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Tumor Biology |
Departments of Surgery [M. S., H. A. R., D. S., P-A. C.] and Medicine [M. A. M.], Duke University Medical Center, Durham, North Carolina 27710; Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina [A. B., Y. A. H.]; and Division of Visceral and Transplantation Surgery, University Hospital Zürich, Zürich, Switzerland [M. S., H. A. R., P-A. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100,000 new cases
diagnosed each year in the United States (1
, 2)
. Less than
20% of patients with metastatic colorectal cancer are candidates for
curative surgery, and conventional chemotherapy is only marginally
effective (3
, 4)
. As a result, this condition has a dismal
prognosis, warranting the development of new therapeutic strategies
(5)
. Induction of apoptosis in tumor cells, a form of physiological death in unwanted or dysfunctional cells, is an appealing therapeutic approach (6) . Escape from apoptotic signals often accompanies tumor progression. For example, Naik et al. (7) showed in a mouse model that progression of islet cell tumors is associated with reduced susceptibility to apoptotic stimuli. Several groups have reported a higher incidence of tumor development in mice with genetic alterations of apoptotic mediators, such as Bcl-2 overexpression or Fas ligand deletion (8) . Furthermore, the response to chemo- or radiation therapy in some cancers correlates with the induction of apoptosis within the tumors (9 , 10) . These findings suggest that dysregulation of the apoptotic pathway might be a pivotal point in the genesis of a variety of cancers. Although the mechanisms and mediators of apoptosis in malignant cells remain under investigation, restoration of the physiological death pathway holds promise as a novel treatment strategy for cancer (11, 12, 13) .
Ceramide, a bioactive lipid arising from the hydrolysis of sphingomyelin or from de novo formation, has been proposed to play important roles in growth arrest, differentiation, and apoptosis in several cell culture models (14, 15, 16, 17) . For example, inhibition of ceramide formation with fumonisin B1 has been shown to prevent apoptosis in response to CPT11 and in response to angiotensin II (18 , 19) . Also, mice knocked out in acid sphingomyelinase show specific defects in endothelial and liver cell apoptosis (20 , 21) . Intracellular targets of ceramide are ceramide-activated phosphatases and ceramide-activated kinases, which in turn activate the apoptotic pathways, SAPK/JNK3 and the caspase cascade (17 , 22) .
Preliminary data have shown that malignant cells with low ceramide levels are resistant to apoptosis (23, 24, 25) . Chmura et al. (23) reported that tumor cell lines with a defect in ceramide generation are resistant to radiation-induced apoptosis. In addition, patients with head and neck cancer resistant to radiotherapy had decreased ceramide levels in their tumor (25) .
In this study, we evaluated the effect of ceramide on viability and growth of human colon cancer. We investigated two ceramide analogues (C2-ceramide and C6-ceramide), which have been shown to mimic the effect of natural ceramide, and two inhibitors of ceramidases (B13 and D-MAPP), critical enzymes involved in ceramide degradation. Ceramidase inhibition leads to elevation of intracellular natural ceramide (26) . Therefore, we hypothesized that ceramide levels are low in human colon cancer, and that restoration of cellular ceramide activity might induce apoptosis and prevent cancer growth in vitro and in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Isolation.
Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were
isolated from male Wistar rats (250 g) as described elsewhere
(27
, 28) . Briefly, hepatocytes were isolated by in
situ perfusion of the liver with 0.013% collagenase (Boehringer
Mannheim, Indianapolis, IN), followed by elutriation. For the
nonparenchymal cell isolation, the rat liver was perfused with 0.013%
collagenase and 0.25% Pronase (Boehringer Mannheim). The different
cells were separated on a discontinuous density gradient. The fraction
of Kupffer cells and endothelial cells was subjected to centrifugal
elutriation (flow rates, 18 and 36 ml/min). The purity of the isolated
cell cultures of hepatocytes, endothelial cells, and Kupffer cells was
>90%, respectively, as assessed by phase-contrast microscopy,
immunocytochemical analysis, and the ability to take up fluorescently
labeled actoacetylated human low-density lipoprotein. The cells were
cultured in Williams medium (Life Technologies, Inc., Gaithersburg, MD)
containing 20% calf serum. The cells were maintained in a humidified
5% CO2 incubator at 37°C, and the medium was
changed 24 h after plating.
Ceramide Analogues.
Short-chain ceramides (C2-ceramide and C6-ceramide) and selected
inhibitors of ceramidase, D-MAPP and B13 (Fig. 1)
, were used in this study. Synthesis and physical
characterization of the above compounds were described previously
(16
, 22)
. C2-ceramide and C6-ceramide were prepared by
acylation reaction of (2S,3R)-sphingosine
obtained in stereoselective synthesis. D-MAPP and B13 were prepared
by acylation of the respective amino alcohols with myristoyl chloride
(26)
. B13 is a newly synthesized analogue of ceramide, and
its molecular structure is similar to D-MAPP. Both compounds are potent
blockers of ceramidase (26)
, a critical scavenging enzyme
of ceramide, thereby elevating natural ceramide content in the
cells.4
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Trypan Blue Staining.
Cells were harvested by trypsinization and stained 1:2 with trypan
blue. The percentage of staining cells was determined with a counting
chamber at x200.
[3
H]Thymidine Assay.
Tumor cells (50,000) were placed in a 96-well plate and grown in serum
containing medium over night. Then, 1 µCi of
[3
H]thymidine was added to each well for
12 h. After the incubation time, the plates were washed, and the
radioactivity of the remaining DNA was counted by a Wallace counter.
TUNEL Assay.
The TUNEL assay was performed as described previously by Gao et
al. (29)
. Briefly, after 4, 12, and 24 h of
treatment with B13, the cells were incubated in freshly prepared 4%
paraformaldehyde in PBS (pH 7.2) for 30 min. The cells were washed with
PBS and finally placed in 100% ethanol. Then the cells were
transferred on glass slides by cytospin (800 rpm). The cells were
treated with terminal deoxynucleotidyl transferase from calf thymus
(Boehringer Mannheim) in the presence of fluorescein-dUTP and
deoxynucleotide triphosphate, according to the supplier’s recommended
protocol. This was followed by poststaining using horseradish
peroxidase-conjugated anti-fluorescein antibody and development using
diaminobenzidine/H2O2.
Positive and negative controls were done using test sections pretreated
with DNase I and staining without deoxynucleotide substrate,
respectively.
Agarose Gel Analysis of DNA Fragmentation.
The cells were harvested by trypsinization. The DNA was extracted using
the Gibco DNA laddering kit (Life Technologies, Inc., Gaithersburg,
MD). The isolated DNA was quantified at 260 nm. Samples (5 µg/lane)
were electrophoresed on a 2% agarose gel at 30V in TAE buffer (40
mM Tris-acetate, 10 mM EDTA, and 20
mM glacial acetic acid, pH 8.4) for 3 h. The DNA was
finally visualized on an UV transilluminator.
Annexin V Assay.
Surface expression of phosphatidyl serine was determined by Annexin V
staining. Cells (106) per milliliter were
harvested and exposed to Annexin V and propidium iodide according to
the recommendation of the manufacturer (R&D, Minneapolis MN). Annexin V
and propidium iodide staining was determined by cell flow cytometry.
Ceramide Measurement.
Lipids were extracted using the Bligh and Dyer procedure
(30)
. This method involves the lysis of cells or tissue
with organic solvent, followed by dilution with chloroform and water to
obtain phase separation. Approximately 100% of the biological
ceramides, diacylglycerols, and sphingosine are extracted into the
organic phase under these conditions. The critical parameters of the
lipid extraction are the ratios of chloroform:methanol:water are
1:1:0.9 (v/v). Efficient lipid extraction into organic phase should be
performed as follows. Cells (1–2 x 106) are separated from medium, washed with cold
1x PBS, pelleted, lysed with 3 ml of chloroform:methanol (1:2, v/v),
and vortexed vigorously. Extraction of neutral lipids into the
chloroform phase is continued by adding 0.8 ml of water, 1 ml of
chloroform, and finally 1 ml of water. After every step, strong
vortexing is recommended. Finally, the organic lower phase (2 ml) is
separated from the upper one (3.8 ml) by centrifugation and is used for
analytical purposes. Lipid extraction from the tissues was performed
after homogenization with buffer (0.25 M sucrose, 25
mM KCl, 50 mM Tris, and 0.5 mM
EDTA, pH 7.4). The right amount of homogenate for diacylglycerol kinase
assay was established through the protein measurement.
The level of the endogenous ceramide was determined by DK kinase
according to the published method (31)
. Briefly, ceramide
standards (50–1000 pmol) and lipid extracts from the experimental
samples are sonicated for 5 min with 20 µl of 3.75% (w/v)
octylglucoside-12.5 mM dioleoylphosphatidylglycerol, made
up in 1 mM DTPA, followed by the addition of 70 µl of the
reaction mixture containing 120 mM HEPES buffer (pH 7.0),
100 mM LiCl, 25 mM MgCl2,
2 mM EGTA, 2 mM DTT, and 5 µl of
diacylglycerol kinase (7 mg/ml). After 10 min, the reaction is started
by adding 10 µl of 10 mM ATP in 20 mM
imidazole buffer (pH 6.6) and 1 mM DTPA. After vigorous
mixing, the reaction mixture is left at room temperature for 0.5 h, and then 3 ml of chloroform:methanol 1:2 (v/v) are added, and the
reaction mixture is mixed vigorously for 1 min. Bligh and Dyer
extraction is continued by adding 700 µl of 1% perchloric acid,
followed by 1 ml of chloroform and 1 ml of 1% perchloric acid. Phases
are separated by centrifugation (3000 rpm for 5 min). 1.5 ml from the
lower phase (total, 2 ml) is transferred to the new vial, and the
organic phase is dried down under nitrogen. Dry lipids are resuspended
in 50 µl of chloroform-methanol (1:1, v/v). Twenty µl of this
solution are applied to a TLC plate, and
[32P]ceramide phosphate is resolved in a
chloroform:acetone:methanol:acetic acid:water (10:4:3:2:1, v/v) solvent
system. The TLC plate is then exposed to X-ray film. The area
corresponding to [32P]ceramide-1-phosphates
(Rf = 0.63–0.68) is scraped
and counted quantitatively by liquid scintillation counting.
Alternatively, visualization and quantitation can be performed on a
Molecular Dynamics Phosphorimager. Quantitation of ceramide mass is
based on the specific activity of [32P]ATP and
on the external standard normalization. Final results are shown as
[32P]ceramide phosphate/total lipid
phosphorous. Pi is determined for an equal amount of phospholipids
present in the lower phase of the lipid extract. The results are then
expressed as ceramide/Pi (pmol/nmol).
Caspase 3-like Activity Assay.
Caspase 3-like activity was determined by measuring the
proteolytic of the specific substrate
N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO; Biomol, Plymouth
Meeting, PA; Ref. 32
). Caspase 3 is distinguished by its
ability to cleave poly(ADP-ribose) polymerase during apoptosis. The
cleavage site in poly(ADP-ribose) polymerase is COOH-terminal to
ASP-216 (33)
. The upstream sequence, DEVD, is the basis
for the substrate and inhibitors used in this assay. Liver tissue was
quickly excised and sonicated in assay buffer [1
mM EDTA, 145 mM NaCl, 100
mM Tris, 0.1 mM DTT, 0.1%
3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, and 10%
glycerol]. The protein content was determined using the Bradford
protein assay. The samples were diluted and incubated at room
temperature with Ac-DEVD-AFC substrate in the presence or absence of
the inhibitor AC-DEVD-CHO. AFC release was measured over 2 h in a
fluorometer, using 400 nm excitation and measuring 505 nm emission. The
AFC release was expressed as arbitrary fluorescence units per mg
protein after subtracting the reading in the inhibited sample from the
noninhibited sample.
In Vivo Model of Liver Metastases.
The tumor cell lines (SW403 and Lovo) were twice washed in 1x PBS and
finally dissolved in 0.2 ml of 1x PBS containing 2 million tumor
cells. The abdominal cavity of the mice was opened by a median
incision. The portal vein was exposed, and the tumor cell suspension
(0.2 ml) was slowly injected into the portal vein. The abdominal cavity
was closed by chromic gut 4.0, and the animals were allowed to awake
with free access to food and water. Two h after tumor cell injection,
the mice either received 75 mg/Kg B13 dissolved in 10% ethanol, 30%
Cremophor, and 60% saline or the solvent alone into the peritoneal
cavity. The injection was repeated every 3 days up to a total number of
five injections.
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RESULTS |
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. The ceramidase inhibitor B13 (100
µmM) was significantly more toxic than the
other compounds with 90% cell death at 24 h. In a second
approach, we investigated the effect of different concentrations (5,
10, 50, and 100 µM) of the most potent compound
(B13) on tumor cell viability. B13 induced death of the tumor cells in
a dose-dependent manner. Similar results were obtained in the
[3
H] incorporation assay using doses of 5, 10,
50, and 100 µM of each compound. DNA synthesis
was significantly impaired by ceramide treatment in a dose- and
time-dependent manner. B13 demonstrated the highest potency for
inhibiting DNA synthesis (20% of the solvent treated control; Fig. 4
). On the basis of these results, only the most potent compound, B13,
was further studied.
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. After 24 h exposure to 100
µM B13, 85% of the colon cancer cells were
TUNEL positive. Consistent with these findings was the appearance of
DNA laddering by gel electrophoresis after 12 h of treatment (Fig. 5B)
. Translocation of phosphatidylserine to the outer
surface of the cytoplasmic membrane, an early feature of apoptosis, was
evaluated by the Annexin V binding assay. To exclude staining of
phosphatidylserine on the inner surface of the cell membrane, which
occurs when the cytoplasmic membrane is disrupted, we counterstained
the cells with propidium iodide. Cells stained for Annexin V alone have
early apoptotic membrane changes with intact cell membranes and are in
an early phase of apoptosis. Cells stained for Annexin V and propidium
iodide have membrane disintegration consistent with necrosis or a late
stage of apoptosis in cell culture. Although Annexin V was minimally
expressed (<12%) in untreated cells at all time points, B13-treated
cells had significantly elevated Annexin V staining at 1 and 4 h
of exposure (40%; Fig. 6
) with only minimal propidium iodide staining. After 4 h
incubation, an increasing portion of cells was also positive for
propidium iodide. Taken together, the data are consistent with B13
inducing apoptosis in these cells with rapid changes in the
distribution of phosphatidylserine, followed by DNA fragmentation. Less
specific features of cell death (propidium iodide staining) are
typically noted at later stage of apoptotic death in cell culture.
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. The release of mitochondrial cytochrome c, a potent
activator of caspase 3, was elevated at 4 and 12 h of B13
treatment (Fig. 8)
. Finally, caspase 3 activity, the executor of the caspase cascade,
demonstrated a 7-fold increase after 4 and 24 h exposure to B13
(Fig. 9A)
. Pretreatment of the cancer cells with a specific caspase
3 inhibitor (Ac-DEVD-CHO; Biomol) significantly prevented cell death at
4 and 12 h after exposure to B13 (Fig. 9B)
. In
contrast, caspase 8 activity, the most upstream cell membrane-related
mediator of the caspase cascade, was unaltered by exposure to B13.
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at a dose associated with >90% cell death in the colon cancer cell
line (see above). To investigate the mechanisms of resistance of normal
hepatocytes against B13, we measured ceramide levels in hepatocyte
cultures exposed to 4 and 12 h of 100 µM B13. No
elevation of ceramide levels was detected in normal hepatocytes,
despite maximum treatment with the ceramidase inhibitor B13. The
baseline ceramide levels of cultured normal hepatocytes were twice as
high as in the colon cancer cell line (data not shown). Similar results
were obtained measuring ceramide levels in human specimens freshly
obtained in the operating room. Liver metastasis from colon cancer had
only half the ceramide levels compared with normal liver tissue (data
not shown).
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. The B13-treated mice, which developed tumor growth, had significantly
lower tumor masses than the control-treated animals (1.2 g ± 0.2 g versus 2.8 g ± 0.8 g; Student’s t test, P = 0.03).
We did not observe any signs of distress, such as a change in behavior
or mutilation, in B13-treated animals. All B13-treated animals had a
similar weight gain when compared with a nontreated control group. On
careful macroscopic examination of the abdominal cavity, no signs of
inflammation or serosal injury were detectable in mice treated with
B13.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ceramide is an emerging lipid mediator, with proapoptotic activities in
cancer cells (15
, 16
, 22)
. Ceramide accumulation in cells
can be induced by a variety of extracellular stimuli, such as TNF-
,
Fas ligand, IFN-, radiation, and chemotherapy (16
, 22
, 39, 40, 41)
. Ceramide activates proapoptotic mechanisms in three
major ways: (a) ceramide acts indirectly to activate
caspases, a well-characterized group of cysteine proteases, which leads
to activation of caspase 3 (22)
, the executor of apoptosis
causing activation of endonucleases and DNA fragmentation;
(b) ceramide activates the stress-activated protein kinases
(SAPK/JNK; Ref. 34
) pathway. SAPK/JNK activation may
result in phosphorylation of c-Jun, which in turn induces apoptosis
by a caspase 3-dependent mechanism (34
, 42)
; and
(c) ceramide up-regulates TNF receptor 1 and Fas receptors
on the cell membrane and, therefore, may enhance the effects of
antitumor T-lymphocytes and other Fas ligand- and TNF-dependent
processes (9)
.
Recent data suggest a role for ceramide in the pathogenesis of cancer and failure of conventional therapy (21 , 23, 24, 25) . For example, resistance to radiation therapy has been associated with a significant decrease in cellular ceramide production (21 , 23, 24, 25) , whereas others have reported that a defect of ceramide production is associated with multidrug resistance (43 , 44) . Another evidence implicating ceramide in the development of cancer is the observation that mice subjected to a diet rich in sphingomyelin, which is cleaved into ceramide and sphingosine inside the gastrointestinal lumen, are resistant to 1,2-dimethylhydralazine-induced colon cancer (21) . Our finding that human colon cancers, primary and metastatic to the liver, contain only half of the ceramide content of normal tissue obtained in the same patient is consistent with a role of this lipid mediator in the pathogenesis of cancer.
We hypothesized that tumors with low ceramide content might be particularly sensitive to the restoration of ceramide. We observed that various ceramide analogues and ceramidase inhibitors induce rapid death by apoptosis of human colon cancer in cell culture. Apoptosis in the cancer cells was mediated by a dramatic activation of the caspase cascade. Whereas caspase 8, the most upstream membrane-linked mediator, remained unaffected by exposure to B13, cytochrome c was rapidly released, resulting in caspase 3 activation. Blocking Caspase 3 by a caspase inhibitor increased tumor cell viability. This indicates that the effects of the ceramidase inhibitor B13 are dependent on the caspase cascade. The exact place of ceramide in this pathway cannot be determined from these experiments, but the lack of caspase 8 activation suggests that ceramide is localized between caspase 8 and the release of cytochrome c. Other effects of ceramide, e.g., directly on caspase 3, cannot be excluded.
B13 has emerged from these studies as a particularly active analogue of ceramide that inhibits ceramide metabolism and leads to accumulation of cellular ceramide. B13 is significantly more active in this experimental model than either short-chain ceramides or D-MAPP, a previously described inhibitor of alkaline ceramidase. The reasons for this enhanced potency are not fully determined but may relate to the enhanced solubility of B13 compared with the other ceramidase inhibitors or ceramide analogues.
An important finding related to the possible clinical use of B13 was the lack of toxicity to normal cells in vitro and to the animals treated with B13. Because our interest is in colon cancer metastatic to the liver, we focused on the effects of B13 on normal hepatic cells including parenchymal (hepatocyte) and nonparenchymal (sinusoidal endothelial and Kupffer cells) cells. Both in vitro and in vivo administration of high-dose B13 was not associated with detectable toxic effects. Increased ceramide levels might be less toxic to cells with an intact sphingosine/ceramide pathway compared with tumor cells with disrupted ceramide generation. Additionally, normal hepatocytes, Kupffer cells, and sinusoidal endothelial cells have a low mitotic rate in cell culture and in vivo. Although i.p. injection of B13 did not induce bone marrow suppression or intra-abdominal inflammation in our model, the systemic use of B13 might have adverse effects on cells with a high mitotic rate. The rodent model alone is insufficient to fully determine the potentially toxic effects of B13, and large animal experiments are necessary prior to clinical use.
In our in vivo model, we found that the development of tumor
metastases can be prevented by the ceramidase inhibitor B13. Several
possible mechanisms exist for the in vivo efficacy of B13.
Apoptosis could be induced in tumor cells within the hepatic sinusoids
similar to our in vitro model of tumor cell death.
Furthermore, up-regulation of proapoptotic membrane receptors, such as
Fas or TNF-, could facilitate the induction of apoptosis by
extracellular mediators. Finally, tumor cell adhesion in the hepatic
sinusoids might be negatively affected by the exposure to the
ceramidase inhibitor B13. Although our in vitro experiments
demonstrated that B13 induces cell death by activation of the caspase
cascade and inhibits tumor cell growth, additional mechanisms might
also be active in vivo. Future studies are necessary to
determine the effects of ceramidase blockage on established metastases.
Several aspects of ceramide as a therapeutic agent have to be clarified in the future. It is unclear at this point if ceramidase inhibitors are effective only in preventing metastases or if established metastatic disease can be treated successfully. The effect of several chemotherapeutic agents are linked to the induction of apoptosis and associated with an increase of cellular ceramide (10) . A defect in ceramide generation might be associated with resistance to chemotherapy. A combination of chemotherapy and regulation of the level of the endogenous ceramide might have synergistic effects on selected tumors.
These findings have profound implications for the treatment of cancer. Our results emphasize that defects in the apoptotic pathway exist in colon cancer. The use of mediators of apoptosis to restore the deficient pathway is therefore a new and attractive strategy to selectively attack the cancer cells. Increasing the ceramide levels by blocking ceramidase was highly effective in inducing apoptosis and preventing growth of colon cancer. B13 appears to be a promising compound for the adjuvant treatment of primary or metastatic colon cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grants DK54048-01A1
(to P-A. C.) and GM 43285 (to Y. A. H.) from the NIH. 
2 To whom requests for reprints should be
addressed, at Division of Visceral and Transplant Surgery, University
Hospital Zürich, 100 Raemistrasse, 8091 Zürich,
Switzerland. Phone: 411-255-33-00; Fax: 411-255-4999; E-mail: clavien{at}chir.unizh.ch
3 The abbreviations used are: SAPK/JNK,
stress-activated protein kinase/c-Jun NH2-terminal kinase;
TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP-digoxigenin nick end labeling.
4 S. El Bawab, Y. Hannun, and A. Bielawska,
unpublished observations.
Received 4/18/00. Accepted 11/28/00.
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F. Scarlatti, C. Bauvy, A. Ventruti, G. Sala, F. Cluzeaud, A. Vandewalle, R. Ghidoni, and P. Codogno Ceramide-mediated Macroautophagy Involves Inhibition of Protein Kinase B and Up-regulation of Beclin 1 J. Biol. Chem., April 30, 2004; 279(18): 18384 - 18391. [Abstract] [Full Text] [PDF]
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J. A. Shabbits, Y. Hu, and L. D. Mayer Tumor Chemosensitization Strategies Based on Apoptosis Manipulations Mol. Cancer Ther., August 1, 2003; 2(8): 805 - 813. [Full Text] [PDF]
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I. Silins, M. Nordstrand, J. Hogberg, and U. Stenius Sphingolipids suppress preneoplastic rat hepatocytes in vitro and in vivo Carcinogenesis, June 1, 2003; 24(6): 1077 - 1083. [Abstract] [Full Text] [PDF]
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K. J. Claycombe, D. Wu, M. Nikolova-Karakashian, H. Palmer, A. Beharka, K. E. Paulson, and S. N. Meydani Ceramide Mediates Age-associated Increase in Macrophage Cyclooxygenase-2 Expression J. Biol. Chem., August 16, 2002; 277(34): 30784 - 30791. [Abstract] [Full Text] [PDF]
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Y. A. Hannun and L. M. Obeid The Ceramide-centric Universe of Lipid-mediated Cell Regulation: Stress Encounters of the Lipid Kind J. Biol. Chem., July 12, 2002; 277(29): 25847 - 25850. [Full Text] [PDF]
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C. Mao, R. Xu, Z. M. Szulc, A. Bielawska, S. H. Galadari, and L. M. Obeid Cloning and Characterization of a Novel Human Alkaline Ceramidase. A MAMMALIAN ENZYME THAT HYDROLYZES PHYTOCERAMIDE J. Biol. Chem., July 6, 2001; 276(28): 26577 - 26588. [Abstract] [Full Text] [PDF]
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, 5 µM; 
, 10
µM; 
, 50 µM; 
, 100 µM;
compared with the control receiving solvent (0.1% ethanol) only. The
two ceramide analogues C2 and C6 and the two ceramidase inhibitors
D-MAPP and B13 reduced the amount of [3H]thymidine
incorporation in a dose-dependent manner, with B13 being the most
potent compound (Student’s t test: *,
P < 0.05; n = 5). Bars, SD.
