soy protein isolate protects against ethanol- …...research article soy protein isolate protects...

Research Article

Soy Protein Isolate Protects Against Ethanol-Mediated Tumor Progression inDiethylnitrosamine-Treated Male MiceKelly E. Mercer1,2, Casey Pulliam2, Leah Hennings3, Keith Lai3, Mario Cleves2, Ellen Jones4,Richard R. Drake4, and Martin Ronis5

Abstract

In this study, diethylnitrosamine-treated male mice wereassigned to three groups: (i) a 35% high fat ethanol liquid diet(EtOH) with casein as the protein source, (ii) the same EtOHliquid diet with soy protein isolate as the sole protein source(EtOH/SPI), (iii) and a chow group. EtOH feeding continued for16 weeks. As expected, EtOH increased the incidence and mul-tiplicity of basophilic lesions and adenomas compared with thechow group, P < 0.05. Soy protein replacement of casein in theEtOHdiet significantly reduced adenoma progression when com-pared with the EtOH and EtOH/SPI group (P < 0.05). Tumorreduction in the EtOH/SPI group corresponded to reduced liverinjury associated with decreased hepatic Tnfa and Cd14 antigen(Cd14) expression and decreased nuclear accumulation ofNF-kB1 protein compared with the EtOH group (P < 0.05).Detection of sphingolipids using high-resolution matrix-assisted

laser desorption/ionization–Fourier transform ion cyclotron res-onance (MALDI-FTICR) imaging mass spectrometry revealedincreased accumulation of long acyl chain ceramide species,and sphingosine-1-phosphate (S1P) in the EtOH group thatwere significantly reduced in the EtOH/SPI group. ChronicEtOH feeding also increased mRNA expression of b-catenintranscriptional targets, including cyclin D1 (Ccnd1), matrixmetallopeptidase 7 (Mmp7), and glutamine synthetase (Glns),which were reduced in the EtOH/SPI group (P < 0.05).We conclude that soy prevents tumorigenesis by reducingproinflammatory and oxidative environment resulting fromEtOH-induced hepatic injury, and by reducing hepatocyteproliferation through inhibition of b-catenin signaling. Thesemechanisms may involve changes in sphingolipid signaling.Cancer Prev Res; 9(6); 466–75. �2016 AACR.

IntroductionHepatocellular carcinoma (HCC) is the third leading cause of

cancer mortality worldwide and is three times more likely to bediagnosed in men (1, 2). Of the known risk factors associatedwith hepatocellular carcinoma, alcohol consumption is thedominant independent factor linked to increased risk. In Chinaand Japan, epidemiologic data suggest that consumption of>80 g/d of alcohol over 10 years results in a 2-fold increase inHCC risk; however, in Western populations, increased HCC riskis 5-fold (3–5). Even at lower consumption rates, alcohol use is

synergistic with other initiating factors, including hepatitis Cand B infections or diabetes mellitus, increasing the overallprevalence of HCC in these populations worldwide (6–8). Inthe United States, binge and heavy drinking has increased inprevalence, with 25% of adults reporting alcohol misuse orabuse, and is a primary contributing risk factor for one-third ofreported cases (6, 9, 10). Moreover, once initiated, HCC riskdoes not decrease with abstinence (9) and there are a fewclinical strategies aimed to reduce risk in alcohol consumers.

Biologic mechanisms involved in alcohol-induced liver cancerinteract at the level of both initiation and promotion. Tumorinitiation results from ethanol (EtOH) metabolism by alcoholdehydrogenase and cytochrome P450 (CYP) 2E1 thus producingacetaldehyde and reactive oxygen species which interfere withDNA synthesis and repair mechanisms, leading to mutagenicityand tumorigenesis (4, 11). In addition to initiating effects, EtOHalso has proliferative and tumor-promoting effects in the liver(7, 8, 12). Our laboratory has demonstrated that EtOH stimulateshepatocyte proliferation in rodents in associationwith liver injuryand hepatic vitamin A depletion (13–15). In our study, hepaticproliferation was associated with increasedWnt/b-catenin signal-ing and tumor multiplicity in male mice receiving EtOH for16 weeks and EtOH-induced tumors were b-catenin positive(13, 16). Likewise, a significant proportion of HCC tumors areb-catenin positive (17), although the extent of b-catenin expres-sion during alcoholic liver disease progression is unknown.

These data suggest that an effective strategy for reducing alcoholpromotion of HCC is to target Wnt signaling. Diets rich in soyproteinor soy-derivedphytochemicals have been reported tohave

1Department of Pediatrics, University of Arkansas for MedicalSciences, Little Rock, Arkansas. 2Arkansas Children's Nutrition Center,Little Rock, Arkansas. 3Department of Pathology, University of Arkan-sas for Medical Sciences, Little Rock, Arkansas. 4Medical University ofSouth Carolina Proteomic Center, Charleston, South Carolina.5Department of Pharmacology & Experimental Therapeutics, Louisi-ana State University Health Sciences Center, New Orleans, Louisiana.

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

Corresponding Authors: Martin J. Ronis, Department of Pharmacology &Experimental Therapeutics, Louisiana State University Health Sciences Center,New Orleans-New Orleans, 1901 Perdido Str., New Orleans, LA 701112. Phone:504-568-4514; Fax: 504-658-4740; E-mail: [email protected]; and Kelly E.Mercer, Department of Pediatrics, University of Arkansas for Medical Sciences,4301WMarkham St, Little Rock, AR 72205. Phone: 501-364-2784; Fax: 501-264-2818; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-15-0417

�2016 American Association for Cancer Research.

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cancer preventative properties in both epidemiologic and exper-imental animal studies (18–26). In addition, in certain tissues,these soy-rich diets are known to block Wnt signaling pathways.For example, in rat mammary epithelial cells, Su and colleagues(27) demonstrated that soy's bioactive isoflavone, genistein,inhibits Wnt signaling through increased expression of cadherin1 and sequestration of b-catenin to the cell membrane. In humancolon epithelial cells, Zhang and colleagues (28) proposed thatgenistein downregulates Wnt signaling through increased expres-sion of soluble inhibitory factors called secreted frizzled relatedproteins (sFRP). Therefore, we hypothesized that dietary inter-vention with soy may prevent EtOH-mediated tumor-promotingeffects by inhibiting b-catenin signaling and associated prolifer-ationmechanisms. To test this hypothesis, we utilized a two-stagemouse model in which tumor initiation by a genotoxic com-pound, diethylnitrosamine (DEN), was followed by the promot-ing agent, an EtOH liquid diet (7, 13, 16), containing either caseinor soy protein isolate (SPI) as the sole protein source. Adminis-tration of these diets continued for 16 weeks, at which livers wereanalyzed for the presence of adenomas.

Materials and MethodsAnimals and experimental design

All experimental procedures involving animals wereapproved by the Institutional Animal Care and Use Committeeat the University of Arkansas for Medical Sciences (UAMS; LittleRock, AR). Mice were housed in an Association Assessment andAccreditation of Laboratory Animal Care–approved animalfacility. Male and female C57Bl/6 mice (Jackson Laboratories)were used to establish a breeding colony to generate malepups, who received either a single intraperitoneal injection of10 mg/kg DEN (n ¼ 54) or saline (n ¼ 25) on postnatal day 14.Mice were weaned to and maintained on rodent chow (HarlanLaboratories) until postnatal day 60, at which the DEN-injectedmice were randomly assigned to three weight-matched dietgroups: a chow diet (n ¼ 10, chow), an EtOH-containing liquiddiet (n ¼ 21, EtOH), and an EtOH-containing liquid dietcontaining soy protein isolate, SPI, (n ¼ 23, EtOH/SPI). Allgroups had access to water ad libitum. Liquid diets were for-mulated according to the Lieber De Carli diet of 35% of energyfrom fat, 18% from protein, and 47% from carbohydrates(Dyets, Inc.), with SPI (Dupont Nutrition & Health) replacingcasein as the sole protein source in the EtOH/SPI liquid diet.EtOH was added to the Lieber-De-Carli liquid diet slowly bysubstituting EtOH for carbohydrate calories in a stepwisemanner until 28% total calories were reached as describedpreviously (13). This dose constitutes a final EtOH concentra-tion of 4.9% (v/v), respectively, and was maintained untilsacrifice (4 months), at which livers were fixed in formalin forpathologic evaluation. At postnatal day 60, an additional groupof saline-injected mice were randomized into three liquid dietgroups, a chow diet (n ¼ 5), an EtOH (n ¼ 10), an EtOH/SPI(n ¼ 10). Diets were maintained for 4 months as describedabove. At sacrifice, liver pieces were flash frozen in liquidnitrogen and stored at �70�C. Total serum isoflavone concen-trations were extracted and analyzed as described previously(29). Serum alanine aminotransferase (ALT) levels wereassessed as a measure of liver damage by using the InfinityALT liquid stable reagent (Thermo Electron) according tomanufacturer's protocols.

Pathologic evaluationAll formalin-fixed liver lobes from DEN-treated mice receiving

chow, EtOH, or EtOH/SPI diets were embedded in paraffin,sectioned (4 mm), stained, and examined in a blinded fashionunder a light microscope as described previously (13) by twoindependent pathologists (L. Hennings and K. Lai). Preneoplasticfoci were counted at 40� magnification. Preneoplastic foci candevelop into adenomas, which were defined as a compressivelesion of any size without evidence of invasion or other criteria ofmalignancy, and may develop into hepatocellular carcinoma,which was defined as a compressive and invasive lesion withcriteria of malignancy (30).

IHCArchival liver tissue sections from deidentified patients diag-

nosed with alcoholic steatohepatitis (n ¼ 5) or alcoholiccirrhosis (n ¼ 7) were obtained from the UAMS Departmentof Pathology. b-Catenin expression was assessed in liver sec-tions by IHC using standard procedures and a monoclonalb-catenin antibody (1:75) detecting the active, dephosphory-lated (Ser37 or Thr41) form (anti-active-b-catenin, clone 8E7,EMD Millipore), as described previously (13). Hepatic prolif-erating cell nuclear antigen (PCNA) staining was assessed byIHC in nontumor tissue in chow, EtOH, and EtOH/SPI fed miceas described previously (13). Stained slides were evaluatedunder a light microscope. Nuclei of S-phase cells stained darkbrown; a total of 10 observations (100� field counted) werescreened per liver sample. Data are expressed as a percentage ofPCNA-stained nuclei in S-phase.

Gene expressionLiver RNA was isolated from DEN- and saline-treated mice

receiving chow, EtOH, or EtOH/SPI diets as described previously.All RNA was reverse transcribed using IScript cDNA Synthesis(Bio-Rad Laboratories) according to the manufacturer's instruc-tions, and subsequent real-time PCR analysis was carried outusing SYBR Green and an ABI 7500 Sequence Detection System(Applied Biosystems). Results were quantified using the DCt

method relative to 18s or Gapdh. Primer sequences are presentedin Supplementary Table S1.

Protein isolation and Western blottingNuclear and cytosolic protein fractions were isolated from

TEN control and EtOH-treated mouse livers using NE-PERNuclear and Cytoplasmic Extraction Reagent Kit (Fisher Scien-tific). Membrane fractions were obtained using a modifiedprotocol as described previously (31). Proteins (30 mg) wereseparated by SDS-PAGE using standard methods. Blotted cyto-solic proteins were incubated with the following antibodies,pAKT, total AKT, pGSK3b (phospho-GSK-3a/b (Ser21/9) andtotal GSK3b, from Cell Signaling Technology using a 1:1,000dilution. Antibodies toward b-catenin (1:1,000, Milipore), andpNF-kB (Cell Signaling Technology) were used on blottednuclear proteins. Membrane fractions were probed with ananti-SPHK1 antibody (1:1,000, Cell Signaling Technology).Secondary antibodies were diluted (1:5,000 to 1:10,000) andincubated at room temperature before chemiluminescencedetection. Protein bands were quantified using a densitometerand band densities were corrected for total protein loaded bystaining the membrane with 0.1% amido black.

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Matrix-assisted laser desorption/ionization imaging massspectrometry

Matrix-assisted laser desorption/ionization (MALDI)-Fouriertransform ion cyclotron resonance (FTICR) imaging mass spec-trometry was used to qualitatively detect sphingolipid species inliver sections from saline-treated mice receiving an EtOH orEtOH/SPI diet using methods as described previously (32). Brief-ly, the left lateral liver lobe (n ¼ 1 per diet group) was rapidlyfrozen in liquid nitrogen for 2 minutes and stored at�80�C untiluse. Liver tissues were sectioned at 10 mm, thaw mounted onindium tinoxide–coated slides (BrukerDaltonics), anddesiccatedat room temperature for 5 minutes. A 2,5-dihydroxybenzoic acidmatrix was added using an ImagePrep spray station (BrukerDaltonics), at a concentration of 0.2 mol/L in 50% methanoland 0.01% trifluoroacetic acid. MALDI imaging mass spectrom-etry analysis was performed using a Bruker Solarix 7T FTICRmassspectrometer, equipped with a SmartBeam II laser operating at1,000 Hz, collecting spectra across the entire tissue in positive ionmode between (m/z 200–2000). A laser spot size of 25 mm, and aresolution or raster width of 200 mm was utilized for analysis,collecting 800 shots per pixel. Data were reduced to 0.98 ICRreduction and loaded into FlexImaging 4.0 software (BrukerDaltonics) for data analysis, and generation of lipid images ofinterest. Within FlexImaging, all data was normalized using rootmean square and intensities were thresholded appropriately.Because of the high mass accuracy and resolution capability ofa 7T FTICRmass spectrometer used in this study, lipid specieswereidentified by mass accuracy in reference to an internal ceramidedatabase (32) and to an external database known as Lipid Maps.

Data and statistical analysisData are presented as means� SE. Comparisons between three

groups were accomplished by one-way ANOVA followed by aStudent–Newman–Keuls post hoc analysis or by Kruskal–Wallisone-way ANOVA on ranks followed by Dunn post hoc analysis.Comparisons between two groups were accomplished usingeither Student t test or Mann–Whitney U rank sum test. Numberof lesions was compared across groups using negative binomialregression which generalizes Poisson regression to account foroverdispersion of the count data. This model included groupmembership as a set of indicator (dummy) variables. The pro-portion of new adenomas (incidence) was compared acrossgroups using Fisher exact test. Statistical analysis was performedusing the SigmaPlot software package 11.0 (Systat Software, Inc.)and Stata statistical software 13.1 (Stata Corporation). Statisticalsignificance was set at P < 0.05.

ResultsStudy design and observations

Male mice were assigned to an EtOH or EtOH/SPI liquid diet,and a chow-fed diet 43 days post-DEN injection. Startingweights for DEN-treated mice were 22.8 � 0.47 g, 22.8 �0.28 g, and 23.0 � 0.28 g for chow, and EtOH and EtOH/SPI,respectively. EtOH feeding continued for 16 weeks. Diet intakesbetween the EtOH and EtOH/SPI groups did not differ statis-tically, 15.4 � 0.12 mL and 15.2 � 0.09 mL, respectively, whichcorresponded to an average daily intake of 18.6 g/kg/d of EtOH.In the saline-treated mice, starting weights were 23.0 � 0.46,24.4 � 0.44, and 23.7 � 0.50, for chow, EtOH, and EtOH/SPI,respectively. In these animals, diet intakes were 15.8 � 0.23 mL

and 16.0 � 0.14 mL for EtOH and EtOH/SPI groups, respec-tively, and corresponded to an average daily intake of 19.7 g/kg/d of EtOH, which resulted in a mean (�SE) blood alcoholconcentration of 29 � 7.2 mg/dL and 23 � 4.3 mg/dL forEtOH and EtOH/SPI groups, respectively. Total circulatingisoflavone concentrations (aglyconeþconjugates) were mea-sured in serum of DEN- and saline-treated mice receiving theEtOH/SPI diet, as described previously (28). Equol, daidzein,genistein, and o-desmethylangolensin were present at the fol-lowing concentrations, 1.15 � 0.14, 0.13 � 0.05, 0.05 � 0.02,and 0.08 � 0.02 mmol/L, respectively, and did not differbetween the two groups.

SPI decreases adenoma incidence and multiplicity associatedwith EtOH feeding in DEN-treated mice

Liver lobes taken from DEN-treated chow, EtOH, and EtOH/SPI mice were assessed for presence of lesions, which encom-passed basophilic foci and adenomas. Basophilic incidence in theEtOH, 0.90 (19/21), and EtOH/SPI, 0.95 (22/23), groups wereincreased by 2-fold compared with the chow, 0.5 (5/10) fed mice(Fisher exact test, P < 0.05). Likewise, basophilic multiplicity inthe EtOH and the EtOH/SPI groups were also significantlyincreased compared with chow, 4.6 (88/21), 3.8 (88/23), and1.7 (17/10), respectively (one-way ANOVA followed by Mann–WhitneyU rank sum test, P < 0.05).We also observed the presenceof adenomas in the DEN-treated EtOH, 0.67 (14/21), which wereabsent in the DEN-treated chow, 0.0 (0/10) group (Fisher exacttest, P < 0.05). When compared with the EtOH group, SPI feedingreduced adenoma incidence, 0.23 (6/23) in the DEN-treatedEtOH/SPI (Fisher exact test, P < 0.05). Likewise, we observed asignificant decrease in adenoma multiplicity in the ETOH/SPI,0.43 (10/23) compared with the EtOH, 1.62 (32/21) fed mice(Mann–Whitney U rank sum test, P < 0.05).

Reduced tumor progression is associated with decreased liverpathology in EtOH/SPI mice

SPI substitution for casein protein in the EtOH diet had amarked impact on liver pathology in the DEN-treated mice. Asseen in Fig. 1, chronic consumption of the DEN/EtOH dietresulted in elevated serum ALT concentrations which corre-sponded to histologic evidence of steatosis and inflammationand increased hepatocyte proliferation in nontumorigenic tissuewhen compared with the DEN/chow–fed group (P < 0.05). Incontrast, EtOH/SPI feeding resulted in a significant reductionin serum ALT, decreased overall pathology, and a reduction inproliferation in comparison with the EtOH-treated mice (Fig. 1).Mechanisms associated with decreased liver pathology wereassessed in hepatic tissue taken from saline-treatedmice receivingchow, EtOH, and EtOH/SPI diets concurrently with the DEN-treated mice. Similar to the DEN-treated mice, the EtOH/SPI dietdecreased liver injury as measured by ALT [37.6 � 3.6 vs. 8.6 �1.58 and 8.6 � 0.89, for the saline-treated EtOH, EtOH/SPI, andchow-fed groups, respectively (P < 0.05)]. Likewise, in Fig. 2, weobserved a 47%and 68%decrease in hepatic Tnfa and Interleukin6 (Il6) mRNA expression in the EtOH/SPI group compared withthe EtOH group. Hepatic liver transcripts of Cd14 and chemokine(C-X-Cmotif) ligand 2 (Cxcl2) were also decreased 1.5- to 2-fold,respectively, in the EtOH/SPI–fed mice (Fig. 2C and D; P < 0.05).These findings were associated with reduced protein accumula-tion of the proinflammatory transcription factor, NF-kB, in thenucleus in the EtOH/SPI group (Fig. 2E, P < 0.05). In addition,

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Cancer Prev Res; 9(6) June 2016 Cancer Prevention Research468




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Figure 1.The effect of EtOH and EtOH/SPI liquid diets on liver pathology in DEN-treated male mice. A, representative hematoxylin and eosin–stained liver sections in DEN-treated male mice receiving chow (DEN/Chow), n ¼ 10, Lieber De Carli EtOH diet (DEN/EtOH), n ¼ 21, or Lieber De Carli EtOH diet with SPI as sole proteinsource (DEN/EtOH/SPI), n ¼ 23; pathology score, liver injury (B), steatosis (C), immune cell infiltration (D), and hepatocyte proliferation (E) were determined asdescribed in the Materials and Methods. Statistical analysis was performed by one-way ANOVA followed by Student–Newman–Keuls post hoc analysis. Meansthat do not share a letter are significantly different at P < 0.05, a < b, a < c, c < b.






fibrosis markers were also decreased in the livers of EtOH/SPI–treated mice. Alpha-smooth muscle actin (a-Sma) and platelet-derived growth factor receptor (Pdgfr) were significantly sup-pressed in comparison with both the EtOH-treated mice andchow controls (3.48 � 1.17 vs. 0.31 � 0.07 and 0.27 � 0.04 fora-Sma in EtOH, EtOH/SPI, and chow controls, respectively, and1.48 � 0.10 vs. 0.89 � 0.13 and 0.88 � 0.1 for Pdgfr, in EtOH,EtOH/SPI, and chow controls, respectively; one-way ANOVA,Student–Newman–Keuls post hoc analysis; P < 0.05).

Immunohistochemical analysis of b-catenin expression andlocalization in livers from chronic alcoholics

Clinically, a significant percentage of liver tumors from alco-holics are positive for b-catenin expression (17). In Fig. 3, welooked for the presence of b-catenin in archival liver tissue takenfrom patients diagnosed with alcoholic steatohepatitis and cir-rhosis by IHC. In these tissues, we observed a pattern of increasedmembrane staining of b-catenin and translocation of b-catenin tothe nucleus coinciding with disease progression. These data areconsistent with our rodentmodel demonstrating thatWnt/b-cate-nin signaling during early stages of steatohepatitis (13), and in theclinical setting, increased activation in cirrhotic livers, where HCCgenerally occurs in humans.

SPI inhibits EtOH-mediated Wnt/b-catenin signaling inhepatocytes

Previously, we have reported chronic EtOH feeding in ratspromotes hepatocyte proliferation through increased b-cateninactivation via the Wnt signaling pathway (13). As seen in Table1, we observed a 3-fold increase in wingless-type MMTVintegration site family, member 2 (Wnt2) mRNA expression,corresponding to approximately a 2-fold increase in the ratioof active (phosphorylated) GSK3b to total GSK3b cytosolicprotein expression, and upregulation of mRNA expression ofknown b-catenin targets, Ccnd1, Glns, and Mmp7 in saline-treated EtOH mice compared with chow controls; SPI blocksthese increases in the EtOH/SPI group (P < 0.05), includingnuclear accumulation of b-catenin (P < 0.05). Consistent withthese findings, in the DEN-treated mice receiving EtOH, weobserved increased mRNA expression of other soluble Wnts,Wnt2b, and Wnt7a (Table 1), and SPI reduced transcriptexpression 16% (P ¼ 0.02) and 34% (P ¼ 0.08), respectively,in the EtOH/SPI group. Moreover, we observed increasedmRNA expression of soluble Wnt inhibitors and dickkopf-3(Dkk3) in the EtOH/SPI group compared with DEN-treatedEtOH mice. These findings are indicative of inhibition of thecanonical Wnt signaling pathway. In our model, we did not see

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Figure 2.Changes in hepatic mRNA expression of proinflammatory cytokines Tnfa (A), Il6 (B),Cxcl2 (C), andCd14 (D) in saline-treatedmalemice receiving EtOH (n¼ 10) andEtOH/SPI (n ¼ 10) diets compared with chow (n ¼ 5) controls. Western blot analysis of nuclear expression (E) of NF-kB expression liver tissue from EtOHand EtOH/SPI–fed mice. Data are expressed as mean � SE. For real-time RT-PCR, statistical analysis was determined by Kruskal–Wallis one-way ANOVA on ranksfollowed by Dunn post hoc analysis, and groups with different letters are significantly different from each other. For the Western blot analysis, statisticalsignificance was determined by Student t test. Means that do not share a letter are significantly different at P < 0.05, a < b, a < c, c < b.

Mercer et al.





any changes in phosphorylation status of the serine/threoninekinase AKT between saline-treated EtOH and chow-fed micethat would suggest that the survival pathway PI3K/AKT/GSK3b

was responsible for EtOH-mediated activation of Wnt signal-ing (Table 1).

Effects of SPI on hepatic ceramide accumulation and ER stressduring chronic EtOH feeding

In humans and in rodents, alcohol exposure has been shown toincrease ceramide accumulation in the liver, thereby enhancingendoplasmic reticulum stress and contributing to liver injury(33). Consistent with these reports, we observed a significantincrease in the mRNA expression of serine palmitoyl transfeasesubunit (Sptlc1), a key enzyme involved in de novo ceramidebiosynthesis, in the EtOH-treated mice compared with chow-fedmice (Fig. 4A). Ceramide synthase (Cers) gene transcripts werealso upregulated; of note, Cers2 and Cers4 were highly expressed(6-fold) in EtOH-treated mice (Fig. 4B; P < 0.05). In addition,chronic EtOH exposure also significantly increasedmRNA expres-sion of acid ceramidase (Asah1), and sphingosine kinase 1(Spkh1), key enzymes in the synthesis of sphingosine-1P (S1P),and sphingosine-1P receptors (Spr) 2 and 3 (Fig. 4C–E). In theEtOH/SPI–treated mice, Sptlc1 mRNA expression was increasedcompared with the EtOH mice, but did not reach significance(P ¼ 0.079). However, we did see a significant decrease in Cers1mRNA expression in response to EtOH/SPI diets, but no othersignificant decreases with respect to Cers2, 4, 5, and 6 expression(Fig. 4B; P < 0.05). In contrast, SPI did shut down sphingosine-1Pproduction, including the mRNA expression and translocation ofSphk1 to the membrane which is necessary for enzymatic activa-tion (Fig. 4F; P < 0.05). Mechanistically, Cers1 predominatelygenerates C18:0-ceramide, Cers2 produces longer acyl chain cer-amides (C20:0—C26:0 ceramides), and Cers5, 6 synthesizes theshorter C14:0, C16:0 ceramide species. In Fig. 5, using a novelMALDI-FTICR imaging mass spectrometry workflow used for on-tissue detection and spatial localization of ceramides and othersphingolipids (32), we qualitatively confirmed the presence ofshort and long acyl chain ceramides and S1P in livers from micereceiving EtOH. We also observed a subsequent loss of ceramideC18 and sphingosine-1P in the EtOH/SPI livers, which is consis-tent with the mechanistic data presented in Fig. 4.

Figure 3.Representative liver sections showing the expression and localization ofb-catenin in archival liver tissue from patients with normal liver pathology orfrom patients diagnosed with alcoholic steatohepatitis and cirrhosis. IHC wasperformed as described in the Materials and Methods.

Table 1. Hepatic Wnt/b-catenin signaling in saline-treated mice receiving EtOH diets

Saline-treated miceChow EtOH EtOH/SPI

Gene expressionWnt2 0.12 � 0.01a 0.40 � 0.09b 0.22 � 0.03a,b

Glns 1.40 � 0.33a 2.79 � 0.43b 1.28 � 0.24a

Mmp7 0.83 � 0.17a 2.48 � 0.72b 0.56 � 0.11a

Ccnd1 0.69 � 0.11a 1.97 � 0.28b 0.92 � 0.11a

Protein expressionpAKT:totalAKT 1.65 � 0.40a 0.71 � 0.12a 1.16 � 0.27a

pGSK3b:totalGSK3b 0.65 � 0.01a 1.25 � 0.12b 0.94 � 0.05a

Nuclear b-catenin — 1.23 � 0.18 0.71 � 0.05�

DEN-treated miceChow EtOH EtOH/SPI

Gene expressionWnt2b 0.21 � 0.03a 1.03 � 0.04b 0.86 � 0.04c

Wnt5a 0.24 � 0.06a 0.51 � 0.04b 0.46 � 0.02b

Wnt7b 0.65 � 0.19a 1.71 � 0.24b 1.12 � 0.16a,b

Dkk3 0.25 � 0.09a 0.48 � 0.01b 0.58 � 0.03c

Wif1 0.10 � 0.03a,b 0.17 � 0.02b 0.06 � 0.01a

NOTE: Data are expressed asmean�SE for saline-treated chow (n¼ 5), EtOH (n¼ 10), andEtOH/SPI (n¼ 10), DEN-treated chow (n¼ 5), EtOH (n¼ 21) andEtOH/SPI(n¼ 23) groups. Gene expression was measured by real-time RT-PCR, and values calculated by 2�DDCt method as described in Materials and Methods. Proteinsinvolved in Wnt/b-catenin signaling were assessed by Western blot analyses as described in Materials and Methods; blots were developed usingchemiluminscence detection and band densities were corrected for total protein load by staining with 0.1% amido black. Statistical significance wasdetermined by one-way ANOVA, P < 0.05, a < b < c, or Student t test; � , P < 0.05, EtOH vs. EtOH/SPI.






DiscussionEpidemiologic data from Asian populations eating traditional

soy-based diets have suggested a variety of health benefits whichinclude a reduction in the risk of different cancer types (21, 22, 25,26, 34). In this study, we examined the possibility that dietaryintervention with SPI would also prevent EtOH-mediated hepatictumor promotion in a rodent model of chemical carcinogenesis(13, 16). In response to alcoholic liver injury, most rodent studieshave reported a corresponding increase in hepatocyte prolifera-tion (14, 15, 35, 36). This appears tobe in response toproliferativesignals such as a reduction in retinoid receptor activation andactivation of b-catenin transcriptional activity downstream of

WNT expression and secretion (8, 13). In the current study,EtOH-mediated hepatocyte proliferation was dependent onWnt–b-catenin signaling. Moreover, we observed increasedb-catenin staining in livers from alcoholics with steatohepatitisand cirrhosis, which supports the role of Wnt–b-catenin sig-naling in tumor growth and progression. Feeding SPI had ahighly significant inhibitory effect on EtOH-mediated adenomatumor progression. Inhibition of b-catenin activation by SPIfeeding was accompanied by decreased phosphorylation ofGSK3b, decreased expression of Wnt mRNAs, and increasedexpression of mRNA for the Wnt signaling inhibitor Dkk3.These data suggest that, at least in part, SPI components blockEtOH-induced Wnt signaling.

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Figure 4.Changes in hepaticmRNAexpressionof de novo ceramide synthesis andsphingosine-1P production. Sptlc1(A), CerS (B), Asah1 (C), Spkh1 (D),and S1p receptors 1, 2, and 3 (E), insaline-treated male mice receivingEtOH (n¼ 10) and EtOH/SPI (n¼ 10)diets compared with chow (n ¼ 5)controls. Western blot analysis ofmembrane protein expression (F) ofSpkh1 in liver tissue from EtOH andEtOH/SPI–fed mice. Data areexpressed as mean � SE. For real-time RT-PCR, statistical analysis wasdetermined by Kruskal–Wallis one-way ANOVA on ranks followed byDunn post hoc analysis, and groupswith different letters are significantlydifferent from each other. Statisticalsignificance forWestern blot analysiswas determined by Student t test.Means that do not share a letter aresignificantly different at P < 0.05,a < b, a < c, c < b.

Mercer et al.





The detailed molecular mechanisms underlying EtOH induc-tion and SPI suppression of hepatic Wnt pathways remain to beelucidated. It has been suggested that Wnt secretion by nonpar-enchymal cells is involved in regulation of hepatocyte prolifera-

tion and in some forms of liver cancer. Studies of hepatocyteproliferation following 2/3 partial hepatectomy have implicat-ed Wnt secretion by Kuppfer cells in this process (37). Recentstudies of cholangiocarcinoma have demonstrated characteris-tic enhancement of Wnt signaling via inflammatory macro-phages (38). EtOH promotion of hepatic tumorigenesis hasalso been demonstrated to be linked to induction of TGFbsignaling and stellate cell activation (7) and enhanced tumor-igenesis has also been observed in other experimental modelsof fibrosis (39). Additional studies are underway to identifywhether Kupffer cells, other inflammatory cell types, stellatecells, or endothelial cells are the cellular source of EtOH-induced Wnts.

SPI feeding also significantly lowered necroinflammatory inju-ry and Kupffer cell recruitment in the EtOH/SPI group. Thehepatoprotective effects of SPI were coincident with reducedmRNA expression of Il6, decreased hepatocyte proliferation, andevidence of reduced b-catenin activation. These data support amechanistic link between soy's inhibitory effects on EtOH-depen-dent tumor promotion, reductions in Wnt-b-catenin signaling,and reduced liver injury. Genistein, the major isoflavone phyto-estrogen, is considered the biologically active anticancer compo-nent in soy foods (27, 28, 40, 41). Recently, using the samemodelreported here, we have found increased EtOH-mediated hepaticadenomas in male mice receiving genistein in the liquid EtOHdiet (42). Our findings suggest that SPI contains additionalanticancer components. Supporting evidence for this hypothesiscomes from a recent report demonstrating feeding of an isofla-vone-free SPI diet specifically inhibitedNF-kB–dependent expres-sion of inflammatory cytokines, including Tnfa in the hyperlipi-demic apoE�/� mouse model, thus reducing the presence ofatherosclerotic lesions (43). These findings suggest that soyhydrolysates and putative peptides derived from digestion of themajor soy storage protein b-conglycinin in the gut may have anti-inflammatory properties.

Recent reports have implied that chronic alcohol consump-tion influences hepatic ceramide generation (33, 44). In thisstudy, we observed significant elevations of hepatic ceramidesynthesis, particularly C18 generation, and S1P production inresponse to chronic EtOH consumption. These data are con-sistent with those reported by Longato and colleagues (33) inchronic alcoholics and from our laboratory using an intragas-tric EtOH treatment in a rat model associated with hepaticinsulin resistance (44). In this study, we did not explore thealternative ceramide generation pathways, including complexsphingolipid hydrolysis or through a salvage pathway involvingceramide deacylation (45). However, Longato and colleagues(33) observed increased sphingomyelin hydrolysis in thehepatic tissue of chronic alcoholics, suggesting that alcoholmay affect additional pathways of ceramide synthesis.

Interestingly, EtOH-mediated increases in C18 ceramide andS1P were reversed in the EtOH/SPI group. Different ceramidespecies have diverse effects on cell death and proliferation (46).Ceramide is converted to S1P, an important proinflammatory andprofibrotic lipid mediator, via the actions of ceramidases andsphingosine kinases. S1P acts via 5 receptors to stimulate inflam-mation both up- and downstream of TNFa and to stimulatefibrosis via activation of stellate cells (24, 46, 47). Moreover,S1P has antiapoptotic actions and paracrine proliferative effectson hepatocytes and pancreatic cancer cells mediated via endo-thelial and stellate cells (46). It is possible that SPI inhibition of

Figure 5.Analysis of ceramide species in a sectioned liver from saline-treated malemice receiving EtOH and EtOH/SPI diets; hematoxylin and eosin stain of serialliver sections (A), qualitative confirmation of short and long acyl chainceramides and sphingosine-1P using MALDI-FTICR imaging massspectrometry previously described for on-tissue detection, spatiallocalization, and structural confirmation of bioactive sphingolipids (ref. 32; B).Representative images were acquired using a high-resolution massspectrometer, Bruker Solarix 7T FTICR. Images were normalized using rootmean squares and thresholded to each other. The intensity of each lipiddetected is shown as a reflection of its color intensity with areas of greaterdistribution of a particular lipid being pink or red, compared with lower signaldetection shown as blue or green. All lipidmasseswere crossed referenced toan internal database (32) and an external database Lipid Maps.






ceramide/S1P synthesis is involved in blockade of Wnt/b-cateninsignaling, particularly if activated stellate cells are a source ofWnts. Alternatively, SPI may affect other Wnt-independent path-ways impacting on tumor promotion such as expansion of tumor-initiating stem cell populations or on apoptosis/proliferationbalance in transformed cell populations. The role of ceramidesand S1P in EtOH-induced hepatic tumor promotion and protec-tion by feeding of SPI are also the subject of ongoing studies.

In conclusion, our data demonstrate that EtOH acts as a tumorpromoter in the mouse DEN model as a result of activation ofWnt/b-catenin signaling. Protection against EtOH-inducedtumorigenesis by switching from casein to SPI as the dietaryprotein source appears to be due to a component other thangenistein which acts to block steatosis and progression of liverinjury, reverses EtOH activation of b-catenin, and prevents EtOH-induced changes in ceramide/sphingosine metabolism.

Disclosure of Potential Conflicts of InterestM. Ronis received speakers bureau honoraria from and is a consultant/

advisory board member for DuPont Nutrition and Health. No potential con-flicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K.E. Mercer, M. RonisDevelopment of methodology: K.E. Mercer, L. Hennings, R.R. Drake, M. RonisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.E. Mercerm, K. Lai, E. Jones, R.R. DrakeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.E. Mercer, C. Pulliam, L. Hennings, K. Lai,M. Cleves, R.R. Drake, M. RonisWriting, review, and/or revision of the manuscript: K.E. Mercer, L. Hennings,K. Lai, R.R. Drake, M. RonisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. PulliamStudy supervision: R.R. Drake, M. Ronis

Grant SupportThis work was funded in part byNIH grant R21CA169389 (toM. Ronis) and

by the Arkansas Children's Hospital Research Institute and the Marion B. LyonNew Scientist Development Award (to K.E. Mercer).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedDecember 10, 2015; revisedMarch 1, 2016; acceptedMarch 2, 2016;published OnlineFirst March 22, 2016.

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2016;9:466-475. Published OnlineFirst March 22, 2016.Cancer Prev Res Kelly E. Mercer, Casey Pulliam, Leah Hennings, et al. Progression in Diethylnitrosamine-Treated Male MiceSoy Protein Isolate Protects Against Ethanol-Mediated Tumor

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