Vitamin C inhibits FAS-induced apoptosis in monocytes and U937 cells
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PHAGOCYTES
Vitamin C inhibits FAS-induced apoptosis in monocytes and U937 cells
Isabel Perez-Cruz, Juan M. Carcamo, and David W. Golde
The FAS receptor–FAS ligand system is a sis in the monocytic U937 cell line and in activation of caspase-8 with no effect on
key apoptotic pathway for cells of the fresh human monocytes. Cells were it enzymatic activity. An independent ac-
immune system. Ligation of the FAS- loaded with vitamin C by exposure to tion of high intracellular concentrations
receptor (CD95) induces apoptosis by dehydroascorbic acid (DHA), thereby cir- of vitamin C on mitochondrial membrane
activation of pro–caspase-8 followed by cumventing in vitro artifacts associated stabilization was also detected. These
downstream events, including an in- with the poor transport and pro-oxidant studies illuminate the nature of redox-
crease in reactive oxygen species (ROS) effects of ascorbic acid (AA). Vitamin C dependent signaling in FAS-induced apo-
and the release of proapoptotic factors inhibition of FAS-mediated apoptosis was ptosis of human monocytes and suggest
from the mitochondria, leading to associated with reduced activity of that vitamin C can modulate the immune
caspase-3 activation. We investigated the caspase-3, -8, and -10, as well as dimin- system by inhibiting FAS-induced mono-
role of vitamin C in FAS-mediated apopto- ished levels of ROS and preservation of cyte death. (Blood. 2003;102:336-343)
sis and found that intracellular accumula- mitochondrial membrane integrity. Mecha-
tion of pharmacologic concentrations of nistic studies indicated that the major
vitamin C inhibited FAS-induced apopto- effect of vitamin C was inhibition of the © 2003 by The American Society of Hematology
Introduction
Apoptosis is a suicidal mode of cell death used to eliminate cells in involved in receptor-mediated signaling,13-15 and antioxidants can
physiologic and pathologic settings.1,2 While diverse events can inhibit important signaling pathways in immune cells.16,17 Vitamin
induce apoptosis, such as radiation and lack of growth factors, C is a critical dietary nutrient in humans, since humans cannot
apoptosis in the immune system is largely regulated by signaling synthesize it as do most nonprimates. Deficiency of vitamin C
through death receptors, including the receptor for tumor necrosis seriously impairs immune function, and there is literature pointing
factor (TNF) and the FAS receptor (FAS-R), also known as CD95.1 to the role of vitamin C in enhanced host defense and the
Apoptosis generally occurs in 2 phases: the commitment to cell modulation of inflammatory reactions.18-22 Vitamin C is generally
death, including activation of procaspases, followed by an execu- thought to enhance immunity and is widely taken as a supplement.
tion phase resulting in cell structure alterations.3 Whereas vitamin C is found in the plasma in its reduced form
Upon FAS-R ligation a set of intracellular signaling proteins is (ascorbic acid [AA]), it is transported by most cells in its oxidized
recruited to the death-inducing signaling complex. The cytosolic form, dehydroascorbic acid (DHA), through facilitative glucose
pro–caspase-8 is recruited and believed to oligomerize and autoac- transporters.23 Inside the cell, DHA is reduced to AA.24 Certain
tivate.4 Caspase-10 is closely related to caspase-8 and it has been specialized cells transport AA directly through cell-surface Na-
proposed that both caspases can be recruited to the death-inducing ascorbate cotransporters.25 We sought to determine the role of
signaling complex, where they are activated and initiate signaling vitamin C in FAS-induced apoptosis in monocytes and used DHA
pathways resulting in apoptosis.5 Active caspase-8 induces translo- to load cells with vitamin C, thereby avoiding the pro-oxidant
cation of the proapoptotic protein BID to the mitochondria, causing effects of AA in the presence of free transition metals ubiquitously
an increase in mitochondrial permeability and a loss in mitochon- found in tissue culture.26 We found vitamin C to be a potent
drial membrane potential (⌬).6 Oxidative phosphorylation is inhibitor of FAS-induced apoptosis in fresh monocytes and in U937
uncoupled and the mitochondria release reactive oxygen species cells. Our data indicate the primary action of vitamin C to be the
(ROS), resulting in elevated levels of ROS in cells stimulated via inhibition of activation of caspase-8 and a separate effect on
the FAS pathway.7,8 The increase in cellular ROS is a key event in downstream oxidative events.
programmed cell death and oxidative molecules themselves can
induce apoptosis, independent of ligation of death receptors.9,10
Release of cytochrome C (Cyt C) and other mitochondrial mol- Materials and methods
ecules, such as Apaf-1, leads to the downstream activation of
caspase-9 and the effector caspase-3.11 Cells lines and monocytes
Cellular ROS generation is a natural result of aerobic metabo- The monocytic cell line U937 was obtained from the American Type
lism and is amplified by cellular activation.12 ROS are widely Culture Collection (Manassas, VA) and cultured in complete medium:
From the Program in Molecular Pharmacology and Chemistry and Clinical Reprints: David W. Golde, Memorial Sloan-Kettering Cancer Center, 1275
Chemistry and Medicine, Memorial Sloan-Kettering Cancer Center, New York Ave, New York, NY 10021; e-mail: d-golde@ski.mskcc.org.
York, NY.
The publication costs of this article were defrayed in part by page charge
Submitted November 25, 2002; accepted February 24, 2003. Prepublished online
payment. Therefore, and solely to indicate this fact, this article is hereby
as Blood First Edition Paper, March 6, 2003; DOI 10.1182/blood-2002-11-3559.
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by a grant from the National Institutes of Health (CA 30388) and the
Lebensfeld Foundation. © 2003 by The American Society of Hematology
336 BLOOD, 1 JULY 2003 䡠 VOLUME 102, NUMBER 1BLOOD, 1 JULY 2003 䡠 VOLUME 102, NUMBER 1 VITAMIN C INHIBITS FAS-INDUCED APOPTOSIS 337
RPMI containing 100 U penicillin, 100 U streptomycin (Gemini Bioprod- cell samples were incubated with several concentrations of DHA from a
ucts, Woodland, CA), L-glutamate, and 10% fetal calf serum (Omega solution containing 0.5 Ci (0.0185 MBq) L-14C-AA (specific activity 8.0
Scientific, CA). Human monocytes were isolated from peripheral blood mCi/mmol (296 MBq/mmol); Perkin-Elmer Life Sciences, MA), 1.0 to 4.0
obtained from the New York Blood Center (NY). The following day, mM AA (Sigma), and 86 U/mL ascorbate oxidase (Sigma) in incubation
peripheral blood mononuclear cells were separated by Ficoll gradient buffer (15.0 mM HEPES [N-2-hydroxyethylpiperazine-N⬘-2-ethanesul-
(Accu-Prep; Accurate Chemicals and Scientific, Westbury, NY). Monocytes fonic acid], 135.0 mM NaCl, 5.0 mM KCl, 1.8 mM CaCl2, and 0.8 mM
were immediately purified by negative selection using the magnetic- MgCl2 [pH 7.4]). Cells were then washed twice with cold PBS and lysed
activated cell separation monocyte isolation kit (Miltenyi Biotec, Auburn, with 0.5 mL sodium dodecyl sulfate (SDS)–lysis solution (10.0 mM Tris
CA) following the manufacturer’s instructions. Monocytes were incubated [tris(hydroxymethyl)aminomethane]–HCl [pH 8.0] and 0.2% SDS). Cell-
in RPMI containing 100 U penicillin, 100 U streptomycin, L-glutamate, and associated radioactivity was quantified by scintillation spectrometry. To
20% human AB serum (Irvine Scientific, Santa Ana, CA). Purity was determine the accumulation of vitamin C supplied as AA, cells were
assessed by staining with anti-CD3 (RPE-Cy5; DAKO, Glostrup, Den- incubated with varying concentrations of AA from a solution prepared with
mark), anti-CD56 (phycoerythrin [PE]; Becton Dickinson, San Diego, CA), 0.5 Ci (0.0185 MBq) L-14C-AA, 1.0 to 4.0 mM L-AA, and 0.1 mM
anti-CD20 (peridinin chlorophyll protein [PerCP]; Becton Dickinson), and 1,4-dithiothreitol in incubation buffer and cell-associated radioactivity
anti-CD14 (fluorescein isothiocyanate [FITC]; Becton Dickinson) fluores- measured by scintillation spectrometry. Additionally, accumulation of
cent antibodies. The cells were suspended in staining buffer consisting of vitamin C supplied as DHA was determined by high-performance liquid
1% heat-inactivated fetal calf serum and 0.1% (wt/vol) sodium azide chromatography (HPLC) electrochemical detection (ESA, Chelmsford,
(Sigma, St Louis, MO) in phosphate-buffered saline (PBS), pH 7.4, MA).27 Cells incubated with DHA were washed twice in cold PBS and then
containing the fluorescent antibody (Ab) for 30 minutes at 4°C. After lysed in 60% methanol. Lysates were centrifuged, the supernatants filtered
staining, the cells were washed with buffer, fixed in 1% paraformaldehyde through 0.2-m nylon membrane filters (Nalgene, Rochester, NY) and
and analyzed by flow cytometry (FACScalibur; Becton Dickinson) within equal amounts of each sample injected onto a modified C18 column (no.
24 hours of staining. The results were analyzed using the CELLQuest 70-4160; ESA). The column was equilibrated at 30°C with a mobile phase
software (Becton Dickinson). Monocyte preparations were always higher buffer consisting of 50 M sodium phosphate, 50 M sodium acetate, 189
than 95% purity. M dodecyltrimethylammonium chloride, and 3.66 M tetraoctylammo-
nium bromide in 30:70 methanol-water (vol/vol, HPLC grade; J. T. Baker,
Detection of CD95 Phillipsburg, NJ), pH 4.8. The flow rate was 0.3 mL/min. Under these
conditions, the retention time of AA was 4.9 minutes. Peak areas for AA
The antihuman CD95-RPE Ab (DAKO) was used to detect the CD95 were determined using a dominant potential for AA of 100 mV, and AA was
antigen in cell lines and monocytes by flow cytometry. Cell-surface staining quantified from a calibration curve.
and analysis were performed as described in the previous section.
Cell volume determination
Induction of apoptosis
Estimation of cell volume was performed as previously described.28 Briefly,
An anti-FAS Ab and soluble FAS ligand (FASL) were used to induce 5 ⫻ 106 cells were incubated for 60 minutes at RT in 200 L incubation
apoptosis. For the induction of apoptosis in monocytes, an anti-FAS Ab buffer containing 1.0 mM 3-oxy-methyl-D-glucose and 5 Ci (0.185 MBq)
(immunoglobulin G1 [IgG1], clone DX2; R and D Systems, Minneapolis, 3H-3-oxy-methyl-D-glucose. Uptake was stopped by adding 2 L of 2.0
MN) or an isotype control Ab (R and D Systems) were plate-immobilized in mM cytochalasin B (Sigma), which blocks glucose transport and prevents
a 12–flat well plate overnight. The plate was washed twice in serum-free the efflux of trapped glucose. The cells were then washed twice in ice-cold
RPMI and blocked with RPMI containing 10% human type AB serum for PBS containing 20 M cytochalasin B and cell-associated radioactivity
30 minutes at 37°C. The plate was then washed and the cells were added determined by scintillation spectrometry. Glucose reached equilibrium after
immediately. For induction of apoptosis in U937 cells, 10 L protein G (2.5 60 minutes of incubation, therefore the amount of radioactivity accumu-
mg/mL suspension in agarose beads; Boehringer Mannheim, Mannheim, lated inside the cells is in direct proportion to the intracellular volume. The
Germany) per g anti-FAS Ab or control Ab was mixed in cold serum-free cell volume estimated for monocytes was 0.15 L per 106 cells and 1.0 L
RPMI for 5 minutes at room temperature (RT) before addition to the cell per 106 U937 cells.
suspension. To induce apoptosis with soluble FASL, Super FASL (Alexis
Biochemicals, Lausen, Switzerland) was added to U937 cells or to freshly
Vitamin C loading
isolated monocytes. Cells treated with anti-FAS Ab, FASL, or control cells
were incubated at 37°C in an environment of 5% CO2 in air. Cells were washed twice in incubation buffer at pH 7.4, and DHA (Aldrich,
Milwaukee, WI) was added. After incubation with DHA for up to 60
Detection of apoptosis minutes at 37°C, the cells were washed twice in serum-free RPMI.
The annexin V–FITC/propidium iodine (PI) apoptosis detection kit (Pharm- Detection of ROS
ingen, San Diego, CA) was used to determine the frequency of apoptosis in
U937 cells, following the manufacturer’s instructions. Alternatively, PI Intracellular ROS in U937 were estimated by 2⬘7⬘ dichlorofluorescein
staining of DNA was used to analyze the sub-G1 region by flow cytometry. diacetate (DCFH-DA) fluorescence.8 Cells were washed twice in Krebs-
For PI staining, cells were washed twice with PBS and fixed with 70% Ringer buffer (20.0 mM HEPES, 10.0 mM dextrose, 127.0 mM NaCl, 5.5
ethanol for at least 30 minutes at 4°C. Cells were then washed twice with mM KCl, 1.0 mM CaCl2, and 2.0 mM MgSO4 [pH 7.4]) and stained with
PBS and resuspended in 50 L of a 100 U/mL RNAse-A solution 0.1 g/mL DCFH-DA (Molecular Probes, Eugene, OR) and 1.0 mM maleic
(Boehringer Mannheim) and 20 L PI (0.1 mg/mL; Sigma) per 1 ⫻ 105 acid diethyl ester (Sigma) in Krebs-Ringer buffer. Fluorescence was
cells for 30 minutes at RT in the dark and kept at 4°C until flow cytometry determined by flow cytometry after incubation at 37°C, and the data were
acquisition. Apoptosis in monocytes was determined by analysis of the analyzed using CELLQuest software.
sub-G1 region by DNA staining and flow cytometry. For PI staining, the
monocytes were incubated with trypsin for 5 minutes at 37°C, washed twice Assessment of mitochondrial membrane potential (⌬)
with PBS, and then stained as described in the previous section.
U937 cells were washed twice with PBS, stained with 40 nM 3,3⬘-
dihexyloxacarbocyanine iodide (DiOC(6)(3); Molecular Probes) with or
Vitamin C uptake
without 100 M carbonyl cyanide m-chlorophenylhydrazone (Sigma) as a
Vitamin C uptake was measured as intracellular accumulation after control for membrane potential disruption, and incubated at 37°C in the
incubation of cells with DHA or AA at 37°C, as previously described.23,24 dark for 15 minutes before determination of fluorescence by flow cytom-
To determine the accumulation of vitamin C supplied as DHA, triplicate etry. These experiments and the ROS quenching studies were not performed338 PEREZ-CRUZ et al BLOOD, 1 JULY 2003 䡠 VOLUME 102, NUMBER 1
in monocytes because cell handling and the required trypsinization led to
erratic results in these assays.
Results
Cytochrome C release Vitamin C inhibits FAS-induced apoptosis
Release of CytC into the cytosol was detected with a CytC enzyme-linked FAS-R (CD95) was expressed in U937 cells and monocytes, and
immunosorbent assay (ELISA) kit (MLB, Nagoya, Japan), following the both anti-FAS Ab and FASL induced apoptosis in these cells in a
manufacturer’s instructions. To obtain cytosolic fractions, cells were dose-dependent manner (Figure 1). Incubation of U937 cells for 3
washed twice with ice-cold PBS, resuspended in cold buffer (1.28 M NaCl, hours with FASL induced up to a 3.5-fold increase in apoptosis,
50.0 mM KCl, 50.0 mM MgSO4, 13.0 mM CaCl2, 0.5 M HEPES, 1.0 mM whereas incubation with anti-FAS Ab caused a 2-fold increase
phenylmethylsulfonyl fluoride, 10% vol 2.5 M sucrose, 10.0 mM 1,4-
(Figure 1B-C). In monocytes, 3-hour treatment with FASL resulted
dithiothreitol, 1.0 mM reduced glutathione, and 1% glycerol) and homoge-
nized mechanically. This total cellular extract was centrifuged at 2000 rpm
in up to a 7-fold increase in apoptosis, whereas incubation with
for 3 minutes at 4°C and the cytosolic supernatant recovered and analyzed anti-FAS Ab resulted in a 4-fold increase (Figure 1E-F). An isotype
for the presence of cytochrome C. control Ab did not cause apoptosis in either cell type (data not
shown). FASL was more efficient than anti-FAS Ab in inducing
apoptosis in both U937 cells and monocytes (Figure 1).
Caspase assays
We investigated the effect of loading cells with vitamin C on
Caspase activity was measured using the caspase-3 (Sigma), caspase-8, or FAS-R–dependent apoptosis. U937 cells and monocytes transport
caspase-10 (R and D Systems) colorimetric assay kits, following the vitamin C in the form of DHA through facilitative glucose
manufacturer’s instructions. Briefly, 1 ⫻ 107 cells were lysed and equal transporters.29,30 As measured by 14C-substrate uptake, there was
quantities of protein loaded in MaxiSorp 96-well plates (Nunc, Roskilde, little evidence for direct transport of AA, whereas DHA was readily
Denmark). The caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Sigma)
taken up by the cells (Figure 2A-D). We estimated the internal
or caspase-8 inhibitor benzylloxycarbonyl-Ileu-Glu-Thr-Asp-fluoromethyl
volume of U937 cells at 1.0 L/106 cells and monocytes at
ketone (Z-IEDT-FMK; BioVision Research Products, CA) was added as a
control at 20-M final concentration. The plate was incubated at 37°C 0.15 L/106 cells from tritiated methylglucose equilibrium stud-
and color development quantified by reading with an ELISA plate reader ies.23,28 Based on this internal volume and cell-associated radioac-
at 405 nm. tivity, the intracellular accumulation of AA in U937 cells and
monocytes was estimated. The intracellular AA concentration
calculated using this method was higher than values determined by
Recombinant caspase-8 in vitro assay
HPLC electrochemical detection (Figure 2A,D). The small accumu-
Recombinant caspase-8 (Alexis Biochemicals) was incubated with different lation of vitamin C in cells incubated with AA was probably due to
concentrations of AA and activity measured using the caspase-8 assay kit (R oxidation of AA to DHA in air.
and D Systems). Background was determined from triplicates of each The frequency of apoptosis induced by anti-FAS Ab or FASL in
concentration of vitamin C without the enzyme and subtracted from the
U937 cells and monocytes was prominently reduced by cellular
experimental results. The results were expressed as the percentage of
loading with vitamin C (Figure 2). Data regarding the intracellular
caspase activity in the absence of vitamin C.
AA concentrations reported in these experiments were based on
HPLC electrochemical detection. The frequency of apoptosis
Caspase-8 Western blot
Cells were lysed in a buffer containing 30 mM Tris-HCl (pH 7.5), 150
mM NaCl, 10% glycerol, and 1% triton and protease inhibitors. Cell
lysates were separated by SDS–polyacrylamide gel electrophoresis on
precast Tris-HEPES-SDS-polyacrylamide gradient minigels (4%-20%;
Gradipore, Frenchs Forest, Australia) according to the manufacturer’s
protocols. The gels were then transferred onto a nitrocellulose mem-
brane (Bio-rad Laboratories, Hercules, CA) in a buffer containing 25
mM Tris (pH 8.3), 192 mM glycine, and 20% methanol in a semidry
system (Bio-rad) for 15 minutes (15 V) at 25°C. Residual binding sites
on the membrane were blocked by incubating the membrane in
Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.4] and 0.15 M NaCl),
containing 5% nonfat dry milk and 0.1% Tween-20, for 2 hours at 25°C
(blocking buffer). Membranes were incubated with monoclonal anti–
caspase-8 (12F5; Alexis Biochemicals) or anti-tubulin (H-235; Santa
Cruz Biotechnology, Santa Cruz, CA) Abs (1:1000 dilution) for 16 hours
at 4°C. The primary Ab was removed, and the blots were washed 3 times
in TBS. To detect Ab reaction, the blots were incubated for one hour
with anti–mouse or anti–rabbit IgG horseradish peroxidase (HRP)–
conjugated Abs (Bio-rad), diluted 1:3000 in blocking buffer, washed
with TBS, and the protein bands were revealed using the enhanced
Figure 1. FAS-R ligation induces apoptosis in U937 cells and in monocytes. (A)
chemiluminescence (ECL⫹plus) Western blotting detection system U937 cells express the FAS-R (CD95) as determined by flow cytometry. (B-C) FASL
(Amersham Pharmacia Biotech, Piscataway, NJ) before exposure to and anti-FAS Ab induce apoptosis in U937 cells in a dose-dependent manner. (D)
BioMax film (Kodak, Rochester, NY). Fresh human monocytes express CD95 as determined by flow cytometry. (E-F) FASL
and anti-FAS Ab induce apoptosis in monocytes in a dose-dependent manner. The
frequency of apoptosis for U937 cells was determined by annexin V⫹/PI⫺ staining;
Statistical analysis apoptosis in monocytes was determined by the frequency of events in the sub-G1
region. The results are presented as the fold increase in the frequency of apoptosis
The paired 1-tail distribution Student t test was applied to compare results over control cells left untreated for 3 hours and are representative of at least 5
with a criterion for statistical significance of a P value of .05 or less. independent experiments for each cell type.BLOOD, 1 JULY 2003 䡠 VOLUME 102, NUMBER 1 VITAMIN C INHIBITS FAS-INDUCED APOPTOSIS 339
Figure 2. Vitamin C decreases FAS-induced apopto-
sis in U937 cells and monocytes. (A) Intracellular
accumulation of vitamin C in U937 cells after exposure to
1.0 mM DHA or AA. Results are presented as the
intracellular concentration of accumulated AA, deter-
mined by cell-associated radioactivity. The standard de-
viation is smaller than the symbols in the graphs, and was
always less than 10% of the values for each point. (B-C)
U937 cells loaded with vitamin C were treated for 3 hours
with 200 ng/mL FASL or 1 g/mL anti-FAS Ab, and
apoptosis was measured by annexin V⫹/PI⫺ staining or
by analysis of the sub-G1 region. The intracellular accu-
mulation of AA was estimated by HPLC. (D) Accumula-
tion of AA in monocytes after exposure to 0.1 mM DHA or
AA, calculated by cell-associated radioactivity. (E-F)
Monocytes were loaded with different amounts of vitamin
C estimated by HPLC analysis before treatment with 100
ng/mL FASL or 0.1 g/mL anti-FAS Ab for 3 hours to
measure apoptosis by analysis of the sub-G1 region.
Asterisks indicate statistically significant differences
(P ⱕ .05) using the Student t test between control and
cells loaded with vitamin C before challenge with FASL.
These experiments were repeated 6 times for each cell
type with similar results. A representative experiment
is shown. Error bars represent the SD of triplicate values.
induced by FASL (200 ng/mL) in U937 cells was 12% and in the levels of ROS, which were quenched by loading cells with
decreased to 7.8% in cells loaded with approximately 13 mM vitamin C (Figure 3B). The production of ROS correlated with the
vitamin C (P ⫽ .01) (Figure 2B). U937 cells incubated 3 hours frequency of apoptosis in cells incubated with FASL in these
with 1 g/mL anti-FAS Ab had a frequency of apoptosis of 5.8%, experiments (data not shown).
which decreased to 4.0% when the cells were loaded with
Vitamin C reduces mitochondrial damage induced by
approximately 13 mM vitamin C (P ⫽ .03) (Figure 2C). The
FAS-R ligation
percentage of reduction in apoptosis for U937 cells loaded with 13
mM vitamin C compared with control ranged from 22% to 27% In apoptotic cells, the decrease in mitochondrial membrane differ-
(mean, 25%) when the cells were challenged with anti-Fas Ab and ential potential (⌬) occurs before chromatin condensation and
from 26% to 50% (mean 35%) when the cells were treated with DNA fragmentation,36 and precedes the enhanced release of ROS
FASL. Vitamin C loading did not affect the expression of CD95 in by the mitochondria.7 Since vitamin C prevented the increase in
these cells (data not shown). Monocytes loaded with vitamin C also cellular ROS levels induced by FAS ligation, we studied the effect
had a reduced frequency of apoptosis after treatment with anti-FAS of vitamin C on mitochondrial apoptosis in U937 cells cultured
Ab or FASL. FASL (100 ng/mL) induced 37.1% apoptosis in with FASL by using the dye DiOC(6)(3). Cells with healthy
monocytes, and when these cells were loaded with approximately mitochondria have a high uptake of DiOC(6)(3) and therefore a high
14 mM vitamin C the frequency was reduced to 20.0% (P ⫽ .04) fluorescence signal, whereas cells with damaged mitochondria
(Figure 2E). Monocytes treated with 0.1 g/mL anti-FAS Ab for 3 show reduced fluorescence. Only 1.1% of control U937 cells had
hours showed 7.2% apoptosis, which was reduced to 5.1% in cells dull DiOC(6)(3) fluorescence (Figure 4A, upper left panel), whereas
loaded with approximately 14 mM vitamin C (P ⫽ .047) (Figure
2F). This represents a mean reduction in apoptosis of 31% to
44%. The lowest intracellular concentration of AA used in these
experiments was 4 mM, which approximates the concentration
of AA reported in monocytes obtained from blood of healthy
donors (3 mM).31,32 AA (4 mM) conferred significant protection
from FAS-induced apoptosis in monocytes (P ⫽ .02)
(Figure 2E-F).
Vitamin C quenches ROS produced by FAS-R ligation
ROS levels are elevated in cells stimulated via the FAS pathway,
and it has been suggested that ROS are an important component of
FAS signaling.8,33,34 Since vitamin C is a potent antioxidant and
Figure 3. Vitamin C inhibits ROS generated by FAS-R ligation. (A) U937 cells
quenches ROS in cells under oxidative stress,35 we examined the were loaded with approximately 13 mM vitamin C prior to treatment with 1 g/mL
effect of vitamin C on ROS accumulation after FAS ligation. anti-FAS Ab for 3.5 hours and then stained with DCFH-DA to detect intracellular ROS
Incubation of U937 cells with anti-FAS Ab for 3.5 hours resulted in by detection of dye fluorescence within 90 minutes by flow cytometry. (B) U937 cells
were loaded with vitamin C, treated with 200 ng/mL FASL for 3 hours, and stained
approximately a 2.5-fold increase in cellular ROS, whereas loading
with DCFH-DA for 30 minutes for fluorescence measurement. The results are
the cells with vitamin C reduced ROS accumulation in the cells presented as the mean fluorescence intensity of DCFH-DA in arbitrary fluorescence
(Figure 3A). FASL incubation for 3 hours induced a 2-fold increase units and are representative of 5 independent experiments.340 PEREZ-CRUZ et al BLOOD, 1 JULY 2003 䡠 VOLUME 102, NUMBER 1
vitamin C, however, prevented FASL-induced apoptosis in similar
proportions (Figure 4B).
Apoptotic cells release cytochrome C (Cyt C) from the mitochon-
dria into the cytosol,38 and we sought to determine the effect of
vitamin C on this process. The amount of Cyt C found in the
cytosolic fractions of cells incubated with FASL was nearly twice
that found in control cells (Figure 4C). In cells loaded with vitamin
C, there was a marked reduction of Cyt C in the cytoplasmic
fractions, even below the control levels. These results indicate that
vitamin C loading results in inhibition of the release of Cyt C from
mitochondria after FAS-R ligation.
Vitamin C reduces FAS-induced caspase-3 and
caspase-10 activation
A consequence of mitochondrial damage during apoptosis is
activation of caspase-3,39 and we studied the effect of vitamin C on
the activation of this caspase. Activation of caspase-3 was induced
by FASL incubation in U937 cells and in monocytes (Figure
5A-B). The activity of caspase-3 induced by FASL in U937 cells
loaded with vitamin C was modestly reduced (30%), although the
difference in caspase activity of cells with or without vitamin C was
statistically significant (Figure 5A, P ⫽ .039). In contrast, caspase-3
activity induced by FASL in monocytes loaded with vitamin C was
markedly reduced (Figure 5B, P ⫽ .02).
The results suggested that caspase-3 in U937 could be activated
by pathways independent of mitochondrial signaling, and we
therefore analyzed the effect of vitamin C on alternative mecha-
nisms for caspase-3 activation in U937 cells. Caspase-10 is
Figure 4. Vitamin C prevents mitochondrial damage induced by FAS-R ligation. homologous to caspase-8 and is activated upstream in the apoptosis
(A) U937 cells were loaded with 13 mM vitamin C prior to 3 hours of treatment with
FASL (200 ng/mL) and were stained with 40 nM DiOC(6)(3) for 15 minutes at 37°C.
cascade and can activate caspase-3.40 We explored the effect of
Control cells were treated with 0.2 M Z-IEDT-FMK. DiOC(6)(3) fluorescence was vitamin C on caspase-10 activity in U937 cells treated with FASL.
analyzed by flow cytometry. The intensity fluorescence of DiOC(6)(3) is shown on the The activity of caspase-10 increased 36% after incubation with
x-axis, and the forward scatter is represented on the y-axis. The numbers in the figure
indicate the frequency of the population with low intensity of DiOC(6)(3) fluorescence
FASL and was reduced by about 50% when the cells were loaded
for each treatment and are representative of 7 independent experiments (except for with vitamin C (Figure 6). We did not detect caspase-10 activity in
the experiment with Z-IEDT-FMK, which was performed twice). (B) Cells cultured monocytes after treatment with FASL (data not shown), and to our
under the conditions described in panel A were stained with annexin-PI to assess knowledge caspase-10 activity in monocytes has not been reported.
frequency of apoptosis. These results are the mean of 3 independent experiments,
performed in duplicates, and are presented as the fold increase in apoptosis over
Vitamin C inhibits caspase-8 activation induced via FASL
untreated control cells. (C) U937 cells were loaded with approximately 13 mM vitamin
C prior to treatment with 200 ng/mL FASL. Cytosolic cell extracts were obtained and
analyzed by ELISA for the presence of Cyt C. The results show the normalized Cyt C
ROS production, mitochondrial damage, and Cyt C release are
content for each experimental condition in arbitrary units based on the Cyt C cytosolic downstream consequences of initiator caspases such as caspase-
content of control cells and represent 2 independent experiments. Asterisk indicates 8.37 We therefore addressed the hypothesis that vitamin C inhibited
statistically significant differences (P ⫽ .039) using the Student t test between control
FAS-dependent caspase-8 activation. In U937 cells incubated with
and cells loaded with vitamin C before challenge with FASL. Error bars represent the
SD of triplicate values.
42% of U937 cells cultured with FASL for 3 hours incorporated
little DiOC(6)(3) (Figure 4A, upper right panel). We found that
loading the cells with vitamin C conferred protection from
FASL-induced mitochondrial damage, and these cells maintained
high DiOC(6)(3) fluorescence (Figure 4A, lower left panel). Control
cells treated with cyanide m-chlorophenylhydrazone showed a
marked decrease in mitochondrial ⌬ (data not shown). Caspase-8
activation precedes the reduction in mitochondrial ⌬ and the
change in mitochondrial membrane permeability.37 Thus, the
mitochondrial damage and the mitochondrial collapse should be Figure 5. Effect of vitamin C on caspase-3 activity. (A) U937 cells loaded with
prevented by incubation with the specific caspase-8 inhibitor approximately 13 mM vitamin C prior to treatment with FASL (200 ng/mL) were lysed,
and the activity of caspase-3 in the cell lysates was determined as described in
Z-IETD-FMK. The addition of 0.2 M Z-IETD-FMK inhibited “Materials and methods.” (B) Monocytes loaded with approximately 14 mM vitamin C
approximately 60% of FASL-induced mitochondrial ⌬ loss prior to treatment with FASL (100 ng/mL) were lysed, and the activity of caspase-3 in
compared with nearly 100% inhibited with vitamin C (Figure 4A, the cell lysates analyzed. The results are presented as the percentage increase in
caspase activity compared with cells without treatment and are the mean of triplicate
lower panels). We therefore reasoned that vitamin C was inhibiting
samples. These results are representative of 3 independent experiments for each cell
events at both the mitochondrial level and upstream sites in the type. Asterisks indicate statistically significant differences (in A, P ⫽ .03 and in B,
apoptosis cascade. The caspase-8 inhibitor Z-IETD-FMK and P ⫽ .02) in caspase activity between cells loaded with vitamin C and control.BLOOD, 1 JULY 2003 䡠 VOLUME 102, NUMBER 1 VITAMIN C INHIBITS FAS-INDUCED APOPTOSIS 341
activation of caspase-8. The molecular mechanisms of inhibition of
the activation of caspase-8 are not known. We postulate that the
activity of caspase-8 is dependent on production of ROS at the
level of the FAS-R and that the antioxidant properties of vitamin C
could inhibit this early step of FAS-initiated apoptosis signaling.
There is support for this notion in the recent finding that preloading
HL-60 cells with vitamin C inhibits the processing of pro–
caspase-8 induced by hydrogen peroxide.43 Vitamin C also main-
tains the integrity of the mitochondrial membrane, which is
disrupted during FAS signaling, and inhibits further downstream
effects. Thus, the results suggest that vitamin C inhibits
FAS-R–induced apoptosis in mononuclear phagocytes because of
its antioxidant capabilities.
Figure 6. Effect of vitamin C on caspase-10 activity. U937 cells were loaded with Discussion
13 mM vitamin C prior to FASL treatment (200 ng/mL) and lysed to determine activity
of caspase-10. The results are presented as the percentage increase in caspase Dietary vitamin C is critical for normal host defense, and vitamin C
activity compared with cells without treatment and are the mean of triplicate samples. is widely believed to enhance immune function when used
These results are representative of 2 independent experiments. Asterisk indicates a
statistically significant difference (P ⫽ .033) in caspase-10 activity of cells loaded with pharmacologically.18-20 Vitamin C may also have anti-inflamma-
vitamin C compared with control. tory properties.21,44-46 There is now a substantial body of evidence
defining a central role for oxidative reactions in cell signal-
FASL there was a 65% increase in caspase-8 activity, while vitamin ing12-15,47,48 and showing that antioxidants can inhibit important
C loading resulted in activity reduced to control levels (Figure 7A). signaling pathways in immune cells.16,17 For example, granulocyte-
A caspase-8 Western blot analysis indicated that cellular loading macrophage colony-stimulating factor (GM-CSF) and interleu-
with vitamin C did not modify the expression of pro–caspase-8 in kin-3 (IL-3) signaling involves ROS,17 and our evidence suggests
U937 cells (Figure 7B). In monocytes, FASL induced caspase-8 that vitamin C inhibition of GM-CSF signaling occurs at the level
activity by 130%, whereas vitamin C–loaded cells showed little of JAK-2 kinase activation.46 In this study, we undertook to
activation (Figure 7C). understand the role of vitamin C in FAS-mediated signaling for
The inhibitory effect of vitamin C on the activity of caspase-8 in apoptosis in human monocytes and the monocytic cell line U937.
cells cultured with FASL could be due to a direct effect of vitamin Vitamin C circulates in the plasma as AA; however, it is
C on the enzyme or to inhibition of enzyme activation. It has been generally transported into cells as DHA through the facilitative
reported that vitamin C can directly inhibit certain enzymes.41,42 As glucose transporters.23,24 Inside the cell, DHA is rapidly reduced to
vitamin C is accumulated intracellulary in its reduced form, AA, we AA.24,28 In this way cells can accumulate high intracellular
investigated if AA had a direct inhibitory effect on caspase-8 concentrations of vitamin C. For example, the intracellular concen-
activity. The activity of recombinant caspase-8 was assayed in the tration of AA in mononuclear cells is reported to be about 3 mM,
presence of AA (1.0-20.0 mM). To inhibit the conversion of AA to with circulating concentrations of AA in the range of 50 M.31,32
DHA, 1,4-dithiothreitol was added to the reaction mixture. There Specialized cells can transport AA directly through sodium/
was no significant effect of AA on the observed activity of ascorbate cotransporters.25 AA is often used in in vitro experiments,
recombinant caspase-8 (Figure 7D), while 20 M Z-IEDT-FMK leading to confounding results due to the lack of direct transport of
completely inhibited the activity of caspase-8 (not shown). There- ascorbate and because AA acts as a pro-oxidant in the presence of
fore, the data indicate that vitamin C is a potent inhibitor of the the free transition metals ubiquitously found in tissue culture.26 We
activation of caspase-8 without a direct effect on enzymatic activity. therefore used DHA to load cells with vitamin C24 and analyzed its
These results indicate that vitamin C inhibits apoptosis induced effect on the intracellular events mediating FAS-induced apoptosis
by FAS-R in monocytes and U937 cells largely by preventing the in monocytic cells.
Figure 7. Effect of vitamin C on caspase-8 activity. (A) U937 cells were loaded with 13 mM vitamin C prior to FASL treatment. The cell lysates were analyzed for caspase-8
activity. Error bars represent the SD of triplicate values. (B) A Western blot was performed to analyze the expression of pro–caspase-8 in U937 cells loaded with 13 mM vitamin
C (arrow, upper panel). Detection of -tubulin was used as control for protein loading (arrow, lower panel). (C) Monocytes were loaded with 14 mM vitamin C prior to FASL
treatment and the cell lysates were analyzed for caspase-8 activity. The results are the mean of triplicate samples expressed as the percentage increase in caspase activity
compared with untreated cells and represent 3 (U937) or 2 (monocyte) experiments. (D) The activity of recombinant caspase-8 (1.25 U/well) with and without vitamin C was
determined as detailed in “Materials and methods.” These results are expressed as the percentage of activity in relation to control wells (without vitamin C) and are
representative of 3 independent experiments. Asterisks indicate statistically significant differences (P ⫽ .0005) in caspase activity of cells loaded with vitamin C before
challenge with FASL.342 PEREZ-CRUZ et al BLOOD, 1 JULY 2003 䡠 VOLUME 102, NUMBER 1
The central observation of this work was a significant inhibition did not result in more protection from apoptosis than the selective
of FAS-induced apoptosis by vitamin C loading in monocytes and inhibition of caspase-8. This result is consistent with the notion that
U937 cells. We used pharmacologic concentrations of vitamin C to the major mechanism by which vitamin C prevents apoptosis is
ascertain the effect of vitamin C loading on the apoptotic process. through the inactivation of caspase-8; however, the data also point
The lowest intracellular concentration of AA used in these experi- to an independent effect of vitamin C on mitochondrial membrane
ments was higher than the physiologic intracellular AA reported for stabilization.
normal human monocytes (3 mM).31,32 Loading monocytes with 4 We studied the downstream consequences of inhibition of
mM AA conferred significant protection from FAS-R–induced caspase-8 activation by vitamin C by measuring the liberation of
apoptosis. Viability of U937 cells and monocytes was not impaired Cyt C into the cytosol and the subsequent activation of caspase-3.
by pharmacologic concentrations of vitamin C. The general cellular Cyt C was not detected in the cytosol of FAS-stimulated U937 cells
level of ROS increased after FAS-R ligation, and that increase was loaded with vitamin C, confirming that mitochondrial integrity was
abrogated by loading cells with vitamin C. The liberation of ROS preserved in cells protected with vitamin C. Also, a FAS-induced
during the apoptotic process is largely due to uncoupling of increase in caspase-3 activity in monocytes was not detected in
oxidative metabolism in the mitochondria.7 Others have noted that cells loaded with vitamin C. Vitamin C reduced basal levels of
N-acetyl-L-cysteine reduces ROS in monocytes after FAS liga- caspase-3 activity in these cells likely because there is some degree
tion,8 possibly through an increase in glutathione. The release of of oxidatively-induced apoptosis occurring in vitro.52 In U937
ROS in apoptosis is preceded by a decrease in mitochondrial cells, however, vitamin C did not greatly inhibit caspase-3,
membrane potential (⌬),7 and we found that the mitochondrial ⌬ maintaining 70% of FAS-induced activity. This phenomenon may
was maintained in U937 cells loaded with vitamin C and stimulated be due to the activity of caspase-10, which can activate caspase-353
with FAS, suggesting that vitamin C inhibited events occurring and which was active in U937 cells incubated with FAS (38%
before injury to the mitochondria. above basal).
FASL-induced aggregation of FAS-R is believed to cause Beneficial effects of vitamin C on immune function are
caspase-8 autoactivation, presumably related to proximity. Vitamin frequently cited, and it is widely assumed that vitamin C enhances
C loading, however, prominently inhibited FASL-mediated activa- immunity via its antioxidant function. Blood monocytes migrate to
tion of caspase-8 in U937 cells and in monocytes. In a cell-free tissues where some mature into macrophages, which participate
system AA did not inhibit the enzymatic activity of recombinant importantly in the modulation of the adaptive immune system as
caspase-8, and therefore these results suggest that vitamin C phagocytes and antigen-presenting cells and by producing cyto-
inhibits the activation of caspase-8, rather than its enzymatic kines. Upon maturation into macrophages, monocytes down-
activity. We postulate that local or “micro-oxidation” is required for regulate the expression of caspase-8 and up-regulate the expression
caspase-8 activation and that vitamin C inhibits that process by of FAS-associated death domain–like IL-1 beta-converting enzyme-
quenching ROS. This thesis implies that FAS ligation leads to the inhibitory protein, a natural caspase-8 inhibitor. Macrophages
local generation of ROS, which is required for caspase-8 activa- therefore are resistant to FAS-induced apoptosis.54,55 Before matu-
tion. Indeed, a rapid and transient synthesis of oxygen radicals ration, however, monocytes are highly susceptible to FAS-induced
following FAS ligation has been reported that is independent of apoptosis while resting and during bacterial phagocytosis.52,56 Here
caspase activation,49,50 indicating that the initiation of FAS- we present evidence that the intracellular accumulation of vitamin
mediated signaling is temporally associated with local generation C in monocytes causes resistance to FAS-induced apoptosis by
of ROS. ROS production after receptor ligation has been noted for inhibiting caspase-8 activation. A strong antioxidant such as
the IL-1R, TNF␣-R, and other cytokine receptor systems,12 and vitamin C could prolong the active life of monocytes before they
there is abundant evidence for ROS-mediated signaling for growth reach a FAS-insensitive stage. Maintenance of monocyte viability
factor receptors.13-15,17,51 by vitamin C may be a physiologic mechanism of vitamin C in host
Both vitamin C and the caspase-8 inhibitor Z-IETD-FMK defense. Further, the pharmacologic use of vitamin C administered
inhibited apoptosis to a similar degree. The loss of mitochondrial as DHA57 could be a means to favorably affect host defense in
⌬ as a consequence of FAS-R ligation was inhibited by both pathologic states.
vitamin C and Z-IETD-FMK, although to a different extent, with
almost complete protection provided by vitamin C. The greater
stabilization of the mitochondrial membrane resulting from high Acknowledgments
concentrations of vitamin C in cells challenged with FAS may be
due to vitamin C’s capability to quench ROS generated at the We appreciate the technical help of O. Borquez, C. Tat, and
receptor level and elsewhere in the cell. Nevertheless, this effect A. Pedraza.
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