa degradation in a time-dependent manner (Figure 3gand h). ST induced the degradation of IjBa was observed within 4 hand complete loss of IjBa expression was found within 6 h of STtreatment. Upon nicotine treatment, degradation started as early asin 2 h with no detectable expression observed within 4 h. Notably,pretreatment with GS abrogated the ST-induced IjBa degradation inboth cell lines (Figure 3i). Similarly we observed nicotine-inducedIjBa degradation was abrogated in GS pretreated SCC4 and HSC2 cells

Fig. 3. Continued.

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(Figure 3j). Thus, GS pretreatment abolished ST- and nicotine-inducedNF-jB activation by inhibiting the phosphorylation of IjBa.

GS inhibits upregulation of COX-2 in response to ST and nicotinetreatment

NF-jB activation results in nuclear translocation p65-subunit fol-lowed by increased expression of its target genes, such as COX-2.Therefore, we investigated the effect of GS on COX-2 expression.

A time-dependent decrease in COX-2 expression was observed, withcomplete absence of COX-2 protein at 6 and 12 h of GS treatment(Figure 4a). Treatment with STand nicotine increased the cytoplasmicexpression of COX-2 (Figure 4b). Treatment with tumor necrosisfactor-a [TNF-a (0.1 nM, 1 h)] was used as a positive control forinducing NF-jB-mediated COX-2 upregulation. Notably, GS treat-ment inhibited the ST- and nicotine-induced expression of COX-2in a time-dependent manner (Figure 4b).

Fig. 4. GS inhibits upregulation of COX-2 in response to ST and nicotine treatment. SCC4 cells (2–3 � 106 cells) were treated with (a) GS (50 lM) for 0, 1, 2, 3, 6and 12 h (b) SCC4 cells (2–3 � 106 cells) were pretreated with GS (50 lM) for 6 h followed by treatment with either ST (20 lg/ml) or with nicotine(10 lM) for4 h and whole-cell extracts were prepared. Tumor necrosis factor-a treated cells were used as a positive control for COX-2 overexpression. Sixty microgramsof whole-cell extracts were resolved on 10% SDS–PAGE, electrotransferred to a polyvinylidenedifluoride membrane followed by blocking with 5%non-fat milk overnight. Protein expression was determined using enhanced chemiluminescence method and probed for the COX-2. Western blot for a-tubulin wasdone to show equal loading of protein. (c) Effect of GS, ST or nicotine on the expression of secretory IL-6. SCC4 cells were treated with 50 lM GS, 20 lg/ml of STor 10 lM nicotine for different time points, and cell-free supernatants were collected and filtered through a 0.22 mm low protein-binding polyethylsulfonatemembrane filter. Aliquots of 100 ll were removed, and IL-6 contents were determined by enzyme-linked immunosorbent assay.


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Effect of GS, ST and nicotine on the expression of secretory IL-6

The persistent activation of NF-jB can lead to elevated expressionand secretion of IL-6 in HNSCC and may contribute to inflammatoryand angiogenic responses as well as its progression and metastasis.We determined secreted IL-6 levels in GS, ST- or nicotine-treated

SCC4 cells using enzyme-linked immunosorbent assay. SCC4 cellswere treated with GS (50 lM), ST (20 lg/ml) and nicotine (10 lM)for different time intervals, and cell-free supernatants were collectedand filtered through a 0.22 l low protein-binding polyethylsulfonatemembrane filter. Aliquots of 100 ll were used for IL-6 assay using an

Fig. 5. GS inhibits IL-6 inducible STAT3 phosphorylation in SCC4 cells. (a) SCC4 cells (2–3 � 106 cells) were treated with IL-6 (10 ng/ml) for the indicatedtimes, and whole-cell extracts were prepared. (b) SCC4 cells (2–3 � 106 cells) were pretreated with 50 lM GS for the indicated durations and stimulated withIL-6 (10 ng/ml) for 2 h and whole-cell extracts were prepared. (c) GS-induced inhibition of STAT3 phosphorylation is reversible. SCC4 (2–3 � 106 cells)were treated with 50 lM GS treated for 4 h and washed with PBS two times to remove GS before resuspension in fresh medium. Cells were removed at indicatedtimes and lysed to prepare the whole-cell extract. (d–h) GS inhibits constitutive and ST or nicotine induces STAT3 phosphorylation in SCC4 cells. SCC4 cells(–3 � 106 cells) were treated with (d) GS (50 lM); ST (20 lg/ml); or (e) with nicotine (10 lM) (f) for the indicated times, and whole-cell extracts were prepared.SCC4 cells (–3 � 106 cells) were pretreated with 50 lM GS for the indicated durations and stimulated with (g) ST (20 lg/ml) for 4 h or (h) with nicotine (10 lM)for 2 h and whole-cell extracts were prepared. Sixty micrograms of whole-cell extracts were resolved on 7.5% SDS–PAGE, electrotransferred toa polyvinylidenedifluoride membrane followed by blocking with 5% non-fat milk overnight. Protein expression was determined using enhancedchemiluminescence method and probed for the phosphorylated STAT3 (upper panel) and stripped and reprobed for STAT3 (lower panel). Western blot for GAPDHwas done to show equal loading of protein.

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enzyme-linked immunosorbent assay kit. We observed that GS decreasedIL-6 levels in a time-dependent manner with complete loss of IL-6 within6 h of GS treatment (Figure 4c). However, both STand nicotine increasedIL-6 secretion in a time-dependent manner (Figure 4c). These resultswere in concordance with the expression levels of NF-jB in SCC4 cells.

GS inhibits IL-6 inducible STAT3 phosphorylation in SCC4 cells

IL-6-induced signals are mediated through STAT3 phosphorylation;therefore, we determined the effect of GS on IL-6-induced STAT3phosphorylation. SCC4 cells were treated with IL-6 (10 ng/ml) fordifferent time intervals and whole-cell lysates were prepared. Westernblot analysis showed that IL-6 induced phosphorylation of STAT3 ina time-dependent manner with phosphorylation induction as early as in10 min and maximum phosphorylation at 2 h (Figure 5a). SCC4 cellswere then preincubated with GS for different time intervals, followedby IL-6 treatment for 2 h and effect on STAT3 phosphorylation wasexamined. Figure 5b shows that IL-6-induced STAT3 phosphorylationwas blocked by GS in a time-dependent manner. Exposure of cells toGS for 4 h was sufficient to completely suppress IL-6-induced STAT3phosphorylation. However, GS treatment alone at these time points hadno effect on STAT3 levels in these cells (Figure 5a).

GS induced inhibition of STAT3 phosphorylation is reversible inSCC4 cells

We next determined whether GS-induced inhibition of STAT3 phos-phorylation was reversible. SCC4 cells were first treated with GS for4 h, washed twice with PBS and the cells were then cultured in freshmedium without GS for different time intervals, and the levels of phos-phorylated STAT3 were measured. GS treatment induced the suppres-sion of STAT3 phosphorylation (Figure 5c), but once GS was removed,the levels of phosphorylated STAT3 increased in a time-bound manner(Figure 5c). The reversal of phosphorylation was partial at 18 h, withcomplete reversal at 36 h, without any changes in the STAT3 levels.

GS inhibited ST/nicotine-induced STAT3 phosphorylation in SCC4cells

The aberrant function of transcription factor NF-jB can lead to stim-ulation of STAT3 by an autocrine/paracrine mechanism involving therelease of IL-6, prompting us to determine the effect of GS on themolecular cross talk between these two pathways. We investigatedwhether GS inhibits the constitutive STAT3 phosphorylation in SCC4cells in accordance with its effect on NF-jB expression. SCC4 cellswere incubated either with GS (50 lM) for indicated time intervalsand compared with the untreated control cells. Western blot analysis

showed that GS inhibited the constitutively active STAT3 in time-dependent manner (Figure 5d). GS-induced inhibition was observedas early as 1 h with complete inhibition of STAT3 phosphorylationobserved at 4 h; however, GS treatment did not alter the overallSTAT3 protein expression (Figure 5d). We also observed similar ef-fect of GS treatment on the expression of activated upstream STAT3kinase, pJAK1 (Figure 5d). However, no effect was observed on thetotal JAK1 expression at these time points of GS treatment. Theseresults suggested that GS-induced inhibition of STAT3 phosphoryla-tion was mediated by inhibiting the upstream kinases.

Furthermore, we investigated the effect of GS on ST- and nicotine-induced STAT3 activation in vitro in SCC4 cells by western blotting. BothST- and nicotine- induced STAT3 phosphorylation (Figure 5e and f).Increased STAT3 phosphorylation was observed in a time-dependentmanner with maximum phosphorylation at 6 h by ST and within 4 hby nicotine, respectively. SCC4 cells were then incubated with GSfor different time intervals and examined for ST and nicotine inducibleSTAT3 phosphorylation. Both ST- and nicotine-induced STAT3 phos-phorylation was blocked by GS in a time-dependent manner (Figure 5gand h). Exposure of cells to GS for 4 h completely suppressed ST- andnicotine-induced STAT3 phosphorylation. GS, ST or nicotine, alone orin combination, had no effect on total STAT3 expression levels in thesecells. These results clearly demonstrate that GS abrogated ST- andnicotine-mediated activation of pSTAT3 in SCC4 cells.

GS inhibited ST- and nicotine-induced expression of VEGF andrecruitment of STAT3 to the VEGF promoter

STAT3 is a transcriptional activator of VEGF and STAT3 inhibitioneffectively blocks VEGF expression. STAT3 is being activated byboth ST and nicotine, therefore, we demonstrated the effect of thesecompounds on VEGF expression and effect of GS pretreatment on ST-or nicotine-induced VEGF expression using western blotting. SCC4cells were treated with either GS (50 lM) for different time intervals,or with ST (20 lg/ml)/nicotine (10 lM) for 6 and 12 h. As shown inFigure 6a, GS downregulated VEGF expression in a time-dependentmanner, whereas both ST- and nicotine-induced VEGF expression(Figure 6b). Notably, pretreatment of cells with GS for 6 h followedby either ST or nicotine for 12 h, inhibited both ST- and nicotine-induced VEGF expression (Figure 6b).

To demonstrate the effect of GS on ST- or nicotine-induced inter-action between STAT3 protein and VEGF promoter in vivo, we carriedout chromatin immunoprecipitation assays using an anti-STAT3 anti-body. The occupancy of the promoter was analyzed using specificpairs of primers spanning the STAT3 binding motif of the VEGFpromoter. SCC4 cells were treated with either GS (50 lM) for 6 h;ST (20 lg/ml)/nicotine (10 lM) 12 h or pretreated with GS for 6 h

Fig. 5. Continued.


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followed by ST and nicotine for 12 h. Significantly greater binding ofSTAT3 to the VEGF promoter was observed in ST- or nicotine-treatedcells (Figure 6c). GS treatment significantly inhibited both ST-and nicotine-induced recruitment of STAT3 to the VEGF promoter(Figure 6c).

Discussion

Knowledge of molecular pathways implicated in ST-induced head andneck cancers is critical for development of novel agents that are bettertargeted to ST-induced molecular changes and to ST-related HNSCCfor effective disease management. Herein, we demonstrated that ST ornicotine activates NF-jB in HNSCC cells (SCC4 and HSC2) and thisactivation was mediated through phosphorylation and degradation ofIjBa. ST as well as nicotine-induced IL-6 secretion, which in turnactivated the STAT3 pathway. Therefore, agents that can suppressNF-jB and STAT3 activation might have potential to suppress ST/

nicotine-induced head and neck cancer. In this context, we investi-gated the potential of GS to block ST/nicotine-induced-NF-jB and/orIL-6/STAT3 activation in HNSCC cells (SCC4/HSC2) and deter-mined its effect on the abrogation of expression of genes implicatedin carcinogenesis. Our results clearly showed that GS treatment ab-rogated both ST- and nicotine-induced NF-jB activation by suppress-ing IjBa phosphorylation and degradation, thereby inhibiting thephosphorylation and nuclear translocation of the p65 subunit and sub-sequent inhibition of COX-2 expression. Proteosomal degradation ofIjBa protein is mediated by inhibitor kappa kinase B dependentphosphorylation. Our results clearly demonstrated decreased phos-phorylation of IjBa in head and neck cancer cells on treatment withGS in time-dependent manner, thus reduced degradation. In support ofour study, Shishodia et al. (40) also showed inhibition of tumor ne-crosis factor-a dependent activation of inhibitor kappa kinase B inpresence of GS. This reduced phosphorylation of IjBa and inhibitednuclear translocation of p65 subunit of NF-jB. This aberrant

Fig. 6. GS inhibits ST- and nicotine-induced expression of VEGF and recruitment of STAT3 to the VEGF promoter. SCC4 cells (2–3 � 106 cells) were treatedwith (a) with either GS (50 lM) for different time points (b) ST (20 lg/ml)/nicotine (10 lM) for 6 and 12 h, or pretreatment of GS with for 6 h followed by eitherST or nicotine for 12 h. Whole-cell extracts were prepared and 60 lg of extracts were resolved on 10% SDS–PAGE, electrotransferred toa polyvinylidenedifluoride membrane followed by blocking with 5% non-fat milk overnight. Protein expression was determined using enhancedchemiluminescence method and probed for the VEGF and stripped and reprobed for a-tubulin to show equal loading of protein. (c) Cross-linked,sheared chromatin was prepared from treated SCC4 and subjected to immunoprecipitation with the STAT3 antibody. The precipitates were subjected topolymerase chain reaction analysis using primer pairs spanning the human VEGF promoter. The control was the polymerase chain reaction product of chromatinobtained before immunoprecipitation.

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activation of NF-jB can also stimulate STAT3 by an autocrine/para-crine mechanism involving the release of IL-6, which confers bothproliferative and survival potential in HNSCC (25,41), prompting usto determine the effect of GS on molecular cross talk between thesetwo pathways. We observed GS decreased IL-6 secretion in SCC4cells, whereas both ST and nicotine increased the secretory IL-6 lev-els, and these changes in IL-6 levels were paralleled by changes in p65subunit of NF-jB. We also observed that GS abrogated both consti-tutive and ST/nicotine-induced STAT3 phosphorylation in cancercells, which may be one of the mechanisms for reducing the prolif-erative potential of HNSCC cells.

Interestingly, Arredondo et al. (42) showed multifold increase inSTAT-3 in oral keratinocytes stimulated with aged and diluted side-stream cigarette smoke or nicotine, both at the messenger RNA andprotein levels. In addition, the downregulation of NF-jB, const-itutive and inducible STAT3 by GS in other HNSCC cell lines andxenografts have been reported (28,31,34,35,43,44). Importantly, theseobservations are in accord with clinical studies from our group andothers, showing significant association of pSTAT3 activation with STconsumption habits (26,45). Previously, we have also shown that GSdownregulates the expression of cyclin D1, Bcl2 and Bcl-xL in SCC4cells (46). These genes are known to be regulated by both STAT3 andNF-jB (47,48). Notably, we found that suppression of STAT3 phos-phorylation by GS was reversible, returning to nearly control valueswithin 36 h of GS withdrawal.

Activation of NF-jB and COX-2 by ST was previously reported byour group and others using different HNSCC cell lines, namelyAMOS III and SCC38 (22,24,48). Herein, we showed that ST- andnicotine-induced NF-jB activation was mediated by the phosphory-lation and degradation of IjBa. Notably, other forms of ST, such assnuff extracts, have been shown to stimulate nuclear localization ofp50 and p65 subunits of NF-jB in RAW264.7 cells. Normal humanoral keratinocytes exposed to environmental tobacco smoke or nico-tine also showed a multifold increase in NF-jB at the messenger RNAand protein levels. Importantly, GS inhibited constitutive COX-2 ex-pression and treatment with GS abrogated the expression of both ST-and nicotine-induced COX-2 expression. COX-2 has been implicatedin carcinogenic processes and its overexpression in malignant cellshas been shown to enhance cellular invasion, induce angiogenesis,regulate anti-apoptotic cellular defenses and augment immunologicresistance through production of prostaglandin E2 (22,48–50). Ourresults suggested that nicotine-induced NF-jB activation resulting inincreased expression of COX-2 protein head and neck cancer cells(SCC4), which can be inhibited by treatment with GS.

Our study also showed GS-inhibited ST/nicotine-induced STAT3activation is required for VEGF receptor signaling and neovascula-ture in human oral cancer (36). In accordance with the STAT3 acti-vation, we observed an increase in VEGF expression in SCC4 cellsupon treatment with ST or nicotine and GS downregulated the levelsof VEGF protein. The similar time kinetics of STAT3 and VEGFexpression prompted us to determine if STAT3 regulated VEGF ex-pression by activation of the VEGF promoter. Using co-immunopre-cipitation and chromatin immunoprecipitation assays, we identifiedan increased interaction of STAT3 on the VEGF promoter upon STand nicotine treatment, whereas GS treatment inhibited this interac-tion, suggesting that STAT3 might serve as a transcription regulatorof VEGF promoter. These observations are consistent with an earlierreport demonstrating GS-inhibited STAT3 binding on VEGF pro-moter (36). Thus, ST-induced STAT3 inhibition by GS and the sub-sequent reduction in VEGF expression could represent a potentialclinical therapeutic approach for patients with ST-associatedHNSCC.

In conclusion, our studies demonstrated that ST and nicotine acti-vate both NF-jB and STAT3 pathways in HNSCC cells and theirdownstream targets—COX-2 and VEGF. Importantly, GS not onlyinhibits the constitutively active NF-jB and STAT3 pathways but alsoabrogates ST- and nicotine-induced activation of these pathwaysunderscoring its putative chemopreventive potential. Hence, our find-ings provide a biologic rationale for investigating the chemopreven-

tive and complementary therapeutic potential of GS in ST-associatedhead and neck cancers.

Acknowledgements

M.A.M is recipient of Senior Research Fellowship of Council of Scientific andIndustrial Research, New Delhi, India. A.M. is the recipient of a Mathematicsof Information Technology and Complex Systems Accelerate Fellowship,Ontario, Canada. RR gratefully acknowledges support from the Joseph andMildred Sonshine Center for Head and Neck Diseases, Alex and SimonaShnaider Laboratory of Molecular Oncology, Temmy Latner/Dynacare andthe Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hos-pital, University of Toronto. K.W.M.S. acknowledges funding from the OntarioInstitute for Cancer Research.

Conflicts of Interest: None declared.

References

1.Shafey,O. et al. (2009) The Tobacco Atlas. American Cancer Society,Atlanta, GA.

2.World Health Organization (2009) Tobacco Key Facts. http://www.who.int/topics/tobacco/facts/en/index.html (Accessed March 29, 2010).

3.Centers for Disease Control and Prevention (2009) Smoking & TobaccoUse. [Cited January 15, 2010]; Trends in Current Cigarette SmokingAmong High School Students and Adults, United States, 1965–2007].http://www.cdc.gov/tobacco/data_statistics/tables/trends/cig_smoking/index.htm. Accessed March 29, 2010.

4.Centers for Disease Control and Prevention (2009) Cigarette smokingamong adults and trends in smoking cessation-United States, 2008. MMWRMorb. Mortal. Wkly. Rep., 58, 1227–1232.

5.Viswanath,K. et al. (2010) Tobacco and cancer: an American Associationfor Cancer Research policy statement. Cancer Res., 70, 3419–3430.

6.Secretan,B. et al. (2009) A review of human carcinogens–Part E: tobacco,areca nut, alcohol, coal smoke, and salted fish. Lancet Oncol., 10, 1033–1034.

7. International Agency for Research on Cancer Working Group on the Eval-uation of Carcinogenic Risks to Humans (2007) IARC Monographs on theEvaluation of Carcinogenic Risks to Humans, Volume 89: SmokelessTobacco and Some Tobacco-specific N-Nitrosamines. IARC, Lyon.

8. Jemal,A. et al. (2009) Cancer statistics. CA Cancer J. Clin., 59, 225–249.9.Argiris,A. et al. (2008) Head and neck cancer. Lancet, 371, 1695–1709.

10.Righini,C.A. et al. (2008) [Risk factors for cancers of the oral cavity,pharynx (cavity excluded) and larynx]. Presse. Med., 37, 1229–1240.

11.Lee,P.N. et al. (2009) The relation between smokeless tobacco and cancer inNorthern Europe and North America. A commentary on differences betweenthe conclusions reached by two recent reviews. BMC Cancer, 9, 256.

12.Petersen,P.E. (2009) Oral cancer prevention and control–the approach ofthe World Health Organization. Oral Oncol., 45, 454–460.

13.Cogliano,V. et al. (2004) Smokeless tobacco and tobacco-related nitros-amines. Lancet Oncol., 5, 708.

14.Stepanov,I. et al. (2010) Analysis of 23 polycyclic aromatic hydrocarbonsin smokeless tobacco by gas chromatography-mass spectrometry. Chem.Res. Toxicol., 23, 66–73.

15.Xu,J. et al. (2007) Nicotine inhibits apoptosis induced by cisplatin in hu-man oral cancer cells. Int. J. Oral Maxillofac. Surg., 36, 739–744.

16. Jayasurya,R. et al. (2005) Phenotypic alterations in Rb pathway have moreprognostic influence than p53 pathway proteins in oral carcinoma. Mod.Pathol., 18, 1056–1066.

17.Mishra,R. et al. (2005) Activation of STAT 5-cyclin D1 pathway in chew-ing tobacco mediated oral squamous cell carcinoma. Mol. Biol. Rep., 32,159–166.

18.Warnakulasuriya,K.A. et al. (2007) Clinical, pathological, cellular and mo-lecular lesions caused by oral smokeless tobacco–a review. J. Oral Pathol.Med., 36, 63–77.

19.Kaur,J. et al. (2010) Clinical significance of phosphatidyl inositol synthaseoverexpression in oral cancer. BMC Cancer, 10, 168.

20.Coppe,J.P. et al. (2008) A role for fibroblasts in mediating the effects oftobacco-induced epithelial cell growth and invasion. Mol. Cancer Res., 6,1085–1098.

21.Ondrey,F.G. et al. (1999) Constitutive activation of transcription factorsNF-(kappa)B, AP-1, and NF-IL6 in human head and neck squamous cellcarcinoma cell lines that express pro-inflammatory and pro-angiogeniccytokines. Mol. Carcinog., 26, 119–129.


379

Dow



ecember 2021

http://www.who.int/topics/tobacco/facts/en/index.html

http://www.who.int/topics/tobacco/facts/en/index.html

http://www.cdc.gov/tobacco/data_statistics/tables/trends/cig_smoking/index.htm

http://www.cdc.gov/tobacco/data_statistics/tables/trends/cig_smoking/index.htm

22.Sawhney,M. et al. (2007) Expression of NF-kappaB parallels COX-2 ex-pression in oral precancer and cancer: association with smokeless tobacco.Int. J. Cancer, 120, 2545–2556.

23.Aggarwal,B.B. (2004) Nuclear factor-kappaB: the enemy within. CancerCell, 6, 203–208.

24.Rohatgi,N. et al. (2005) Smokeless tobacco (khaini) extracts modulate geneexpression in epithelial cell culture from an oral hyperplasia. Oral Oncol.,41, 806–820.

25.Squarize,C.H. et al. (2006) Molecular cross-talk between the NFkappaBand STAT3 signaling pathways in head and neck squamous cell carcinoma.Neoplasia, 8, 733–746.

26.Macha,M.A. et al. (2010) Prognostic significance of nuclear pSTAT3 inoral cancer. Head Neck, Jul 22. [Epub ahead of print].

27.Shishodia,S. et al. (2008) The guggul for chronic diseases: ancient medi-cine, modern targets. Anticancer Res., 28, 3647–3664.

28.Shishodia,S. et al. (2004) Guggulsterone inhibits NF-kappaB andIkappaBalpha kinase activation, suppresses expression of anti-apoptotic gene products, and enhances apoptosis. J. Biol. Chem., 279,47148–47158.

29.Singh,S.V. et al. (2005) Caspase-dependent apoptosis induction by guggul-sterone, a constituent of Ayurvedic medicinal plant Commiphora mukul, inPC-3 human prostate cancer cells is mediated by Bax and Bak. Mol. CancerTher., 4, 1747–1754.

30.Samudio,I. et al. (2005) Guggulsterones induce apoptosis and differentia-tion in acute myeloid leukemia: identification of isomer-specific antileuke-mic activities of the pregnadienedione structure. Mol. Cancer Ther., 4,1982–1992.

31.Shishodia,S. et al. (2007) Guggulsterone inhibits tumor cell proliferation,induces S-phase arrest, and promotes apoptosis through activation of c-JunN-terminal kinase, suppression of Akt pathway, and downregulation ofantiapoptotic gene products. Biochem. Pharmacol., 74, 118–130.

32.Singh,S.V. et al. (2007) Guggulsterone-induced apoptosis in humanprostate cancer cells is caused by reactive oxygen intermediate dependentactivation of c-Jun NH2-terminal kinase. Cancer Res., 67, 7439–7449.

33.An,M.J. et al. (2009) Guggulsterone induces apoptosis in colon cancer cellsand inhibits tumor growth in murine colorectal cancer xenografts. CancerLett., 279, 93–100.

34.Ahn,K.S. et al. (2008) Guggulsterone, a farnesoid X receptor antago-nist, inhibits constitutive and inducible STAT3 activation through in-duction of a protein tyrosine phosphatase SHP-1. Cancer Res., 68,4406–4415.

35.Leeman-Neill,R.J. et al. (2009) Guggulsterone enhances head and neckcancer therapies via inhibition of signal transducer and activator oftranscription-3. Carcinogenesis, 30, 1848–1856.

36.Kim,E.S. et al. (2008) Guggulsterone inhibits angiogenesis by blockingSTAT3 and VEGF expression in colon cancer cells. Oncol. Rep., 20,1321–1327.

37.Rohatgi,N. et al. (2006) Novel molecular targets of smokeless tobacco(khaini) in cell culture from oral hyperplasia. Toxicology., 224, 1–13.

38.Macha,M.A. et al. (2010) Clinical significance of TC21 overexpression inoral cancer. J. Oral. Pathol. Med., 39, 477–485.

39.Deeb,D. et al. (2004) Curcumin sensitizes prostate cancer cells to tumornecrosis factor-related apoptosis-inducing ligand/Apo2L by inhibiting nu-clear factor-kappaB through suppression of IkappaBalpha phosphorylation.Mol. Cancer Ther., 3, 803–812.

40.Shishodia,S. et al. (2004) Guggulsterone inhibits NF-kappaB and Ikappa-Balpha kinase activation, suppresses expression of anti-apoptotic geneproducts, and enhances apoptosis. J. Biol. Chem., 279, 47148–47158.

41.Sriuranpong,V. et al. (2003) Epidermal growth factor receptor-independentconstitutive activation of STAT3 in head and neck squamous cell carcinomais mediated by the autocrine/paracrine stimulation of the interleukin6/gp130 cytokine system. Cancer Res., 63, 2948–2956.

42.Arredondo,J. et al. (2006) Receptor-mediated tobacco toxicity: cooperationof the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream ofalpha7 nicotinic receptor in oral keratinocytes. FASEB J., 20, 2093–2101.

43. Ichikawa,H. et al. (2006) Guggulsterone inhibits osteoclastogenesis in-duced by receptor activator of nuclear factor-kappaB ligand and by tumorcells by suppressing nuclear factor-kappaB activation. Clin. Cancer Res.,12, 662–668.

44.Cheon,J.H. et al. (2006) Plant sterol guggulsterone inhibits nuclear factor-kappaB signaling in intestinal epithelial cells by blocking IkappaB kinaseand ameliorates acute murine colitis. Inflamm. Bowel Dis., 12, 1152–1161.

45.Nagpal,J.K. et al. (2002) Activation of Stat-3 as one of the early events intobacco chewing-mediated oral carcinogenesis. Cancer, 94, 2393–2400.

46.Macha,M.A. et al. (2010) 14-3-3 zeta is a molecular target in guggulsteroneinduced apoptosis in Head and Neck cancer cells. BMC Cancer, 10, 655.

47.Sinibaldi,D. et al. (2000) Induction of p21WAF1/CIP1 and cyclin D1 ex-pression by the Src oncoprotein in mouse fibroblasts: role of activatedSTAT3 signaling. Oncogene, 19, 5419–5427.

48.Uddin,S. et al. (2010) Cyclooxygenase-2 inhibition inhibits PI3K/AKTkinase activity in epithelial ovarian cancer. Int. J. Cancer, 126, 382–394.

49.Lei,T. et al. (2007) The potential role of Id1 in COX-2 mediated angiogen-esis in gastric cancer. Zhonghua Yi Xue Za Zhi, 87, 1570–1575.

50.Harris,R.E. (2007) Cyclooxygenase-2 (cox-2) and the inflammogenesis ofcancer. Subcell. Biochem., 42, 93–126.

Received May 19, 2010; revised December 2, 2010;accepted December 8, 2010

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