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Departments of
*
Biochemistry and
Pathology and Animal Resources, Emory University, Atlanta, GA 30322
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • proliferation • sphingomyelin • glycosphingolipids • colon cancer • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The backbone moieties of sphingolipids [ceramide (Cer) and
sphingosine] are potent inhibitors of cell growth, and induce
differentiation and apoptosis (see reviews Spiegel and Merrill 1996
,
Merrill et al. 1997
). Since these processes are deregulated in
carcinogenesis and sphingolipids are abundant in some foods
(Vesper et al. 1999
), dietary sphingolipids are
promising candidates for cancer chemoprevention.
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), inhibits
an early stage of colon carcinogenesis in mice (the formation of
aberrant crypt foci, ACF) and decreases the portion of tumors that
appear as malignant adenocarcinomas vs. benign adenomas
(Dillehay et al. 1994
, Schmelz et al. 1996
). However, it is not known if the other major category of
sphingolipids (glycosphingolipids) has similar effects. To address this
question, glucosylceramide (GluCer), lactosylceramide (LacCer) and
ganglioside GD3 (GD3) were
fed to 1,2-dimethylhydrazine (DMH)-treated CF1 mice; all suppressed ACF
formation and reduced proliferation in the colonic crypts. Thus,
glycosphingolipids also can suppress key stages of colon
carcinogenesis. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female CF1 mice were obtained from Charles Rivers Laboratories (Portage, MI) at 5 wk of age. They were housed in microisolator cages (five mice per cage), and were maintained in a relative humidity of 50–60%, a temperature of 23°C and a 12-h light/dark cycle. The mice had free access to water and were weighed weekly and monitored closely for signs of illness. All protocols involving animals were approved by the Institutional Animal Care and Use Committee and conducted according to National Research Council Guidelines.
Sphingolipids.
The glycosphingolipids (extracted from buttermilk) were purchased from Matreya (Pleasant Gap, PA). The purity was first determined by thin layer chromatography, and only one band on the plates was visible after exposure to iodine or ninhydrin when 100 µg were analyzed (developed in chloroform/methanol/ formic acid/water, 56:30:4:2, v/v/v/v). Next, mass spectrometric analyses (as described below) were employed to evaluate purity and provide detailed information about the structure of the sphingolipids.
Mass spectrometric analyses were performed on a Perkin Elmer-Sciex (Norwalk, CT) API 3000 triple quadrupole mass spectrometer, equipped with a turbo-ionspray source. Solutions of sphingolipids (~0.5 µmol/L in 5 mmol/L of ammonium acetate in methanol and 10 g/L of acetic acid) were infused at a flow rate of 5 µL · min-1. The ionspray needle was held at 5500 V while the inlet voltage was kept at < 50 V to minimize collisional activation of molecular ions prior to entry into the first quadrupole.
Precursor ions spectra were acquired by scanning Q1 over a 200 u mass range around the estimated molecular mass of each sphingolipid in 0.1 u steps with a dwell time of 1.0 ms. Q2 was offset from Q1 by 50, 60 and 90 eV, respectively, for GluCer, LacCer and GD3, to maximize formation of molecularly distinctive product ions of m/z 264.2. Q3 was then set to pass product ions of m/z 264.2. Data were acquired for a total of 5 min; thus, each spectrum is the signal average of 150 scans. Relative ion abundances were calculated using the highest raw signal count from each molecular species.
Experimental diets and carcinogen treatment.
The mice were randomly divided into groups upon arrival. After
acclimation for 5 d, they were injected i.p. with
1,2-dimethylhydrazine·HCl (DMH, 30 mg/kg body weight) in 1
mmol/L of EDTA (Sigma Chemical, St. Louis, MO) once a week for 6 wk.
During this period of tumor initiation, the mice were fed a
semipurified AIN 76A diet (PMI Feeds, Richmond, IN) (American Institute of Nutrition 1977
) that is essentially sphingolipid-free
(Schmelz et al. 1996
). The AIN 76A diet was selected
over newer formulations (AIN 93G and 93M) because these have replaced
corn oil with soy oil, and soy is a rich source of sphingolipids
(Vesper et al. 1999
).
One week after the last injection, the mice were fed the AIN 76A diet alone (control group) or supplemented with 0.025 or 0.1 g/100 g of GluCer, LacCer or GD3. The diets were mixed immediately before the feeding period and kept in closed containers at 4°C. Sphingolipids are stable under these conditions. One group of mice was not injected with the carcinogen (untreated controls). These mice were fed the AIN 76A diet without sphingolipid supplements throughout the study.
Aberrant colonic crypt analysis.
The mice were killed by CO2 asphyxiation after 4 wk of feeding the experimental diets (5 wk after the last carcinogen injection). The abdominal cavity was opened, the colons were removed, rinsed with cold Tris buffer [0.05 mol/L Tris (hydroxymethyl) amino methane-HCl, pH 7.6 at 4°C], opened longitudinally and fixed flat overnight in fresh 100 g/L of neutral buffered formalin. The colonic crypts were stained with 2 g/L of methylene blue in PBS for ~20 min. The number of ACF and aberrant crypt multiplicity were determined by light microscopy at 40- or 100-fold magnification in a blinded manner.
This study was divided into two experiments (due to the number of experimental groups), each with a control group. The control group in the second experiment developed more ACF than in the first experiment (30.2 ± 4.19 vs. 16.8 ± 1.82); therefore, all ACF data are expressed as percentage inhibition of the number of ACF found in the matched control.
5-Bromo-2'-deoxyuridine (BrdU)-staining of proliferating cells in situ.
On the last day of the study, the mice were injected i.p. with a BrdU solution (10 µmol/L, 1 mL per 100 g body weight) (Boehringer Mannheim In Situ Cell Proliferation kit #1758756, Indianapolis, IN) to label proliferating cells. The mice were killed 2 h later by CO2 asphyxiation, and the tissue was processed as described above to determine the number of ACF. The colons were embedded in paraffin, and sections of 3–5 µm were cut longitudinally along the full length of the colon. The sections were deparaffinized with xylene and rehydrated through graded alcohol (100, 95, 80, 50, 30 and 0%). According to the manufacturers instructions, the colonic sections were digested with trypsin (1 g/L in PBS with 1 g/L of CaCl2), denaturated in 4 mol/L of HCl for 10 min and incubated with an alkaline phosphatase-conjugated anti-BrdU monoclonal antibody for 30 min at 37°C. This antibody complex was stained with fast red. The sections were covered with an aqueous mounting solution and analyzed by light microscopy at 10 or 40 times magnification; 50 fully visible colonic crypts per animal were scored in a blinded manner.
For comparison, colonic sections from tissue collected in an earlier
study (feeding purified milk SM, and synthetic SM to DMH-treated CF1 mice; Schmelz et al. 1997
) were included in these
immunohistochemical determinations of proliferation.
Determination of apoptosis by TUNEL assay.
The number of apoptotic cells was evaluated by TUNEL assay (In Situ cell death detection kit, POD, Boehringer Mannheim). CF1 mice were treated as described above and killed by CO2 asphyxiation. The colons were removed, cut into 3-mm segments, rinsed in PBS and fixed overnight in fresh 100 g/L of formalin. The colons were embedded in paraffin; sections of 3–5 µm were deparaffinized and rehydrated (as described above), and incubated with proteinase K (20 mg/L in 10 mmol/L of Tris-HCl, pH 8.0) for 30 min at room temperature. Endogenous peroxidase was blocked with 0.3% H2O2 in methanol for 30 min at room temperature. After treatment with a permeabilization solution (1 g/L Triton X-100 in 1 g/L of sodium citrate) for 2 min on ice, the sections were incubated with terminal deoxynucleotidyl transferase (which labels DNA strand breaks with fluorescein) for 60 min at 37°C. Incorporated fluorescein was detected by an anti-fluorescein antibody, conjugated with horseradish peroxidase (incubation for 30 min at 37°C). This complex was stained for light microscopic analysis with 3,3'-diaminobenzidine (Vector Laboratories, Burlingame, CA) for 10 min and counterstained with Mayer’s hematoxylin for 5 s. The sections were dehydrated through graded alcohol in a reverse order as described for the rehydration, cleared with xylene, and 50 fully visible crypts per mouse (200 per group) were scored by one observer in a blinded manner. Only TUNEL-positive cells with a distinct apoptotic morphology (condensed chromatin, fragmented nuclei or apoptotic bodies) were scored "apoptotic."
Analysis of glycosphingolipid hydrolysis.
Female CF1 mice were killed by CO2 asphyxiation. The abdominal cavity was opened, the colon removed, rinsed with PBS, opened longitudinally and incubated in a mixed micelle solution for the indicated times. Mixed micelles were prepared by mixing 500 µg of the individual sphingolipid (in chloroform/methanol, 1:1; v/v) with an equal amount of phosphatidylcholine (from egg yolk), and sodium cholate (final concentration 30 µmol/L) (both from Sigma), evaporation of the solvents under nitrogen, addition of 3 mL of potassium phosphate buffer (20 mmol/L, pH 6.8) and sonication of the mixture until clear. After the indicated times, 100 µL aliquots were removed, the sphingolipids solubilized with 900 µL of methanol: chloroform (1:1; v/v), sonicated, and analyzed by mass spectrometry for the disappearance of the starting material, and the appearance of the metabolites (as described below).
Mass spectrometric analyses of glycosphingolipid hydrolysis.
Multiple reaction monitoring scans were used to measure the abundances of undigested SM, GluCer, LacCer and GD3, and the appearance of the metabolites: Cer, GluCer (as a metabolite of LacCer and GD3) and LacCer (as a metabolite of GD3). Aliquots of each sample (prepared as described above) were mixed with 890 µL of 5 mmol/L of ammonium acetate in methanol, and 10 µL of glacial acetic acid. Each was injected into the mass spectrometer via a Rheodyne 8125 injector with a 20 µL injection loop at a flow rate of 50 µL min-1. Cer, GluCer and LacCer were monitored in positive ion mode with the source needle at 5500 V, while the orifice voltage was 30 V. GD3 was monitored in negative ion mode with the source needle at -4500 V and an orifice voltage of -50 V. Nitrogen was used as a nebulizing gas at a flow rate of 6 L min-1 and at 50°C. The orifice voltage and the bath gas temperature were kept low to prevent decomposition of intact precursor ions before entry into Q1.
The starting sphingolipids were first examined by mass spectrometry (MS) to determine the major ceramide backbones, which were found to be comprised of sphingosine (d18:1) with the fatty acids 16:0, 22:0, 23:0 and 24:0. Q1 was set to pass m/z 703.7, 787.9, 872.1, 886.2 for SM, m/z 700.7, 784.8, 798.8, 812.9 for GluCer, m/z 862.8, 947, 960.9, 975 for LacCer. Q3 was set to pass m/z 264.2 for the product ion from the sphingoid base (conjugated carbocation) for Cer, GluCer and LacCer. SM and GD3 were analyzed by setting Q3 to pass m/z 184.2 (the phosphocholine headgroup) and m/z 290.1 (the N-acetyl-neuraminic acid). Nitrogen was used to collisionally activate precursor ion decomposition in Q2, which was offset from Q1 by 40 eV for Cer, 50 eV for SM and GluCer, 60 eV for LacCer and 50 eV for GD3. The multiple reaction scan transitions were performed with a dwell time of 200 ms for SM, and Cer, and 50 ms for the glycosphingolipids.
The disappearance of the starting compounds (and the appearance of the metabolites) were expressed relative to the intensity of the starting compound at time zero. Because the appropriate internal standards are not available at this time, no further attempt was made to perform these analyses quantitatively. Each sphingolipid was analyzed in duplicate, with good agreement (i.e., <10% difference) in the results.
Statistical analyses All statistical analyses were executed using the Instat® software (Instat, San Diego, CA).
The animal weight and number of ACF were evaluated by Student’s
t test (ANOVA analysis, followed by Bonferroni multiple
comparison) and are shown as means ± SEM The
correlation of animal weight and number of ACF was evaluated by
regression analysis. Differences were considered significant at
P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mass spectrometric analyses of the milk glycosphingolipids showed
sphingosine as the most abundant sphingoid base backbone; sphinganine
and 4-hydroxysphinganine (phytosphingosine) were not observed in large
relative abundances (<1%). Hence, the glycosphingolipids were
analyzed by tandem MS by selecting for m/z 264 (the sphingosine-derived carbocation referred to as "N" in Adams and Ann, 1993
) as described under "METHODS". A precursor ion scan for
Glu Cer is shown in Figure 2
. The different m/z for the GluCer species are the result of
variations in the amide-linked fatty acid chains: 16:0
(m/z 700.7), 22:0 (m/z 784.9), 23:0
(m/z 798.8), 24:0 carbons (m/z 812.9) and 24:1
(m/z 810.9).
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. Most of the fatty acids were saturated with the exception of a small
amount of nervonic acid, 24:1. As seen in sphingolipids in general (and for the ceramides of milk SM, Schmelz et al. 1996
), there was a bimodal
grouping of ceramides with very-long-chain fatty acids (22 to 24 carbon
atoms) and palmititc acid (16:0). GD3 contained a higher
proportion of 20:0 and 21:0 than the other glycolipids. The ß-hydroxy
group found in some sphingolipids (Vesper et al. 1999
)
was not detected in these dairy glycosphingolipids.
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When fed to DMH-treated CF1 mice, all of the glycosphingolipids
reduced significantly the number of ACF (Fig. 3
). At 0.025 and 0.1% of the diet, the suppression was, respectively, 53
and 51% for GluCer (P < 0.001 and P
< 0.01), 62 and 58% for LacCer (P < 0.001), and
42 and 54% for GD3 (P < 0.05
and P < 0.01). These results are comparable to the
suppression of ACF by milk and synthetic SM (Dillehay et al. 1994
, Schmelz et al. 1996
, 1997
).
There was a trend (P = 0.38 and P = 0.25 for GluCer, P = 0.12 and P = 0.32
for LacCer and P = 0.12 and P = 0.33
for GD3) to a reduced size of ACF (number of
aberrant crypts per focus); however, unlike earlier experiments
(Schmelz et al. 1996
), this did not reach statistical
significance.
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), but there was no correlation for the mice fed the glycosphingolipid
or SM supplements (data not shown). This was not due to differences in
weight because feeding sphingolipids had no significant effect on
weight gain (data not shown).
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DMH treatment caused a large increase in proliferation in the upper
half of the colonic crypts, which was suppressed significantly
(P < 0.001 for all but GluCer at 0.025%, P
< 0.05) by feeding all of the glycosphingolipids (Fig. 5A
). DMH-treatment also increased the rate of proliferation in
the lower half of the crypts, but by only 30% (P < 0.001). Dietary glycosphingolipids reduced proliferation in the lower
half of the crypts (Fig. 5B
), with the exception of 0.1%
GD3, which did not suppress cell proliferation.
This suppression of proliferation is comparable to the effect of milk
SM and synthetic SM (25 to 33% in the lower half, and 80% in the
upper half) (Fig. 5A
and 5B
).
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Exogenous addition of sphingolipids to cells in culture induces
apoptosis (see review Merrill et al. 1997
); therefore, the effect of a
dietary sphingolipid (SM) on the number of apoptotic cells in the
colonic epithelium was examined. There was no difference in the number
of apoptotic cells per crypt between the control group (0.19 ± 0.03) or the groups fed milk SM or synthetic SM for 4 wk (0.21 ± 0.03, and 0.17 ± 0.03, respectively). Hence, the effects of
dietary sphingolipids on apoptosis (if any) are not as easy to detect
as the inhibition of proliferation.
Hydrolysis of sphingolipids by colonic segments.
The proposed mechanism for the suppression of colonic cell
proliferation and ACF formation by dietary sphingolipids is that they
are hydrolyzed to ceramide and other "growth suppressive"
metabolites (Schmelz et al. 1996
, Vesper et al. 1999
). To determine if the colon contains the requisite
hydrolytic activity(ies), the sphingolipids were incubated with colonic
segments from mice. As shown in Figure 6
, all of the sphingolipids (including SM) disappeared during this
incubation: by 2 h, only 50 to 70% of the starting compound
remained; after 8 h, 25 to 40% remained.
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, open
circles). However, increases in other intermediate metabolites (GluCer
for LacCer, GluCer and LacCer for GD3) were not
found, which suggests that hydrolysis of the metabolites is
"coupled" (i.e., once hydrolysis begins, it continues rapidly until
Cer is formed) or that the cleavage of these complex sphingolipids is
effected by endoglycosidases (i.e., that cleave between the Cer
backbone and the headgroups).
There was no apparent preference for sphingolipids with a particular
Cer backbone because all of the subspecies of each sphingolipid (i.e.,
with 16:0, 22:0, 23:0, 24:0 and 24:1) disappeared in parallel (data not
shown). However, the Cer detected in all sphingolipid preparations
after hydrolysis contained a higher proportion of the 16:0 fatty acid
(Fig. 7
), which suggests that the fate of the Cer may differ for various
subspecies.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Schmelz et al. 1996
, 1997
). It is not clear why the inhibition
was not much different when the lower (0.025 g/100 g) and higher (0.1
g/100 g) amounts of sphingolipids were fed (It is possible, for
example, that sphingolipids only "correct" the phenotype of cells
with some (or multiple) genetic mutations produced by DMH.). It is
possible, though, that at 0.025 g/100g, we have already reached the
maximum response in this model. Nonetheless, these studies have proven
that the effects of dietary sphingolipids on these early stages of
colon carcinogenesis are not limited to compounds with a particular
(phosphorylcholine) headgroup.
This headgroup independence is consistent with the hypothesis that the
"active" component(s) are most likely the digestion products
ceramide and/or sphingosine (Dillehay et al. 1994
;
Schmelz et al. 1996
, 1997
). This is
difficult to prove in vivo, however, both of these metabolites inhibit
proliferation of transformed colonic cells in culture (Schmelz et al. 1998
). This study also established that all of
these complex sphingolipids can be hydrolyzed to Cer by colonic enzymes
of tissue (specific intracellular enzymes have been identified: Brady et al. 1965
, Kobayashi and Suzuki 1981
, Leese and Semenza 1973
) or,
perhaps, microbial origin (yet to be identified in regular intestinal
microflora). Previous studies have also shown that Cer (as well as SM
and GluCer) are digested, and the release metabolites such as sphingoid
bases absorbed throughout the upper and lower intestinal tract of
rodents (Nilsson 1968
, 1969
;
Schmelz et al. 1994
). Hence, ingestion of complex
sphingolipids delivers these bioactive metabolites to colonic cells.
Sphingoid bases and ceramides are known from experiments with cells in
culture to cause cell cycle arrest (Jayadev et al. 1995
)
by shifting retinoblastoma protein (Rb) to the dephospho- state
(Chao et al. 1992
, Lee et al. 1998, Pushkareva et al. 1995
, Dbaibo et al. 1995
), apparently due to up-regulation of endogenous
inhibitors (p21CIP1/WAF1 and p27 Kip1) of cyclin-dependent protein kinases
(Ciacci-Zanella et al. 1998
). These effects may occur
downstream of p53 (Dbaibo et al. 1998
). This may be the
mechanism for the lower proliferation of colonic crypt cells (and lower
ACF formation) in mice fed sphingolipids.
Hyperproliferation is a common property of tumors and can have several
causes: an increased rate of cell growth, a reduced rate of cell death
(apoptosis) or a combination of both. In addition to inhibiting growth,
sphingoid bases have been shown to be highly cytotoxic (Merrill et al. 1996
, Stevens et al. 1990
), and ceramides
(and sphingoid bases) are potent inducers of apoptosis (Obeid et al. 1993
, Ohta et al. 1995
, Sakakura et al. 1998
, Sweeney et al. 1996
). For one of the
compounds tested in this study (SM), immunohistochemical methods were
used to determine if sphingolipid feeding altered both proliferation
and the number of apoptotic cells in the colonic crypts. SM feeding for
4 wk did not increase the number of apoptotic cells, which suggests
that the induction of apoptosis is not the predominant cause of reduced
proliferation (and suppression of ACF formation) in this model. This
question warrants further investigation because apoptotic cells of the
colonic epithelium are short-lived and often difficult to detect.
It warrants mention that the amounts of sphingolipids used in these
studies did not alter the overall fat (or energy) intake of the mice
significantly, since high-fat diets (and high-energy intake)
have been associated with a higher risk of cancer (Hocman 1988
, Kumar et al. 1990
, MacLennan 1997
, Shivapurkar et al. 1992
). This association
might account, however, for the observation in this study that control
mice with the higher body weights developed more ACF.
In summary, both the SM and glycosphingolipids of milk have been shown
to suppress early stages of chemically induced colon cancer in female
CF1 mice. Milk contains 39 to 115 mg of SM per liter, which (based on
nonaqueous mass) is close to amounts of SM (0.025 to 0.1 g/100 g) that
inhibit proliferation, ACF formation and carcinogenesis in this model
(Dillehay et al. 1994
, Schmelz et al. 1996
and this study). The amounts of glycosphingolipids in milk
are also substantial (per liter: 6 to 11 mg of GluCer, 6.5 to 15 mg of
LacCer and ca. 11 mg of gangliosides) (Jensen 1995
);
hence, for the purpose of considering the effect of milk sphingolipids
on colon carcinogenesis, the total content is 70 to 150 mg per liter.
Whether this is effective in humans remains to be examined.
The inhibition of colon cancer by these glycosphingolipids also raises
interesting possibilities with respect to other foods. Plants contain
mostly cerebrosides (GlcCer), which could be effective inhibitors of
colon cancer. Nonetheless, the glycosphingolipids of plants, fungi,
yeast and many other organisms are composed of a variety of different
sphingoid base backbones and fatty acids (Vesper et al. 1999
), and little is known about the bioactivity of these
species. An understanding of the impact of sphingolipid consumption by
humans on colon cancer risk awaits additional studies of such
structure/function relationships and the use of additional cancer
models.
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FOOTNOTES |
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3 Supported by Dairy Management, Inc., and National Institutes of Health grant CA-61820.
4 Abbreviations used: ACF, aberrant crypt foci; BrdU, 5–2'-bromodeoxyuridine; Cer, ceramide; DMH, 1,2-dimethylhydrazine; GD3, ganglioside GD3; GluCer, glucosylceramide; LacCer, lactosylceramide; MS, mass spectrometry; SM, sphingomyelin.
Manuscript received August 2, 1999. Initial review completed September 13, 1999. Revision accepted November 16, 1999.
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