Apoptosis (2007) 12:225–234DOI 10.1007/s10495-006-0475-0

Caspase-8 dependent trail-induced apoptosis in cancer cell linesis inhibited by vitamin C and catalaseIsabel Perez-Cruz · Juan M. Carcamo · David W. Golde

Published online: 6 October 2006C! Springer Science + Business Media, LLC 2006

Abstract TNF-related apoptosis-inducing ligand (TRAIL/Apo-2L) is a member of the TNF family of apoptosis-inducing proteins that initiates apoptosis in a variety of neo-plastic cells while displaying minimal or absent cytotoxicityto most normal cells. Therefore, TRAIL is currently con-sidered a promising target to develop anti-cancer therapies.TRAIL-receptor ligation recruits and activates pro-caspase-8, which in turn activates proteins that mediate disruptionof the mitochondrial membranes. These events lead to thenuclear and cytosolic damage characteristic of apoptosis.Here we report that TRAIL-induced apoptosis is mediatedby oxidative stress and that vitamin C (ascorbic acid), a po-tent nutritional antioxidant, protects cancer cell lines fromapoptosis induced by TRAIL. Vitamin C impedes the ele-vation of reactive oxygen species (ROS) levels induced by

This work was supported by grants from the National Institutes ofHealth (CA 30388), the New York State Department of Health(M020113) and the Lebensfeld Foundation.

I. Perez-Cruz (!) · J. M. Carcamo · D. W. GoldeProgram in Molecular Pharmacology and Chemistry, MemorialSloan-Kettering Cancer Center, 1275 York Avenue,New York, NY 10021, USAe-mail: iperez@saturn.med.nyu.edu

J. M. CarcamoDepartment of Clinical Laboratories, Memorial Sloan-KetteringCancer Center, 1275 York Avenue,New York, NY 10021, USACurrent addressEnzo Life Sciences, 60 Executive Boulevard, Farmingdale, NewYork, NY 11735

I. Perez-CruzCurrent addressNew York University Cancer Center,Smilow Building, Lab. 12-06. 522 First Avenue, New York,NY 10016, USA

TRAIL and impairs caspase-8 activation. We found that theremoval of hydrogen peroxide by extracellular catalase dur-ing TRAIL-induced apoptosis also impairs caspase-8 activa-tion. These data suggest that hydrogen peroxide is producedduring TRAIL-receptor ligation, and that the increase of in-tracellular ROS regulates the activation of caspase-8 duringapoptosis. Additionally we propose a mechanism by whichcancer cells might resist apoptosis via TRAIL, by the intakeof the nutritional antioxidant vitamin C.

Keywords TRAIL . Apoptosis . Ascorbic acid .Caspase-8 . Catalase . ROS . Hydrogen peroxide

1 Introduction

TNF-!, FAS-ligand (FAS-L) and TNF-related apoptosisinducing ligand (TRAIL) are members of the TNF-! familyof ligands that induce apoptosis in a variety of transformedcells [1, 2]. Although TNF-! and FAS-L can induce the deathof transformed cells in vitro, these ligands are toxic when ad-ministered systematically, which precludes their clinical use[3, 4]. In contrast, it has been shown that TRAIL inducesapoptosis preferentially in transformed cells [5], and the sys-temic administration of TRAIL in mice and non-human pri-mate models of cancer reduces the growth of tumors withoutthe toxic side effects of TNF-! and FAS-L [6, 7]. Subse-quently, TRAIL is now considered to be a promising anti-cancer reagent.

Trimeric TRAIL binds to the TRAIL-receptor (TRAIL-R) –1 and TRAIL-R2, an event followed by recruitmentof cytosolic adapter molecules and pro-caspase-8 to theTRAIL–R [8], forming the death-inducing signaling com-plex (DISC). The activation of caspase-8 by TRAIL inducesthe translocation of other cytosolic pro-apoptotic proteins to

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the mitochondria, causing a dissipation of the mitochondrialmembrane potential ("#) [9]. Consequently, mitochondriarelease reactive oxygen species (ROS) and pro-apoptotic pro-teins into the cytoplasm thus inducing cellular and DNAdamage [10].

The activation of pro-caspase-8 is believed to be depen-dent solely on proximity to other pro-caspase-8 units duringrecruitment to the DISC [11]. However, we have observedthat intracellular anti-oxidants modulate pro-caspase-8 acti-vation after death receptor-engagement, which suggests thatROS in the vicinity of the DISC assist in initiation of apopto-sis signaling [12]. These observations indicate that whereasproximity is required for activation of pro-caspase-8, ROScan modulate the initiation of signaling. The production ofROS during apoptosis has been described amply (for reviewsee: [13]). Hydrogen peroxide (H2O2) per se is able to in-duce apoptosis [14] and is capable of directly modulating thein vitro enzymatic activity of apoptosis related-enzymes (Aktand protein phosphatases) [15, 16]. Nevertheless, the mech-anisms by which H2O2 and ROS modulate apical-enzymeactivation during receptor-induced signaling and apoptosisare not well understood.

We have found that the powerful nutritional anti-oxidantvitamin C (ascorbic acid, (AA)) can prevent TRAIL-inducedapoptosis in cancer cell lines, by preventing the rise inintracellular ROS levels and the activation of caspase-8induced by TRAIL. We have found additionally that theremoval of extracellular H2O2 by catalase reduces TRAIL-induced apoptosis by inhibiting the activation of pro-caspase-8. These results suggest that H2O2 is produced as a conse-quence of TRAIL-R binding to its cognate ligand and thatintracellular AA, which does not directly scavenge H2O2,quenches the intracellular ROS that can be derived fromH2O2 during TRAIL-induced apoptosis. Thus, we proposethat oxygen radicals modulate apoptosis signaling by assist-ing in the activation of initiator caspases. Our results alsosuggest that cancer cells may have an intrinsic resistancemechanism to TRAIL-induced apoptosis by accumulatingAA. We believe that these observations should be consid-ered when designing TRAIL-based therapeutics for cancer.

2 Materials and methods

2.1 Cells and induction of apoptosis

Cell lines were obtained from the American Type CultureCollection, VA. The cell lines DU-145 and PC-3 (of ep-ithelial origin) were grown adherent to plates and K562and U937 (of myeloid origin) were cultured in suspension.All cell lines were cultured in RPMI containing 100 Upenicillin, 100 U streptomycin (Gemini-bioproducts, CA)and 2 mM L-glutamate (Omega Scientific, CA). The com-

plete medium included 10% fetal calf serum (Omega Sci-entific) for DU-156, K562 and U937 cells, and 7% fetalcalf serum for PC-3 cells. Apoptosis in these cells was in-duced by incubation with human recombinant TRAIL (R& D Systems, MN). The caspase-8 irreversible inhibitorbenzylloxycarbonyl-Ileu-Glu-Thr-Asp-fluoromethyl ketone(Z-IEDT-FMK; MP Biomedicals, OH) was included in someof the experiments. To detect apoptosis, cells in suspen-sion were fixed in 60% ice-cold methanol for 30 min at4"C, washed twice in PBS and resuspended in 50 µl of a100 U/ml RNAse-A solution (Roche, IN) and 20 µl pro-pidium iodine (PI, Alexis Biochemicals, CA). Apoptosiswas analyzed following 24 hr in a FACScalibur utilizingCellQuest (Beckton Dickinson, CA). Apoptosis was definedas the frequency of events in the sub-G1 region of the cellcycle.

2.2 Treatment with vitamin C

Cells were loaded with AA by exposure for one hour todifferent amounts of DHA (Sigma, MO) dissolved in an in-cubation buffer (15.0 mM HEPES, 135.0 mM NaCl, 5.0 mMKCl, 1.8 mM CaCl2, 0.8 mM MgCl2, pH 7.4). Accumu-lation of intracellular AA was measured as previously de-scribed [17]. Briefly, to determine accumulation after ex-posure to DHA, triplicate cell samples were incubated in asolution prepared by mixing 0.5 µCi of L-14C-AA (specificactivity, 8.0 mCi/mmol; Perkin-Elmer Life Sciences, MA),1.0–4.0 mM AA (Sigma) and 86 U/ml ascorbate oxidase(Sigma) in an incubation buffer. Cells were then washedtwice in PBS and lysed with an SDS-lysis buffer (10.0 mMTris-HCl pH 8.0, 0.2 % SDS). Cell-associated radioactivitywas determined by scintillation spectrometry. The accumu-lation of vitamin C in cells exposed to AA was measured byincubation in a solution prepared with 0.5 µCi of L-14C-AA,1.0–4.0 mM L-AA and 0.1 mM 1,4-dithiothreitol, in an incu-bation buffer. Cell-associated radioactivity was determinedby scintillation spectrometry, and accumulation of AA wascalculated based on these results and known cellular volumesas previously described [17].

2.3 Cell volume determination

Estimation of cell volume was performed as previously de-scribed [17]. Glucose uptake of 5 # 106 cells was initiatedby incubation in 200 µl of a buffer containing 1.0 mM 3-oxy-methyl-D-glucose and 5 µCi of 3H-3-oxy-methyl-D-glucose. Uptake was stopped after 60 min by adding 2 µlof 2.0 mM cytochalasin B (Sigma) to the cells. The cellswere then washed twice in PBS containing 20 µM cytocha-lasin B. Cell-associated radioactivity was determined byscintillation spectrometry. The cell volume was estimated

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for DU-145 (2.3 µl/106 cells) PC-3 (2.4 µl/106 cells) U937(1.0 µl/106 cells) and K562 cells (1.8 µl/106 cells).

2.4 Detection of ROS

Intracellular ROS in K562 cells was estimated by oxida-tion of 2$7$ dichlorofluorescein diacetate acetyl ester (CM-H2DCFDA, Molecular Probes, OR). Cells were washedtwice in a Krebs-Ringer buffer (20.0 mM HEPES, 10.0 mMdextrose, 127.0 mM NaCl, 5.5 mM KCl, 1.0 mM CaCl2and 2.0 mM MgSO4 pH 7.4) and stained with 20 µM CM-H2DCFDA in a Krebs-Ringer buffer. After stimulation withTRAIL at 37"C, fluorescence was determined by flow cytom-etry and the data were analyzed using CellQuest software.

2.5 Caspase-8 activity assay

Caspase-8 activity was measured using the caspase-8 colori-metric assay kit (R & D systems), following the manufac-turer’s instructions.

2.6 Caspase-8 Western Blot

Cells were lysed in a buffer containing 30 mM TRIS-HCl(pH 7.5), 150 mM NaCl, 10% glycerol, 1% triton and pro-tease inhibitors. Equal amounts of protein were separatedby SDS-PAGE and subsequently transferred to a nitrocel-lulose membrane (Bio-Rad Laboratories). The membranewas blocked with 5% nonfat dry milk in TBS-Tween-20 andthen incubated with an antibody that recognizes pro-activeand active caspase-8 forms (12F5, Alexis Biochemicals). Ahorseradish peroxidase-conjugated secondary antibody wasadded and the protein bands were detected by chemilumi-nescence. The membranes were stripped and re-blotted for$ actin detection as a control for protein loading.

2.7 Mitochondrial membrane potential ("#)

K562 cells were stained with 40 nM 3,3$-dihexyloxacarbocyanine iodide, DiOC(6)(3) (MolecularProbes) in PBS for 15 min at 37"C in the dark and thenanalyzed by flow cytometry utilizing CellQuest software.

2.8 Statistics

Tests for statistical significance were performed using a two-tailed, paired Student’s t-test. Samples were considered sig-nificantly different if p < 0.05.

3 Results

3.1 Induction of apoptosis by TRAIL in transformed celllines

TRAIL-induced apoptosis was studied in cell lines de-rived from solid tumors (prostatic cancer: DU-145 and PC-3 cells) and non-solid tumors (myeloid cancer: K562 andU937 cells). The frequency of cell death in these lines wasdependent on the concentration of TRAIL and maximumapoptosis was reached with approximately 500 ng/ml (Fig.1). Apoptosis was also dependent on the time of incuba-tion with TRAIL (Fig. 1, inserts). 500 ng/ml TRAIL wassufficient to initiate apoptosis induction in the cell cul-tures after one hour of incubation, and 15% to 50% ofthe cultures were apoptotic after three hours of incubation.All cell lines showed an exponential kinetics of responseto TRAIL. However, sensitivity to TRAIL varied amongcells: the most sensitive cell line being PC-3, followed byK562. DU-145 and U937 cells had similar sensitivities toTRAIL.

Fig. 1 Induction of apoptosis by TRAIL in cancer cell lines. DU-145, PC-3, K562 and U937 cell lines were incubated for three hourswith different concentrations of TRAIL and apoptosis was measured.Inserts show apoptosis in cell lines incubated with 500 ng/ml TRAIL at

different time points. The experiments were performed at least twoseparate times for each cell line, with similar results. Here one repre-sentative example is shown. Each experimental point was performed intriplicate. The results represent the mean percentage of apoptosis

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Fig. 2 Intracellular AA confers resistance to TRAIL induced apopto-sis. A. DU-145, PC-3, K562 and U937 cell lines preferentially transportthe oxidized form of vitamin C (DHA) over AA. Cells were exposed to0.1 mM 14C-DHA (black circles) or 0.1 mM 14C-AA (white circles) fordifferent periods of time. The experiments where repeated twice withsimilar results, and one representative experiment is shown. The resultsare expressed as accumulated intracellular AA (mM). B. Cells pre-loaded with different amounts of AA were incubated with 500 ng/mlTRAIL for 3 hr and apoptosis was measured. The results present the

absolute percentage of apoptosis (left) and the normalized values withrespect to the maximum apoptosis obtained for each cell line, withoutAA loading (right). The experiments were performed three times withsimilar results, and one representative example is shown. Each experi-mental point was performed in triplicate. The results represent the meanpercentage of apoptosis. Asterisks (%) indicate the lowest concentrationof AA that provided a statistically significant protection from apoptosiswith respect to controls

3.2 Vitamin C inhibits TRAIL-induced apoptosis byquenching excess of ROS induced by TRAIL and byinhibition of caspase-8 activation

Vitamin C is an antioxidant of major importance in hu-man nutrition. We have shown that vitamin C inhibits FAS-induced apoptosis by preventing cellular oxidation [12]. Herewe studied its effect on TRAIL-induced apoptosis. VitaminC is found in human plasma in its reduced form, AA. How-ever, it is transported by most cells in its oxidized form,dehydroascorbic acid (DHA), through facilitative glucosetransporters [17]. Inside the cell, DHA is reduced and ac-cumulates as AA [17]. By exposing cell lines to either 14C-labeled DHA or 14C-labeled AA, we found that DU-145,PC-3, K562 and U937 cell lines preferentially transportedDHA over AA as expected, and significant accumulationof AA was only achieved when the cells were exposed toDHA (Fig. 2A). Therefore, in subsequent experiments, cellswere exposed to extracellular DHA, to allow for intracellularaccumulation of AA.

After loading cells with intracellular AA, they were in-cubated with TRAIL and the frequency of apoptosis wasdetermined. The frequency of apoptosis induced by TRAILin all cell lines studied here was prominently reduced bycellular loading with vitamin C (Fig. 2B). This apoptoticeffect was dose-dependent. Data on intracellular AA con-centrations in these experiments were based on a correlation

between cellular volumes and the uptake of radioactive vita-min C. A reduction in the frequency of apoptosis was seenwith doses of intracellular AA as low as 3 mM in DU-145cells, 6 mM in U937 cells and 8 mM in PC-3 cells. K562cells required 15 mM AA to acquire protection. However,only concentrations of intracellular AA above 15 mM inall cell lines provided a statistically significant resistance toTRAIL-induced apoptosis (Fig. 2B). Control cells (loadedwith vitamin C but not challenged with TRAIL) did notundergo apoptosis at any of the concentrations shown inFig. 2B (data not shown). The reduction in apoptosis in celllines derived from hematopoietic diseases was the greatest,reaching a 60% reduction in apoptosis in K562 cells andaround 50% in U937 cells (Fig. 2B, right axis). In cells de-rived from solid tumors (DU-145 and PC-3), the protectionwas lower, with a reduction in apoptosis between 20% and30%.

Occupancy of death receptors like FAS and TNF! bytheir ligands induces an increase in the levels of intracellu-lar ROS levels, which participates in signaling [18, 19]. Ourexperiments with vitamin C indicated that oxidative stress isa component of TRAIL-induced apoptosis and it has beensuggested that ROS participate on TRAIL-mediated apop-tosis in HeLa cells [20]. Thus, we sought to study the ele-vation of ROS levels as a consequence to TRAIL stimula-tion. To measure the increase in intracellular ROS in K562cells incubated in TRAIL, we performed flow cytometry in

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Fig. 3 AA quenches ROSproduced upon TRAIL-stimulation. A. K562 cells wereincubated in 500 ng/ml TRAILfor different times beforeloading them withCM-H2DCFDA. Cells were thenacquired in a flow cytometer andfluorescence was assessed. Thefigure shows an increment inarbitrary fluorescent units inintracellular ROS. B. K562 cellswere loaded with 25 mM AAbefore incubation in 500 ng/mlTRAIL for 20 min. IntracellularROS levels were measured withCM-H2DCFDA fluorescence asdescribed in A

these cells after loading them with CM-H2DCFDA, a dyethat becomes fluorescent upon oxidation with H2O2, hy-droxyl radical (•OH), peroxyl radical and peroxynitrite an-ion. We found that TRAIL stimulation increased the amountof intracellular ROS on K562 cells gradually, and that theamount of ROS returns to normal levels after approximatelyone hour, although the levels continue to decrease thereafter(Fig. 3). However, by loading cells with AA before TRAILstimulation, the levels of ROS did not increase and whereslightly below control levels (Fig. 3). Thus, TRAIL stimula-tion induces oxidative stress in the cell, which is preventedby intracellular AA.

Caspase-8 mediates TRAIL-induced apoptosis [8]. Wehave previously reported that the antioxidant activity ofvitamin C prevents FAS-induced apoptosis by inhibitingcaspase-8 activity [12]. After death-receptor engagementwith its ligand, pro-caspase-8 (p55/54) is recruited to thedeath receptors via the DISC and subsequently cleaved intothe p18/10 kD heterodimeric active caspase-8. Therefore, we

sought to study the effect of intracellular AA on caspase-8 activation and activity during TRAIL-induced apopto-sis. Intracellular AA reduced TRAIL-dependent caspase-8 activity in DU-145 cells in a dose-dependent manner(Fig. 4A). We demonstrated previously that AA does not di-rectly inhibit the activity of caspase-8 [12], so we investigatedweather AA interferes with the processing of pro-caspase-8.We found that AA reduced TRAIL-dependent pro-caspase-8 activation in DU-145 and K562 cells and that this effectwas dependent on the concentration of intracellular AA(Fig. 4B).

3.3 Catalase reduces TRAIL-induced apoptosis byinhibiting caspase-8 activity

It has been found that extracellular catalase reduces FAS-induced apoptosis [18]. Additionally, recent work from ourgroup indicates that H2O2 is produced as a consequenceof ligand-receptor binding [21]. Therefore, we formulated

Fig. 4 Intracellular AA inhibits TRAIL-mediated caspase-8 activa-tion. A. DU-145 cells were pre-loaded with AA and then incubatedwith 500 ng/ml TRAIL. The activity of caspase-8 in cell lysates wasdetermined and is expressed as fold increase of activity over control.Asterisks (%) indicate a statistically significant difference between AA

loaded and not loaded cells. B. DU-145 and K562 cells were pre-loadedwith AA before incubation with TRAIL for 30 (DU-145) or 20 (K562)minutes. Total cell extracts were separated by SDS-PAGE and pro-caspase-8 (p55) and active caspase-8 (p18) were detected by westernblot. $-actin detection was used to confirm equal protein loading

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Fig. 5 Catalase inhibits TRAIL-induced apoptosis by preventing pro-caspase-8 activation. A. DU-145 and K562 cells were co-incubatedwith different amounts of catalase (cat) and 500 ng/ml TRAIL andapoptosis was measured after 2 hr. The results are normalized with re-spect to the maximum frequency of apoptosis obtained in the absence ofcatalase and Asterisks (%) indicate a statistically significant difference

with respect to cells incubated in the absence of catalase. B. DU-145cells were co-incubated with catalase and TRAIL for 30 min. Total cellextracts were separated by SDS-PAGE and pro-caspase-8 (p55), andactive caspase-8 (p18) were detected by western blot. $-actin detectionwas used to confirm equal protein loading

the hypothesis that part of the oxidative stress seen duringTRAIL stimulation is due to the production of H2O2. To testthis hypothesis, stimulation of DU-145 and K562 cells withTRAIL was performed in the presence of different concen-trations of extracellular catalase. Apoptosis was reduced bycatalase in these cell lines, and this effect was concentration-dependent (Fig. 5A). The maximal reduction in apoptosiswas approximately 50% in both cell types with respect tocontrols without catalase, and was obtained using 100 U/mlcatalase. Incubation with catalase alone (up to 3 hr in 10 to25 000 U/ml catalase) did not change cell viability (data notshown).

To assess the biochemical consequences of removal ofH2O2 by catalase, we studied the activation of caspase-8

Fig. 6 DMSO inhibits TRAIL induced apoptosis. K562 cells wereloaded with AA or exposed to DMSO for 5 min before incubation with500 ng/ml TRAIL for 1 hr and the percentage of apoptosis was deter-mined. The results of three independent experiments, each containingtriplicates, were normalized with respect to the maximum apoptosisobtained with TRAIL in the absence of anti-oxidants. Asterisks (%)indicate a statistically significant difference from cells incubated withTRAIL alone

by detecting the active p18 subunit. We found that whereasp18 was detected in DU-145 cells exposed to TRAIL for1 hr, co-incubation with 100 U/ml of extracellular catalaseinhibited the production of active caspase-8 (Fig. 5B). Theseexperiments indicate that binding of TRAIL to its recep-tor involves the production of H2O2, which participates incaspase-8 activation.

3.4 Participation of several ROS during TRAIL-inducedapoptosis

Intracellular AA is unable to quench H2O2 directly. However,since both catalase and intracellular AA inhibit apoptosis sig-naling, we concluded that different oxygen species must par-ticipate in TRAIL-induced signaling. It has been proposedthat •OH can be formed at the endoplasmic reticulum, in airon-dependent manner, from H2O2 by the Fenton reaction[22]. Because AA can scavenge •OH, we explored the possi-bility that •OH is one ROS participating in TRAIL-inducedsignaling. We investigated if DMSO, a specific scavenger for•OH [23], could inhibit TRAIL-induced apoptosis in K562cells. We found that 0.1% DMSO inhibited TRAIL-inducedapoptosis by approximately 30% (Fig. 6). These results sug-gest the possibility that •OH participates on TRAIL-inducedapoptosis.

3.5 Vitamin C stabilizes the mitochondrial membranepotential in cells stimulated with TRAIL

Active caspase-8 cleaves pro-apoptotic proteins such as t-Bidwhich in turn translocate to the mitochondria to mediatedissipation of the mitochondrial membrane proton potential("#) [9]. Destabilization of "# precedes the uncoupling of

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Fig. 7 AA reduces the dissipation of membrane potential induced byTRAIL. A. K562 cells were pre-loaded with different concentrationsof AA before incubation with 500 ng/ml TRAIL, and the dissipationof "# was determined by flow cytometry. The mean percentage oflow "# fluorescent cells and the standard deviation is indicated for theexperiment shown here B. Cells pre-loaded with AA or Z-EITD-FMKwere incubated with TRAIL. The dissipation of "# and the frequency

of apoptosis were determined. The results of three independent experi-ments, each containing triplicates, were normalized with respect to themaximum apoptosis (black bars) or maximum "# dissipation (hatchedbars) obtained with TRAIL. The means values and standard deviationsare shown. Asterisks (%) indicate statistically significant differences be-tween cells incubated with TRAIL alone or with AA or Z-EITD-FMK

the electron transport chain and the release of ROS and pro-apoptotic proteins to the cytoplasm [24, 25]. We previouslyfound that intracellular AA confers stabilization of "# incells stimulated with FAS-L [12]. We sought to investigateif there was a protective effect of intracellular AA on mito-chondria of TRAIL-stimulated cells. We exposed K562 cellsto the fluorescent dye DiOC(6)(3) to estimate the dissipationof "# . The percentage of K562 cells with low DiOC(6)(3)fluorescence under TRAIL stimulation was two to four foldhigher from basal conditions. We found that intracellularAA prevented TRAIL-induced dissipation of "# in K562cells in a dose-dependent manner (Fig. 7A). The caspase-8 irreversible inhibitor Z-IETD-FMK also protected againstthe TRAIL-mediated dissipation of "# in a dose-dependentmanner (data not shown). A similar inhibition of TRAIL-induced apoptosis was obtained using 1.5 µM Z-IETD-FMKor 25 mM AA in K562 cells (Fig. 7B), and under these con-ditions, intracellular AA conferred more stabilization to "#

than Z-IETD-FMK. Therefore, the protective action of AAduring receptor-mediated apoptosis occurs early in the sig-naling cascade and additional protection of the mitochondrialproton gradient does not alter the fate of the cell stimulatedwith TRAIL.

4 Discussion

The principal finding in this study is that intracellular AAinhibited TRAIL-induced apoptosis in all cancer cell linesanalyzed here. These cell lines, as do most cells [17], trans-

port AA in the oxidized form DHA, via glucose transporters.Once transported in the form of DHA, the vitamin C insideis reduced to AA, and is accumulated in this form only. In-tracellular AA content in normal tissues ranges from 1 to10 mM [12, 26, 27], but a higher content of intracellularAA in tumor tissues compared with normal tissues has beenreported [28]. This might be due to the higher capacity ofcancer cells to transport glucose [29] and therefore to accu-mulate AA when transporting DHA, thus acquiring protec-tion against an oxidative high metabolism. Here, cancer celllines of different origins acquired protection from TRAIL-induced apoptosis through accumulation of AA. The celllines utilized in this study can efficiently transport DHA andaccumulate milimolar concentrations of intracellular AA. Ithas been reported that in transformed B cells from chroniclymphocytic leukemia patients, the intracellular concentra-tion of AA is as high as 15 mM [28]. How cells in vivoacquire vitamin C physiologically is an apparent paradox,since the plasma concentrations of DHA do not exceed 1–2%of ascorbate concentrations [30]. However, AA can be con-verted to DHA at the level of the cellular membrane andtherefore transported trough glucose transporters by the ac-tion of superoxide [31], a product of immune and endothelialcells [31–33]. Once DHA is available, its higher uptake bytransformed cells as compared to normal counterparts [34],occurs due to the upregulation in malignant cells of the ex-pression of the ubiquitous glucose transporter Glut1 [35].In this study the minimum concentration of intracellular AAthat provided protection varied among cells from 3 to 15 mM.The extent of protection also varied, from a 20% of reduction

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in apoptosis in PC3 cells with 30 mM AA, to an impressive60% in K562 cells, achieved with 25 mM AA. These findingsindicate that tumors can successfully avoid apoptosis inducedby TRAIL by uptake of the nutritional antioxidant vitamin C.

Our data indicate that ROS are produced after stimulationwith TRAIL and that they participate in signaling, in par-ticular H2O2. Our finding that TRAIL-mediated caspase-8activation was impaired in cells loaded with AA suggeststhat caspase-8 activation is sensitive to the oxidative stateof the milieu. There are two caspases with similar struc-tures, which have active cysteine sites: caspases 8 and 3.It has been proposed that these cysteine sites are suscepti-ble to oxidation, and several reports indicate that exposureto H2O2 induces caspase-3 activation [36–39]. Remarkably,pro-caspase-8 can be activated in cells exposed to H2O2

in the absence of re-localization induced by death-receptorligation: these cells exhibit total or partial processing ofpro-caspase-8 [37, 39], Furthermore, exposure to H2O2 en-hances FAS-induced caspase-8 activation [40]. Our exper-iments with catalase indicate that H2O2 produced duringTRAIL-R engagement is necessary for the activation of pro-caspase-8. Thus ROS, and particularly H2O2, have a directeffect on caspase-8 activation and mutual proximity of pro-caspase-8 is only one of the requirements for its activation.This suggests that ROS stimulate caspase-8 auto-cleavage,producing an extra level of regulation in this important cel-lular process.

Recent direct evidence from our laboratory indicates thatthe interaction between receptor and its ligand can produceH2O2 that facilitates signaling [21]. H2O2 is a small, highlydiffusible molecule with limited toxicity, which can be de-stroyed rapidly and efficiently by the cellular anti-oxidant de-fenses (peroxiredoxin, catalase and glutathione peroxidase).It has therefore been considered to possess “second messen-ger’s” characteristics [41]. In particular, cellular exposure tomilimolar concentrations of H2O2 can directly activate apop-tosis signaling [14] whereas the intracellular over-expressionof catalase confers resistance to FAS-induced apoptosis [40].However, AA does not directly quench H2O2. Therefore,several oxygen species should participate in modulation ofapoptosis signaling. AA quenches several ROS, includinghighly oxidative molecules such as •OH, peroxyl radical,superoxide anion and water-soluble peroxyl radicals [42].From these ROS, the intracellular production of •OH hasbeen documented. Perinuclear iron deposits in close prox-imity to the endoplasmic reticulum have been found, and ithas been shown that •OH is formed by the Fenton reaction inthat organelle [22]. Extracellular H2O2 can diffuse throughthe cell membrane and can react intracellularly with iron andcopper ions to form •OH, among other oxygen species, beforebeing transformed into water and oxygen [22]. Therefore,H2O2 may be a precursor molecule of other ROS involved inTRAIL-induced signaling. We hypothesized that •OH is one

ROS that participates in TRAIL signaling. In experimentsusing DMSO (a •OH scavenger [23]), we found that it re-duced the frequency of apoptosis induced by TRAIL. Ourexplanation of this observation is that intracellular AA in-hibits H2O2-induced apoptosis [39, 43] by quenching H2O2-derived •OH . However, the specificity of DMSO intracel-lularly can be overestimated in a biological system at theconcentrations used here, since •OH could react with severaltargets before DMSO could reach it. This might be the rea-son why the reduction of apoptosis achieved by DMSO ismore modest that the one achieved by vitamin C. Therefore,the contribution of other ROS in apoptosis signaling has tobe considered [44]. The participation of several ROS duringreceptor-mediated apoptosis explains why the anti-oxidantsused here did not completely abrogate apoptosis signaling,as none of them can quench all ROS.

Active caspase-8 mediates the dissipation of the mito-chondrial membrane potential, "# [9]. In our experimentswe found that intracellular AA stabilized "# in cells in-cubated in TRAIL. At concentrations of 25 mM AA and1.5 µM Z-EITD-FMK, both compounds provided similarprotection from TRAIL-induced apoptosis. However, AAwas more effective at stabilizing "# . We previously ob-tained similar results in our study of FAS-induced apoptosis[12]. It has been proposed that during apoptosis, ROS derivedfrom the mitochondria can cause further damage to mito-chondrial membranes by forming a loop of oxidation [45,46]. But our results suggest that whereas AA can scavengeintracellular ROS produced during apoptosis, quenching ofROS at the mitochondria level does not affect the courseof apoptosis in a TRAIL-stimulated cell. This may be theconsequence of a TRAIL signaling pathway that achievesindependence from the mitochondrial signaling pathway, asit has been described for the cells referred to as type-I cells[47]. Thus, the prevention of total mitochondrial depolar-ization does not delay apoptosis when a critical amount ofcaspase-8 has been activated.

5 Conclusion

In conclusion, we propose that production of ROS is as-sociated with the initiation of TRAIL-induced apoptosis incancer cells. Our results indicate that whereas TRAIL inter-action with its receptor induces the production of H2O2,several oxygen species must participate in signaling. In-tracellular AA can quench some of these ROS, reduc-ing early TRAIL-induced signaling (caspase-8 activation)and downstream events and therefore drastically reducingthe frequency of apoptosis. Our results suggest that tumorcells have a means of resisting TRAIL-induced apopto-sis through the accumulation of the nutritional antioxidantvitamin C.

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Acknowledgments We appreciate the technical help of Mr. GeorgiosStratis in the determination of cell volume and thank Ms. Mary AnneMelnick and Mr. Richard Stout for reading the manuscript.

Dr. D. W. Golde, our mentor and the inspiration behind this work,died on August 9th, 2004.

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