Carcinogen-Altered Genes in Rat Esophagus PositivelyModulated to Normal Levels of Expression by Both BlackRaspberries and Phenylethyl Isothiocyanate
Gary D. Stoner1, Alan A. Dombkowski3, Rashmeet K. Reen1, Daniela Cukovic3, SrideviSalagrama3, Li-Shu Wang2, and John F. Lechner21Department of Internal Medicine, The Ohio State University College of Medicine, Columbus,Ohio2Comprehensive Cancer Center, The Ohio State University College of Medicine, Columbus, Ohio3Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan
AbstractOur recent study identified 2,261 dysregulated genes in the esophagi of rats that received a 1-weekexposure to the carcinogen N-nitrosomethylbenzylamine (NMBA). We further reported that 1,323of these genes were positively modulated to near-normal levels of expression in NMBA-treatedanimals that consumed dietary phenylethyl isothiocyanate (PEITC), a constituent of cruciferousvegetables. Herein, we report our results with companion animals that were fed a diet containing5% freeze-dried black raspberries (BRB) instead of PEITC. We found that 462 of the 2,261NMBA-dysregulated genes in rat esophagus were restored to near-normal levels of expression byBRB. Further, we have identified 53 NMBA-dysregulated genes that are positively modulated byboth PEITC and BRB. These 53 common genes include genes involved in phase I and IImetabolism, oxidative damage, and oncogenes and tumor suppressor genes that regulate apoptosis,cell cycling, and angiogenesis. Because both PEITC and BRB maintain near-normal levels ofexpression of these 53 genes, their dysregulation during the early phase of NMBA-inducedesophageal cancer may be especially important in the genesis of the disease.
IntroductionWorldwide, esophageal squamous cell carcinoma (ESCC) is the sixth most prevalent cancerin humans. Due to a lack of symptoms during the early stages of the disease, ESCC is rarelydetected until it has metastasized (1). Tobacco products, alcohol, and inadequate diet areprimary causes of ESCC; thus, in addition to promoting the lifestyle changes of smokingcessation and alcohol moderation, there is a need to identify foods and food constituents thatinhibit or prolong the onset of clinical disease (2,3). The F344 rat esophagus bioassay model(3), in which tumors can be induced by nitrosamine carcinogens such as N-nitrosomethylbenzylamine (NMBA), has long been used to identify foods and foodconstituents with anticarcinogenic activity. These studies have identified two dissimilar food
groups, cruciferous vegetables and berries, to be potent inhibitors of rat esophageal tumorsinduced by NMBA (4,5). The former contains numerous chemopreventive agents, includingthe isothiocyanates, and the latter contains high amounts of polyphenols and ellagitannins.The most extensively studied isothiocyanate is phenylethyl isothiocyanate (PEITC), whichowes its prevention properties, at least in part, to inhibition of carcinogen-activatingcytochrome P450 enzymes and induction of carcinogen-detoxifying phase II enzymes (6,7).Regarding the polyphenols, we originally found that a diet supplemented with ellagic acidwas preventative in the rat esophagus (8). Subsequently, we identified blackberries,raspberries, black raspberries (BRB), strawberries, and cranberries as being exceptionallyrich in ellagic acid, with dry weight concentrations ranging from 630 to 1,500 µg/g (9).Further analyses showed that the ellagic acid was most abundant in the pulp and seeds ofberries, with little in the juice. The pulp and seeds also contain many other knownchemopreventive agents, including vitamins A, C, and E and folic acid, calcium andselenium, β-carotene, α-carotene, lutein, gallic acid, ferulic acid, p-coumaric acid, quercetin,several anthocyanins, β-sitosterol, stigmasterol, and kaempferol (4,10,11). Importantly, wealso found that the active constituents can be concentrated 9- to 10-fold relative to the freshberry by freeze drying (11). BRBs for the present study were obtained from a single Ohiofarm, freeze dried, and ground into a powder as described (11). Typically, the berries are notgrown organically; however, no pesticides or fungicides are applied to the berries within 10days before harvest. This results in nondetectable levels of any pesticides or fungicides inthe berry powder due to biodegradation of these compounds during the 10-day period beforeharvest.
The conversion of a normal cell into a tumorigenic cell is driven by numerous molecularaberrations that arise over time that endow the ultimate cell with resistance to apoptosis,insensitivity to growth-inhibitory signals, limitless replicative potential, sustainedangiogenesis, and tissue invasion/metastasis capabilities (12). The time line of these eventsin the rat esophagus model covers several months. We recently began delineating the genesdysregulated early in this carcinogenesis process and have reported that treatment of ratswith three s.c. injections (0.5 mg/kg body weight) of NMBA over 1 week led to thedysregulation of 2,261 genes (13). We also reported that the expression of 1,323 of thesegenes was positively modulated if the animals were fed PEITC 2 weeks before and duringthe week of NMBA treatment. Herein, we have evaluated the esophagi of companionanimals in the same protocol whose diet was supplemented with 5% freeze-dried BRBs,instead of PEITC, for 2 weeks before and during the week the animals were treated withNMBA. We found that 462 of the NMBA-dysregulated genes were restored to near-normallevels of expression by BRB. Interestingly, we found that 53of the 2,261 genes dysregulatedby NMBA were expressed at control levels of transcription if the animals received eitherPEITC or BRB. These common genes include those involved in phase I and II metabolism,oxidative damage, and oncogenes and tumor suppressor genes that regulate apoptosis, cellcycling, and angiogenesis. Overall, our study has identified a unique collection of 53 geneswhose early dysregulation may be especially important in the carcinogenesis of rat ESCC.
Materials and MethodsThe procedures for determining the effects of dietary PEITC and BRB on gene expression inNMBA-treated rat esophagus have been described in detail in our companion report (13);thus, they are discussed only briefly here. Four- to 5-wk-old male Fischer F344 rats wererandomized into three experimental groups of 18 animals each. Rats in group 1 receivedcontrol AIN-76A diet. Those in group 2 were given AIN-76A + PEITC (5 µmol) and thosein group 3wer e given AIN-76A + 5% BRB. After 2 wk, one half of the animals in group 1received three s.c. injections of 20% DMSO/water (vehicle control) spaced every other day,and the other half received three s.c. injections of NMBA (0.5 mg/kg body weight in 20%
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DMSO/water). Similarly, one half of the animals in groups 2 and 3wer e divided into PEITCcontrol and PEITC+NMBA subgroups and BRB control and BRB+NMBA subgroups,respectively. PEITC, at 5 µmol in the diet, reduces NMBAinduced tumors in the ratesophagus by ~ 100% and BRB, at 5% of the diet, causes an approximate 50% reduction inesophageal tumors (5). All rats were sacrificed 24 h after the last NMBA injection. Theesophagus from each animal was excised, opened longitudinally, and cut into two parts. Onepart was fixed in 10% buffered formalin for routine histopathology, and the other was frozenin liquid nitrogen and stored at −80° C for analysis of transcription changes using ratmicroarrays (41,000 transcripts). Triplicate microarrays were completed for each of the sixgroups of nine rats for a total of 18 microarrays.
Normalized data for each microarray were imported into Rosetta Resolver (14) for analysis(version 5.1.0.1.23; Rossetta Biosoftware). ANOVA was performed on log ratios using theRosetta Resolver error model and weighting, as described by Weng and colleagues (15).One-way ANOVA was used to identify genes with a significant treatment effect. A P valuecutoff of 0.001 was used to determine statistical significance in each treatment comparison.Statistically significant genes were further filtered to identify those with a minimum 1.5-foldchange (16,17). Gene ontology and pathway analysis of differentially expressed genes wasdone using the DAVID program.4 Microarray results were confirmed by comparison withmRNA levels obtained by quantitative reverse transcription-PCR (RT-PCR) using selectedgene-specific primer pairs for a total of eight genes as described in our companion report(13). For each of the eight genes evaluated, the RT-PCR results confirmed gene expressionchanges that were observed by microarray.
Results and DiscussionEffect of BRB on esophageal histopathology
We previously reported that the cellular morphology of esophagi from rats treated with 5µmol dietary PEITC alone or PEITC+NMBA was not significantly different from that ofanimals on control diet. In contrast, the esophagi of rats treated with NMBA only exhibitedsignificant hyperplasia and low-grade dysplasia with evident cytotoxicity and markedinfiltration of inflammatory cells. Essentially identical results were observed in the esophagiof rats treated with BRB and BRB+NMBA [i.e., the esophagi from both the BRB alone andBRB+NMBA animals were indistinguishable (P < 0.05) from the controls; Fig. 1].
NMBA-dysregulated genes affected by BRBIn our companion report (13), we noted that the expression of 2,261 genes was either up-regulated or down-regulated more than ≥1.5-fold (P ≤ 0.001) in the esophagi of rats treatedfor 1 week with NMBA, and PEITC modulated 1,323 of these genes to near-normal levelsof expression. In contrast, only 462 of the NMBA-affected genes were modulated by BRB.Of these, 203exhibited partially restored expression levels and 259 were expressed at controllevels (±33%; see Fig. 2 and Supplementary Table S1). Two hundred and fourteen of the259 genes were up-regulated by NMBA and 45 were down-regulated. We hypothesize thatan important mechanism of chemoprevention by BRB is the correction of NMBA-dysregulated genes to homeostatic levels of expression; thus, we have focused onunderstanding the possible roles of the 259 genes returned to near-normal levels oftranscription by the BRB diet. Not all of these genes have known functions, as denoted bythe DAVID program; however, known among them were those involved in signaltransduction, cell proliferation, cell cycle progression, chromosome partitioning,inflammation, differentiation, cell junctions, cytoskeleton, apoptosis, and angiogenesis
4http://david.niaid.nih.gov/
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(Table 1). As in our companion report (13), we also observed effects of BRB on theexpression of genes involved in carcinogen metabolism and DNA adduct formation. Theseand other effects of BRB on gene expression in NMBA-treated rat esophagus are describedbelow, in brief.
NMBA-dysregulated DNA adduct formation genes normalized by BRB and PEITCExposure of rats to NMBA causes an elevation in O6-methylguanine adducts in esophagealDNA (3,11). Formation of O6-methylguanine adducts is considered to be an important earlyevent in NMBA-induced esophageal carcinogenesis because these adducts produceGC→AT transition mutations in the second base of codon 12 of the Hras1 oncogene, a genethat is activated early in esophageal tumorigenesis (18). Both BRB and PEITC have beenshown to reduce O6-methylguanine adduct levels in the esophagi of NMBA-treated rats(11,19). In our companion report (13), we noted that the phase I enzymes, CYP2a2 andCYP3a13, were overexpressed in NMBA-treated rat esophagus, whereas their expressionwas normal in the esophagus of NMBA+PEITC rats. In the present study, CYP2a2expression was also found to be near normal in BRB+NMBA–treated rat esophagus;however, CYP3a13 remained up-regulated. Thus, our data suggest that CYP2a2 may beimportant in the bioactivation of NMBA in rat esophagus and that down-regulation ofCYP2a2 by both BRB and PEITC may be responsible for the observed ability of thesechemopreventives to inhibit O6-methylguanine adduct formation in NMBA-treatedesophagus.
Phase II enzymes lower the level of DNA adducts because they inactivate carcinogens (and/or their metabolites) by conjugating them to acceptor molecules (e.g., glutathione,glucuronic acid, and sulfates; ref. 6). The specifics of phase II enzymes involved in ratesophageal metabolism of NMBA have not been fully elucidated, although the formation ofglucuronide conjugates has been reported (20). As we previously reported (13), PEITCcorrected the NMBA-reduced expression levels of some glutathione S-transferases (i.e.,Gsta1, Gsta2, and Gstt2). In contrast, expression of these genes remained low in theesophagi of BRB+NMBA rats, suggesting that overexpression of these phase II enzymesmay not be sentinel for the inactivation of NMBA in rat esophagus. In keeping with thisinterpretation is the finding that BRB also antagonized NMBA-induced up-regulation ofTaldo1, a key transaldolase in the pentose phosphate pathway that provides the NADPHneeded for reducing glutathione (21).
NMBA-dysregulated inflammation genes normalized by BRBInducible nitric oxide synthase (iNOS) is overexpressed in many human cancers, includingESCC (22). We have reported that this radical generating enzyme is elevated in the esophagiof rats 15 and 25 weeks after NMBA treatment, whereas companion rats that also receivedthe BRB diet had normal levels of this enzyme (23). In contrast, iNOS was not elevated inthe esophagi of rats in the present study. Cyclooxygenase-2 (COX-2) expression and enzymeactivity are also up-regulated in human ESCC (24), with a corresponding increased level ofprostaglandin E2 and associated inflammatory processes. We have found that COX-2 wasoverexpressed in dysplastic lesions 15 and 25 weeks after NMBA treatment (23), but theexpression level of COX-2 in the esophagi recovered from corresponding NMBA-treatedanimals in the present study was not elevated. Thus, iNOS and COX-2 dysregulation doesnot seem to be involved with the early phase of NMBA-caused esophageal carcinogenesisand the chemopreventive effects of BRB regarding the regulation of these genes areseemingly restricted to later stages of tumor development.
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NMBA-dysregulated signal transduction genes normalized by BRBAmong the DAVID clustering of the genes dysregulated by NMBA, listed in Table 1, are 37genes involved in signal transduction. Of interest is the observation that several of thesegenes are associated with ras activity. Among these are Map2k3 and Apc1, whosedysregulation affects Hras1 activity. As noted above, ~ 100% of rat esophageal tumorsinduced by NMBA have a GC → AT transition mutation in the second base of codon 12 ofthe Hras1 gene (18). Map2k3is a mitogen-activated protein kinase (MAPK) kinase familymember that is activated by stress to phosphorylate MAPK14/p38-MAPK, and elevatedexpression of the Hras1 oncogene results in the accumulation of the active form ofMAPK14/p38-MAPK in breast cancer cells (25). Apc1, which is transcribed at increasedlevels in breast and colon cancers, has transforming capability, possibly through tyrosinedephosphorylation of the EphA2 receptor (26). Apc1 also interacts with several otherreceptor tyrosine kinases and docking proteins, including platelet-derived growth factorreceptor and β-catenin. It is considered to be a negative regulator of growth factor–inducedcell proliferation, but in Hras1-transformed cells, its overexpression increases cellproliferation (27). Thus, reducing NMBA up-regulated transcription of Map2k3 and Apc1 byBRB would antagonize the pro-oncogenic activities of activated Hras1. Other ras-relatedNMBA-dysregulated genes are Rab1 and H2A histone family member Z (H2afz). Rab1 is amember of the Ras oncogene superfamily and is up-regulated in tongue SCC (28). H2afz isinteresting because it is an adaptor protein that interacts preferentially with the active formof Hras1 and augments cell growth. Thus, several genes associated with the ras signaltransduction pathway were returned to homeostasis by the BRB diet.
NMBA-dysregulated differentiation and morphogenesis genes normalized by BRBEighteen transcripts were classified as cellular differentiation and morphogenesis genes(Table 1). Two notable ones were Grem1 and the 14-3-3 family chaperone protein Ywhah.Grem1, which interacts with the Ywhah protein, encodes a secreted antagonist of the bonemorphogenetic protein pathway, which in turn plays a crucial role in regulating the balancebetween expansion and cell differentiation (29). In addition to its interaction with Grem1,Ywhah mediates signal transduction via activation of protein kinase C and calcium/calmodulin-dependent protein kinase II. Loss of the chaperone activity of Ywhah may alsoplay a role in oxidative signaling underlying oxidative damage (29). Another interestingtranscript is sciellin, which encodes a precursor to the cornified envelope of terminallydifferentiated cells (30). Its down-regulation by NMBA could disrupt the normaldifferentiation program of esophageal squamous cells and promote cell transformation.Thus, the observations that BRB restore near-normal expression of Grem1, Ywhah, andsciellin could be mechanisms by which the berries impede esophageal cell transformation.
NMBA-dysregulated cell junction, adhesion, or motility genes normalized by BRBTwenty transcripts were classified by the DAVID program as cell junction, adhesion, ormotility genes (Table 1). Among these is CD44 antigen, which encodes a cell surfaceglycoprotein involved in cell-cell interactions, cell adhesion, and migration. The v6 alternatetranscript of CD44 is up-regulated in human ESCC (31). Another is Actn4, which encodes anonmuscle actinin and is localized to moving structures and is significantly elevated in cellsexhibiting enhanced motility. The level of this protein progressively increases from early- tolate-stage ESSC (32). Another NMBA-caused overexpression normalized by BRB wasmoesin. Elevated moesin has been associated with oral squamous cell carcinomas (33). Theprotein is a membrane-cytoskeleton linker in microvilli, ruffles, and cleavage furrows andthus plays a key role in cell morphology, adhesion, and motility. Lastly, the up-regulation ofCdc42 by NMBA was also normalized by BRB. Thus, another possible chemopreventivemechanism of BRB may be to interfere with the formation of the Cdc42/Rac1 complex (34),which promotes cell migration. Overall, the data support the conclusion that an important
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mechanism of BRB chemoprevention is correcting expression of NMBA-dysregulated celljunction, adhesion, and motility genes.
NMBA-dysregulated apoptosis/cell death genes normalized by BRBPrevious studies have shown that the BRB diet increased the level of apoptosis/cell death intumors and tumor cell lines (4). The DAVID program analysis identified 14 genes (Table 1)that were expressed at normal levels in the NMBA+BRB rats compared with the NMBAanimals. One is Hmox1; its increased expression is associated with resistance to induction ofapoptosis by oxidative stress caused by a wide range of chemical injuries (35). Another isMcl1, a member of the Bcl-2 family, which was regulated to near-normal levels by the BRBdiet. Mcl1 protects mitochondrial integrity though suppression of cytochrome c release (36).Therefore, correction of Mcl1 transcription by BRB should augment apoptotic activity inNMBA-initiated cells. Expression of the gene Pip5k1a (predicted) was also corrected by theBRB diet. Overexpression of this gene can rescue cells from stress-induced apoptosismediated by the activation of extracellular signal-regulated kinase (ERK) 1/2 signaling (37).Thus, appropriate regulation of apoptosis seems to be a mechanism of BRBchemoprevention.
NMBA-dysregulated angiogenesis genes normalized by BRBThe BRB diet also normalized the expression of several genes the DAVID programclustered with angiogenesis activities that were dysregulated by NMBA (Table 1). Amongthese was Klf5, which was down-regulated by NMBA. It is a Kruppel-like zinc fingertranscription factor that modulates cell proliferation, differentiation, cell cycle, apoptosis,and angiogenesis. Klf5 seems to be a tumor suppressor for breast cancer (38). Thus, onemechanism of BRB may be to restore expression of this tumor suppressor factor. Fgfbp1was up-regulated by NMBA; it is a secreted protein thought to enhance fibroblast growthfactor activity and drive tumor angiogenesis. Fgfbp1 is up-regulated early during woundhealing of mouse and human skin as well as during the initiation of skin neoplasia bychemical carcinogens (39). Thus, its normalization by BRB should depress angiogenesis.Interestingly, previous studies have shown that a 5% BRB diet down-regulates theexpression levels of vascular endothelial growth factor-1 (VEGF-1) in the esophagus of ratswhen given for several weeks after treatment of rats with NMBA (40). VEGF-1 was notoverly expressed in the present study, indicating that this gene, along with iNOS and COX-2,does not seem to be involved in the early stages of tumor initiation in the rat esophagus.
Genes dysregulated by NMBA and normalized by PEITC or BRBAnother purpose of our study was to identify gene expression changes in NMBA-treated ratesophagus that were returned to homeostasis by both PEITC and BRB. We identified53genes modulated by both treatments (Table 2). These genes are of special interest becausethey may be sentinel genes of NMBA-induced carcinogenesis and, as such, primary targetsfor chemoprevention. One of the most interesting among these genes is Pls3. Itsoverexpression increases cell proliferation and invasion of tumor cells, probably due tosuppression of E-cadherin. Expression of this gene is increased in the lung tissue of micetreated with carcinogens, and its dysregulation is corrected by a diet containing indole-3-carbinol (41). Thus, Pls3 overexpression is antagonized by three different forms ofchemopreventive diets (i.e., BRB, PEITC, and indole-3-carbinol) in both esophagus andlung rodent cancer models.
Other up-regulated genes that have been associated with cancer are Bub1, Crk, Map2k3,Psck9, PVR, Rab1, PDCD10, H2afz, and Cyp2a2. Map2k3, PVR, Rab1, H2afz, and Cyp2a2are discussed above. Mutations in spindle checkpoint function kinase gene Bub1 have beenassociated with aneuploidy (42). The oncogene Crk is increased in several human cancers
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and its overexpression in cultured epithelial cells causes them to exhibit an alteredmorphology, to proliferate in soft agar, and to grow as massive tumors in nude mice (43).Psck9 is a Ca2+-dependent apoptosis-regulated convertase that stimulates tumor cellproliferation, motility, and invasiveness (44). PDCD10 encodes a protein with similarity toproteins that participate in apoptosis. It interacts with MST4 kinase to promote cellproliferation via modulation of the ERK pathway when overexpressed (45). Thus, returningall of these genes to near-normal levels of expression might be mechanisms by whichPEITC and BRB impede esophageal cell transformation.
Also among the 53 genes are some that were not associated with a DAVID-identified cancerontology. On closer review of the literature, however, they are of interest because they haveactivities that, if dysregulated, could have carcinogenic effects. Several of these genes wereup-regulated by NMBA. Among these is Adamts6_predicted, a member of a family of 20genes involved in tissue organization during embryogenesis and angiogenesis. Some of theproteins encoded by members of this family have matrix-degrading activity and may beinvolved in cell invasion (46). The gene Ddx5 is a transcriptional coactivator and/orcorepressor, depending on the context of the promoter and the transcriptional complex inwhich it associates (47). Thus, its dysregulation may cause inappropriate expression ofgenes with oncogenic or tumor suppressor activities. The transcript NS5A (hepatitis C virus)transactivated protein 9 (Ns5atp9) is the rat homologue of human KIAA0101 whose functionis unknown. However, it binds with proliferating cell nuclear antigen and is overexpressedin hepatocellular carcinomas (48). Transfection of this gene enhanced cancer cell growthand transformed NIH3T3 cells, whereas its inhibition caused attenuation of proliferation.
Both BRB and PEITC diets normalized the expression of seven genes that were down-regulated by NMBA. Of special note is Rbbp6, which encodes a 250-kDa ring finger-containing protein that is frequently up-regulated in human ESCC. This protein binds tounderphosphorylated but not phosphorylated retinoblastoma protein (Rb). Thephosphorylated Rb gene product binds the nuclear transcription factor E2F and prevents itsability to function in the S phase of the cell cycle (49). Thus, Rbbp6 competes with E2F forbinding to the underphosphorylated form of Rb and high concentrations of the protein, as arefound in human ESCC cells, would free E2F to stimulate cell proliferation. Rbbp6 alsobinds to p53, thereby enhancing Mdm2-mediated ubiquitination and degradation of p53,leading to decreased apoptosis. Consequently, down-regulation of Rbbp6 by BRB or PEITCshould slow cell growth and increase the rate of apoptosis.
Another NMBA-dysregulated gene restored to normal levels of expression by BRB andPEITC was homologue of zebra fish ES1 (RGD1303003), which is the rat homologue ofhuman C21orf33. This gene is required for the growth-inhibitory effect of all-trans retinoicacid on MCF-7 breast cancer cells (50). Its down-regulation by NMBA suggests amechanism whereby the carcinogen promotes cancer by repressing terminal differentiationpathways. Mss4 protein (Mss4) binds to the membrane proximal conserved region of α-integrin chains and regulates the activation of inactive pro– matrix metalloproteins. Theactive forms of metalloproteins are important in tumor invasion and metastasis (51). Thus, itwill be interesting to determine if and how down-regulation of Mss4 by NMBA contributesto esophageal carcinogenesis.
In summary, in this and our companion report (13), we have found that a short exposure ofthe rat esophagus to NMBA causes dysregulation of 2,261 transcripts that affect a multitudeof cellular functions. Diets containing 5 µmol PEITC or 5% BRB restored 1,323 or 462 ofthese 2,261 transcripts to near-normal levels of expression, respectively. Thus, as might beexpected, there is a direct correlation between the number of transcripts modulated to near-normal levels of expression and the inhibitory potential of PEITC and BRB in this model
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because PEITC is a more potent inhibitor of NMBA-induced rat esophageal tumorigenesisthan BRB (3). In the present study, we discuss 53 transcripts that are dysregulated byNMBA and restored to homeostatic levels of expression by both BRB and PEITC. Wespeculate that these 53 genes are sentinel early changes in the process of NMBA-inducedtumorigenesis in the rat esophagus, and future investigations will focus on elucidating theirmechanistic roles.
Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.
AcknowledgmentsGrant support: NIH grants RO1 CA103180 and R01CA96130 (G.D. Stoner). The microarray and bioinformaticswork was facilitated by the Microarray and Bioinformatics Facility Core of the Environmental Health SciencesCenter at Wayne State University (National Institute of Environmental Health Sciences Center grant P30 ES06639).
We thank Ronald Nines for his excellent technical assistance.
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Figure 1.Effect of BRB on NMBA-induced preneoplastic lesions in rat esophagus. Microscopicesophageal tissue sections (magnification, ×100) stained with H&E. A, vehicle controlesophagus. B, NMBA-treated esophagus (note extensive cytotoxicity and inflammatoryresponse). C, BRB-treated esophagus. D, BRB+NMBA–treated esophagus.
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Figure 2.BRB modulates NMBA-induced changes in gene expression. Data points reflect mean valueof the three replicate arrays. Y axis, normalized gene expression levels in which theexpression value for each treatment of a gene was divided by the average of all treatmentsfor that gene.
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Table 1
Gene ontology clusters of genes that were dysregulated by NMBA and adjusted to near-normal levels oftranscription (±33%) by the 5% BRB diet in rat esophagus
Gene ontology/biological process No. genes Official symbol
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