Soy Protein Isolate Protects Against Ethanol-Mediated Tumor Progression in Diethylnitrosamine-Treated Male Mice

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and is three times more likely to be diagnosed in men (1, 2). Of the known risk factors associated with hepatocellular carcinoma, alcohol consumption is the dominant independent factor linked to increased risk. In China and Japan, epidemiologic data suggest that consumption of >80 g/d of alcohol over 10 years results in a 2-fold increase in HCC risk; however, in Western populations, increased HCC risk is 5-fold (3–5). Even at lower consumption rates, alcohol use is synergistic with other initiating factors, including hepatitis C and B infections or diabetes mellitus, increasing the overall prevalence of HCC in these populations worldwide (6–8). In the United States, binge and heavy drinking has increased in prevalence, with 25% of adults reporting alcohol misuse or abuse, and is a primary contributing risk factor for one-third of reported cases (6, 9, 10). Moreover, once initiated, HCC risk does not decrease with abstinence (9) and there are a few clinical strategies aimed to reduce risk in alcohol consumers.

Biologic mechanisms involved in alcohol-induced liver cancer interact at the level of both initiation and promotion. Tumor initiation results from ethanol (EtOH) metabolism by alcohol dehydrogenase and cytochrome P450 (CYP) 2E1 thus producing acetaldehyde and reactive oxygen species which interfere with DNA synthesis and repair mechanisms, leading to mutagenicity and tumorigenesis (4, 11). In addition to initiating effects, EtOH also has proliferative and tumor-promoting effects in the liver (7, 8, 12). Our laboratory has demonstrated that EtOH stimulates hepatocyte proliferation in rodents in association with liver injury and hepatic vitamin A depletion (13–15). In our study, hepatic proliferation was associated with increased Wnt/β-catenin signaling and tumor multiplicity in male mice receiving EtOH for 16 weeks and EtOH-induced tumors were β-catenin positive (13, 16). Likewise, a significant proportion of HCC tumors are β-catenin positive (17), although the extent of β-catenin expression during alcoholic liver disease progression is unknown.

These data suggest that an effective strategy for reducing alcohol promotion of HCC is to target Wnt signaling. Diets rich in soy protein or soy-derived phytochemicals have been reported to have cancer preventative properties in both epidemiologic and experimental 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 cadherin 1 and sequestration of β-catenin to the cell membrane. In human colon epithelial cells, Zhang and colleagues (28) proposed that genistein downregulates Wnt signaling through increased expression of soluble inhibitory factors called secreted frizzled related proteins (sFRP). Therefore, we hypothesized that dietary intervention with soy may prevent EtOH-mediated tumor-promoting effects by inhibiting β-catenin signaling and associated proliferation mechanisms. To test this hypothesis, we utilized a two-stage mouse model in which tumor initiation by a genotoxic compound, diethylnitrosamine (DEN), was followed by the promoting agent, an EtOH liquid diet (7, 13, 16), containing either casein or soy protein isolate (SPI) as the sole protein source. Administration of these diets continued for 16 weeks, at which livers were analyzed for the presence of adenomas.

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences (UAMS; Little Rock, AR). Mice were housed in an Association Assessment and Accreditation of Laboratory Animal Care–approved animal facility. Male and female C57Bl/6 mice (Jackson Laboratories) were used to establish a breeding colony to generate male pups, who received either a single intraperitoneal injection of 10 mg/kg DEN (n = 54) or saline (n = 25) on postnatal day 14. Mice were weaned to and maintained on rodent chow (Harlan Laboratories) until postnatal day 60, at which the DEN-injected mice were randomly assigned to three weight-matched diet groups: a chow diet (n = 10, chow), an EtOH-containing liquid diet (n = 21, EtOH), and an EtOH-containing liquid diet containing soy protein isolate, SPI, (n = 23, EtOH/SPI). All groups had access to water ad libitum. Liquid diets were formulated according to the Lieber De Carli diet of 35% of energy from fat, 18% from protein, and 47% from carbohydrates (Dyets, Inc.), with SPI (Dupont Nutrition & Health) replacing casein as the sole protein source in the EtOH/SPI liquid diet. EtOH was added to the Lieber-De-Carli liquid diet slowly by substituting EtOH for carbohydrate calories in a stepwise manner until 28% total calories were reached as described previously (13). This dose constitutes a final EtOH concentration of 4.9% (v/v), respectively, and was maintained until sacrifice (4 months), at which livers were fixed in formalin for pathologic evaluation. At postnatal day 60, an additional group of saline-injected mice were randomized into three liquid diet groups, a chow diet (n = 5), an EtOH (n = 10), an EtOH/SPI (n = 10). Diets were maintained for 4 months as described above. At sacrifice, liver pieces were flash frozen in liquid nitrogen and stored at −70°C. Total serum isoflavone concentrations were extracted and analyzed as described previously (29). Serum alanine aminotransferase (ALT) levels were assessed as a measure of liver damage by using the Infinity ALT liquid stable reagent (Thermo Electron) according to manufacturer's protocols.

All formalin-fixed liver lobes from DEN-treated mice receiving chow, EtOH, or EtOH/SPI diets were embedded in paraffin, sectioned (4 μm), stained, and examined in a blinded fashion under a light microscope as described previously (13) by two independent pathologists (L. Hennings and K. Lai). Preneoplastic foci were counted at 40× magnification. Preneoplastic foci can develop into adenomas, which were defined as a compressive lesion of any size without evidence of invasion or other criteria of malignancy, and may develop into hepatocellular carcinoma, which was defined as a compressive and invasive lesion with criteria of malignancy (30).

Archival liver tissue sections from deidentified patients diagnosed with alcoholic steatohepatitis (n = 5) or alcoholic cirrhosis (n = 7) were obtained from the UAMS Department of Pathology. β-Catenin expression was assessed in liver sections by IHC using standard procedures and a monoclonal β-catenin antibody (1:75) detecting the active, dephosphorylated (Ser37 or Thr41) form (anti-active-β-catenin, clone 8E7, EMD Millipore), as described previously (13). Hepatic proliferating cell nuclear antigen (PCNA) staining was assessed by IHC in nontumor tissue in chow, EtOH, and EtOH/SPI fed mice as described previously (13). Stained slides were evaluated under a light microscope. Nuclei of S-phase cells stained dark brown; a total of 10 observations (100× field counted) were screened per liver sample. Data are expressed as a percentage of PCNA-stained nuclei in S-phase.

Liver 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 instructions, and subsequent real-time PCR analysis was carried out using SYBR Green and an ABI 7500 Sequence Detection System (Applied Biosystems). Results were quantified using the ΔC_t method relative to 18s or Gapdh. Primer sequences are presented in Supplementary Table S1.

Nuclear and cytosolic protein fractions were isolated from TEN control and EtOH-treated mouse livers using NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Fisher Scientific). Membrane fractions were obtained using a modified protocol as described previously (31). Proteins (30 μg) were separated by SDS-PAGE using standard methods. Blotted cytosolic proteins were incubated with the following antibodies, pAKT, total AKT, pGSK3β (phospho-GSK-3α/β (Ser21/9) and total GSK3β, from Cell Signaling Technology using a 1:1,000 dilution. Antibodies toward β-catenin (1:1,000, Milipore), and pNF-κB (Cell Signaling Technology) were used on blotted nuclear proteins. Membrane fractions were probed with an anti-SPHK1 antibody (1:1,000, Cell Signaling Technology). Secondary antibodies were diluted (1:5,000 to 1:10,000) and incubated at room temperature before chemiluminescence detection. Protein bands were quantified using a densitometer and band densities were corrected for total protein loaded by staining the membrane with 0.1% amido black.

Matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) imaging mass spectrometry was used to qualitatively detect sphingolipid species in liver sections from saline-treated mice receiving an EtOH or EtOH/SPI diet using methods as described previously (32). Briefly, the left lateral liver lobe (n = 1 per diet group) was rapidly frozen in liquid nitrogen for 2 minutes and stored at −80°C until use. Liver tissues were sectioned at 10 μm, thaw mounted on indium tin oxide–coated slides (Bruker Daltonics), and desiccated at room temperature for 5 minutes. A 2,5-dihydroxybenzoic acid matrix was added using an ImagePrep spray station (Bruker Daltonics), at a concentration of 0.2 mol/L in 50% methanol and 0.01% trifluoroacetic acid. MALDI imaging mass spectrometry analysis was performed using a Bruker Solarix 7T FTICR mass spectrometer, equipped with a SmartBeam II laser operating at 1,000 Hz, collecting spectra across the entire tissue in positive ion mode between (m/z 200–2000). A laser spot size of 25 μm, and a resolution or raster width of 200 μm was utilized for analysis, collecting 800 shots per pixel. Data were reduced to 0.98 ICR reduction and loaded into FlexImaging 4.0 software (Bruker Daltonics) for data analysis, and generation of lipid images of interest. Within FlexImaging, all data was normalized using root mean square and intensities were thresholded appropriately. Because of the high mass accuracy and resolution capability of a 7T FTICR mass spectrometer used in this study, lipid species were identified by mass accuracy in reference to an internal ceramide database (32) and to an external database known as Lipid Maps.

Data are presented as means ± SE. Comparisons between three groups were accomplished by one-way ANOVA followed by a Student–Newman–Keuls post hoc analysis or by Kruskal–Wallis one-way ANOVA on ranks followed by Dunn post hoc analysis. Comparisons between two groups were accomplished using either Student t test or Mann–Whitney U rank sum test. Number of lesions was compared across groups using negative binomial regression which generalizes Poisson regression to account for overdispersion of the count data. This model included group membership as a set of indicator (dummy) variables. The proportion of new adenomas (incidence) was compared across groups using Fisher exact test. Statistical analysis was performed using the SigmaPlot software package 11.0 (Systat Software, Inc.) and Stata statistical software 13.1 (Stata Corporation). Statistical significance was set at P < 0.05.

Male mice were assigned to an EtOH or EtOH/SPI liquid diet, and a chow-fed diet 43 days post-DEN injection. Starting weights 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 intakes between the EtOH and EtOH/SPI groups did not differ statistically, 15.4 ± 0.12 mL and 15.2 ± 0.09 mL, respectively, which corresponded 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, respectively, and corresponded to an average daily intake of 19.7 g/kg/d of EtOH, which resulted in a mean (±SE) blood alcohol concentration of 29 ± 7.2 mg/dL and 23 ± 4.3 mg/dL for EtOH and EtOH/SPI groups, respectively. Total circulating isoflavone concentrations (aglycone+conjugates) were measured in serum of DEN- and saline-treated mice receiving the EtOH/SPI diet, as described previously (28). Equol, daidzein, genistein, and o-desmethylangolensin were present at the following concentrations, 1.15 ± 0.14, 0.13 ± 0.05, 0.05 ± 0.02, and 0.08 ± 0.02 μmol/L, respectively, and did not differ between the two groups.

Liver lobes taken from DEN-treated chow, EtOH, and EtOH/SPI mice were assessed for presence of lesions, which encompassed basophilic foci and adenomas. Basophilic incidence in the EtOH, 0.90 (19/21), and EtOH/SPI, 0.95 (22/23), groups were increased by 2-fold compared with the chow, 0.5 (5/10) fed mice (Fisher exact test, P < 0.05). Likewise, basophilic multiplicity in the EtOH and the EtOH/SPI groups were also significantly increased compared with chow, 4.6 (88/21), 3.8 (88/23), and 1.7 (17/10), respectively (one-way ANOVA followed by Mann–Whitney U rank sum test, P < 0.05). We also observed the presence of adenomas in the DEN-treated EtOH, 0.67 (14/21), which were absent in the DEN-treated chow, 0.0 (0/10) group (Fisher exact test, P < 0.05). When compared with the EtOH group, SPI feeding reduced adenoma incidence, 0.23 (6/23) in the DEN-treated EtOH/SPI (Fisher exact test, P < 0.05). Likewise, we observed a significant 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).

SPI substitution for casein protein in the EtOH diet had a marked impact on liver pathology in the DEN-treated mice. As seen in Fig. 1, chronic consumption of the DEN/EtOH diet resulted in elevated serum ALT concentrations which corresponded to histologic evidence of steatosis and inflammation and increased hepatocyte proliferation in nontumorigenic tissue when compared with the DEN/chow–fed group (P < 0.05). In contrast, EtOH/SPI feeding resulted in a significant reduction in serum ALT, decreased overall pathology, and a reduction in proliferation in comparison with the EtOH-treated mice (Fig. 1). Mechanisms associated with decreased liver pathology were assessed in hepatic tissue taken from saline-treated mice receiving chow, EtOH, and EtOH/SPI diets concurrently with the DEN-treated mice. Similar to the DEN-treated mice, the EtOH/SPI diet decreased 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, and chow-fed groups, respectively (P < 0.05)]. Likewise, in Fig. 2, we observed a 47% and 68% decrease in hepatic Tnfα and Interleukin 6 (Il6) mRNA expression in the EtOH/SPI group compared with the EtOH group. Hepatic liver transcripts of Cd14 and chemokine (C-X-C motif) 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 accumulation of the proinflammatory transcription factor, NF-κB, in the nucleus in the EtOH/SPI group (Fig. 2E, P < 0.05). In addition, fibrosis markers were also decreased in the livers of EtOH/SPI–treated mice. Alpha-smooth muscle actin (α-Sma) and platelet-derived growth factor receptor (Pdgfr) were significantly suppressed in comparison with both the EtOH-treated mice and chow controls (3.48 ± 1.17 vs. 0.31 ± 0.07 and 0.27 ± 0.04 for α-Sma in EtOH, EtOH/SPI, and chow controls, respectively, and 1.48 ± 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).

Clinically, a significant percentage of liver tumors from alcoholics are positive for β-catenin expression (17). In Fig. 3, we looked for the presence of β-catenin in archival liver tissue taken from patients diagnosed with alcoholic steatohepatitis and cirrhosis by IHC. In these tissues, we observed a pattern of increased membrane staining of β-catenin and translocation of β-catenin to the nucleus coinciding with disease progression. These data are consistent with our rodent model demonstrating that Wnt/β-catenin signaling during early stages of steatohepatitis (13), and in the clinical setting, increased activation in cirrhotic livers, where HCC generally occurs in humans.

Previously, we have reported chronic EtOH feeding in rats promotes hepatocyte proliferation through increased β-catenin activation via the Wnt signaling pathway (13). As seen in Table 1, we observed a 3-fold increase in wingless-type MMTV integration site family, member 2 (Wnt2) mRNA expression, corresponding to approximately a 2-fold increase in the ratio of active (phosphorylated) GSK3β to total GSK3β cytosolic protein expression, and upregulation of mRNA expression of known β-catenin targets, Ccnd1, Glns, and Mmp7 in saline-treated EtOH mice compared with chow controls; SPI blocks these increases in the EtOH/SPI group (P < 0.05), including nuclear accumulation of β-catenin (P < 0.05). Consistent with these findings, in the DEN-treated mice receiving EtOH, we observed increased mRNA expression of other soluble Wnts, Wnt2b, and Wnt7a (Table 1), and SPI reduced transcript expression 16% (P = 0.02) and 34% (P = 0.08), respectively, in the EtOH/SPI group. Moreover, we observed increased mRNA expression of soluble Wnt inhibitors and dickkopf-3 (Dkk3) in the EtOH/SPI group compared with DEN-treated EtOH mice. These findings are indicative of inhibition of the canonical Wnt signaling pathway. In our model, we did not see any changes in phosphorylation status of the serine/threonine kinase AKT between saline-treated EtOH and chow-fed mice that would suggest that the survival pathway PI3K/AKT/GSK3β was responsible for EtOH-mediated activation of Wnt signaling (Table 1).

In humans and in rodents, alcohol exposure has been shown to increase ceramide accumulation in the liver, thereby enhancing endoplasmic reticulum stress and contributing to liver injury (33). Consistent with these reports, we observed a significant increase in the mRNA expression of serine palmitoyl transfease subunit (Sptlc1), a key enzyme involved in de novo ceramide biosynthesis, in the EtOH-treated mice compared with chow-fed mice (Fig. 4A). Ceramide synthase (Cers) gene transcripts were also 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 increased mRNA expression 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 the EtOH/SPI–treated mice, Sptlc1 mRNA expression was increased compared with the EtOH mice, but did not reach significance (P = 0.079). However, we did see a significant decrease in Cers1 mRNA expression in response to EtOH/SPI diets, but no other significant decreases with respect to Cers2, 4, 5, and 6 expression (Fig. 4B; P < 0.05). In contrast, SPI did shut down sphingosine-1P production, including the mRNA expression and translocation of Sphk1 to the membrane which is necessary for enzymatic activation (Fig. 4F; P < 0.05). Mechanistically, Cers1 predominately generates C18:0-ceramide, Cers2 produces longer acyl chain ceramides (C20:0—C26:0 ceramides), and Cers5, 6 synthesizes the shorter C14:0, C16:0 ceramide species. In Fig. 5, using a novel MALDI-FTICR imaging mass spectrometry workflow used for on-tissue detection and spatial localization of ceramides and other sphingolipids (32), we qualitatively confirmed the presence of short and long acyl chain ceramides and S1P in livers from mice receiving EtOH. We also observed a subsequent loss of ceramide C18 and sphingosine-1P in the EtOH/SPI livers, which is consistent with the mechanistic data presented in Fig. 4. 

Epidemiologic data from Asian populations eating traditional soy-based diets have suggested a variety of health benefits which include a reduction in the risk of different cancer types (21, 22, 25, 26, 34). In this study, we examined the possibility that dietary intervention with SPI would also prevent EtOH-mediated hepatic tumor promotion in a rodent model of chemical carcinogenesis (13, 16). In response to alcoholic liver injury, most rodent studies have reported a corresponding increase in hepatocyte proliferation (14, 15, 35, 36). This appears to be in response to proliferative signals such as a reduction in retinoid receptor activation and activation of β-catenin transcriptional activity downstream of WNT expression and secretion (8, 13). In the current study, EtOH-mediated hepatocyte proliferation was dependent on Wnt–β-catenin signaling. Moreover, we observed increased β-catenin staining in livers from alcoholics with steatohepatitis and cirrhosis, which supports the role of Wnt–β-catenin signaling in tumor growth and progression. Feeding SPI had a highly significant inhibitory effect on EtOH-mediated adenoma tumor progression. Inhibition of β-catenin activation by SPI feeding was accompanied by decreased phosphorylation of GSK3β, decreased expression of Wnt mRNAs, and increased expression of mRNA for the Wnt signaling inhibitor Dkk3. These data suggest that, at least in part, SPI components block EtOH-induced Wnt signaling.

The detailed molecular mechanisms underlying EtOH induction and SPI suppression of hepatic Wnt pathways remain to be elucidated. It has been suggested that Wnt secretion by nonparenchymal cells is involved in regulation of hepatocyte proliferation and in some forms of liver cancer. Studies of hepatocyte proliferation following 2/3 partial hepatectomy have implicated Wnt secretion by Kuppfer cells in this process (37). Recent studies of cholangiocarcinoma have demonstrated characteristic enhancement of Wnt signaling via inflammatory macrophages (38). EtOH promotion of hepatic tumorigenesis has also been demonstrated to be linked to induction of TGFβ signaling and stellate cell activation (7) and enhanced tumorigenesis has also been observed in other experimental models of fibrosis (39). Additional studies are underway to identify whether Kupffer cells, other inflammatory cell types, stellate cells, or endothelial cells are the cellular source of EtOH-induced Wnts.

SPI feeding also significantly lowered necroinflammatory injury and Kupffer cell recruitment in the EtOH/SPI group. The hepatoprotective effects of SPI were coincident with reduced mRNA expression of Il6, decreased hepatocyte proliferation, and evidence of reduced β-catenin activation. These data support a mechanistic link between soy's inhibitory effects on EtOH-dependent tumor promotion, reductions in Wnt-β-catenin signaling, and reduced liver injury. Genistein, the major isoflavone phytoestrogen, is considered the biologically active anticancer component in soy foods (27, 28, 40, 41). Recently, using the same model reported here, we have found increased EtOH-mediated hepatic adenomas in male mice receiving genistein in the liquid EtOH diet (42). Our findings suggest that SPI contains additional anticancer components. Supporting evidence for this hypothesis comes from a recent report demonstrating feeding of an isoflavone-free SPI diet specifically inhibited NF-κB–dependent expression of inflammatory cytokines, including Tnfα in the hyperlipidemic apoE^−/− mouse model, thus reducing the presence of atherosclerotic lesions (43). These findings suggest that soy hydrolysates and putative peptides derived from digestion of the major soy storage protein β-conglycinin in the gut may have anti-inflammatory properties.

Recent reports have implied that chronic alcohol consumption influences hepatic ceramide generation (33, 44). In this study, we observed significant elevations of hepatic ceramide synthesis, particularly C18 generation, and S1P production in response to chronic EtOH consumption. These data are consistent with those reported by Longato and colleagues (33) in chronic alcoholics and from our laboratory using an intragastric EtOH treatment in a rat model associated with hepatic insulin resistance (44). In this study, we did not explore the alternative ceramide generation pathways, including complex sphingolipid hydrolysis or through a salvage pathway involving ceramide deacylation (45). However, Longato and colleagues (33) observed increased sphingomyelin hydrolysis in the hepatic tissue of chronic alcoholics, suggesting that alcohol may affect additional pathways of ceramide synthesis.

Interestingly, EtOH-mediated increases in C18 ceramide and S1P were reversed in the EtOH/SPI group. Different ceramide species have diverse effects on cell death and proliferation (46). Ceramide is converted to S1P, an important proinflammatory and profibrotic lipid mediator, via the actions of ceramidases and sphingosine kinases. S1P acts via 5 receptors to stimulate inflammation both up- and downstream of TNFα and to stimulate fibrosis via activation of stellate cells (24, 46, 47). Moreover, S1P has antiapoptotic actions and paracrine proliferative effects on hepatocytes and pancreatic cancer cells mediated via endothelial and stellate cells (46). It is possible that SPI inhibition of ceramide/S1P synthesis is involved in blockade of Wnt/β-catenin signaling, particularly if activated stellate cells are a source of Wnts. Alternatively, SPI may affect other Wnt-independent pathways impacting on tumor promotion such as expansion of tumor-initiating stem cell populations or on apoptosis/proliferation balance in transformed cell populations. The role of ceramides and S1P in EtOH-induced hepatic tumor promotion and protection by feeding of SPI are also the subject of ongoing studies.

In conclusion, our data demonstrate that EtOH acts as a tumor promoter in the mouse DEN model as a result of activation of Wnt/β-catenin signaling. Protection against EtOH-induced tumorigenesis by switching from casein to SPI as the dietary protein source appears to be due to a component other than genistein which acts to block steatosis and progression of liver injury, reverses EtOH activation of β-catenin, and prevents EtOH-induced changes in ceramide/sphingosine metabolism.

M. Ronis received speakers bureau honoraria from and is a consultant/advisory board member for DuPont Nutrition and Health. No potential conflicts of interest were disclosed by the other authors.

Conception and design: K.E. Mercer, M. Ronis

Development of methodology: K.E. Mercer, L. Hennings, R.R. Drake, M. Ronis

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.E. Mercerm, K. Lai, E. Jones, R.R. Drake

Analysis 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. Ronis

Writing, review, and/or revision of the manuscript: K.E. Mercer, L. Hennings, K. Lai, R.R. Drake, M. Ronis

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Pulliam

Study supervision: R.R. Drake, M. Ronis

This work was funded in part by NIH grant R21 CA169389 (to M. Ronis) and by the Arkansas Children's Hospital Research Institute and the Marion B. Lyon New Scientist Development Award (to K.E. Mercer).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.