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J. Biol. Chem., Vol. 277, Issue 51, 49531-49537, December 20, 2002
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From the
Received for publication, September 29, 2002
Stress stimuli can mediate apoptosis by
generation of the lipid second messenger, ceramide. Herein we
investigate the molecular mechanism of ceramide signaling in
endothelial apoptosis induced by fenretinide
(N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic
derivative of retinoic acid that induces ceramide in tumor cell lines,
has been shown to have antiangiogenic effects, but the molecular
mechanism of these is largely unknown. We report that 4-HPR was
cytotoxic to endothelial cells (50% cytotoxicity at 2.4 µM, 90% at 5.36 µM) and induced a
caspase-dependent endothelial apoptosis. 4-HPR (5 µM) increased ceramide levels in endothelial cells
5.3-fold, and the increase in ceramide was required to achieve the
apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was
suppressed by inhibitors of ceramide synthesis, fumonisin B1, myriocin, and L-cycloserine, and 4-HPR
transiently activated serine palmitoyltransferase, demonstrating that
4-HPR induced de novo ceramide synthesis. Sphingomyelin
levels were not altered by 4-HPR, and desipramine had no effect on
ceramide level, suggesting that sphingomyelinase did not contribute to
the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor,
t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide
accumulation, suggesting that ceramide is upstream of caspases. Our
results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects.
Stress stimuli such as irradiation, tumor necrosis factor- Fenretinide (N-(4-hydroxyphenyl)retinamide
(4-HPR))1 is a synthetic
derivative of all-trans-retinoic acid that induces
apoptosis in cancer cell lines and is in clinical trials for adult
and pediatric cancers (reviewed in Refs. 11-13). 4-HPR has been shown
to possess antiendothelial activity in tissue culture and the chick
chorioallantoic membrane model (14, 15). However, whereas Pienta
et al. (14) demonstrated that 4-HPR was cytotoxic to CPAE
bovine artery endothelial cells, Ribatti et al. (15) found
that it inhibited proliferation but was not cytotoxic to human adrenal
gland capillary endothelial cells. These data support an antiangiogenic
role for 4-HPR, yet the molecular mechanism of the effects of 4-HPR in
endothelial cells remains largely unknown.
To date, the cytotoxic mechanism of 4-HPR has been studied almost
exclusively in tumor cell lines, where it appears to function by more
than one mechanism (12, 13). In leukemia cells, 4-HPR-mediated apoptosis was associated with decreased levels of bcl-2
mRNA and was diminished by inhibitors of tyrosine kinases, by
inhibitors of RNA and protein synthesis, by activators of protein
kinase C, and by antioxidants (16, 17). 4-HPR-mediated apoptosis in
leukemia cells was also associated with activation of caspase-3 via a
mechanism separate from induction of reactive oxygen species (18) and
with increased de novo synthesis of ceramide (19). In
neuroblastoma cells, 4-HPR induced a mixed caspase-mediated apoptosis
and caspase-independent necrosis that was associated with increased
reactive oxygen species and increase in intracellular ceramide via
de novo synthesis (20). Combining 4-HPR with agents that
inhibit intracellular ceramide metabolism further increased ceramide
levels and was associated with increased cytotoxicity, suggesting that
ceramide is a mediator of 4-HPR-induced tumor cell cytotoxicity
(21).
In light of the antiendothelial effects of 4-HPR, its potential for
antiangiogenic activity (14, 15), and the role of ceramide as an
important mediator of endothelial cell apoptosis (5, 22-24), we were
interested in the role of ceramide signaling in mediating the
antiendothelial effect of 4-HPR. In the work reported here we
demonstrate that 4-HPR induces a caspase-dependent endothelial apoptosis. We also show that 4-HPR increased endothelial cell ceramide by stimulation of de novo synthesis and that
de novo generated ceramide has a causal role in
4-HPR-mediated apoptosis of human brain microvascular endothelial cells
(human BMEC). Last, we establish that ceramide functions upstream of
caspases in the ordering of ceramide and caspases in endothelial cells.
These results provide the first evidence for endothelial apoptosis by 4-HPR and show that activation of the ceramide pathway is required for
this apoptosis, thus presenting a molecular mechanism for the
antiangiogenic effects of 4-HPR.
Cell Culture--
Human brain microvascular endothelial cells
(human BMEC (25)) and large-T antigen-transfected bovine microvascular
endothelial cells (bovine TBMEC (26)) were a gift from Dr. K. S. Kim, Johns Hopkins School of Medicine (Baltimore, MD). Two different
isolates of human BMEC were used. Cells were maintained in RPMI 1640 supplemented with L-glutamine, sodium pyruvate, 10%
heat-inactivated fetal bovine serum (FBS), and 10% Nu-SerumTM IV
Culture Supplement (Collaborative Biomedical Products, Becton Dickinson
Labware, Bedford, MA) as described (5, 25, 26), with the
addition of 20 mM HEPES buffer for the human BMEC. Human
umbilical vein endothelial cells (HUVEC) (ATCC CRL-1730; passages
16-20) were maintained according to the supplier's recommendations.
For experiments, cells were plated and allowed to attach and spread for
4-18 h prior to beginning the experiment. In all cases, floating and
attached cells in each sample were both combined for processing at the
end of incubation. Both high passage (passage 56) and low passage
(passage 16) human BMEC showed characteristic endothelial morphology
comparable with primary human BMEC (passage 5). Factor VIII-reactive
antigen (Dako, Carpinteria, CA) (25) was expressed at all passages as
determined by flow cytometry, although levels decreased in the higher
passages. Uptake of fluorescent
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (BTI, Stoughton, MA) remained similar for all passages and further confirmed the endothelial phenotype (25). High and low passage human BMEC were functionally similar, as demonstrated by equivalent expression of intercellular adhesion molecule in response to tumor necrosis factor- Reagents--
4-HPR was kindly provided by Dr. Sherry Ansher
(NCI, National Institutes of Health). Stock solution (10 mM) in ethanol was stored protected from light at
Apoptosis Assays--
Apoptosis was assessed by staining
ethanol-fixed, RNase-treated cells with propidium iodide (50 µg/ml in
phosphate-buffered saline containing 5 mM EDTA, 10 min on
ice) and identifying cells with a sub-G0/G1 DNA
content, indicative of apoptosis, using a Coulter Epics ELITE flow
cytometer (Coulter, Miami, FL). For morphological assessment of DNA
condensation and/or apoptotic bodies, cells were grown on chamber
slides and then incubated with the supravital DNA stain Hoechst 33342 (10 µg/ml for 30 min at 37 °C). When using fixed cells
(cytospins), the dye used was Hoechst-bisbenzamide 33258 according to
the manufacturer's instructions (Sigma). For analysis of apoptosis by
Hoechst staining, ~300-500 cells from a total of 5-10 fields in
each sample were counted under UV filter at ×400 magnification.
Endothelial Viability and Cytotoxicity--
Cell viability was
assessed by uptake of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(thiazolyl blue (MTT)) (5, 28) and confirmed by trypan blue exclusion
and a fluorescence-based assay using fluorescein diacetate that
selectively accumulates in live cells and is measured by digital
imaging microscopy (DIMSCAN) (20, 21).
Radiolabeling and Analysis of Cellular Ceramide, Sphingomyelin,
and Glucosylceramide--
Ceramide metabolism was studied as described
(5, 20) with some modifications. Briefly, endothelial cells (3 × 106 cells/10-cm dish) were allowed to attach and spread in
medium containing 10% heat-inactivated FBS. Cells were radioactively labeled with [3H]palmitic acid (1 µCi/ml, 10 ml per
10-cm dish), and 4-HPR was added either simultaneously with
[3H]palmitic acid or following 6-24 h labeling, as
indicated. For experiments to measure sphingomyelin, glucosylceramide,
or ceramide in the absence of de novo synthesis, cells were
prepared by prelabeling with [3H]palmitic acid for
24 h, washing with phosphate-buffered saline, and incubating them
for 2 h in fresh growth medium containing 0.1% FBS and lacking
isotope. After 2 h, the medium was replaced, and 4-HPR or vehicle
control was added for the indicated time.
At the end of incubation, adherent and detached cells were trypsinized,
combined, and washed with phosphate-buffered saline (4 °C). Total
cellular lipids were extracted using equal volumes of methanol 2%
acetic acid (v/v), water, and chloroform. After phase separation by
centrifugation, the lower phase was dried under N2 and
stored at High Performance Liquid Chromatography (HPLC) for Measurement of
Cellular 4-HPR Content--
4-HPR content in endothelial cell pellets
was determined by HPLC using UV absorbance detection in a modification
of the method used by Le Doze et al. (32). Weighed pellets
of treated cells were homogenized in 1 ml of acetonitrile and
centrifuged, and the supernatant was analyzed by HPLC.
Isolation of Microsomal Membranes--
Human BMEC cultured
in 10-cm dishes were placed on ice, rinsed twice (ice-cold
phosphate-buffered saline), and scraped into 0.5 ml of homogenization
buffer (20 mM HEPES, pH 7.4, 5 mM
dithiothreitol, 5 mM EDTA, 2 µg/ml leupeptin, 20 µg/ml
aprotinin). Cell suspensions were sonicated over ice at 20% output,
alternating a 15-s sonication with 20-s pause for four cycles, using a
Micro Ultrasonic Cell Disrupter from Kontes (Vineland, NJ). Lysates
were centrifuged at 10,000 × g for 10 min. The
postnuclear supernatant was isolated and centrifuged at 100,000 × g for 60 min at 4 °C. The microsomal membrane pellet was
resuspended in 100 µl of homogenization buffer by sonication for
5 s and was frozen at Serine Palmitoyltransferase Assay--
Enzymatic activity was
determined by measuring the incorporation of [3H]serine
into 3-ketosphinganine (34, 35). Each tube (0.1-ml final volume)
contained 100 µg of microsomal protein in 0.1 M HEPES, pH
8.3, 2.5 mM EDTA, 50 µM pyridoxal phosphate,
5 mM dithiothreitol, and 1.0 mM
L-serine. After preincubation at 37 °C for 10 min, the
reaction was initiated by the simultaneous addition of palmitoyl-CoA (0.2 mM) and 1.0 µCi of [3H]serine. Control
samples contained either boiled microsomes or no protein. After
incubation at 37 °C for 7 min, the reaction was terminated by the
addition of 0.2 ml of 0.5 N NH4OH.
Organic-soluble products were isolated by the addition of 3 ml of
chloroform/methanol (2:1), 25 µg of sphingosine carrier, and 2.0 ml
of 0.5 N NH4OH. The washed organic phase was
isolated, and 1.0 ml was dried under a stream of nitrogen and analyzed
by liquid scintillation counting (33).
Ceramide Synthase Assay--
Enzymatic activity was determined
by measuring the incorporation of [3H]sphinganine into
[3H]dihydroceramide (34, 35). Sphinganine in
chloroform/methanol (2:1) was dried under nitrogen and dissolved to 10 µM with sonication, in 25 mM HEPES, pH 7.4, 2 mM MgCl2, 0.5 mM dithiothreitol,
prior to the addition of microsomal protein (100 µg) to a final
reaction volume of 0.1 ml. Assays were initiated by the simultaneous
addition of palmitoyl-CoA (0.1 mM) and 0.5 µCi of
[3H]sphinganine followed by incubation at 37 °C for 40 min with gentle shaking. The reaction was terminated by lipid
extraction. [3H]dihydroceramide was isolated by TLC and
quantitated by liquid scintillation counting.
Statistical Analysis--
Statistical analyses were performed
using GraphPad Prism version 3.0c for MacIntosh (GraphPad Software, San
Diego, CA). Values are given as means ± S.E. When two means were
compared, p values were based on the t test
(unpaired or paired, depending on the experimental design). When three
or more means were compared, the overall p value was based
on the F-test from an analysis of variance (ANOVA). If the means were
based on doses or times (i.e. on a continuum), then the
p value was based on the test for trend using linear
regression. p < 0.05 was considered significant.
4-HPR Was Cytotoxic to Endothelial Cells--
Since only one
of two reports found 4-HPR to be
cytotoxic to endothelial cells, we determined the cytotoxicity of 4-HPR
in human BMEC (Fig. 1). 4-HPR was
cytotoxic to human BMEC in a dose-dependent manner as
measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with 50% cytotoxicity at 2.4 ± 0.2 µM (S.E.) and 90% cytotoxicity at 5.3 ± 0.5 µM 4-HPR when incubated in medium containing 0.1% FBS
(Fig. 1A). Assessment by trypan blue exclusion and DIMSCAN
(digital imaging microscopy (20)) revealed similar results.2 Cytotoxicity was
similar in low passage (passages 14-30) and high passage (passages
40-57) human BMEC, with 50% cytotoxicity at a mean 4-HPR of 2.2 ± 0.3 and 2.6 ± 0.2 µM, respectively
(n = 10 and 11; p = 0.32 by unpaired
t test). With HUVEC, 50 and 90% cytotoxicity was achieved
at 3.1 and 9.0 µM 4-HPR, respectively (n = 10, p < 0.001 by one-way ANOVA), and in bovine
TBMEC, 5 µM 4-HPR induced 98% cytotoxicity
(n = 10, p < 0.001 by unpaired t test compared with control cells). Thus, 4-HPR was
cytotoxic to both human and bovine BMEC as well as in HUVEC.
Some growth factors can activate survival signaling pathways such as
the phosphatidylinositol-3-kinase/Akt cascade to protect them from
apoptosis, as shown for insulin- and insulin-like growth factor-1-mediated inhibition of anoikis (36). Therefore, we examined
whether FBS could rescue endothelial cells from 4-HPR-mediated cytotoxicity. Including 1-10% FBS in the medium diminished
4-HPR-induced cytotoxicity in human BMEC, bovine TBMEC, and HUVEC
compared with 0.1% FBS when measured at 24 h (Fig. 1,
B and C).2 The addition of bovine
serum albumin to the medium did not suppress the cytotoxic effect of
4-HPR (Fig. 1C), indicating that protection by FBS was not
due to binding of the drug to the albumin in the FBS.
4-HPR Induced Apoptosis in Endothelial Cells--
Protection by
serum suggested that 4-HPR could be inducing apoptosis in the
endothelial cells. Indeed, human BMEC exposed to 4-HPR demonstrated an
increase in the sub-G0/G1 DNA content indicative of apoptosis as revealed by flow cytometry (representative experiments shown in Fig. 2, A
and B).
We then examined whether serum protected the cells from
4-HPR-induced apoptosis. At 24 h, 10% FBS completely abrogated
the 4-HPR-mediated apoptosis even in the presence of 7.5 µM 4-HPR (Fig. 2C). Since in some tumor cell
lines induction of apoptosis by 4-HPR requires longer exposures (37,
38), we also examined the apoptotic effect of 4-HPR in the presence of
10% FBS at 48 and 72 h. Indeed, in 1-10% FBS, the apoptotic
effect of 4-HPR was restored with longer incubations (Fig. 2,
D and E). To examine the possibility that the
delay in apoptosis was due to binding of 4-HPR to the FBS, we
determined 4-HPR content in human BMEC in medium containing 0.1-10%
FBS. Fig. 2F demonstrates that cellular 4-HPR content was
similar in cells treated in culture medium with 0.1 and 1% FBS and was
decreased in 10% FBS. In order to maintain controlled conditions and
minimize overconfluence of cells at the high FBS concentrations during
longer incubations, subsequent experiments were performed in medium
containing 0.1-1% FBS.
4-HPR Increased Endogenous Ceramide in Endothelial
Cells--
Ceramide is thought to mediate endothelial apoptosis by
stress stimuli (5, 22-24), and exogenous C2-ceramide
itself can induce endothelial apoptosis (5). Since we observed that
4-HPR-induced apoptosis in endothelial cells, we determined whether
this was associated with increased ceramide. 4-HPR effectively
increased ceramide levels in human BMEC (Fig.
3) as well as in bovine TBMEC and HUVEC
(Table I). In the human BMEC, mean
ceramide increase was 5.25-fold ± 0.72 following a 17-h
incubation with 5 µM 4-HPR (n = 12 experiments in triplicate, p < 0.001). The increase in ceramide was observed as early as 4-8 h from the start of exposure to
4-HPR, before cytotoxicity or apoptosis could be detected, and
continued to increase up to 24 h (Fig. 3, B and
C). A decrease in several unidentified faster and slower
migrating lipid bands was observed in parallel to the increase in
[3H]ceramide (Fig. 3C). Whereas it is possible
that some of these are the lipids that contribute to the increase in
ceramide (higher Rf in particular), it is difficult to evaluate the
origin due to the large amount of radioactivity.
In most experiments, 4-HPR was added to the medium simultaneously with
[3H]palmitic acid (Fig. 3). [3H]Palmitic
acid rapidly entered both control human BMEC and those incubated with
4-HPR (72 ± 4 and 77 ± 1%, respectively, at 6 h, and
85 ± 2 and 83 ± 5% at 24 h, p = not
significant with versus without 4-HPR, by unpaired
t tests, n = 3). Thus, the increase in
ceramide in the presence of 4-HPR was not due to stimulation of uptake
of [3H]palmitic acid into the cells. The 4-HPR-induced
increase in [3H]ceramide was also observed in human and
bovine BMEC and in HUVEC when incubation with
[3H]palmitic acid began 17-24 h prior to the addition of
4-HPR and when 10% FBS was included in the medium (Table I). These
data indicate that 4-HPR induced generation of ceramide in
endothelial cells.
4-HPR Stimulated de Novo Ceramide Synthesis--
Sphingomyelinase
activation has been thought to be the main pathway for ceramide
generation in apoptosis following stress stimuli (1-4). However, later
reports demonstrate that some stimuli can induce apoptosis via de
novo ceramide synthesis (6, 8, 10). To determine which of these
pathways was activated by 4-HPR in human BMEC, we first examined
whether 4-HPR-induced ceramide formation could be suppressed by
inhibitors of de novo ceramide synthesis (Fig.
4). Fumonisin B1 (25 µM), an inhibitor of ceramide synthase, potently
inhibited ceramide generation in endothelial cells exposed to 4-HPR
(Fig. 4A). Similarly, L-cycloserine (30 µM) and myriocin (0.05 µM), both inhibitors
of the rate-limiting enzyme in the de novo ceramide
synthesis pathway, serine palmitoyltransferase (SPT) (10), potently
suppressed the 4-HPR-induced generation of ceramide (Fig. 4,
B and C). In order to more directly demonstrate the stimulation of de novo ceramide synthesis by 4-HPR, we
measured activity of SPT in microsomes isolated from endothelial cells that had been exposed to the drug or to vehicle control (Fig. 4D). Activity of SPT in the absence of 4-HPR was 29.8 ± 0.2 pmol/mg of protein/min and increased by 1.75-fold after 2 h
of incubation with 10 µM 4-HPR (Fig. 4D),
confirming stimulation of de novo ceramide synthesis by
4-HPR. By 6 h of incubation with 4-HPR, SPT activity returned to
base-line levels (Fig. 4D). 4-HPR did not induce activation
of ceramide synthase, an enzyme downstream of SPT, and at 10 µM even suppressed its activity by up to 25% (data not
shown). However, since SPT is the rate-limiting enzyme for de
novo ceramide synthesis and the base-line activity of ceramide synthase (138 ± 5 pmol/mg protein/min) in the human BMEC was more than 4 times higher than that of SPT (29.8 ± 0.2 pmol/mg of
protein/min), such change in ceramide synthase was not expected to
affect the increase in ceramide induced by 4-HPR. These experiments
demonstrate that 4-HPR activated de novo ceramide synthesis
in endothelial cells.
Effect of 4-HPR on Sphingomyelin Metabolism in Human BMEC--
To
assess whether sphingomyelin hydrolysis contributed to ceramide
increase, we prelabeled human BMEC with [3H]palmitic acid
and measured its decay following incubation with 4-HPR (Fig.
5). Under these conditions, sphingomyelin
levels in human BMEC treated with 4-HPR either remained unchanged or
increased slightly but did not decrease compared with controls
following the 24-h incubation with the drug (Fig. 5A).
Incubation with desipramine, a nonspecific inhibitor of sphingomyelin
hydrolysis, starting 2 h before the addition of 4-HPR, did not
alter sphingomyelin levels in the presence of 4-HPR, further suggesting
that 4-HPR did not activate sphingomyelinase (Fig. 5A).
Under these conditions, there was also no change in levels of cellular
glucosylceramide in the presence of 4-HPR (Fig. 5C).
Interestingly, despite the removal of the remaining exogenous
[3H]palmitic acid from the medium prior to the addition
of 4-HPR in these experiments, [3H]ceramide levels
increased in the presence of 4-HPR (Fig. 5B). The increase
in ceramide was not inhibited by desipramine (Fig. 5B),
further suggesting that sphingomyelinase activation did not contribute to the 4-HPR-induced ceramide increase.
Inhibition of 4-HPR-induced Ceramide Generation Suppressed
Endothelial Apoptosis--
To determine whether activation of de
novo ceramide synthesis has a causal role in 4-HPR-induced
endothelial apoptosis, we examined whether suppression of the
increase in ceramide could prevent 4-HPR-induced apoptosis (Fig.
6). Incubation of human BMEC with the
ceramide synthase inhibitor, fumonisin B1, at a concentration that effectively suppressed de novo-generated
ceramide increase (25 µM; Fig. 4A), completely
prevented apoptosis induced by 4-HPR (Fig. 6, A and
B). These data provide evidence for a causal role of
4-HPR-induced de novo-generated ceramide in endothelial apoptosis.
4-HPR-induced Ceramide Increase Was Independent of Caspase
Activation in Endothelial Cell Apoptosis--
To determine whether
4-HPR-induced apoptosis in BMEC was mediated by caspases, we examined
whether apoptosis could be inhibited by caspase inhibitors. When human
BMEC were incubated with the pancaspase inhibitor,
BOC-D-FMK, and exposed to 4-HPR, apoptosis was inhibited to
base-line levels compared with control cells (Fig.
7, A-C).
Benzyloxycarbonyl-VAD-FMK, another caspase family inhibitor, also
suppressed 4-HPR-induced apoptosis, and similar inhibition by
BOC-D-FMK was observed in bovine TBMEC (data not shown).
The effect of the caspase inhibitors further confirmed the apoptotic
nature of 4-HPR toxicity toward the human BMEC (Fig. 7,
A-C). In order to determine the ordering of caspase
activation and 4-HPR-induced ceramide generation, we incubated human
BMEC with BOC-D-FMK in the presence of 4-HPR and analyzed
ceramide levels (Fig. 7D). Whereas BOC-D-FMK
effectively inhibited 4-HPR-induced apoptosis (Fig. 7,
A-C), it had no effect on the 4-HPR-induced ceramide
increase in human BMEC (Fig. 7D). Similar results were obtained using bovine TBMEC (data not shown). These data place ceramide
upstream of caspases in 4-HPR-induced endothelial apoptosis. Collectively, these data provide evidence for a signaling role for
ceramide in 4-HPR-mediated endothelial apoptosis.
Stress stimuli such as irradiation, lipopolysaccharide, serum
starvation, and tumor necrosis factor- To date, induction of apoptosis by 4-HPR has only been described in
tumor cell lines (12, 13, 20). Our data now provide evidence for
apoptosis by 4-HPR in cultured endothelial cells, achieved at
concentrations similar to those that induce apoptosis in tumor
cell lines (20, 37-40). Decrease in capillary formation in the chick
chorioallantoic membrane by 4-HPR has been demonstrated by two groups,
indicating its in vivo antiangiogenic activity (14, 15).
However, only one of these groups found 4-HPR (2.5-5 µM,
48 h) to be cytotoxic to endothelial cells (14), whereas the other
did not detect cytotoxicity even after 72-h incubation with 10 µM 4-HPR (15). In our experiments, 4-HPR was cytotoxic to
the three types of endothelial cell preparations studied in tissue
culture. Thus, 4-HPR-mediated endothelial cytotoxicity occurs in four
of the five endothelial preparations studied to date, suggesting that
its effects may differ depending on the culture conditions and the
cells used. Clinical trials demonstrate that 4-HPR does not induce
generalized vascular damage in humans (12). This suggests that its
antiendothelial effect may manifest selectively in angiogenic
endothelium, which is thought to be biologically different from
endothelium in stable vasculature, and thus presents a selective
target for antiangiogenic therapies (41).
Ceramide increase in cells undergoing apoptosis has mostly been
described to occur independently of caspases (1). However, this pathway
may be cell-specific, stimulus-dependent, and affected by
culture conditions (1). For some stimuli such as CD95 (Fas/APO-1), ceramide accumulation occurs upstream of effector caspases but is
downstream of the initiator caspases (42). Similar to 4-HPR-mediated apoptosis in neuroblastoma and HL-60 cell lines (20, 39), 4-HPR-induced
apoptosis in BMEC was dependent on caspase activation. Initial
molecular ordering of ceramide in the BMEC utilizing a pancaspase
inhibitor placed ceramide upstream of caspase activation, as has been
shown for 4-HPR-induced apoptosis in HL-60 cells (39). However, it is
still not known whether there is a requirement for the initiator
caspases in 4-HPR-mediated ceramide increase in endothelial cells.
Fenretinide caused no detectable sphingomyelin hydrolysis (Fig. 5).
Additionally, sphingomyelin and ceramide levels in 4-HPR-treated cells
were not altered by desipramine (Fig. 5). Thus, it is unlikely that
sphingomyelinase, an important component of ceramide-mediated apoptosis
by several other stimuli (9), contributed to 4-HPR-induced generation
of ceramide in endothelial cells. Instead, the de novo ceramide synthesis pathway was implicated, as was found in
neuroblastoma and leukemia cells (19, 21, 35). This was shown by the
efficient suppression of 4-HPR-induced increase in
[3H]ceramide in human BMEC by inhibitors of de
novo ceramide synthesis (Fig. 4, A-C). Additionally,
4-HPR activated SPT, the first enzyme in this pathway (Fig.
4D), as was found in neuroblastoma cells (35). In
neuroblastoma, a downstream enzyme, ceramide synthase, was also
activated by 4-HPR (35), whereas in the human BMEC it was not. Lack of
stimulation of ceramide synthase, or even its mild inhibition, is not
expected to prevent an increase in de novo generated
ceramide by 4-HPR, since in the human BMEC ceramide synthase activity
was over 4 times higher than that of SPT at base line (138.0 and 29.8 pmol/mg protein/min, respectively). This is similar to the findings in
breast cancer cells, where PSC 833 induced a robust increase in
de novo ceramide synthesis by stimulation of SPT, the
rate-limiting enzyme, without effect on ceramide synthase activity,
which was about 3 times higher than SPT at base line (34). This
supports a role for increased SPT activity in 4-HPR-mediated
up-regulation of ceramide in endothelial cells. In the human BMEC, the
activation of SPT was early and transient, whereas ceramide continued
to increase for many hours, suggesting that other lipid metabolic
pathways may contribute to 4-HPR-induced increase in ceramide.
Supporting existence of additional mechanism(s) for 4-HPR-induced
ceramide increase, our data show that 4-HPR increased ceramide levels
even in BMEC prelabeled with [3H]palmitic acid (Fig.
5B), conditions where de novo synthesis of
[3H]ceramide directly from exogenous
[3H]palmitic acid was unlikely. Taken together, these
data demonstrate that 4-HPR stimulates de novo ceramide
synthesis and suggest that other ceramide-generating pathways may also
be affected by 4-HPR under the conditions used in these experiments.
Our results further show that fumonisin B1 efficiently
suppressed the 4-HPR-mediated increase in endothelial ceramide, in parallel to prevention of the associated apoptosis (Figs. 4 and 6).
This demonstrates a causal role for ceramide in 4-HPR-mediated endothelial apoptosis. This is similar to the mechanism of
fenretinide-induced cytotoxicity in leukemia, where inhibitors of
de novo ceramide synthesis were shown to inhibit apoptosis
(39). The concentrations of fumonisin B1 required to
inhibit the 4-HPR-induced increase in ceramide and apoptosis in the
BMEC (25 µM) were similar or lower than those used in
some tumor cell lines (25-100 µM) (39, 43). It is
possible that other proapoptotic mechanisms not mediated via ceramide
are also activated by 4-HPR (12) and contribute to its apoptotic effect
in the endothelial cells. One such mechanism is 4-HPR-induced
generation of reactive oxygen species described in
neuroblastoma and in HL-60 myeloid leukemia, which itself may be part of the signaling cascade to ceramide-mediated apoptosis (16,
17).
Taken together, our study establishes a causal role for de
novo-generated ceramide in the molecular mechanism of
4-HPR-induced endothelial apoptosis and supports examination of 4-HPR
as part of a combined antiangiogenic and anti-tumor approach to cancer therapy.
We thank Dr. Kwang Sik Kim for the generous
gift of the human BMEC and bovine TBMEC and Dr. Tomas Frgala for
assistance and helpful discussions. We thank Paul Alfaro for
expert technical assistance.
*
This work was supported by National Institutes of Health
Grant CA 81403, the Michael Hoefflin Children's Cancer Research Fund, and the My Brother Joey Foundation. It was also supported in part by
the Neil Bogart Memorial Fund of the T. J. Martell Foundation for
Leukemia, Cancer, and AIDS Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Childrens Hospital Los
Angeles, 4650 Sunset Blvd., Mailstop 57, Los Angeles, CA 90027. Tel.:
323-669-4613; Fax: 323-664-9455; E-mail: epstein@usc.edu.
Published, JBC Papers in Press, October 17, 2002, DOI 10.1074/jbc.M209962200
2
A. Erdreich-Epstein, unpublished data.
The abbreviations used are:
4-HPR, fenretinide;
bovine TBMEC, bovine large-T brain microvascular endothelial cells;
FBS, fetal bovine serum;
human BMEC, human brain microvascular
endothelial cells;
HPLC, high performance liquid chromatography;
HUVEC, human umbilical vein endothelial cells;
SPT, serine
palmitoyltransferase;
ANOVA, analysis of variance;
FMK, fluoromethyl
ketone;
BOC-D-FMK, butyloxycarbonyl-Asp-fluoromethyl ketone.
Ceramide Signaling in Fenretinide-induced Endothelial Cell
Apoptosis*
§,,,
,,,, and
Division of Hematology-Oncology,
Childrens Hospital Los Angeles, Department of Pediatrics and the
Department of Preventive Medicine, Keck
School of Medicine, University of Southern California, Los Angeles,
California 90027, the ¶ John Wayne Cancer Institute at Saint
John's Health Center, Santa Monica, California 90404, the
Section of Hematology/Oncology, Department of Pediatrics, Herman
B Wells Center for Pediatric Research, Indiana University School of
Medicine, Indianapolis, Indiana 46202, and the
** Division of Infectious Diseases, Department of
Pediatrics, Johns Hopkins School of Medicine,
Baltimore, Maryland 21205
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
lipopolysaccharide, and some chemotherapy drugs such as doxorubicin mediate apoptosis by generation of the lipid second messenger, ceramide
(1-4). We have recently shown that another stress response, endothelial anoikis (apoptosis resulting from the loss of matrix adhesion) is also associated with increased ceramide (5). Ceramide can
be generated by hydrolysis of membrane sphingomyelin by acid and/or
neutral sphingomyelinase and via activation of the de novo ceramide synthesis pathway, both of which can promote apoptosis (1, 2,
4, 6-11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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(10 ng/ml) with or without interferon-
pretreatment and in response to other stimuli (e.g. gp120 (0.5 µg/ml), bacterial exposure
(Escherichia coli E44), or lipopolysaccharide (50 ng/ml)) as
determined by enzyme-linked immunosorbent assay (27). In addition,
tumor necrosis factor-- and lipopolysaccharide-induced lactate
dehydrogenase release (Sigma kit) were also similar in both the high
and low passage human BMEC (data not shown). Experiments were performed using both low passage (passages 12-25) and higher passage (passages 25-55) human BMEC, yielding similar results for cytotoxicity, apoptosis, and ceramide increase.
20 °C. BOC-D-FMK were from Enzyme Systems Products,
Livermore, CA, and myriocin (ISP-1), and L-cycloserine were
from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).
Benzyloxycarbonyl-VAD-FMK was from BioVision Inc. (Mountain View, CA).
[9,10-3H]Palmitic acid (50 Ci/mmol) was from
PerkinElmer Life Sciences. L-[3H(G)]Serine
(20 Ci/mmol) and [5,6-3H]sphinganine (60 Ci/mmol) were
from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Sphinganine
(D-erythro-dihydrosphingosine) was purchased
from Matreya (Pleasant Gap, PA). Lipid standards were from Avanti Polar
Lipids, Inc. (Alabaster, AL). Uniplate Silica gel G TLC plates were
from Analtech, Inc. (Newark, DE). EN3HANCE®
spray was from PerkinElmer Life Sciences. Ecolume scintillation mixture
was from ICN Biomedicals, Inc. (Costa Mesa, CA). All other reagents
were purchased from Sigma.
20 °C. Lipids were solubilized in chloroform/methanol (2:1, v/v) and analyzed by TLC utilizing commercial lipid standards as
markers visualized in iodine vapors, as described (5, 20, 29,
30). The solvent systems used were chloroform/acetic acid (9:1, v/v)
for ceramide (5, 20, 29, 30), chloroform/methanol/acetic acid/double-distilled H2O (50:30:7:4, v/v/v/v) for
sphingomyelin (29), and chloroform/methanol/ammonium hydroxide
(70:20:4, v/v/v) for glucosylceramide (31). Tritium in the TLC-resolved
lipid band and total tritium in equal aliquots of the extracted
cellular lipids were quantitated by liquid scintillation counting. The amount of ceramide, sphingomyelin, or glucosylceramide was expressed as
percentage of cpm in these classes of lipids out of the total lipid
tritium in the sample. For radiographs, the TLC plates were sprayed
with EN3HANCE® according to the
manufacturer's instructions and exposed to film (Hyperfilm; Amersham
Biosciences) at 80 °C for 3-7 days.
80 °C (33).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
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Fig. 1.
Fenretinide was cytotoxic to human
BMEC. Human BMEC (6 × 103 cells/well in a
96-well plate) were incubated with 4-HPR for 24 h. Cell viability
was assessed using a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. A, cells were incubated in medium
containing 0.1% FBS. Data points are means ± S.E. of 10-21
experiments, performed in 5-10 replicates. p < 0.001 by one way ANOVA for 0-10 µM 4-HPR. B, cells
were incubated in culture medium containing 10% ( 
) or 0.1% (
)
FBS. Data points are means ± S.E. of 3-6 experiments performed
in 5-10 replicates. p < 0.001 by two-tailed paired
t test comparing mean survivals in 10% and 0.1% FBS at
each 4-HPR concentration in each of the experiments. C,
cells were incubated with the indicated concentrations of FBS with
(
) or without (
) 4-HPR (5 µM) in the presence or
absence of 1% heat-inactivated fatty acid-free bovine serum albumin
(BSA). Bars represent means ± S.E. of five
replicate samples. *, p = 0.0023 for cells in 2%
compared with 0.1% FBS; **, p = 0.013 for cells in
10% compared with 2% FBS, by unpaired t tests.
p = not significant in the presence compared with the
absence of bovine serum albumin at each of the FBS concentrations, by
unpaired t tests.
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Fig. 2.
Fenretinide-induced apoptosis in human
BMEC. A-E, apoptosis (cells with a
sub-G0/G1 DNA content) was assessed by flow
cytometry of permeabilized propidium iodide-stained human BMEC
(106 cells/10-cm dish) that were incubated with 4-HPR as
indicated below. A, cells were incubated for 24 h with
4-HPR in culture medium containing 0.1% FBS. UV-irradiated cells
harvested 24 h following irradiation (25 mJ; UV) were
used as positive control. Shown are means ± S.E. of a
representative experiment from over 10 performed in triplicates with
similar results. p < 0.001 by one-way ANOVA for 0-5
µM 4-HPR. B, flow cytometry tracings from a
representative experiment of cells incubated for 24 h with 4-HPR
(5 µM) or vehicle control or following UV irradiation as
described for A. C and D, cells were
incubated with 4-HPR for 24 h (C) or 72 h
(D) in medium containing 0.1% ( ), 1% (
), or 10%
() FBS. C, representative experiment of three performed
under similar conditions; D, mean ± S.E. of nine
independent experiments. E, cells were incubated for 24 (), 48 (
), or 72 h () with 0-7.5 µM 4-HPR
in medium containing 10% FBS. Shown are means ± S.E. of three
independent experiments. F, human BMEC (3 × 106 cells/10-cm plate) were incubated for 6 h with 5 µM 4-HPR in medium containing the indicated
concentrations of FBS. Cells were trypsinized and 4-HPR content was
determined by HPLC. Bars, -fold change of 4-HPR content/g of
cells (mean ± S.E.) from three independent experiments.
p = 0.017 comparing 1 and 10% FBS by unpaired
t tests.
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Fig. 3.
4-HPR increased endothelial ceramide in a
dose- and time-dependent manner. A, 4-HPR
and [3H]palmitic acid were added simultaneously to human
BMEC (3 × 106 cells/10-cm dish) in medium containing
0.1% FBS. After 16 h, cells were harvested, lipids were
extracted, and ceramide was determined by TLC. Results are presented as
cpm in [3H]ceramide as a percentage of the total lipid
tritium. Shown are means ± S.E. from a representative experiment
of multiple repeats performed in triplicates. p < 0.001 by one-way ANOVA for 0, 3, and 5 µM 4-HPR.
B, human BMEC (3 × 106 cells/10-cm dish)
were incubated in medium containing [3H]palmitic acid and
0.1% FBS. Vehicle control ( ) or 4-HPR (; 5 µM)
were added either at the time of the addition of
[3H]palmitic acid (for the 24-h time point) or 6, 12, 16, or 20 h later (to achieve incubations with 4-HPR of 24, 18, 12, 8, and 4 h, respectively). Cells were harvested 24 h after the
addition of [3H]palmitic acid. The x axis
denotes length of incubation with 4-HPR or control. Lipids were
processed as described. Shown are means ± S.E. from a
representative experiment of three performed in triplicates.
p = 0.0060 in cells with 5 µM 4-HPR at
4-24 h, by simple linear regression. C, human BMEC were
labeled with [3H]palmitic acid and treated with 4-HPR (5 µM) as in B. Lipids were extracted, equal
amounts (cpm) of total labeled lipids were separated on TLC, and
radiographs were developed. The time indicated is length of incubation
with 4-HPR.
Ceramide increase by 4-HPR occurred in three different endothelial
preparations and in the presence of 10% FBS
View larger version (28K):
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Fig. 4.
4-HPR stimulated activity of SPT and
increased de novo synthesis of ceramide in human BMEC.
A-C, human BMEC (3 × 106 cells/10-cm
dish) were incubated in medium containing 0.1% FBS with inhibitor
(fumonisin B1, 25 µM (A),
L-cycloserine, 30 µM (B), and
myriocin, 0.05 µM (C); ) or vehicle control
(Me2SO; ) beginning 2 h before the addition of
[3H]palmitic acid and 4-HPR (0, 3, or 5 µM). After overnight incubation with 4-HPR, cells were
harvested, lipids were extracted, and ceramide was determined by TLC.
Results are presented as cpm in [3H]ceramide, as a
percentage of the total lipid tritium. p < 0.001 in
cells exposed to 4-HPR with inhibitors () compared with without them
(), by unpaired t tests for each inhibitor. Shown are
means ± S.E. from representative experiments of three, performed
in triplicates. D, human BMEC were incubated with 4-HPR (10 µM) for the indicated times. Microsomes were isolated,
and enzymatic activity of SPT was determined as described under
"Experimental Procedures." Results are expressed as -fold change in
SPT activity (mean ± S.E.). SPT activity in the absence of 4-HPR
was 29.8 pmol/mg of protein/min. Shown is one of two similar
experiments, performed in duplicates. p = 0.022 by
one-way ANOVA.
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Fig. 5.
Effect of 4-HPR on sphingomyelin levels in
endothelial cells. A-C, human BMEC (3 × 106 cells/10-cm dish) were preloaded with
[3H]palmitic acid for 24 h, and the isotope was
washed out for 2 h. Desipramine (5 µM;
horizontally hatched bars) or vehicle
control (filled bars) were added in fresh medium
lacking isotope and containing 0.1% FBS, and 4-HPR was added 2 h
later for 24 h. Cells were harvested, lipids were extracted, and
sphingomyelin (A; SM), ceramide (B),
and glucosylceramide (C) were determined by TLC using the
solvent systems described under "Experimental Procedures." Results
are presented as cpm in either [3H]ceramide,
[3H]sphingomyelin, or [3H]glucosylceramide,
as a percentage of the total lipid tritium. For sphingomyelin
(A), there was no effect of 4-HPR or desipramine
(p = not significant, by two-way ANOVA). However, for
ceramide (B) there was a strong 4-HPR effect
(p < 0.001) but no desipramine effect
(p = 0.13 for the overall effect and p = 0.98 for the interaction; all based on two-way ANOVA).
Bars, means ± S.E. of one of ten experiments with
similar results, and comparison with desipramine represents one of two
experiments, all performed in triplicate.
View larger version (17K):
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Fig. 6.
Prevention of 4-HPR-induced generation of
ceramide suppressed endothelial cell apoptosis. Fumonisin
B1 or vehicle control were added to human BMEC
(106 cells/10-cm dish) incubated in medium containing 1%
FBS, followed 2 h later by 4-HPR or vehicle control. 72 h
later, cells were permeabilized, stained with propidium iodide, and
analyzed for apoptosis by flow cytometry. A, 4-HPR (3 µM) ( ), control (), and UV (25 mJ) (
), in the
presence of 0-25 µM FB1. Data points
represent mean percentage of apoptosis of duplicate samples. Error bars
(S.E.) are smaller than the symbols. p < 0.0001 for cells with 4-HPR and 0-25 µM fumonisin
B1, by one-way ANOVA. B, fumonisin
B1 (25 µM) (filled
bars) and Me2SO (horizontally
hatched bars) in the presence of 0-3
µM 4-HPR. p = 0.016 between cells treated
with 4-HPR with fumonisin or without it, by paired t
test.
View larger version (26K):
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Fig. 7.
4-HPR-induced ceramide generation was
independent of caspases in endothelial cells. Human BMEC
(105 cells/well in two-well chamber slide (A),
106 cells/10-cm dish (B and C), and
3 × 106 cells/10-cm dish (D)) were
preincubated in medium containing 0.1% FBS with 25 µM
BOC-D-FMK ( ) or Me2SO () for 2 h.
4-HPR or vehicle control were then added for 24 h
(A-C) or 16 h (D). Shown are means ± S.E. of triplicate samples. A, cells were fixed, stained
with Hoechst-bisbenzamide 33258, and scored for apoptosis (cells with
condensed and fragmented nuclei) in a blinded manner. *,
p = 0.017; **, p = 0.001 by unpaired
t test. B, apoptosis was assessed by flow
cytometry of propidium iodide-stained fixed cells. Shown are means ± S.E.; *, p < 0.001 comparing cells with 5 µM 4-HPR with or without BOC-D-FMK by
unpaired t test. Shown is one of two experiments with
similar results, performed in triplicate. C, representative
flow cytometry tracings of propidium iodide-stained fixed cells from
the experiment shown in B. UV-irradiated (25 mJ) human BMEC
serve as positive control. D, cells were harvested, lipids
were extracted, and ceramide was quantitated by TLC. Results are
presented as cpm in [3H]ceramide, as a percentage of the
total lipid tritium. p = 0.42 comparing cells in 3 µM 4-HPR with or without BOC-D-FMK by
unpaired t test. Shown is one of two experiments with
similar results.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mediate endothelial apoptosis
by increasing ceramide (22-24). We have recently shown that
endothelial anoikis, the apoptosis resulting from the loss of matrix
adhesion, is also associated with increased ceramide (5). Our current
results demonstrate that 1) 4-HPR increased endothelial ceramide by
de novo, nonsphingomyelinase-mediated ceramide synthesis, 2)
4-HPR induced caspase-dependent endothelial apoptosis
mediated by ceramide, and 3) ceramide was upstream of caspases in 4-HPR
signaling to endothelial apoptosis. These data are the first
investigation of the molecular signaling events mediating 4-HPR-induced
endothelial cell apoptosis.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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  P. G. Sreekumar, J. Zhou, J. Sohn, C. Spee, S. J. Ryan, B. J. Maurer, R. Kannan, and D. R. Hinton N-(4-hydroxyphenyl) Retinamide Augments Laser-Induced Choroidal Neovascularization in Mice Invest. Ophthalmol. Vis. Sci., March 1, 2008; 49(3): 1210 - 1220. [Abstract] [Full Text] [PDF]
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 W. Wrasidlo, A. Mielgo, V. A. Torres, S. Barbero, K. Stoletov, T. L. Suyama, R. L. Klemke, W. H. Gerwick, D. A. Carson, and D. G. Stupack The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8 PNAS, February 19, 2008; 105(7): 2313 - 2318. [Abstract] [Full Text] [PDF]
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 C. Guilbault, J. B. De Sanctis, G. Wojewodka, Z. Saeed, C. Lachance, T. A. A. Skinner, R. M. Vilela, S. Kubow, L. C. Lands, M. Hajduch, et al. Fenretinide Corrects Newly Found Ceramide Deficiency in Cystic Fibrosis Am. J. Respir. Cell Mol. Biol., January 1, 2008; 38(1): 47 - 56. [Abstract] [Full Text] [PDF]
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 A. M. Abdel Nour, D. Ringot, J.-L. Gueant, and A. Chango Folate receptor and human reduced folate carrier expression in HepG2 cell line exposed to fumonisin B1 and folate deficiency Carcinogenesis, November 1, 2007; 28(11): 2291 - 2297. [Abstract] [Full Text] [PDF]
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 R. Vene, G. Arena, A. Poggi, C. D'Arrigo, M. Mormino, D. M. Noonan, A. Albini, and F. Tosetti Novel cell death pathways induced by N-(4-hydroxyphenyl)retinamide: therapeutic implications Mol. Cancer Ther., January 1, 2007; 6(1): 286 - 298. [Abstract] [Full Text] [PDF]
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M. Moussavi, K. Assi, A. Gomez-Munoz, and B. Salh Curcumin mediates ceramide generation via the de novo pathway in colon cancer cells Carcinogenesis, August 1, 2006; 27(8): 1636 - 1644. [Abstract] [Full Text] [PDF]
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 J. G. Villablanca, M. D. Krailo, M. M. Ames, J. M. Reid, G. H. Reaman, and C. P. Reynolds Phase I Trial of Oral Fenretinide in Children With High-Risk Solid Tumors: A Report From the Children's Oncology Group (CCG 09709) J. Clin. Oncol., July 20, 2006; 24(21): 3423 - 3430. [Abstract] [Full Text] [PDF]
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 P.-L. Liu, Y.-L. Chen, Y.-H. Chen, S.-J. Lin, and Y. R. Kou Wood smoke extract induces oxidative stress-mediated caspase-independent apoptosis in human lung endothelial cells: role of AIF and EndoG Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L739 - L749. [Abstract] [Full Text] [PDF]
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 A. Erdreich-Epstein, L. B. Tran, O. T. Cox, E. Y. Huang, W. E. Laug, H. Shimada, and M. Millard Endothelial apoptosis induced by inhibition of integrins {alpha}v{beta}3 and {alpha}v{beta}5 involves ceramide metabolic pathways Blood, June 1, 2005; 105(11): 4353 - 4361. [Abstract] [Full Text] [PDF]
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 T. C. Stover, A. Sharma, G. P. Robertson, and M. Kester Systemic Delivery of Liposomal Short-Chain Ceramide Limits Solid Tumor Growth in Murine Models of Breast Adenocarcinoma Clin. Cancer Res., May 1, 2005; 11(9): 3465 - 3474. [Abstract] [Full Text] [PDF]
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D. Serrano, L. Baglietto, H. Johansson, F. Mariette, R. Torrisi, M. Onetto, M. Paganuzzi, and A. Decensi Effect of the Synthetic Retinoid Fenretinide on Circulating Free Prostate-Specific Antigen, Insulin-Like Growth Factor-I, and Insulin-Like Growth Factor Binding Protein-3 Levels in Men with Superficial Bladder Cancer Clin. Cancer Res., March 1, 2005; 11(5): 2083 - 2088. [Abstract] [Full Text] [PDF]
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 E. Dmitrovsky Fenretinide Activates a Distinct Apoptotic Pathway J Natl Cancer Inst, September 1, 2004; 96(17): 1264 - 1265. [Full Text] [PDF]
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P. E. Lovat, F. Di Sano, M. Corazzari, B. Fazi, R. P. Donnorso, A. D. J. Pearson, A. G. Hall, C. P. F. Redfern, and M. Piacentini Gangliosides Link the Acidic Sphingomyelinase-Mediated Induction of Ceramide to 12-Lipoxygenase-Dependent Apoptosis of Neuroblastoma in Response to Fenretinide J Natl Cancer Inst, September 1, 2004; 96(17): 1288 - 1299. [Abstract] [Full Text] [PDF]
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 T. Matsunaga, S. Kotamraju, S. V. Kalivendi, A. Dhanasekaran, J. Joseph, and B. Kalyanaraman Ceramide-induced Intracellular Oxidant Formation, Iron Signaling, and Apoptosis in Endothelial Cells: PROTECTIVE ROLE OF ENDOGENOUS NITRIC OXIDE J. Biol. Chem., July 2, 2004; 279(27): 28614 - 28624. [Abstract] [Full Text] [PDF]
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A. Decensi, B. Bonanni, L. Baglietto, A. Guerrieri-Gonzaga, F. Ramazzotto, H. Johansson, C. Robertson, I. Marinucci, F. Mariette, M. T. Sandri, et al. A Two-by-Two Factorial Trial Comparing Oral with Transdermal Estrogen Therapy and Fenretinide with Placebo on Breast Cancer Biomarkers Clin. Cancer Res., July 1, 2004; 10(13): 4389 - 4397. [Abstract] [Full Text] [PDF]
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V. Dolgachev, M. S. Farooqui, O. I. Kulaeva, M. A. Tainsky, B. Nagy, K. Hanada, and D. Separovic De Novo Ceramide Accumulation Due to Inhibition of Its Conversion to Complex Sphingolipids in Apoptotic Photosensitized Cells J. Biol. Chem., May 28, 2004; 279(22): 23238 - 23249. [Abstract] [Full Text] [PDF]
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 R. J. Perry and N. D. Ridgway The role of de novo ceramide synthesis in the mechanism of action of the tricyclic xanthate D609 J. Lipid Res., January 1, 2004; 45(1): 164 - 173. [Abstract] [Full Text] [PDF]
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N. Ferrari, M. Morini, U. Pfeffer, S. Minghelli, D. M. Noonan, and A. Albini Inhibition of Kaposi's Sarcoma in Vivo by Fenretinide Clin. Cancer Res., December 1, 2003; 9(16): 6020 - 6029. [Abstract] [Full Text] [PDF]
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