Resveratrol-Induced Apoptosis Is Mediated by Early …cancerpreventionresearch.aacrjournals.org/content/canprevres/4/1/... · Research Article Resveratrol-Induced Apoptosis Is Mediated

Research Article

Resveratrol-Induced Apoptosis Is Mediated by Early GrowthResponse-1, Kr€uppel-Like Factor 4, and ActivatingTranscription Factor 3

Nichelle C. Whitlock1, Jae Hoon Bahn1, Seong-Ho Lee1, Thomas E. Eling2, and Seung Joon Baek1

AbstractResveratrol, a dietary phytoalexin readily available in the diet, is reported to possess antitumorigenic

properties in several cancers, including colorectal. However, the underlying mechanism(s) involved is not

completely understood. In the present study, we investigated the effect of resveratrol treatment on gene

modulation in human colorectal cancer cells and identified activating transcription factor 3 (ATF3) as the

most highly induced gene after treatment. We confirmed that resveratrol upregulates ATF3 expression, both

at the mRNA and protein level, and showed resveratrol involvement in ATF3 transcriptional regulation.

Analysis of the ATF3 promoter revealed the importance of early growth response-1 (Egr-1; located at �245

to �236) and Kr€uppel-like factor 4 (KLF4; located at �178 to �174) putative binding sites in resveratrol-

mediated ATF3 transactivation. Specificity of these sites to the Egr-1 and KLF4 protein was confirmed by

electrophoretic mobility shift and chromatin immunoprecipitation assays. Resveratrol increased Egr-1 and

KLF4 expression, which preceded ATF3 expression, and further suggests Egr-1 and KLF4 involvement in

resveratrol-mediated activity. We provide evidence for Egr-1 and KLF4 interaction in the presence of

resveratrol, which may facilitate ATF3 transcriptional regulation by this compound. Furthermore, we

demonstrate that induction of apoptosis by resveratrol is mediated, in part, by increased ATF3 expression.

Taken together, these results provide a novel mechanism by which resveratrol induces ATF3 expression and

represent an additional explanation of how resveratrol exerts its antitumorigenic effects in human

colorectal cancer cells. Cancer Prev Res; 4(1); 116–27. �2011 AACR.

Introduction

Current research suggests that various dietary phyto-chemicals function as chemopreventive and/or adjuvantchemotherapeutic agents, adding to the paradigm that adiet high in fruit and vegetable content confers protectionagainst chronic disease (1). One such phytochemical isresveratrol (3,4,5’-trihydroxystilbene), a naturally occur-ring phytoalexin readily available in the diet and to whicha plethora of health-promoting effects have been ascribed(2). Resveratrol has elicited much attention as a potentialanticancer agent since the inhibitory effect of this com-

pound on carcinogenic processes was first reported in 1997(3). Subsequently, numerous studies have illustrated theantiproliferative effect of resveratrol on cancer cells, whichis believed attributable to induction of cell-cycle arrest inthe G1/S or G2/M phase and induction of apoptosis andrelated proteins (4, 5). More importantly, treatment withresveratrol inhibited tumorigenesis in vivo (6, 7). However,the underlying mechanism(s) involved in the antitumori-genic/carcinogenic activities of resveratrol remain poorlydefined due to its capacity to modulate a multitude ofsignaling pathways.

Activating transcription factor 3 (ATF3), a member of theATF/CREB family of bZIP transcription factors, is charac-terized as a stress-inducible or adaptive response gene (8).Much controversy exists as to the physiological role of ATF3in tumorigenesis and is demonstrated to be a positive ornegative modulator of tumor progression. Recently, adichotomous role was reported for ATF3 in cancer devel-opment; the authors concluded that its role as a tumorsuppressor or oncogene is largely dependent on cellularcontext and extent of malignancy (9). Yet, several lines ofevidence suggest that ATF3 may function as a tumor sup-pressor gene in colorectal carcinogenesis. First, ATF3expression is markedly reduced in cancer tissues, includingcolon, when compared to normal adjacent tissue (10, 11).

Authors' Affiliations: 1Laboratory of Environmental Carcinogenesis,Department of Pathobiology, College of Veterinary Medicine, Universityof Tennessee, Knoxville, Tennesse; and 2Laboratory of Molecular Carci-nogenesis, National Institute of Environmental Health Sciences, NIH,Research Triangle Park, North Carolina

Note: Supplementary data for this article are available at Cancer Preven-tion Research Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Seung Joon Baek, Department of Pathobiology,College of Veterinary Medicine, University of Tennessee, 2407 River Dr.,Knoxville, TN 37996. Phone: 865-974-8216; Fax: 865-974-5616; E-mail:[email protected].

doi: 10.1158/1940-6207.CAPR-10-0218

�2011 American Association for Cancer Research.

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Second, ATF3 is reported to mediate or enhance inductionof apoptosis by compounds demonstrated to have anti-tumor properties (12–16). Finally, ATF3 overexpressionelicits a number of cellular responses, including inductionof cell-cycle arrest and inhibition of proliferation (17),induction of apoptosis in vitro and in vivo (12, 18-20),inhibition of invasion (21-23), and retardation of tumorformation in vivo (20, 22). Thus, we believe that ATF3 mayplay an antitumorigenic role in colorectal tumorigenesis.In the present study, we examined the effect of resveratrol

treatment on gene modulation in HCT-116 human color-ectal cancer cells. We identified ATF3 as the most highlyinduced gene after treatment and sought to investigate thetranscriptional mechanism and biological consequence ofATF3 expression in response to resveratrol. Here, we reportthat early growth response-1 (Egr-1) and Kr€uppel-likefactor 4 (KLF4) mediate ATF3 transactivation by resvera-trol. We show Egr-1 and KLF4 interaction in the presence ofresveratrol, which may facilitate ATF3 transcriptional reg-ulation by this compound. Furthermore, we demonstratethat induction of apoptosis by resveratrol is mediated, atleast in part, by ATF3.

Materials and Methods

Cell lines and reagentsAll human cancer cell lines were purchased from Amer-

ican Type Culture Collection unless otherwise stated;authentication occurred via short tandem repeat profiling,monitor of cell morphology, and karyotyping. SqCC/Y1head and neck squamous cell carcinoma cell line wasgenerously provided by Dr. Dong M. Shin (Emory Uni-versity) and characterized previously (24). Cell lines weremaintained according to established protocol. Resveratrolwas purchased from Alexis Biochemicals, 3,30-diindoyl-methane (DIM) from Sigma Aldrich, and cycloheximidefrom Fisher Scientific. All chemicals were dissolved indimethylsulfoxide (DMSO). ATF3, Egr-1, KLF4, and actinantibodies were purchased from Santa Cruz Biotechnology.

Construction of plasmidsFor deletion analysis of the ATF3 promoter, pATF3

�1850/þ34 (12) was serially deleted using the Erase-a-Base System (Promega) according to manufacturer’s proto-col. The pATF3 �514 del Egr-1 reporter construct waspreviously described (25). Putative binding sitesof KLF4 within the �514 bp region of the promoter weredeleted using Stratagene’s QuikChange II Site DirectedMutagenesis Kit with the following primers: del KLF4-A (F, 50-CCCCCTCTCTTTCGGCCCCGCCTTGGCCCC-30

and R, 5’-CGGGGCCGAAAGAGAGGGGGCACTGGT-GATG-30) and del KLF4-B (F, 50-GGCCCCTCCTCCTT-CCTCCGCTCCGTTCGG-30 and R, 50-CGGAGGAAGG-AGGAGGGGCCAAGGCGGGGC-30). pATF3 �514/þ34del Egr-1 KLF4 reporter construct was generated usingpATF3 �514 del Egr-1 and KLF4-A deletion primers asdescribed. Deletions were confirmed by DNA sequen-cing. ATF3 (pCG-ATF3) expression vector was kindly

provided by Dr. T. Hai (Ohio State University). KLF4(pcDNA3.1/His/V5/KLF4) expression vector was gener-ated using the primers F, 50-CGAATTCTATGGCTGT-CAGCGACGCG-30 and R, 50-CCCAAGCTTTTAAAAATG-CCTCTTCATGTGTAAGGC-30. Egr-1 (pcDNA3.1/NEO/Egr-1), Sp1 (pCMV-Sp1), and p53 (pcDNA3.1/myc/His/p53) expression vectors were previously described(26–28).

Reverse transcription polymerase chain reactionTotal RNA was isolated from HCT-116 cells treated with

DMSO or resveratrol (50 mmol/L) using 5 Prime PerfectRNACell/Tissue Kit and 1mg of RNAwas reverse transcribedusing Verso cDNA Synthesis Kit (Thermo Fisher Scienti-fic). PCR was performed as described (25) using ATF3 (F,50-GTTTGAGGATTTTGCTAACCTGAC-30 and R, 50-AGC-TGCAATCTTATTTCTTTCTCGT-30) and GADPH (F, 50-TCAACGGATTTGGTCGTATT-30 and R, 50-CTGTGGTCAT-GAGTCCTTCC-30) human gene specific primers.

Western blot analysisCells were grown to 60% to 80% confluence in 60-mm

plates, serum starved overnight, and treated with resvera-trol as indicated in serum-free media. Protein lysates wereisolated in RIPA buffer containing 1mmol/L of PMSF, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 0.1 nmol/L of Na3VO4,and 25 mmol/L of NaF. Total protein was subjected toWestern blot analysis as previously described (25).

De novo protein synthesisHCT-116 cells were grown to 60% to 80% confluence in

60-mm plates and pretreated with cycloheximide (10 mg/mL) for 30 minutes in serum-free media. After pretreat-ment, DMSO or resveratrol (50 mmol/L) was added directlyto the media and incubated for 24 hours. Total RNA wasisolated and reverse transcribed. Real-time RT-PCR wasperformed according to Fast SYBR Green Master Mix pro-tocol (Applied Biosystems) and analyzed using MyIQSingle Color Real-time PCR Detection System (Bio-RadLaboratories).

Transfection using luciferase reporter systemTransient transfections were performed using LipofectA-

MINE (Invitrogen) according to manufacturer’s instruc-tions. HCT-116 cells were seeded in 12-well plates at aconcentration of 2.0 � 105 cells per well. The next day,plasmid mixtures containing ATF3 promoter (0.5 mg) andpRL-null vector (0.05 mg) were cotransfected for 5 hours inserum-free media. For cotransfection experiments, 0.25 mgof the ATF3 promoter and 0.25 mg of expression vectorwere cotransfected with 0.05 mg pRL-null vector. For com-petition assays, plasmid mixtures were prepared as indi-cated in Fig. 5A. After transfection, cells were treated withDMSO or resveratrol (50 mmol/L) in serum-free media for24 hours. Cells were harvested in 1� passive lysis buffer,and luciferase activity was measured and normalized topRL-null luciferase activity using DualGlo Luciferase AssayKit (Promega).

ATF3 Mediates Resveratrol-Induced Apoptosis

www.aacrjournals.org Cancer Prev Res; 4(1) January 2011 117



RNA interferenceEgr-1 sense and antisense oligonucleotides were pre-

viously described (25). ATF3 and KLF4 siRNA were pur-chased from Santa Cruz Biotechnology; control siRNA waspurchased from Ambion. HCT-116 cells were transfectedwith either 100 nmol/L (ATF3 and KLF4) or 200 nmol/L(Egr-1) of each construct using TransIT-TKO transfectionreagent (Mirus). After transfection for 24 hours, cells wereserum starved overnight and treated as indicated. Totalprotein was subjected toWestern blot analysis as described.

Electrophoretic mobility shift assayHCT-116 cells were grown to 80% confluence. After

overnight serum starvation, cells were treated with resver-atrol (50 mmol/L) for 24 hours. Cells were then washedwith PBS and nuclear extracts were prepared usingNuclear Extract Kit (Active Motif) according to protocol.Double-stranded oligonucleotides corresponding to theEgr-1 (F, 50-GTGAGCGAGGGCGGGG-30) and KLF4 (F, 50-TCCACCCCTTCCACCCCT-30) binding sites were synthe-sized and end-labeled with biotin (Operon). Mutant oli-gonucleotides of Egr-1 (F, 50-GTGAGCGAAAACGGGG-30)and KLF4 (F, 50-TCCAAAACTTCCAAAACT-30) were alsosynthesized. To ensure specific binding of Egr-1 and KLF4,recombinant proteins were generated using TNT QuickCoupled Transcription/Translation System (Promega).EMSA was performed as previously described (29).

Chromatin immunoprecipitationHCT-116 cells were grown to 80% confluence and treated

with DMSO or resveratrol (50 mmol/L). After 24 hours, cellswere fixed with 1% formaldehyde at 37�C for 10 minutesand chromatin immunoprecipitation (ChIP) was performedas previously described (29). The region between �298 and�114 bp of the human ATF3 promoter was amplified byreal-time and RT-PCR using F, 50-CGGCTCCGGTCCTGA-TATGG-30 and R, 50-AGAACCGGCCGAACGGAGCG-30. The184-bp product was resolved on 2% agarose gel and visua-lized under UV light.

Mammalian 2-hybrid system assayThe pM/Egr-1 and pVP16/KLF4 vectors for mam-

malian 2-hybrid were generated. PCR fragments wereamplified from pcDNA3.1/NEO/Egr-1 and pcDNA3.1/His/V5/KLF4 using Egr-1 (F, 50-TTGAATTCGCCGC-GGCCAAGGCCGAG-30 and R, 50-TTAAGCTTGCAAAT-TTCAATTGTCC-30) and KLF4 (F, 50-CGAATTCTATGGC-TGTCAGCGACGCG-30 and R, 50-CCAAAGCTTTTAA-AAATGCCTCTTCATGTGTAAGGC-30)-specific primers,respectively. After PCR, fragments were cloned intopCR2.1/TOPO vector (Invitrogen), digested with EcoRIand HindIII restriction enzymes, and cloned into pMor pVP16 vectors. Deletion clones of pM/Egr-1 weregenerated as described using the primers: del AD(F, 50-CATCACCTATGGCCTAGTGAGCATGACCAACC-CAC-30and R, 50-TCACTAGGCCATAGGTGATGGGGGG-CAGTCGAGTG-30) and del ZD2 (F, 50-GCCCTCCCAG-TTCGCCTGCGACATCTGTGGAAGAA-30 and R, 50-CG-

CAGGCGAACTGGAAGGGCTTCTGGCCTGTGTGG-30).Mammalian 2-hybrid assay was performed according toMatchmaker Mammalian Assay Kit 2 protocol (Clon-tech). Transfection occurred as described using plasmidmixtures containing 0.2 mg of pM/Egr-1 or deletionconstruct, 0.2 mg of pVP16/KLF4, 0.2 mg of pG5Luc,and 0.06 mg of pRL-null vector.

Immunoprecipitation of Egr-1 and KLF4For immunoprecipitation of Egr-1 (WT and deletions,

generated as described previously) and KLF4, each expres-sion vector was transfected into HCT-116 cells as indicatedin Fig. 5D and grown to confluence. Cells were then serumstarved overnight and treated with resveratrol (50 mmol/L)for 2 hours. After washing with ice-cold PBS, cells wereharvested in RIPA buffer containing inhibitors and mixedat 4�C for 15 minutes. Cell suspensions were centrifugedand protein concentrations were measured. Immunopre-cipitation was performed as previously described (12).

Caspase 3/7 enzymatic activityEnzyme activity of caspase 3/7 was analyzed by Apo-

ONE Homogenous Caspase-Glo 3/7 Assay (Promega)according to manufacturer’s protocol. The cells were har-vested in RIPA buffer containing protease and phophataseinhibitors. The same volume of caspase-Glo 3/7 reagentwas added to cell lysates (30 mg protein) in 96-well platesand incubated at room temperature in the dark for 1 hour.Luminescence was measured using FLX800 microplatereader (BioTek).

Statistical analysisSAS for Windows (v9.2; SAS Institute, Inc.) statistical

analysis software was used. For multiple group compar-isons, analysis of variance with Tukey’s multiple com-parison test was used to compare mean values. TheStudent’s t test was used to analyze differences betweensamples. Results were considered statistically significantat *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

Resveratrol increases ATF3 expression in cancer cellsThemechanism(s) underlying the antitumorigenic prop-

erties of resveratrol in colorectal cancer remain mostlyunclear. To investigate how resveratrol alters gene expres-sion in colorectal cancer cells, microarray analysis wasperformed using HCT-116 cells treated with resveratrol(50 and 100 mmol/L). Among the genes upregulated bythe treatment, ATF3 was identified as most highly induced(Table S1 and S2). We confirmed resveratrol-induced ATF3transcript using RT-PCR (Fig. 1A). We next determinedwhether resveratrol increases ATF3 expression at theprotein level. As shown in Fig. 1B and 1C, resveratrolincreased ATF3 in a concentration- and time-dependentmanner in HCT-116 cells. Furthermore, increased ATF3expression was observed in other colorectal (Fig. 1D)and non-colorectal (Fig. 1E) cancer cells after treatment;

Whitlock et al.

Cancer Prev Res; 4(1) January 2011 Cancer Prevention Research118



however, we did not observe ATF3 induction in LoVo andSqCC/Y1 cells. Absence of ATF3 induction by resveratrol inthese cells is most likely due to high endogenous expressionof ATF3 (LoVo) or the lack of proper signaling pathway(s)needed for ATF3 increase by resveratrol (SqCC/Y1).Because resveratrol increased ATF3 expression at the mRNAand protein level, we sought to characterize ATF3 as amolecular target of this compound at the transcriptionlevel.

Egr-1 and KLF4 involved in resveratrol-inducedATF3 expressionTo identify the transcriptional binding site responsible

for resveratrol-mediated ATF3 expression, HCT-116 cellswere transfected with different promoter constructs span-ning the �1850 to þ34 bp promoter region (Fig. 2A).Resveratrol treatment resulted in a significant increasein ATF3 promoter activity for all reporter constructs;

however, the greatest induction was observed withinthe �514 and þ34 bp region, suggesting that a majorresponse element(s) may be present. This region wasfurther analyzed using 3 independent transcription factorsearch engines (Genomatix-MatInspector, TFSearch, andTranscription Element Search System), and 5 possiblefactors were commonly identified: p53, Sp1, Egr-1,KLF4, and ATF/CREB (Fig. 2B).

To investigate the role of identified cis-acting elementsin resveratrol-mediated transcriptional regulation ofATF3, cotransfection experiments were performed utiliz-ing expression vectors of the aforementioned factorsand ATF3 promoter constructs as indicated (Fig. 2Cand D). Expression of each vector, with the exceptionof ATF3, resulted in increased pATF3 �514/þ34 lucifer-ase activity after resveratrol treatment compared topcDNA3.1 empty vector control (Fig. 2C). Interestingly,cells overexpressing KLF4 and Egr-1 showed the greatest

Figure 1. Resveratrol increasedATF3 expression in cancer cells.A, HCT-116 cells were treatedwith vehicle or resveratrol (Resv) inserum-free media for 24 hours.RT-PCR was performed usingATF3 andGAPDH human primers,the latter served as a loadingcontrol. Representative gelpictures (top) and geldensitometry (bottom) of 3independent experiments areshown. Values are expressed asfold induction relative to vehicle-treated cells adjusted to GAPDH.*, P < 0.05. B–E, cancer cells wereseeded and grown to 60% to 80%confluence, serum starvedovernight, and treated with Resvas indicated in serum-free media.Protein lysates were harvestedand subjected to Western blotanalysis using ATF3 and actinantibodies. B, HCT-116 cells weretreated with 0, 10, 50, and 100mmol/L Resv for 24 hours. C, HCT-116 cells were incubated withResv for the indicated times. D,colorectal (HT-29, Caco-2, LoVo,and SW480) and (E) non-colorectal (NCI-H292 lung, MCF7breast, PC3 prostate, and SqCC/Y1 head and neck) cancer cellswere treated with vehicle or Resvfor 24 hours.

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increase in resveratrol-induced ATF3 promoter activity,resulting in a 4.1- and 4.3-fold induction, respectively. Asimilar pattern was observed for the �132 to þ34 bpregion (Supplementary Fig. S1); however, cotransfectionof these expression vectors with pATF3 �84/þ34 reporterconstruct had no effect on promoter activity after treat-ment (Fig. 2D). These results identify putative resveratrolresponse-elements located within the �514 to �84 bpregion of the ATF3 promoter and suggest that both Egr-1and KLF4 may play a role in ATF3 regulation byresveratrol.

To clarify the importance of Egr-1 and KLF4 in resvera-trol-mediated activation of ATF3, internal deletion clones

lacking the Egr-1 and KLF4 binding sites were generatedand transfected into HCT-116 cells. Deletion of Egr-1 orKLF4-A binding site markedly reduced ATF3 promoteractivity in response to resveratrol treatment when com-pared with wild type (Fig. 2E). Deletion of the KLF4-Bbinding site did not change resveratrol-induced transacti-vation. Because deletion of binding sites corresponding toeither Egr-1 or KLF4-A resulted in significant decrease inresveratrol-induced promoter activity, a deletion clonelacking both these sites was generated. Resveratrol-inducedactivity was dramatically reduced in cells transfected withthe double deletion promoter construct compared to wildtype (Fig. 2E). Moreover, suppression of either endogenous

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Figure 2. Egr-1 and KLF4 are involved in resveratrol induction of ATF3. A, each indicated construct of the ATF3 promoter (0.5 mg) and pRL-null vector(0.05 mg) were transiently transfected into HCT-116 cells and treated with vehicle or Resv for 24 hours. The promoter activity was measured as a ratio offirefly luciferase signal/renilla luciferase signal. The x-axis shows relative luciferase unit of each construct. The results are the mean � SD of 3 replicates. *,P < 0.05; **, P < 0.01; and ***,P < 0.001, on the basis of Student's t test. B, nucleotide sequence of the �514 to þ34 bp regions of the ATF3 promoter.Predicted binding sites of identified transcription factors are capitalized and underlined with name located underneath. C and D, empty vector(pCDNA3.1/NEO) or the indicated expression vector (0.25 mg each) was cotransfected with pATF3 �514/þ34 (C) or pATF3 �84/þ34 (D) and pRL-nullvector (0.05 mg) into HCT-116 cells. Cells were then treated with vehicle or Resv in serum-free media for 24 hours. The x-axis shows fold induction relativeto pcDNA3.1 control. Data analyzed using Tukey's multiple comparison test; mean with same letters indicate no significance (P < 0.05). E, pATF3�514/þ34 or its internal deletion clones (0.5 mg each) were transfected into HCT-116 cells followed by treatment with vehicle or Resv in serum-free mediafor 24 hours. Values were normalized to vehicle treatment. The x-axis represents fold induction relative to pATF3 �514/þ34 control. Data analyzedusing Tukey's multiple comparison test; mean values with same letters indicate no significance (P < 0.05). F, HCT-116 cells were transfected withEgr-1 sense/antisense oligonucleotides or control/KLF4 siRNA using TransIT-TKO transfection reagent. Cells were serum starved overnight and treatedwith vehicle or Resv for 6 hours (KLF4) or 24 hours (Egr-1). Protein lysates were harvested and subjected to Western blot analysis for Egr-1 or KLF4, ATF3,and Actin.

Whitlock et al.




Egr-1 or KLF4 decreased ATF3 expression in the presence ofresveratrol (Fig. 2F), confirming that both Egr-1 and KLF4contribute to resveratrol-induced ATF3 expression.

Egr-1 and KLF4 bind to the ATF3 promoterTo determine whether Egr-1 and KLF4 bind to the ATF3

promoter, EMSA was performed using biotin-labeled oli-gonucleotides containing 1 to 2 copies of the correspondingpromoter binding site. First, we examined the effect ofresveratrol on promoter binding by these factors using

nuclear extracts prepared as described in Materials andMethods. Preincubation of resveratrol-treated nuclearextracts with Egr-1 and KLF4 oligonucleotides resulted inDNA/protein complex formation that was competed outwith addition of 10� and 100� molar excess of unlabeledwild-type oligonucleotides (Fig. 3A and B, left, lanes 3–4),whereas preincubationwithunlabeledmutant oligonucleo-tides had no affect on DNA/protein complex formation(Fig. 3A and B, left, lanes 5–6). This suggests that both Egr-1and KLF4 are able to bind to the ATF3 promoter. Secondly,

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Figure 3. Egr-1 and KLF4 bind to the ATF3 promoter. A and B, gel shift assays were performed using nuclear extracts (NE) from Resv (50 mmol/L)-treatedHCT-116 cells for 24 hours or in vitro translated (IVT) proteins as described in Materials and Methods. Competitions were done in the presence of10� and 100� excess of unlabeled oligonucleotides (lanes 3–4 and 10–11). Specificity of the DNA/protein complex was confirmed by the absence ofcompetition with an excess of unlabeled mutated oligonucleotide (lanes 5–6 and 12–13). A, Egr-1; and B, KLF4. Arrows, DNA/protein complexes. C, HCT-116cells were treated with vehicle or Resv for 24 hours. The chromatin/protein complexes were cross-linked by formaldehyde treatment, and chromatinpellets were extracted and sonicated. The associated Egr-1 and KLF4 DNA was isolated as described. The sequence of the human ATF3 promoter(�298/�114) was amplified by PCR primer pairs (arrows). The input represents PCR products obtained from 1% aliquots of chromatin pellets beforeimmunoprecipitation. Left, ChIP real-time RT-PCR. The x-axis shows enrichment relative to input. The results are the mean � SD of 3 experiments.Right, RT-PCR; representative gel picture of 3 experiments is shown.





we verified the specificity of the binding sites for Egr-1 andKLF4 using in vitro translated proteins mixed with theirrespective oligonucleotides. As shown in Fig. 3A and B,both Egr-1 and KLF4 bind to their specific binding sites asevidenced by the formation of shift bands that were com-peted out by 10� and 100� molar excesses of unlabeledoligonucleotides (Fig. 3A and B, right, lanes 10–11). Addi-tion of mutant oligonucleotides did not affect binding ofEgr-1 or KLF4 to the ATF3 promoter (Fig. 3A and B, right,lanes 12–13). Finally, a ChIP assaywas performed to furtherconfirm that Egr-1 and KLF4 bind to the ATF3 promoterafter resveratrol treatment. As shown in Fig. 3C, immuno-precipitation of the chromatin/protein complex with Egr-1and KLF4 resulted in the enrichment of these proteins atthe ATF3 promoter (left) and the visualization of a 184-bpbandof the amplifiedpromoter (right). In conjunctionwithprevious results shown in Fig. 2, these data suggest that bothEgr-1 and KLF4 bind to the ATF3 promoter and activatetranscription in the presence of resveratrol. However, Egr-1may contribute more to resveratrol-induced ATF3 expres-sion, since resveratrol enhances Egr-1 binding capacityto the ATF3 promoter.

De novo synthesis is required to increase ATF3expression

We then examined requirement of de novo protein synth-esis for resveratrol-mediated ATF3 expression because Egr-1and KLF4 appear to be required for induction of ATF3.HCT-116 cells were treated with cycloheximide and resver-atrol (50 mmol/L). In the presence of cycloheximide, resver-atrol was unable to increase ATF3 expression, suggestingthat ATF3 increase by resveratrol is dependent on de novoprotein synthesis (Fig. 4A). We next determined whetherresveratrol could alter expression of Egr-1 and KLF4. Resver-atrol treatment increased Egr-1 and KLF4 mRNA transcript

(Supplementary Fig. S2) and protein expression (Fig. 4B). Itshould be noted that induction of both Egr-1 and KLF4,which occurred at approximately 1 hour of treatment andcontinued until 3 hours, preceded that of ATF3, whichbegan after 6 hours. Together, these results are compatiblewith the notion that observed increase in ATF3 expressionby resveratrol requires synthesis of Egr-1 and KLF4.

Egr-1 and KLF4 interaction facilitates resveratrol-mediated ATF3 induction

Because both Egr-1 and KLF4 are involved in resveratrol-mediated activity, we examined whether these factors coor-dinate or compete in ATF3 expression. HCT-116 cells werecotransfected with pATF3�514/þ34 and expression vectorsand treated as described. As observed previously, both Egr-1 and KLF4 increased ATF3 promoter activity in the pre-sence of resveratrol (Fig. 5A). Moreover, coexpression ofthese factors resulted in enhanced transactivation of ATF3at both the basal level and after resveratrol treatment.

Previous studies have shown interaction between 2 Znfinger transcription factors (30) and that this interactionfacilitates transcriptional activation (31). To investigate thepotential for Egr-1and KLF4 interaction, mammalian 2-hybrid assay was performed using pM/Egr-1 and pVP16/KLF4 vectors. Egr-1 and KLF4 bind to each other at basallevel and this interaction is enhanced after resveratroltreatment (7.2-fold vs. 52-fold increase; Fig. 5B). IncreasedEgr-1 and KLF4 interaction was also observed after DIMtreatment. DIM increases ATF3 expression (13). To excludethe potential for direct binding of pVP16/KLF4 to the DNAbinding domain of the pM empty vector, the vectors werecotransfected and mammalian 2-hybrid assay was per-formed; no interaction was detected, allowing one to inferthe interaction of Egr-1 and KLF4 is genuine (Supplemen-tary Fig. S3).

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Whitlock et al.




Wenext sought to tentatively identify the region that mayfacilitate this interaction and generated pM/Egr-1 deletionconstructs lacking (1) 133 to �159 amino acids (aa) of theactivation domain and (2) 370 to 395 aa of the Zn fingerdomain (Fig. 5C, top). Both the partial deletion of theactivation domain and deletion of the second Zn fingerdomain of Egr-1 resulted in decreased interaction withKLF4 (Fig. 5C, bottom). We confirmed mammalian 2-hybrid assay by Egr-1 and KLF4 immunoprecipitationexperiments (Fig. 5D). Egr-1 and KLF4 are coimmunopre-cipitated with either antibody, whereas Egr-1 deletion

clones had diminished binding capacity to KLF4. Together,these data suggest that Egr-1 and KLF4 physically interact,which is increased by resveratrol, and that the Egr-1 activa-tion or Zn finger domain may mediate this interaction.

Knockdown of ATF3 suppresses resveratrol-inducedapoptosis

We have identified ATF3 as a molecular target of resver-atrol; however, the biological consequence(s) of ATF3induction by resveratrol in colorectal tumorigenesis is un-known. As described in the introduction, pharmaceuticals

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Figure 5. Egr-1 and KLF4 interaction facilitates ATF3 activation by resveratrol. A. the pATF3 �514/þ34 reporter construct (0.25 mg) was cotransfectedwith empty, Egr-1, and KLF4 expression vectors as indicated in the presence of pRL-null vector (0.05 mg). Cells were treated with vehicle or Resv in serum-freemedia for 24 hours. The y-axis shows fold induction relative to pcDNA3.1 control. The results are the mean � SD of 3 replicates Data analyzed usingTukey's multiple comparison test; mean values with same letters indicate no significance (P < 0.05). B, mammalian 2-hybrid system was performed asdescribed in Materials and Methods. HCT-116 cells were cotransfected with pM and pVP16 empty vectors (0.2 mg each) and pM/Egr-1 and pVP16/KLF4vectors (0.2 mg each) in the presence of pG5luc vector (0.2 mg) and pRL-null vector (0.06 mg). Cells were treated with vehicle, Resv, or DIM for 24 hoursin serum-free media. Promoter activity was measured as a ratio of firefly luciferase signal/renilla luciferase signal, and the results are expressed as themean � SD of 3 replicates. The y-axis shows fold induction over control vectors. Data analyzed using Tukey's multiple comparison test; mean values withsame letters indicate no significance (P < 0.05). C top,schematic diagram of Egr-1 protein structure and locations used to generate internal deletions.Bottom, HCT-116 cells were transfected with pM/Egr-1 internal deletion constructs, pVP16/KLF4 vector, and pG5luc vector. Cells were treated with vehicle orResv in serum-free media for 24 hours. Values are normalized to vehicle treatment. The y-axis represents fold induction relative to pM/Egr-1 and pVP16/KLF4control. Data analyzed using Tukey's multiple comparison test; mean values with same letters indicate no significance (P < 0.05). D, HCT-116 cellswere transfected with empty, Egr-1 (WT or deletion), or cotransfected with KLF4 expression vectors as indicated and grown to 60% to 80% confluence. Cellswere serum starved overnight, treated with Resv (50 mmol/L) for 2 hours, and harvested as described. The cell extracts were immunoprecipitated andimmunoblotted with antibody against Egr-1and KLF4.





and phytochemicals’ increase of ATF3 mediated orenhanced apoptosis by these compounds (12-16). To deter-mine if this is true for resveratrol, we measured caspase 3/7enzymatic activity for resveratrol-treated HCT-116 andHT29 colorectal cancer cells. As depicted in Fig. 6A, resver-atrol treatment increased apoptosis in a dose-dependentmanner. Furthermore, ATF3 overexpression in these cellsalso increased apoptosis, which depend on caspase activa-tion (Fig. 6B). Together, these data suggest that both resver-atrol and ATF3 expression induce apoptosis in our modelsystem. Next, to investigate whether resveratrol-inducedapoptosis is mediated by ATF3, ATF3 expression wasknocked down by siRNA followed by resveratrol treatmentfor 24 hours. As depicted in Fig. 6C, knockdown of ATF3

significantly abrogated caspase 3/7 enzymatic activity byresveratrol.This suggests thatATF3playsa role in resveratrol-induced apoptosis.

Discussion

Resveratrol, a dietary phytoalexin readily available inthe diet, has garnered much attention as a potential che-mopreventive and/or chemotherapeutic agent. Numerousstudies, utilizing in vitro and in vivo model systems, haveillustrated resveratrol’s capacity to inhibit the stages ofcarcinogenesis (initiation, promotion, and progression)and modulate a multitude of signaling pathways associatedwith cellular growth and division, apoptosis, angiogenesis,

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Figure 6. Relevance of ATF3 in resveratrol-induced apoptosis. Caspase 3/7 enzymatic activity was measured as described in Materials and Methods. A,HCT-116 and HT29 cells were treated with 0, 10, 50, and 100 mmol/L Resv for 24 hours. Data analyzed using Tukey's multiple comparison test; mean valueswith same letters indicate no significance (P < 0.05). B, HCT-116 and HT29 cells were transfected with empty or ATF3expression vector and grown to60% to 80% confluence. Cells were serum starved overnight and treated with the pan caspase inhibitor z-vad-fmk ( ZVF, 1 mmol/L) for 3 hours. ATF3overexpression was validated (top). C, HCT-116 cells were transfected with control/ATF3 siRNA using TransIT-TKO transfection reagent and treated withResv 24 hours. Validation of ATF3 knockdown is indicated (top). *, P < 0.01; ***,P < 0.001, on the basis of Student's t test. D, schematic diagram of resveratrol'saction in colorectal cancer cells: Egr-1 and KLF4 are involved in the induction of ATF3 by resveratrol. Resveratrol increases the expression of both Egr-1and KLF4; this in turn results in the interaction of Egr-1 with KLF4, potentially facilitated by the activation or Zn finger domain of Egr-1, leading to promoterbinding and transactivation of ATF3. Increased ATF3 expression results in increase of apoptosis in colorectal cancer cells.

Whitlock et al.




and metastasis (32). However, the underlying molecularmechanism(s) involved in the antitumorigenic/carcinogenicactivities of resveratrol, especially in colorectal cancer, arecomplex and remain poorly defined. This study sought toinvestigate the effect of resveratrol on gene modulationin HCT-116 human colorectal cancer cells in order toidentify a novel target that may mediate the anticanceractivities of this compound and determine the mechanisminvolved in its regulation. Our data showed ATF3 was in-deed upregulated at both the mRNA and protein (Fig. 1)level by the compound.Recently, a dichotomous role in cancer development

was reported for ATF3; the authors of that study demon-strated both a tumor suppressive (early-stage tumorigen-esis) and oncogenic (late-stage tumorigenesis) role inbreast cancer cell lines derived from similar genetic back-grounds with varying degrees of malignancy (9). Similarly,a duality of function was demonstrated for ATF3 in cancerstudies of the prostate (19, 33) and of the colon (22, 34).Yet, as described in the introduction, several lines ofevidence suggest ATF3 behaves as a negative regulator oftumorigenesis. ATF3 expression is induced by several com-pounds demonstrated to possess antitumorigenic proper-ties (12–16). The results presented here add to the list ofcompounds that induce ATF3 with mechanistic data.To elucidate the molecular mechanism by which resvera-

trol induced ATF3 expression, the promoter region span-ning �1850 to þ34 bp was assessed by luciferase assay inresponse to resveratrol. We found that resveratrol transacti-vated the ATF3 promoter and identified putative response-elements located within the �514- to �84 bp region(Fig. 2A). Cotransfection experiments suggested involve-ment of the transcription factors Egr-1 and KLF4 (Fig. 2C).Egr-1 and KLF4 participation in resveratrol-mediated activa-tion ofATF3was confirmed by deletion (Fig. 2E) and knock-down (Fig. 2F) experiments. Interestingly, simultaneousdeletion of both Egr-1 and KLF4-A binding sites from theATF3 promoter dramatically reduced transactivation byresveratrol, indicating that the combination of these 2 factorsis important for ATF3 transcriptional regulation by resvera-trol. Yet, we cannot exclude the involvement of other cof-actors, such as p53 and Sp1 (Fig. 2C), in the compound’smediated activity. ATF3 regulation by p53 is documented inthe literature (35), which is increased by resveratrol (4) andsuggests a potential role for p53 in resveratrol-mediatedATF3activation. Furthermore, Sp1 was recently demonstratedto regulate ATF3 expression in colon cancer cells (36); how-ever, we did not observe Sp1 induction by resveratrol (datanot shown). Here, we focused on Egr-1 and KLF4 becauseboth increased resveratrol-mediated ATF3 activation andwere modulated by the compound.Egr-1 and KLF4 belong to a family of immediate early

response genes whose expression is transiently induced inresponse to various environmental stimuli (37, 38). Eachencodes a transcription factor containing 3 carboxyl C2H2-type Zn finger motifs that coordinate the expression ofgenes associated with cell proliferation, differentiation, andapoptosis (38–40). In addition, Egr-1 and KLF4 are sug-

gested to act as master regulatory proteins involved in cellfate decisions (41, 42). Nonetheless, as with ATF3, muchcontroversy exists as to the role of Egr-1 and KLF4 incancer development and their biological function appearslargely context dependent. Several studies have demon-strated that Egr-1 and KLF4 expression facilitates tumorprogression in vivo (43–45); however, there is ampleevidence supporting a tumor-suppressive role for bothtranscription factors (29, 46–51).

Our data indicates that Egr-1 and KLF4 are able to bindto the ATF3 promoter (Fig. 3) and that their biosynthesisis necessary for resveratrol-mediated activity (Fig. 4).Moreover, these transcription factors cooperate in ATF3transactivation (Fig. 5A). From this, 2 inferences can bemade: (1) synergism between Egr-1 and KLF4 promoteATF3 activation, which is enhanced by resveratrol, and(2) interaction between the 2 facilitates transcriptionalregulation of ATF3 by the compound. Indeed, the resultsascertained by mammalian 2-hybrid assay and immuno-precipitation experiments corroborate Egr-1 and KLF4interaction (Fig. 5B and D). Furthermore, resveratrol andDIM increased Egr-1 and KLF4 interaction. These resultsimply that a similar mechanism may contribute to induc-tion of ATF3 expression by these and possibly other phyto-chemicals that increase ATF3 expression.

Current research suggests the importance of C2H2 typeZn finger domains in protein–protein interaction (52).Consistently, we found the Zn finger domain of Egr-1 asa possible region responsible for interaction with KLF4.Interestingly, our data also identified the activation domainof Egr-1 as responsible for mediating the interaction withKLF4 (Fig. 5C and D). To our knowledge, this is the firstreport identifying the involvement of the activationdomain in protein–protein interaction of 2 Zn fingertranscription factors. Thus, the activation domain contri-butes not only to the initiation of transcription but alsocontribute to protein–protein interactions with other tran-scription factors; interaction via the activation domain orthe Zn finger domain leads to enhanced compound-induced transcription.

Results identify ATF3 as a molecular target of resveratrolwhose regulation ismediatedbyEgr-1 andKLF4 interaction.However, the biological consequence(s) of ATF3 inductionby the compound is unknown in colorectal tumorigenesis.To answer this question, we examined induction of apop-tosis by resveratrol and ATF3 in colorectal cancer cellsbecause, as described, ATF3 mediated or enhanced apop-tosis by compounds with known antitumor activities. Asdepicted in Figure 6A and B, resveratrol and ATF3 over-expression increased caspase 3/7 enzymatic activity inHCT-116 and HT29 cancer cells. Furthermore, knockdown ofATF3 expression resulted in reduced apoptosis by resvera-trol and suggests that ATF3 plays a role in resveratrol-induced apoptosis. However, further study is required todetermine whether ATF3 contributes to resveratrol antic-ancer effects in vivo.

In conclusion, we found the stress-inducible and/oradaptive response gene ATF3 as most highly induced gene





after resveratrol treatment in human colorectal cancer cells.Here, we report for the first time the involvement ofresveratrol in transcriptional regulation of ATF3 and thatthis regulation is mediated by the C2H2 type Zn fingertranscription factors Egr-1 and KLF4. We demonstrate thatEgr-1 and KLF4 interact with each other in the presence ofresveratrol, which may facilitate ATF3 transcriptional reg-ulation by this compound. Furthermore, increased ATF3expression by resveratrol facilitates induction of apoptosisby the compound (Fig. 6D).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Misty Bailey for her critical reading of this manuscript. We alsothank Dr. Luis Miguel Lembcke for his technical assistance.

Grant Support

American Cancer Society grant CNE-111611, NIH grant RO1CA108975, TheUniversity of Tennessee Center of Excellence in Livestock Diseases and HumanHealth (S.J. Baek), and in part by NIEHS/NIH intramural research program (T.E.Eling).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 30, 2010; revised October 21, 2010; accepted October25, 2010; published online January 4, 2011.

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2011;4:116-127. Cancer Prev Res Nichelle C. Whitlock, Jae Hoon Bahn, Seong-Ho Lee, et al. Factor 3Response-1, Krüppel-Like Factor 4, and Activating Transcription Resveratrol-Induced Apoptosis Is Mediated by Early Growth

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