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,3
* Program in Nutrition and Health Science and
Department of Pathology and Division of Animal Resources, Emory University, Atlanta, GA 30322;
School of Biology and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332; and
** Karmanos Cancer Institute, Detroit, MI 48201
3To whom correspondence should be addressed. E-mail: Al.Merrill{at}biology.gatech.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-hydroxy-palmitic acid], which might affect their bioactivity. Soy GlcCer was added to the AIN-76A diet (which contains <0.005% sphingolipid) to investigate whether it would also suppress tumorigenesis in these mouse models. Soy GlcCer reduced colonic cell proliferation in the upper half of the crypts in mice treated with DMH by 50 and 56% (P < 0.05) at 0.025 and 0.1% of the diet (wt/wt), respectively, and reduced the number of aberrant colonic crypt foci (an early marker of colon carcinogenesis) by 38 and 52% (P < 0.05). Min mice fed diets containing 0.025 and 0.1% (wt/wt) soy GlcCer developed 22 and 37% fewer adenomas (P < 0.05), respectively. The effects of dietary sphingolipids on gene expression in the intestinal mucosal cells of Min mice were analyzed using Affymetrix GeneChip microarrays. Soy GlcCer affected the expression of 96 genes by 
2-fold in a dose-dependent manner, increasing 32 and decreasing 64. Decreases in the mRNA expression of two transcription factors associated with cancer, hypoxia-induced factor 1 (HIF1) and transcription factor 4 (TCF4), were confirmed by quantitative RT-PCR. In conclusion, soy GlcCer suppressed colon tumorigenesis in two mouse models; hence, plant sphingolipids warrant further investigation as inhibitors of colon cancer. Because soy contains relatively high amounts of GlcCer, sphingolipids may partially account for the anticancer benefits attributed to soy-based foods.
KEY WORDS: • sphingolipids • soy • gene expression • colon cancer • mouse models
Sphingolipids are found in many foods, and it is estimated that individuals consume 
0.3 to 0.4 g/d (1). Digestion of sphingolipids releases the backbone ceramides and sphingoid bases (2–5), bioactive molecules that inhibit the growth of cancer cells and induce differentiation and apoptosis in vitro (6). Sphingolipids might therefore serve as bioactive dietary components to suppress carcinogenesis (7). The cellular targets of the ceramide and sphingoid sphenoid bases are numerous, and include intracellular signaling pathways [reviewed in Cuvillier (8) and Pettus et al. (9)] involving protein kinase C (PKC)4 (10–12), phosphoinositol-3 kinase (13–16), and cell cycle regulatory proteins (17,18).
Milk sphingolipids inhibit both the early and late stages of colon carcinogenesis in mice in which tumorigenesis is chemically induced by 1,2-dimethylhydrazine (DMH) (19–23) or caused by an inherited genetic defect (24), as in mice of the multiple intestinal neoplasia (Min) strain, which have a mutation in the adenomatous polyposis coli (APC) gene (25). A wide spectrum of milk sphingolipids that differ in the polar headgroup (sphingomyelin, glucosylceramide, lactosylceramide, and ganglioside GD3) suppress tumorigenesis; however, there are no reports on sphingolipids with ceramide backbones not found in mammals. For example, many plant sphingolipids, such as soy glucosylceramide (GlcCer; Fig. 1), have a backbone of 4,8-sphingadiene rather than sphingosine (4-sphingenine), and incorporate an -hydroxy-fatty acid (26). Because studies with cell cultures report that the biological activity of sphingolipids varies with the backbone structure (27), it is not known a priori whether plant sphingolipids will also inhibit colon carcinogenesis. To address this question, soy GlcCer was evaluated in two models of colon carcinogenesis: DMH-induced carcinoma in CF1 mice and spontaneous tumorigenesis in Min mice.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experimental diets and carcinogen treatment. The mice were randomly assigned to the experimental groups upon arrival (10 mice/group). After 1 wk of acclimatization, the mice were fed a semipurified AIN-76A diet (Dyets) (28) and were administered weekly i.p. injections of DMH (30 mg/kg body wt in 1 mmol/L EDTA; Sigma) for 6 wk. The AIN-76A diet is essentially sphingolipid-free (19). After a washout period of 1 wk, the mice in the experimental groups were fed diets supplemented with 0.025 or 0.1% (wt/wt) soy GlcCer (Avanti Polarlipids), and the control group was fed the AIN 76A diet without supplementation. The soy GlcCer was certified to be >99% pure; this was confirmed by mass spectrometry (26).
Aberrant colonic crypt analysis.
After 4 wk of dietary treatment, the mice were killed by CO2 asphyxiation, and the colons were excised, opened longitudinally, rinsed, and fixed overnight in fresh 10% buffered formalin. The aberrant crypt foci (ACF) were stained with methylene blue solution (20 g/L) for 5 min and evaluated by light microscopy at 40X magnification in a blind scoring procedure.
Bromodeoxyuridine staining of proliferating cells in situ. On the last day of the study, 5 mice/group (treated as described above) were injected i.p. with 10 mL/kg body wt of bromodeoxyuridine (BrdU) solution (In Situ Cell Proliferation Kit 1758756; Boehringer Mannheim) to label proliferating cells during the S phase. After 2 h, the mice were killed by CO2 asphyxiation, and the colons were removed, opened longitudinally, flushed with cold PBS, and fixed overnight in 10% neutral buffered formalin. The number and size of the ACF were recorded, and the tissues were embedded in paraffin. Sections of 3 to 5 µm were deparaffinized with fresh xylene and rehydrated with graded alcohol (100, 95, 80, 50, and 30%). Per the kit manufacturer’s instructions, the colon sections were incubated with an alkaline phosphatase-conjugated anti-BrdU monoclonal antibody to detect incorporated BrdU. This antibody was made visible with Fast Red, which forms an insoluble colored precipitate at the site of the immunocomplex. The sections were covered with an aqueous mounting solution and examined by light microscopy at 10 or 40X in a blind scoring procedure (30 fully visible crypts were scored per mouse). The proliferation index was calculated as the percentage of BrdU-positive cells in the crypt, measured by counting the positive cells.
Analysis of adenomas in Min mice. The mice were separated into experimental groups to achieve a similar initial mean weight and housed in microisolator cages (5 mice/cage). The control group was fed the AIN-76A diet, and the experimental groups were fed the same diet supplemented with 0.025 or 0.1% soy GlcCer. The diets were mixed fresh every 2 wk and stored at 4°C in airtight containers. At 100 d of age (after 8 wk of sphingolipid treatment), the mice were killed by CO2 asphyxiation. Blood was immediately drawn by heart puncture, collected in heparin-treated Eppendorff tubes, and snap-frozen for later analyses. The intestines were excised, opened longitudinally, flushed with ice-cold PBS, and fixed flat overnight in 10% neutral buffered formalin. All sections of the intestine were examined by light microscopy, and tumor number and size were documented in a blind scoring procedure. Analysis of gene expression.
After 8 wk of sphingolipid treatment, the Min mice were killed by CO2 asphyxiation. The intestines were immediately excised, opened longitudinally, and flushed with ice-cold PBS to remove any intestinal contents. An RNase-free glass microscope slide was used to gently scrape intestinal mucosa. Samples were transferred by pipette into TRIzol Reagent (Life Technologies) to isolate total RNA, then purified using Rneasy columns (Qiagen). The total RNA from 5 mice in each group was then pooled in equal amounts for further analysis. Double-stranded cDNA was synthesized with the Superscript Choice System for cDNA (Kit #18090–019; Invitrogen Life Technologies) using a T7-(dT)24 primer (GenSet). Using the BioArray High Yield RNA transcription kit with T7 RNA polymerase (Enzo Diagnostics), the cDNA was transcribed in vitro to produce biotin-labeled cRNA. The cRNA was then hybridized with the GeneChip Murine Genome U74A Array (Affymetrix) according to the Affymetrix protocol. The microarray data were analyzed with Microarray Suite 5.0 (Affymetrix) and Gene Microarray Pathway Profiler (Gladstone Institute).
Quantitative RT-PCR confirmation of gene expression.
Complementary DNA was synthesized with TaqMan Reverse Transcription reagents (Applied Biosystems), using random hexamers in a total volume of 40 µL containing 0.5 µg total RNA. The HIF1 gene was amplified by real-time PCR. The real-time quantitative PCR procedure involves continuous optical monitoring of the reaction with a fluorescent signal. The signal was generated by the double-strand DNA interchelator SYBR Green I dye (Applied Biosystem). The fluorogenic PCR procedure was done in a total reaction volume of 25 µL, containing 2 µL cDNA, 1.5 µL (5 µmol/L) each of the forward [HIF1:(5'-ATGGTAGCCACAATTGCAC-3') TCF4:(5'-AATGTTACCACTGTGCCTTC-3') VEGF (5'-CTGCTGTCTTGGGTGCATTG-3') ß-actin:(5'-TCCTGTGGCATCCACGAAACT-3')] and reverse [HIF1(5'-CTTCATGATCCAGGCTTAAC-3') TCF4(5'-CACACTACTTCGGCTACACA) VEGF (5'-TTCACATTTGTTGTGCTGTAG-3') ß-actin (5'-GAAGCATTTGCGGYGGACGAT)] primers (Qiagen-Operon), 12.5 µL SYBR Green I PCR master mix (Applied Biosystems), and 7.5µL RNase-free H2O. The mixtures were preheated at 95°C for 10 min, then cycled 40 times at 95°C for 20 sec, 55°C (58°C for ß-actin) for 40 sec, and 72°C for 40 sec in an iCycler iQ Real-Time Detection System (Bio-Rad). All data were normalized using ß-actin.
Statistical methods. Instat software (Instat) was used for all statistical analyses. Differences in the number and size of ACF and weight were determined by ANOVA and post-hoc multiple t tests. Linear correlation analysis (parametric Pearson correlation) was used to evaluate correlations. All data were expressed as means ± SEM. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Inhibition of ACF formation in DMH-treated CF1 mice. Dietary soy GlcCer supplementation significantly reduced the number of ACF in DMH-treated CF1 mice (Table 1). The suppression levels were 38% (P < 0.05) and 52% (P < 0.01) for 0.025 and 0.1% GlcCer, respectively. Crypt multiplicity did not differ between the control and experimental mice (Table 1).
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30% (P < 0.001). The sphingolipid treatment had a greater effect on the upper half of the crypt; the 0.025 and 0.1% GlcCer supplementation reduced the proliferation index by 50 and 56%, respectively, compared to the control group (P < 0.001; Table 1). There was a smaller but significant reduction in the proliferative index in the lower half of the crypt (19 and 16% reduction for 0.025 and 0.1% GlcCer, respectively; P < 0.05; Table 1). Inhibition of adenoma formation in Min mice. Min mice fed the AIN-76A diet alone developed 44.2 ± 2.5 tumors per mouse. Supplementation with soy GlcCer reduced the number of tumors by 27% (P < 0.05) and 40% (P < 0.01) in the mice fed 0.025 and 0.1% GlcCer, respectively (Table 2).
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This survey was intended to identify candidates for further study, not to characterize all changes in gene expression. Two of the genes that appeared to change were relevant to carcinogenesis (HIF1 and TCF4), and the changes in these genes were confirmed by quantitative RT-PCR (QRT-PCR; Fig. 2). According to the Affymetrix GeneChip analysis, HIF1 expression decreased by 62 and 53% in mice fed 0.025 and 0.1% soy GlcCer, respectively; by QRT-PCR analysis the decreases were remarkably similar (60 ± 10 and 53 ± 20%, respectively, compared to the control mice; P < 0.05, n = 5). Expression of TCF4 decreased by 71 and 78% in mice fed 0.025 and 0.1% soy GlcCer, respectively, according to the Affymetrix GeneChip analysis; by QRT-PCR analysis, the decreases were 56 ± 20 and 59 ± 7%, respectively; P < 0.05).5
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expression was reduced, the expression of a related gene that also regulates angiogenesis, the vascular endothelial growth factor (VEGF) gene, was also examined by QRT-PCR. Expression of VEGF decreased by 24 ± 2 and 33 ± 4% in mice fed 0.025 and 0.1% soy GlcCer, respectively, although these decreases were only marginally significant (P = 0.09). The data from the Affymetrix GeneChip microarray were analyzed by GenMAPP to identify any recognizable patterns in the changes in gene expression related to major biochemical pathways. This program groups genes by various principles, including metabolic pathway, signal transduction cascade, gene family, and subcellular component. There were no detectable differential changes or patterns of related pathways or gene families.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-hydroxy-fatty acid of soy GlcCer (26), affect the biologic activity. The findings with soy GlcCer in 2 mouse models established that sphingolipids of plant origin can suppress colon cancer.
Sphingolipids with three different sphingoid-base backbones [sphinganine (d18:0), sphingosine (
4d18:1), and sphingadiene (4,8d18:2)] inhibit colon carcinogenesis in vivo. In addition, a synthetic analogue without the 1-hydroxyl group of sphinganine suppressed ACF formation (29). These findings are intriguing because studies with cells in vitro report that only ceramides with a 4 double bond (27) or 4-hydroxyl (i.e., phytoceramides) (30) induce apoptosis. In contrast, free sphingoid bases of a wide range of structural types also are cytotoxic (31); therefore, it is possible that the sphingoid bases that are produced by digestion of sphingolipids, rather than ceramides, were responsible for the suppression of tumorigenesis in the in vivo models used in these studies.
The specific targets of the dietary sphingolipids are unclear, because sphingolipids affect several cellular signaling pathways that might account for their anticarcinogenic properties, such as PKC (which is inhibited) (10–12); phosphoprotein phosphatases PP2A and PP1 (which are activated) (15,16,32); retinoblastoma protein (which undergoes dephosphorylation) (33,34); cell cycle regulatory proteins p21, p27, and p57 (which are upregulated) (17,18); cyclin-dependent kinases (which are downregulated) (15); and telomerase (which is inhibited) (35); as well as many components of cellular apoptotic pathways, such as cytochrome c release (36), caspase activation (37,38), and modulation of bcl-2 family members (39,40). In addition to their role in signaling, it is also possible that sphingolipids reduce the absorption of cocarcinogens or increase the delivery of other anticarcinogenic compounds to the colon (41).
Of particular interest with respect to colon cancer is the finding that sphingolipids normalize ß-catenin localization in intestinal epithelial cells in vivo as well as decreasing cytosolic and nuclear ß-catenin in colon cancer cells in vitro (24). One possible explanation for this normalization of ß-catenin is the inhibition of PKC by sphingoid bases (10), especially considering that PKCßII is overexpressed in colon cancer (42) and may contribute to the abnormal turnover of ß-catenin by inhibiting glycogen synthase kinase-3ß, an enzyme responsible for the phosphorylation of ß-catenin to activate ubiquitination and proteosomal degradation (43). Therefore, suppression of PKC by sphingoid bases might increase ß-catenin turnover. However, this is not the only pathway by which sphingoid bases induce ß-catenin turnover, because they also activate caspases (44–46), including caspase 3, which can degrade ß-catenin in early apoptosis (47).
These new studies also found that dietary sphingolipid supplementation reduces the expression of TCF4. This transcription factor is a component of the Wnt signaling pathway and binds ß-catenin to activate the expression of proliferative genes (48). Hence, reductions in ß-catenin are usually presumed to suppress proliferation via loss of the ß-catenin partner of the ß-catenin-TCF4 complex. The effects of sphingolipids apparently occur at two levels, reducing both ß-catenin and TCF4 expression.
Another unexpected finding was that additional genes that are important in carcinogenesis are affected by sphingolipid consumption, namely a reduction in the expression of HIF1, a transcription factor that enables tumors to adapt to hypoxic environments by activating survival genes. The HIF1 gene is frequently overexpressed in colon tumors (49), and regulation often occurs at the level of protein degradation; however, studies document the regulation of HIF1 mRNA amounts (50,51). One of the ways that HIF1 affects angiogenesis is by regulating VEGF expression, which may also be reduced somewhat (25 to 35%) by sphingolipid consumption, although further studies are needed to verify the small changes in VEGF mRNA expression.
The amounts of soy GlcCer that were used in these studies are similar to those naturally found in soybeans (1), as was also the case in the studies of sphingolipids derived from dairy products (19–24); therefore, it is plausible that some diets contain sphingolipids in amounts that could affect colon cancer risk. It is also possible that sphingolipids partially account for the cancer-suppressing effects attributed to foods that are rich in them, such as soybeans. Epidemiologic studies of diet and cancer that include sphingolipid content as one of the analyzed variables are needed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by National Cancer Institute grants CA73327 and CA87525.
4 Abbreviations used: ACF, aberrant crypt foci; APC, adenomatous polyposis coli; BrdU, bromodeoxyuridine; DMH, 1,2-dimethylhydrazine; GlcCer, glucosylceramide; HIF1, hypoxia-induced factor 1; Min, multiple intestinal neoplasia; PKC, protein kinase C; QRT-PCR, quantitative reverse transcriptase–polymerase chain reaction; TCF4, transcription factor 4; VEGF, vascular endothelial growth factor.
5 Because the RNA samples were pooled for analysis on the GeneChip microarray, the results were confirmed by QRT-PCR analysis of the 5 individual samples to obtain the mean ± SEM.
Manuscript received 23 December 2003. Initial review completed 22 January 2004. Revision accepted 17 February 2004.
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