Scientific paper describing the role of oxidation and anti-oxidants during programed cell death in cancer cells lines.

1. Apoptosis (2007) 12:225–234
DOI 10.1007/s10495-006-0475-0
Caspase-8 dependent trail-induced apoptosis in cancer cell lines
is inhibited by vitamin C and catalase
Isabel Perez-Cruz · Juan M. C´ rcamo · David W. Golde
a
Published online: 6 October 2006
C Springer Science + Business Media, LLC 2006
Abstract TNF-related apoptosis-inducing ligand (TRAIL/ TRAIL and impairs caspase-8 activation. We found that the
Apo-2L) is a member of the TNF family of apoptosis- removal of hydrogen peroxide by extracellular catalase dur-
inducing proteins that initiates apoptosis in a variety of neo- ing TRAIL-induced apoptosis also impairs caspase-8 activa-
plastic cells while displaying minimal or absent cytotoxicity tion. These data suggest that hydrogen peroxide is produced
to most normal cells. Therefore, TRAIL is currently con- during TRAIL-receptor ligation, and that the increase of in-
sidered a promising target to develop anti-cancer therapies. tracellular ROS regulates the activation of caspase-8 during
TRAIL-receptor ligation recruits and activates pro-caspase- apoptosis. Additionally we propose a mechanism by which
8, which in turn activates proteins that mediate disruption cancer cells might resist apoptosis via TRAIL, by the intake
of the mitochondrial membranes. These events lead to the of the nutritional antioxidant vitamin C.
nuclear and cytosolic damage characteristic of apoptosis.
Here we report that TRAIL-induced apoptosis is mediated Keywords TRAIL . Apoptosis . Ascorbic acid .
by oxidative stress and that vitamin C (ascorbic acid), a po- Caspase-8 . Catalase . ROS . Hydrogen peroxide
tent nutritional antioxidant, protects cancer cell lines from
apoptosis induced by TRAIL. Vitamin C impedes the ele-
vation of reactive oxygen species (ROS) levels induced by 1 Introduction
TNF-α, FAS-ligand (FAS-L) and TNF-related apoptosis
This work was supported by grants from the National Institutes of
Health (CA 30388), the New York State Department of Health inducing ligand (TRAIL) are members of the TNF-α family
(M020113) and the Lebensfeld Foundation. of ligands that induce apoptosis in a variety of transformed
cells [1, 2]. Although TNF-α and FAS-L can induce the death
I. Perez-Cruz ( ) · J. M. C´ rcamo · D. W. Golde
a
Program in Molecular Pharmacology and Chemistry, Memorial of transformed cells in vitro, these ligands are toxic when ad-
Sloan-Kettering Cancer Center, 1275 York Avenue, ministered systematically, which precludes their clinical use
New York, NY 10021, USA [3, 4]. In contrast, it has been shown that TRAIL induces
e-mail: iperez@saturn.med.nyu.edu
apoptosis preferentially in transformed cells [5], and the sys-
J. M. C´ rcamo
a temic administration of TRAIL in mice and non-human pri-
Department of Clinical Laboratories, Memorial Sloan-Kettering mate models of cancer reduces the growth of tumors without
Cancer Center, 1275 York Avenue, the toxic side effects of TNF-α and FAS-L [6, 7]. Subse-
New York, NY 10021, USA
quently, TRAIL is now considered to be a promising anti-
Current address
Enzo Life Sciences, 60 Executive Boulevard, Farmingdale, New cancer reagent.
York, NY 11735 Trimeric TRAIL binds to the TRAIL-receptor (TRAIL-
R) –1 and TRAIL-R2, an event followed by recruitment
I. Perez-Cruz
of cytosolic adapter molecules and pro-caspase-8 to the
Current address
New York University Cancer Center, TRAIL–R [8], forming the death-inducing signaling com-
Smilow Building, Lab. 12-06. 522 First Avenue, New York, plex (DISC). The activation of caspase-8 by TRAIL induces
NY 10016, USA the translocation of other cytosolic pro-apoptotic proteins to
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2. 226 Apoptosis (2007) 12:225–234
the mitochondria, causing a dissipation of the mitochondrial plete medium included 10% fetal calf serum (Omega Sci-
membrane potential ( ψ) [9]. Consequently, mitochondria entific) for DU-156, K562 and U937 cells, and 7% fetal
release reactive oxygen species (ROS) and pro-apoptotic pro- calf serum for PC-3 cells. Apoptosis in these cells was in-
teins into the cytoplasm thus inducing cellular and DNA duced by incubation with human recombinant TRAIL (R
damage [10]. & D Systems, MN). The caspase-8 irreversible inhibitor
The activation of pro-caspase-8 is believed to be depen- benzylloxycarbonyl-Ileu-Glu-Thr-Asp-fluoromethyl ketone
dent solely on proximity to other pro-caspase-8 units during (Z-IEDT-FMK; MP Biomedicals, OH) was included in some
recruitment to the DISC [11]. However, we have observed of the experiments. To detect apoptosis, cells in suspen-
that intracellular anti-oxidants modulate pro-caspase-8 acti- sion were fixed in 60% ice-cold methanol for 30 min at
vation after death receptor-engagement, which suggests that 4◦ C, washed twice in PBS and resuspended in 50 µl of a
ROS in the vicinity of the DISC assist in initiation of apopto- 100 U/ml RNAse-A solution (Roche, IN) and 20 µl pro-
sis signaling [12]. These observations indicate that whereas pidium iodine (PI, Alexis Biochemicals, CA). Apoptosis
proximity is required for activation of pro-caspase-8, ROS was analyzed following 24 hr in a FACScalibur utilizing
can modulate the initiation of signaling. The production of CellQuest (Beckton Dickinson, CA). Apoptosis was defined
ROS during apoptosis has been described amply (for review as the frequency of events in the sub-G1 region of the cell
see: [13]). Hydrogen peroxide (H2 O2 ) per se is able to in- cycle.
duce apoptosis [14] and is capable of directly modulating the
in vitro enzymatic activity of apoptosis related-enzymes (Akt
2.2 Treatment with vitamin C
and protein phosphatases) [15, 16]. Nevertheless, the mech-
anisms by which H2 O2 and ROS modulate apical-enzyme
Cells were loaded with AA by exposure for one hour to
activation during receptor-induced signaling and apoptosis
different amounts of DHA (Sigma, MO) dissolved in an in-
are not well understood.
cubation buffer (15.0 mM HEPES, 135.0 mM NaCl, 5.0 mM
We have found that the powerful nutritional anti-oxidant
KCl, 1.8 mM CaCl2 , 0.8 mM MgCl2 , pH 7.4). Accumu-
vitamin C (ascorbic acid, (AA)) can prevent TRAIL-induced
lation of intracellular AA was measured as previously de-
apoptosis in cancer cell lines, by preventing the rise in
scribed [17]. Briefly, to determine accumulation after ex-
intracellular ROS levels and the activation of caspase-8
posure to DHA, triplicate cell samples were incubated in a
induced by TRAIL. We have found additionally that the
solution prepared by mixing 0.5 µCi of L-14 C-AA (specific
removal of extracellular H2 O2 by catalase reduces TRAIL-
activity, 8.0 mCi/mmol; Perkin-Elmer Life Sciences, MA),
induced apoptosis by inhibiting the activation of pro-caspase-
1.0–4.0 mM AA (Sigma) and 86 U/ml ascorbate oxidase
8. These results suggest that H2 O2 is produced as a conse-
(Sigma) in an incubation buffer. Cells were then washed
quence of TRAIL-R binding to its cognate ligand and that
twice in PBS and lysed with an SDS-lysis buffer (10.0 mM
intracellular AA, which does not directly scavenge H2 O2 ,
Tris-HCl pH 8.0, 0.2 % SDS). Cell-associated radioactivity
quenches the intracellular ROS that can be derived from
was determined by scintillation spectrometry. The accumu-
H2 O2 during TRAIL-induced apoptosis. Thus, we propose
lation of vitamin C in cells exposed to AA was measured by
that oxygen radicals modulate apoptosis signaling by assist-
incubation in a solution prepared with 0.5 µCi of L-14 C-AA,
ing in the activation of initiator caspases. Our results also
1.0–4.0 mM L-AA and 0.1 mM 1,4-dithiothreitol, in an incu-
suggest that cancer cells may have an intrinsic resistance
bation buffer. Cell-associated radioactivity was determined
mechanism to TRAIL-induced apoptosis by accumulating
by scintillation spectrometry, and accumulation of AA was
AA. We believe that these observations should be consid-
calculated based on these results and known cellular volumes
ered when designing TRAIL-based therapeutics for cancer.
as previously described [17].
2 Materials and methods 2.3 Cell volume determination
2.1 Cells and induction of apoptosis Estimation of cell volume was performed as previously de-
scribed [17]. Glucose uptake of 5 × 106 cells was initiated
Cell lines were obtained from the American Type Culture by incubation in 200 µl of a buffer containing 1.0 mM 3-
Collection, VA. The cell lines DU-145 and PC-3 (of ep- oxy-methyl-D-glucose and 5 µCi of 3 H-3-oxy-methyl-D-
ithelial origin) were grown adherent to plates and K562 glucose. Uptake was stopped after 60 min by adding 2 µl
and U937 (of myeloid origin) were cultured in suspension. of 2.0 mM cytochalasin B (Sigma) to the cells. The cells
All cell lines were cultured in RPMI containing 100 U were then washed twice in PBS containing 20 µM cytocha-
penicillin, 100 U streptomycin (Gemini-bioproducts, CA) lasin B. Cell-associated radioactivity was determined by
and 2 mM L-glutamate (Omega Scientific, CA). The com- scintillation spectrometry. The cell volume was estimated
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3. Apoptosis (2007) 12:225–234 227
for DU-145 (2.3 µl/106 cells) PC-3 (2.4 µl/106 cells) U937 2.7 Mitochondrial membrane potential ( ψ)
(1.0 µl/106 cells) and K562 cells (1.8 µl/106 cells).
K562 cells were stained with 40 nM 3,3 -
2.4 Detection of ROS dihexyloxacarbocyanine iodide, DiOC(6) (3) (Molecular
Probes) in PBS for 15 min at 37◦ C in the dark and then
Intracellular ROS in K562 cells was estimated by oxida- analyzed by flow cytometry utilizing CellQuest software.
tion of 2 7 dichlorofluorescein diacetate acetyl ester (CM-
H2 DCFDA, Molecular Probes, OR). Cells were washed
2.8 Statistics
twice in a Krebs-Ringer buffer (20.0 mM HEPES, 10.0 mM
dextrose, 127.0 mM NaCl, 5.5 mM KCl, 1.0 mM CaCl2 Tests for statistical significance were performed using a two-
and 2.0 mM MgSO4 pH 7.4) and stained with 20 µM CM-
tailed, paired Student’s t-test. Samples were considered sig-
H2 DCFDA in a Krebs-Ringer buffer. After stimulation with
nificantly different if p < 0.05.
TRAIL at 37◦ C, fluorescence was determined by flow cytom-
etry and the data were analyzed using CellQuest software.
3 Results
2.5 Caspase-8 activity assay
3.1 Induction of apoptosis by TRAIL in transformed cell
Caspase-8 activity was measured using the caspase-8 colori- lines
metric assay kit (R & D systems), following the manufac-
turer’s instructions. TRAIL-induced apoptosis was studied in cell lines de-
rived from solid tumors (prostatic cancer: DU-145 and PC-
2.6 Caspase-8 Western Blot 3 cells) and non-solid tumors (myeloid cancer: K562 and
U937 cells). The frequency of cell death in these lines was
Cells were lysed in a buffer containing 30 mM TRIS-HCl dependent on the concentration of TRAIL and maximum
(pH 7.5), 150 mM NaCl, 10% glycerol, 1% triton and pro- apoptosis was reached with approximately 500 ng/ml (Fig.
tease inhibitors. Equal amounts of protein were separated 1). Apoptosis was also dependent on the time of incuba-
by SDS-PAGE and subsequently transferred to a nitrocel- tion with TRAIL (Fig. 1, inserts). 500 ng/ml TRAIL was
lulose membrane (Bio-Rad Laboratories). The membrane sufficient to initiate apoptosis induction in the cell cul-
was blocked with 5% nonfat dry milk in TBS-Tween-20 and tures after one hour of incubation, and 15% to 50% of
then incubated with an antibody that recognizes pro-active the cultures were apoptotic after three hours of incubation.
and active caspase-8 forms (12F5, Alexis Biochemicals). A All cell lines showed an exponential kinetics of response
horseradish peroxidase-conjugated secondary antibody was to TRAIL. However, sensitivity to TRAIL varied among
added and the protein bands were detected by chemilumi- cells: the most sensitive cell line being PC-3, followed by
nescence. The membranes were stripped and re-blotted for K562. DU-145 and U937 cells had similar sensitivities to
β actin detection as a control for protein loading. TRAIL.
Fig. 1 Induction of apoptosis by TRAIL in cancer cell lines. DU- different time points. The experiments were performed at least two
145, PC-3, K562 and U937 cell lines were incubated for three hours separate times for each cell line, with similar results. Here one repre-
with different concentrations of TRAIL and apoptosis was measured. sentative example is shown. Each experimental point was performed in
Inserts show apoptosis in cell lines incubated with 500 ng/ml TRAIL at triplicate. The results represent the mean percentage of apoptosis
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4. 228 Apoptosis (2007) 12:225–234
Fig. 2 Intracellular AA confers resistance to TRAIL induced apopto- absolute percentage of apoptosis (left) and the normalized values with
sis. A. DU-145, PC-3, K562 and U937 cell lines preferentially transport respect to the maximum apoptosis obtained for each cell line, without
the oxidized form of vitamin C (DHA) over AA. Cells were exposed to AA loading (right). The experiments were performed three times with
0.1 mM 14 C-DHA (black circles) or 0.1 mM 14 C-AA (white circles) for similar results, and one representative example is shown. Each experi-
different periods of time. The experiments where repeated twice with mental point was performed in triplicate. The results represent the mean
similar results, and one representative experiment is shown. The results percentage of apoptosis. Asterisks (∗ ) indicate the lowest concentration
are expressed as accumulated intracellular AA (mM). B. Cells pre- of AA that provided a statistically significant protection from apoptosis
loaded with different amounts of AA were incubated with 500 ng/ml with respect to controls
TRAIL for 3 hr and apoptosis was measured. The results present the
3.2 Vitamin C inhibits TRAIL-induced apoptosis by between cellular volumes and the uptake of radioactive vita-
quenching excess of ROS induced by TRAIL and by min C. A reduction in the frequency of apoptosis was seen
inhibition of caspase-8 activation with doses of intracellular AA as low as 3 mM in DU-145
cells, 6 mM in U937 cells and 8 mM in PC-3 cells. K562
Vitamin C is an antioxidant of major importance in hu- cells required 15 mM AA to acquire protection. However,
man nutrition. We have shown that vitamin C inhibits FAS- only concentrations of intracellular AA above 15 mM in
induced apoptosis by preventing cellular oxidation [12]. Here all cell lines provided a statistically significant resistance to
we studied its effect on TRAIL-induced apoptosis. Vitamin TRAIL-induced apoptosis (Fig. 2B). Control cells (loaded
C is found in human plasma in its reduced form, AA. How- with vitamin C but not challenged with TRAIL) did not
ever, it is transported by most cells in its oxidized form, undergo apoptosis at any of the concentrations shown in
dehydroascorbic acid (DHA), through facilitative glucose Fig. 2B (data not shown). The reduction in apoptosis in cell
transporters [17]. Inside the cell, DHA is reduced and ac- lines derived from hematopoietic diseases was the greatest,
cumulates as AA [17]. By exposing cell lines to either 14 C- reaching a 60% reduction in apoptosis in K562 cells and
labeled DHA or 14 C-labeled AA, we found that DU-145, around 50% in U937 cells (Fig. 2B, right axis). In cells de-
PC-3, K562 and U937 cell lines preferentially transported rived from solid tumors (DU-145 and PC-3), the protection
DHA over AA as expected, and significant accumulation was lower, with a reduction in apoptosis between 20% and
of AA was only achieved when the cells were exposed to 30%.
DHA (Fig. 2A). Therefore, in subsequent experiments, cells Occupancy of death receptors like FAS and TNFα by
were exposed to extracellular DHA, to allow for intracellular their ligands induces an increase in the levels of intracellu-
accumulation of AA. lar ROS levels, which participates in signaling [18, 19]. Our
After loading cells with intracellular AA, they were in- experiments with vitamin C indicated that oxidative stress is
cubated with TRAIL and the frequency of apoptosis was a component of TRAIL-induced apoptosis and it has been
determined. The frequency of apoptosis induced by TRAIL suggested that ROS participate on TRAIL-mediated apop-
in all cell lines studied here was prominently reduced by tosis in HeLa cells [20]. Thus, we sought to study the ele-
cellular loading with vitamin C (Fig. 2B). This apoptotic vation of ROS levels as a consequence to TRAIL stimula-
effect was dose-dependent. Data on intracellular AA con- tion. To measure the increase in intracellular ROS in K562
centrations in these experiments were based on a correlation cells incubated in TRAIL, we performed flow cytometry in
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5. Apoptosis (2007) 12:225–234 229
Fig. 3 AA quenches ROS
produced upon TRAIL-
stimulation. A. K562 cells were
incubated in 500 ng/ml TRAIL
for different times before
loading them with
CM-H2 DCFDA. Cells were then
acquired in a flow cytometer and
fluorescence was assessed. The
figure shows an increment in
arbitrary fluorescent units in
intracellular ROS. B. K562 cells
were loaded with 25 mM AA
before incubation in 500 ng/ml
TRAIL for 20 min. Intracellular
ROS levels were measured with
CM-H2 DCFDA fluorescence as
described in A
these cells after loading them with CM-H2 DCFDA, a dye sought to study the effect of intracellular AA on caspase-
that becomes fluorescent upon oxidation with H2 O2 , hy- 8 activation and activity during TRAIL-induced apopto-
droxyl radical (•OH), peroxyl radical and peroxynitrite an- sis. Intracellular AA reduced TRAIL-dependent caspase-
ion. We found that TRAIL stimulation increased the amount 8 activity in DU-145 cells in a dose-dependent manner
of intracellular ROS on K562 cells gradually, and that the (Fig. 4A). We demonstrated previously that AA does not di-
amount of ROS returns to normal levels after approximately rectly inhibit the activity of caspase-8 [12], so we investigated
one hour, although the levels continue to decrease thereafter weather AA interferes with the processing of pro-caspase-8.
(Fig. 3). However, by loading cells with AA before TRAIL We found that AA reduced TRAIL-dependent pro-caspase-
stimulation, the levels of ROS did not increase and where 8 activation in DU-145 and K562 cells and that this effect
slightly below control levels (Fig. 3). Thus, TRAIL stimula- was dependent on the concentration of intracellular AA
tion induces oxidative stress in the cell, which is prevented (Fig. 4B).
by intracellular AA.
Caspase-8 mediates TRAIL-induced apoptosis [8]. We 3.3 Catalase reduces TRAIL-induced apoptosis by
have previously reported that the antioxidant activity of inhibiting caspase-8 activity
vitamin C prevents FAS-induced apoptosis by inhibiting
caspase-8 activity [12]. After death-receptor engagement It has been found that extracellular catalase reduces FAS-
with its ligand, pro-caspase-8 (p55/54) is recruited to the induced apoptosis [18]. Additionally, recent work from our
death receptors via the DISC and subsequently cleaved into group indicates that H2 O2 is produced as a consequence
the p18/10 kD heterodimeric active caspase-8. Therefore, we of ligand-receptor binding [21]. Therefore, we formulated
Fig. 4 Intracellular AA inhibits TRAIL-mediated caspase-8 activa- loaded and not loaded cells. B. DU-145 and K562 cells were pre-loaded
tion. A. DU-145 cells were pre-loaded with AA and then incubated with AA before incubation with TRAIL for 30 (DU-145) or 20 (K562)
with 500 ng/ml TRAIL. The activity of caspase-8 in cell lysates was minutes. Total cell extracts were separated by SDS-PAGE and pro-
determined and is expressed as fold increase of activity over control. caspase-8 (p55) and active caspase-8 (p18) were detected by western
Asterisks (∗ ) indicate a statistically significant difference between AA blot. β-actin detection was used to confirm equal protein loading
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6. 230 Apoptosis (2007) 12:225–234
Fig. 5 Catalase inhibits TRAIL-induced apoptosis by preventing pro- with respect to cells incubated in the absence of catalase. B. DU-145
caspase-8 activation. A. DU-145 and K562 cells were co-incubated cells were co-incubated with catalase and TRAIL for 30 min. Total cell
with different amounts of catalase (cat) and 500 ng/ml TRAIL and extracts were separated by SDS-PAGE and pro-caspase-8 (p55), and
apoptosis was measured after 2 hr. The results are normalized with re- active caspase-8 (p18) were detected by western blot. β-actin detection
spect to the maximum frequency of apoptosis obtained in the absence of was used to confirm equal protein loading
catalase and Asterisks (∗ ) indicate a statistically significant difference
the hypothesis that part of the oxidative stress seen during by detecting the active p18 subunit. We found that whereas
TRAIL stimulation is due to the production of H2 O2 . To test p18 was detected in DU-145 cells exposed to TRAIL for
this hypothesis, stimulation of DU-145 and K562 cells with 1 hr, co-incubation with 100 U/ml of extracellular catalase
TRAIL was performed in the presence of different concen- inhibited the production of active caspase-8 (Fig. 5B). These
trations of extracellular catalase. Apoptosis was reduced by experiments indicate that binding of TRAIL to its recep-
catalase in these cell lines, and this effect was concentration- tor involves the production of H2 O2 , which participates in
dependent (Fig. 5A). The maximal reduction in apoptosis caspase-8 activation.
was approximately 50% in both cell types with respect to
controls without catalase, and was obtained using 100 U/ml 3.4 Participation of several ROS during TRAIL-induced
catalase. Incubation with catalase alone (up to 3 hr in 10 to apoptosis
25 000 U/ml catalase) did not change cell viability (data not
shown). Intracellular AA is unable to quench H2 O2 directly. However,
To assess the biochemical consequences of removal of since both catalase and intracellular AA inhibit apoptosis sig-
H2 O2 by catalase, we studied the activation of caspase-8 naling, we concluded that different oxygen species must par-
ticipate in TRAIL-induced signaling. It has been proposed
that •OH can be formed at the endoplasmic reticulum, in a
iron-dependent manner, from H2 O2 by the Fenton reaction
[22]. Because AA can scavenge •OH, we explored the possi-
bility that •OH is one ROS participating in TRAIL-induced
signaling. We investigated if DMSO, a specific scavenger for
•
OH [23], could inhibit TRAIL-induced apoptosis in K562
cells. We found that 0.1% DMSO inhibited TRAIL-induced
apoptosis by approximately 30% (Fig. 6). These results sug-
gest the possibility that •OH participates on TRAIL-induced
apoptosis.
3.5 Vitamin C stabilizes the mitochondrial membrane
Fig. 6 DMSO inhibits TRAIL induced apoptosis. K562 cells were potential in cells stimulated with TRAIL
loaded with AA or exposed to DMSO for 5 min before incubation with
500 ng/ml TRAIL for 1 hr and the percentage of apoptosis was deter-
mined. The results of three independent experiments, each containing Active caspase-8 cleaves pro-apoptotic proteins such as t-Bid
triplicates, were normalized with respect to the maximum apoptosis which in turn translocate to the mitochondria to mediate
obtained with TRAIL in the absence of anti-oxidants. Asterisks (∗ ) dissipation of the mitochondrial membrane proton potential
indicate a statistically significant difference from cells incubated with
TRAIL alone
( ψ) [9]. Destabilization of ψ precedes the uncoupling of
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7. Apoptosis (2007) 12:225–234 231
Fig. 7 AA reduces the dissipation of membrane potential induced by of apoptosis were determined. The results of three independent experi-
TRAIL. A. K562 cells were pre-loaded with different concentrations ments, each containing triplicates, were normalized with respect to the
of AA before incubation with 500 ng/ml TRAIL, and the dissipation maximum apoptosis (black bars) or maximum ψ dissipation (hatched
of ψ was determined by flow cytometry. The mean percentage of bars) obtained with TRAIL. The means values and standard deviations
low ψ fluorescent cells and the standard deviation is indicated for the are shown. Asterisks (∗ ) indicate statistically significant differences be-
experiment shown here B. Cells pre-loaded with AA or Z-EITD-FMK tween cells incubated with TRAIL alone or with AA or Z-EITD-FMK
were incubated with TRAIL. The dissipation of ψ and the frequency
the electron transport chain and the release of ROS and pro- port AA in the oxidized form DHA, via glucose transporters.
apoptotic proteins to the cytoplasm [24, 25]. We previously Once transported in the form of DHA, the vitamin C inside
found that intracellular AA confers stabilization of ψ in is reduced to AA, and is accumulated in this form only. In-
cells stimulated with FAS-L [12]. We sought to investigate tracellular AA content in normal tissues ranges from 1 to
if there was a protective effect of intracellular AA on mito- 10 mM [12, 26, 27], but a higher content of intracellular
chondria of TRAIL-stimulated cells. We exposed K562 cells AA in tumor tissues compared with normal tissues has been
to the fluorescent dye DiOC(6) (3) to estimate the dissipation reported [28]. This might be due to the higher capacity of
of ψ. The percentage of K562 cells with low DiOC(6) (3) cancer cells to transport glucose [29] and therefore to accu-
fluorescence under TRAIL stimulation was two to four fold mulate AA when transporting DHA, thus acquiring protec-
higher from basal conditions. We found that intracellular tion against an oxidative high metabolism. Here, cancer cell
AA prevented TRAIL-induced dissipation of ψ in K562 lines of different origins acquired protection from TRAIL-
cells in a dose-dependent manner (Fig. 7A). The caspase- induced apoptosis through accumulation of AA. The cell
8 irreversible inhibitor Z-IETD-FMK also protected against lines utilized in this study can efficiently transport DHA and
the TRAIL-mediated dissipation of ψ in a dose-dependent accumulate milimolar concentrations of intracellular AA. It
manner (data not shown). A similar inhibition of TRAIL- has been reported that in transformed B cells from chronic
induced apoptosis was obtained using 1.5 µM Z-IETD-FMK lymphocytic leukemia patients, the intracellular concentra-
or 25 mM AA in K562 cells (Fig. 7B), and under these con- tion of AA is as high as 15 mM [28]. How cells in vivo
ditions, intracellular AA conferred more stabilization to ψ acquire vitamin C physiologically is an apparent paradox,
than Z-IETD-FMK. Therefore, the protective action of AA since the plasma concentrations of DHA do not exceed 1–2%
during receptor-mediated apoptosis occurs early in the sig- of ascorbate concentrations [30]. However, AA can be con-
naling cascade and additional protection of the mitochondrial verted to DHA at the level of the cellular membrane and
proton gradient does not alter the fate of the cell stimulated therefore transported trough glucose transporters by the ac-
with TRAIL. tion of superoxide [31], a product of immune and endothelial
cells [31–33]. Once DHA is available, its higher uptake by
transformed cells as compared to normal counterparts [34],
4 Discussion occurs due to the upregulation in malignant cells of the ex-
pression of the ubiquitous glucose transporter Glut1 [35].
The principal finding in this study is that intracellular AA In this study the minimum concentration of intracellular AA
inhibited TRAIL-induced apoptosis in all cancer cell lines that provided protection varied among cells from 3 to 15 mM.
analyzed here. These cell lines, as do most cells [17], trans- The extent of protection also varied, from a 20% of reduction
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8. 232 Apoptosis (2007) 12:225–234
in apoptosis in PC3 cells with 30 mM AA, to an impressive ROS that participates in TRAIL signaling. In experiments
60% in K562 cells, achieved with 25 mM AA. These findings using DMSO (a • OH scavenger [23]), we found that it re-
indicate that tumors can successfully avoid apoptosis induced duced the frequency of apoptosis induced by TRAIL. Our
by TRAIL by uptake of the nutritional antioxidant vitamin C. explanation of this observation is that intracellular AA in-
Our data indicate that ROS are produced after stimulation hibits H2 O2 -induced apoptosis [39, 43] by quenching H2 O2 -
with TRAIL and that they participate in signaling, in par- derived • OH . However, the specificity of DMSO intracel-
ticular H2 O2 . Our finding that TRAIL-mediated caspase-8 lularly can be overestimated in a biological system at the
activation was impaired in cells loaded with AA suggests concentrations used here, since •OH could react with several
that caspase-8 activation is sensitive to the oxidative state targets before DMSO could reach it. This might be the rea-
of the milieu. There are two caspases with similar struc- son why the reduction of apoptosis achieved by DMSO is
tures, which have active cysteine sites: caspases 8 and 3. more modest that the one achieved by vitamin C. Therefore,
It has been proposed that these cysteine sites are suscepti- the contribution of other ROS in apoptosis signaling has to
ble to oxidation, and several reports indicate that exposure be considered [44]. The participation of several ROS during
to H2 O2 induces caspase-3 activation [36–39]. Remarkably, receptor-mediated apoptosis explains why the anti-oxidants
pro-caspase-8 can be activated in cells exposed to H2 O2 used here did not completely abrogate apoptosis signaling,
in the absence of re-localization induced by death-receptor as none of them can quench all ROS.
ligation: these cells exhibit total or partial processing of Active caspase-8 mediates the dissipation of the mito-
pro-caspase-8 [37, 39], Furthermore, exposure to H2 O2 en- chondrial membrane potential, ψ [9]. In our experiments
hances FAS-induced caspase-8 activation [40]. Our exper- we found that intracellular AA stabilized ψ in cells in-
iments with catalase indicate that H2 O2 produced during cubated in TRAIL. At concentrations of 25 mM AA and
TRAIL-R engagement is necessary for the activation of pro- 1.5 µM Z-EITD-FMK, both compounds provided similar
caspase-8. Thus ROS, and particularly H2 O2 , have a direct protection from TRAIL-induced apoptosis. However, AA
effect on caspase-8 activation and mutual proximity of pro- was more effective at stabilizing ψ. We previously ob-
caspase-8 is only one of the requirements for its activation. tained similar results in our study of FAS-induced apoptosis
This suggests that ROS stimulate caspase-8 auto-cleavage, [12]. It has been proposed that during apoptosis, ROS derived
producing an extra level of regulation in this important cel- from the mitochondria can cause further damage to mito-
lular process. chondrial membranes by forming a loop of oxidation [45,
Recent direct evidence from our laboratory indicates that 46]. But our results suggest that whereas AA can scavenge
the interaction between receptor and its ligand can produce intracellular ROS produced during apoptosis, quenching of
H2 O2 that facilitates signaling [21]. H2 O2 is a small, highly ROS at the mitochondria level does not affect the course
diffusible molecule with limited toxicity, which can be de- of apoptosis in a TRAIL-stimulated cell. This may be the
stroyed rapidly and efficiently by the cellular anti-oxidant de- consequence of a TRAIL signaling pathway that achieves
fenses (peroxiredoxin, catalase and glutathione peroxidase). independence from the mitochondrial signaling pathway, as
It has therefore been considered to possess “second messen- it has been described for the cells referred to as type-I cells
ger’s” characteristics [41]. In particular, cellular exposure to [47]. Thus, the prevention of total mitochondrial depolar-
milimolar concentrations of H2 O2 can directly activate apop- ization does not delay apoptosis when a critical amount of
tosis signaling [14] whereas the intracellular over-expression caspase-8 has been activated.
of catalase confers resistance to FAS-induced apoptosis [40].
However, AA does not directly quench H2 O2 . Therefore,
several oxygen species should participate in modulation of 5 Conclusion
apoptosis signaling. AA quenches several ROS, including
highly oxidative molecules such as • OH, peroxyl radical, In conclusion, we propose that production of ROS is as-
superoxide anion and water-soluble peroxyl radicals [42]. sociated with the initiation of TRAIL-induced apoptosis in
From these ROS, the intracellular production of • OH has cancer cells. Our results indicate that whereas TRAIL inter-
been documented. Perinuclear iron deposits in close prox- action with its receptor induces the production of H2 O2 ,
imity to the endoplasmic reticulum have been found, and it several oxygen species must participate in signaling. In-
has been shown that •OH is formed by the Fenton reaction in tracellular AA can quench some of these ROS, reduc-
that organelle [22]. Extracellular H2 O2 can diffuse through ing early TRAIL-induced signaling (caspase-8 activation)
the cell membrane and can react intracellularly with iron and and downstream events and therefore drastically reducing
copper ions to form •OH, among other oxygen species, before the frequency of apoptosis. Our results suggest that tumor
being transformed into water and oxygen [22]. Therefore, cells have a means of resisting TRAIL-induced apopto-
H2 O2 may be a precursor molecule of other ROS involved in sis through the accumulation of the nutritional antioxidant
TRAIL-induced signaling. We hypothesized that •OH is one vitamin C.
Springer

9. Apoptosis (2007) 12:225–234 233
Acknowledgments We appreciate the technical help of Mr. Georgios IL-1alpha induce apoptosis in subconfluent rat mesangial cells.
Stratis in the determination of cell volume and thank Ms. Mary Anne Evidence for the involvement of hydrogen peroxide and lipid
Melnick and Mr. Richard Stout for reading the manuscript. peroxidation as second messengers. Cytokine 12:986–991
Dr. D. W. Golde, our mentor and the inspiration behind this work, 20. Lee MW, Park SC, Kim JH et al. (2002) The involvement of
died on August 9th, 2004. oxidative stress in tumor necrosis factor (TNF)-related apoptosis-
inducing ligand (TRAIL)-induced apoptosis in HeLa cells. Cancer
Lett 182:75–82
21. DeYulia GJ Jr, Carcamo JM, Borquez-Ojeda O, Shelton CC,
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