Resveratrol Inhibits Drug-Induced Apoptosis in Human Leukemia Cells byCreating an Intracellular Milieu Nonpermissive for Death Execution Kashif Adil Ahmad,1Marie-Veronique Clement,2Ismai
Trang 1Resveratrol Inhibits Drug-Induced Apoptosis in Human Leukemia Cells by
Creating an Intracellular Milieu Nonpermissive for Death Execution
Kashif Adil Ahmad,1Marie-Veronique Clement,2Ismail Muhamad Hanif,1and Shazib Pervaiz1
Departments of 1 Physiology and 2 Biochemistry, Faculty of Medicine, National University of Singapore, Singapore
ABSTRACT
Efficient apoptotic signaling is a function of a permissive intracellular
milieu created by a decrease in the ratio of superoxide to hydrogen
peroxide and cytosolic acidification Resveratrol (RSV) triggers apoptosis
in some systems and inhibits the death signal in others In this regard, the
inhibitory effect on hydrogen peroxide-induced apoptosis is attributed to
its antioxidant property We provide evidence that exposure of human
leukemia cells to low concentrations of RSV (4 – 8M ) inhibits caspase
activation, DNA fragmentation, and translocation of cytochrome c
in-duced by hydrogen peroxide or anticancer drugs C2, vincristine, and
daunorubicin Interestingly, at these concentrations, RSV induces an
in-crease in intracellular superoxide and inhibits drug-induced acidification.
Blocking the activation of NADPH oxidase complex neutralized
RSV-induced inhibition of apoptosis Furthermore, our results implicate
intra-cellular hydrogen peroxide as a common effector mechanism in
drug-induced apoptosis that is inhibited by preincubation with RSV.
Interestingly, decreasing intracellular superoxide with the NADPH
oxi-dase inhibitor diphenyliodonium reversed the inhibitory effect of RSV on
drug-induced hydrogen peroxide production These data show that low
concentrations of RSV inhibit death signaling in human leukemia cells via
NADPH oxidase-dependent elevation of intracellular superoxide that
blocks mitochondrial hydrogen peroxide production, thereby resulting in
an intracellular environment nonconducive for death execution.
INTRODUCTION
The effector components of apoptotic death signaling and their
intricate networking have been unraveled during the past couple of
decades (1–3) Consequently, it is now well established that
depend-ing on the level of activation of the initiator caspase, such as
caspase-8, the death signal can recruit directly downstream effector
caspases or engage the mitochondria with the resultant release of
death amplification factors, such as cytochrome c, apoptosis inducing
factor, and Smac/DIABLO (2, 4, 5) Death signaling by anticancer
drugs generally relies on positive input from the mitochondria, as is
evidenced by the resistance of tumor cells overexpressing the
death-inhibitory protein Bcl-2 that is localized to the membranes of
mito-chondria, endoplasmic reticulum, and nucleus (6 – 8) Therefore, by
implication, an intracellular milieu permissive for caspase activation/
activity and recruitment of mitochondria-derived amplification factors
is critical for efficient apoptotic execution To that end, we have
demonstrated the critical role of cellular redox status in the regulation
of death signaling (9 –12) Whereas an overwhelming accumulation of
intracellular reactive oxygen species could create an oxidatively
stressed environment leading to necrosis, a slight increase is a
stim-ulus for cellular proliferation (13, 14) Pro-oxidant intracellular milieu
is a hallmark of many tumor cells and is believed to endow tumor cells
with a survival advantage over their normal counterparts (15, 16)
Furthermore, we have demonstrated that a slightly elevated intracel-lular concentration of superoxide (O2⫺) inhibited apoptotic signaling, irrespective of the trigger (17, 18) Contrarily, our data and that of others have highlighted the critical role of intracellular H2O2 in rendering the cytosolic milieu permissive for efficient apoptotic exe-cution (19 –21) Thenceforth, we hypothesize that a critical balance between intracellular H2O2 and O2⫺dictates the response of tumor cells to apoptotic stimuli (9, 10, 12), and any stimulus/signal that inhibits the ability of intracellular H2O2, triggered on drug exposure,
to reduce the intracellular environment could potentially favor the acquisition of the resistant phenotype
One area of recent interest in cancer biology is the chemopreventive potential of natural products Among the compounds being evaluated for their cancer inhibiting activity is a phytoalexin, resveratrol (RSV), found in grapes and wines and known for its diverse biological activities, including antioxidant property (22–24) We reported previ-ously that the chemopreventive activity of RSV could be the result of its ability to induce apoptotic death in human leukemia and breast carcinoma cells (25) However, depending on the cell type and the concentration used, RSV has been shown to induce or inhibit cellular proliferation and death signaling (25–29) Our present study was stimulated by a recent report that H2O2-induced apoptosis was inhib-ited in the presence of RSV and the implication that this could be a function of its antioxidant activity (30) Given our recent findings that drug exposure of human leukemia cells resulted in H2O2-dependent apoptosis, we set out to investigate the mechanism by which RSV inhibited apoptotic signaling triggered by exogenous H2O2 or by exposure to three anticancer agents, namely, C2, vincristine, or dauno-rubicin, that induce apoptotic death in cancer cells (21)
MATERIALS AND METHODS
Determination of Cell Viability and DNA Fragmentation Human
pro-myelocytic leukemia (HL60) cell line was purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supple-mented with 10% fetal bovine serum, 1%L-glutamine, and 1% S-penicillin In
a typical survival assay, HL60 cells (1⫻ 105cells/well) plated in 96-well plates were preincubated with RSV (4 – 8M) for 2 h and then treated with 100
M of H2O2, 50 g/ml of C2, 1.25 g/ml of vincristine, or 0.2 g/ml of
daunorubicin for 18 h Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously or
by the-galactosidase (-gal) survival assay described below (31)
Propidium iodide staining was performed for analyzing DNA fragmentation
as described previously (25) Stained cells were analyzed by flow cytometry (Coulter EPICS Elite ESP; Beckman Coulter, Fullerton, CA) with the excita-tion and emission wavelengths at 488 nm and 610 nm, respectively At least 10,000 events were analyzed by WinMDI software
Determination of Caspase-3 and -9 Activity Caspase-3 and -9 activity
was determined using the Bio-Rad fluorescent assay kit (Hercules, CA) for the two caspases Cells (1⫻ 106) were preincubated for 2 h with RSV (4 – 8M) and then incubated for 12 h with 100MH2O2, 50g/ml of C2, 1.25 g/ml
of vincristine, or 0.2g/ml of daunorubicin Fifty l of 2⫻ reaction buffer
with 10 mM DTT and 5 l of the conjugate substrate (DEVD-AFC for
caspase-3 and LEHD-AFC for caspase-9) were added to cell lysates Caspase activity was determined by the relative fluorescence intensity at 505 nm
Received 8/5/03; revised 12/9/03; accepted 12/10/03.
Grant support: Research grants from the NMRC, Singapore (R-185-000-032-213) to
S Pervaiz and from the BMRC, Singapore (R-185-000-048-305) to S Pervaiz and
M-V Clement.
The costs of publication of this article were defrayed in part by the payment of page
charges This article must therefore be hereby marked advertisement in accordance with
18 U.S.C Section 1734 solely to indicate this fact.
Trang 2activity relative to untreated cells (1⫻) In addition, cleavage of the caspase-3
substrate, poly(ADP ribose) polymerase (PARP), was assessed by Western
blot analysis using anti-PARP (clone C-2–10; PharMingen, San Diego, CA) as
described previously (9)
Measurements of Intracellular O 2ⴚand pH Intracellular O2⫺was
as-sayed by a lucigenin-based chemiluminescence assay as described previously
(11) Data are shown as percentage change (% change) in the intracellular O2⫺
concentration compared with untreated cells and are the mean⫾ SD of three
independent measurements
For measurement of cytosolic pH, cells were loaded with 10M2
⬘,7⬘-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM; Sigma, St Louis, MO),
and the fluorescence ratio of 525:610 nm was used to derive cytosolic pH using
a standard pH calibration curve as described previously (11)
Flow Cytometric Analysis of Intracellular H 2 O 2 Cells were exposed to
the apoptotic triggers for 4 –12 h, loaded with 5-(and-6)-chloromethyl-2
⬘,7⬘-dichlorofluorescin diacetate (CM-H2DCFDA; Molecular Probes, Eugene, OR;
5M) (32) at 37°C for 15 min, and analyzed by flow cytometry (Coulter
EPICS Elite ESP) using an excitation wavelength of 488 nm as described
previously (21) At least 10,000 events were analyzed
Determination of Mitochondrial ⌬⌿m and Cytosolic Cytochrome c.
Potential-sensitive probe 3,3⬘dihexyloxacarbocyanine iodide was used to
measure mitochondrial⌬mas described previously (31) Briefly, 1⫻ 106
cells were incubated with 3,3⬘dihexyloxacarbocyanine iodide (40 nM) for 15
min at 37°C As a positive control, cells were incubated separately with an
uncoupling agent, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (100
M) At least 10,000 events were analyzed by flow cytometry with excitation
set at 488 nm
Cytochrome c was detected in cytosolic extracts from 30⫻ 106cells by
Western blot analysis using anti-cytochrome c (7H8.2C12; PharMingen) as
described previously (33)
Transient Transfection with pIRESRacN17 and -Gal Survival Assay.
Transient transfections of CEM cells were performed using the SuperFect
transfection reagents from Qiagen (Hilden, Germany), and survival of
trans-fected cells was assessed by the-gal survival assay as described recently (34)
Cell survival was calculated as [(-gal activity g⫺1protein of transfected
cells incubated with the apoptotic trigger)/(-gal activity g⫺1 protein of
transfected cells incubated without the trigger)].-Gal activity was measured
using the Galacto-Star mammalian reporter kit (Applied Biosystems, Foster
City, CA) Protein concentration was determined using the Coomasie Plus
protein assay reagent from Pierce (Rockford, IL)
Data Analysis Data presented are mean⫾ SD of at least three independent
experiments performed in triplicate, unless otherwise indicated Statistical
significance was determined by the Student’s t test.
RESULTS Low Doses of RSV Inhibit H 2 O 2 -Induced Apoptosis Upstream
of the Mitochondria Corroborating earlier findings on the
death-inducing activity of H2O2in cancer cells, exposure of HL60 cells to
H2O2(100M) resulted in a significant decrease in cell survival (Fig
1A) The cytotoxic activity of H2O2was a function of activation of the apoptotic death pathway as evidenced by the significant increase in caspase activity, cleavage of the caspase-3 substrate PARP, and appearance of the sub-G1fraction (Fig 1, B–D) Interestingly,
prein-cubation of cells with 4 – 8 MRSV for 2 h before the addition of
H2O2 for 18 h resulted in an increase in cell survival (Fig 1A),
significant inhibition of caspase-3 activity and PARP cleavage (Fig 1,
B and C), and inhibition of DNA fragmentation (Fig 1D) Moreover,
preincubation with RSV prevented H2O2-induced decrease in mito-chondrial ⌬m (Fig 2A) and significantly blocked H2O2-induced
cytosolic translocation of cytochrome c from the mitochondria (Fig 2B) One possible explanation for the observed inhibitory effect of
RSV could be that RSV functioned as an efficient scavenger of H2O2 However, addition of RSV and H2O2simultaneously to the culture medium (RSV/H2O2) had no effect on H2O2-induced cell death (Fig
2C), whereas previous incubation with RSV for 2 h (RSV⫹ H2O2)
significantly (P ⬍ 0.05) inhibited apoptotic signaling (Fig 2C) These
data indicate that the death-inhibitory effect of RSV is not simply a function of its ability to scavenge H2O2but involves mechanism(s) upstream of the mitochondria or signals that engage the mitochondrial death machinery
RSV Inhibits H 2 O 2 -Induced Decreases in Intracellular O 2ⴚand Cytosolic pH We have shown that H2O2induces a decrease in O2⫺ and cytosolic acidification, thereby creating an intracellular milieu permissive for apoptotic execution (18, 34, 35) Therefore, we ques-tioned if the inhibitory effect on H2O2-induced apoptosis was caused
by the ability of RSV to create an intracellular environment
noncon-Fig 1 Preincubation with resveratrol (RSV)
in-hibits H2O2-induced caspase activation and DNA
fragmentation in HL60 cells A, HL60 cells
(1 ⫻ 10 6 cell/ml) were incubated with 100 M
H2O2for 18 h with or without preincubation for 2 h
with RSV (4 and 8 M ) Cell viability was
deter-mined by the MTT assay B, caspase-9 and -3
activity was determined by fluorimetric assays, and
(C) poly(ADP ribose) polymerase cleavage was
assessed by Western blot analysis D, DNA
frag-mentation was determined by propidium iodide
staining and the appearance of sub-G1fraction.
1453
Trang 3ducive for death execution Preincubation with RSV not only induced
a slight increase in intracellular O2⫺but also blocked the inhibitory
effect of H2O2 on intracellular O2⫺levels (Fig 3A) Interestingly,
preincubation with RSV before the addition of H2O2 consistently
resulted in higher intracellular O2⫺than RSV alone In addition, the
significant decrease in cytosolic pH (P⬍ 0.01) triggered by H2O2was
inhibited by previous incubation with RSV (Fig 3B).
RSV-Induced Inhibition of Apoptosis Can Be Reverted by
Blocking NADPH Oxidase Activation Considering the reports on
the antioxidant potential of polyphenolic compounds, such as RSV,
we were intrigued by our observation that low concentrations of RSV
induced an increase, rather than a decrease, in intracellular O2⫺
concentration (30, 36 –38) Therefore, we investigated the effect of
blocking NADPH oxidase complex, one of the major sources of
intracellular O2⫺, on RSV-induced increase in O2⫺ Pharmacologic
inhibition of NADPH oxidase with diphenyleneiodonium chloride
(DPI) completely blocked RSV-induced increase in intracellular O2⫺
in HL60 cells (Fig 3C) Furthermore, incubation of cells for 1 h with
DPI before the addition of RSV significantly (P⬍ 0.004) restored the
sensitivity of HL60 and CEM leukemia cells to H2O2(Fig 3D; data
not shown) To provide additional evidence that this was a function of
NADPH oxidase activation, CEM cells were transfected transiently
with a dominant negative mutant of Rac (RacN17), which inhibits
NADPH oxidase-dependent increase in intracellular O2⫺(34) Similar
to DPI, transient transfection with RacN17 completely neutralized the
death-inhibitory activity of RSV and restored the sensitivity of
leu-kemia cells to H2O2(Fig 3E).
RSV Inhibits Apoptosis Triggered by Anticancer Drugs C2,
Vincristine, and Daunorubicin In our earlier reports, we
demon-strated that exposure of human leukemia and melanoma cells to C2, a
purified photoproduct of MC540, triggered mitochondrial generation
of H2O2 that was responsible for the release of cytochrome c and
downstream activation of the caspase cascade (21) Similar to the data
obtained with H2O2, preincubation of cells with RSV before the
addition of C2 resulted in an increase in cell survival compared with
the cells treated with C2 alone (Fig 4A) Whereas exposure of
leukemia cells to C2 resulted in robust increases in caspase-3 and -9
activity, preincubation with RSV significantly inhibited both caspases
and the cleavage of the caspase-3 substrate PARP (Fig 4B) That this
translocation of cytochrome c in cells preincubated with RSV before the addition of C2 (Fig 4C) In addition, similar to the results obtained
with H2O2, previous incubation with RSV (RSV⫹ C2) was required
for the inhibitory effect of RSV on C2-induced death signaling, whereas simultaneous exposure to RSV and C2 (RSV/C2) had no effect on C2 signaling (data not shown)
We next questioned whether the inhibitory effect of RSV on
C2-induced apoptosis also was mediated by its ability to (a) create a pro-oxidant intracellular milieu; and (b) inhibit cytosolic acidification.
Results indicate that preincubation of HL60 cells with RSV inhibited
H2O2production triggered by exposure to C2 (Fig 5A) Similar to the
results obtained with H2O2, preincubation of HL60 cells with RSV for
2 h resulted in an increase in intracellular O2⫺concentration, which
was even more pronounced on subsequent addition of C2 (Fig 5B) It
should be noted that a slight increase in intracellular O2⫺also was observed on exposure of cells to C2 alone; however, unlike RSV, this was accompanied by a surge in intracellular H2O2 production In addition, cytosolic acidification triggered on exposure to C2 was
inhibited completely on previous exposure to RSV (Fig 5C)
Further-more, incubation of cells with DPI before the addition of RSV and C2 resulted in a decrease of intracellular O2⫺ (data not shown) and
restored death signaling in response to C2 (Fig 5D).
To gain additional insight into the death-inhibitory effect of RSV and its potential clinical implications, we next investigated the effect
of RSV on apoptosis induced by two chemotherapeutic agents, vin-cristine and daunorubicin (39) Corroborating the results obtained with H2O2 and C2, results indicate a dose-dependent (up to 8M)
inhibitory effect of RSV on vincristine-induced cell death (Fig 6A)
together with significant inhibition of caspase-9 and -3 activity (Fig
6B) That the apoptosis inhibitory effect of RSV was linked to its
ability to create a pro-oxidant intracellular milieu was supported additionally by the ability of DPI or transient transfection with RacN17 to revert the sensitivity of leukemia cells in the presence of
RSV to vincristine-induced apoptosis (Fig 6, C and D).
The inhibitory effect of low concentrations of RSV on drug-induced apoptosis was not exclusive to C2 or vincristine Preincuba-tion of leukemia cells to RSV for 2 h resulted in a significant increase
in cell survival (Fig 7A), inhibition of caspase activity (Fig 7B), and
Fig 2 Inhibitory effect of resveratrol (RSV) on
H2O2-induced death signaling is upstream of the
mitochondria A, HL60 (1⫻ 10 6 ) cells were
ex-posed to 100 M H2O2for 4 h with or without
previous incubation for 2 h with RSV (8 M ), and
⌬ m was determined as described in “Materials
and Methods.” B, cytosolic cytochrome c was
as-sessed by Western blot analysis
Staurosporin-treated HL60 cytosolic extract was used as positive
control C, HL60 cells (1⫻ 10 6 /ml) were incubated
simultaneously with RSV (8 M ) and H2O2(100
M) for 18 h (RSV/H 2 O 2) or preincubated for 2 h
with RSV (8 M ) before the addition of 100 M
H2O2(RSV ⫹ H 2 O2) Cell viability was
deter-mined by MTT assay.
Trang 4Fig 3 Inhibition of NADPH oxidase activation prevents resveratrol (RSV)-induced increase in intracellular O2⫺and overcomes its death-inhibitory effect A, cytosolic pH was
determined with the pH-sensitive probe 2⬘,7⬘-bis(2-carboxyethyl)-5,6-carboxyfluorescein B, 2 ⫻ 106 cells were treated with RSV (8 M ) for 2 h or H2O2(100 M ) for 4 h with or without 2 h of previous incubation with RSV (8 M ) Intracellular O2⫺was measured by a lucigenin-based chemiluminescence assay C, HL60 (2⫻ 10 6 ) cells were incubated with
8 M RSV for 4 h in the presence or absence of DPI (1.25 M ), and intracellular O2⫺was measured as described previously D, HL60 cells (1⫻ 10 6 /ml) were preincubated with DPI (1.25 M ) or with DPI (1.25 M ) ⫹ RSV (8 M ) for 4 h before the addition of 100 M H2O2for 18 h Cell survival was assessed by the MTT assay E, CEM cells (1⫻ 10 6 /ml) cotransfected with pCMV--galactosidase (-gal) and pIRES-RacN17 were exposed to 100 M H2O2for 18 h with or without previous incubation for 2 h with 8 M RSV Cell survival was assessed by the -gal survival assay Mean of two independent transfections done in duplicate is shown.
1455
Trang 5Decrease in Intracellular O 2ⴚOverrides the Inhibitory Effect
of RSV on Drug-Induced H 2 O 2 Production Thus far, we have
shown that the inhibitory activity of RSV on drug-induced apoptosis
was linked to its ability to increase NADPH oxidase-dependent
intra-cellular O2⫺production Interestingly, all of the drugs used in this
study resulted in an intracellular increase in H2O2 production (Fig
NADPH oxidase complex had no effect on intracellular H2O2increase (21) Similar to the results reported with C2, preincubation of HL60 cells with DPI did not affect intracellular H2O2production on expo-sure to vincristine and daunorubicin, thus strongly suggesting mito-chondria as the cellular source on drug exposure (data not shown) More importantly, decreasing intracellular O2⫺with DPI restored the ability of C2, vincristine, and daunorubicin to trigger intracellular
H2O2production even in the presence of RSV (Fig 7C).
DISCUSSION RSV Inhibits Apoptosis by Altering Cellular Redox Status.
Taken together, our data provide strong evidence that contrary to its proapoptotic activity atⱖ25M, low micromolar concentrations (4 – 8
M) inhibit apoptotic signaling (25, 40, 41) This divergent signaling
by RSV is intriguing and could be explained by our earlier observa-tions that at concentraobserva-tions of ⱖ32M, apoptosis induced in HL60 cells is mediated by up-regulation of CD95 (Fas/Apo1)-CD95L inter-action (25) This effect on up-regulation of the death receptor ligand
is not observed at concentrations of RSV⬍16M, hence the inability
to trigger apoptosis in these cells (data not shown) Contrarily, at these concentrations, RSV inhibits apoptotic signaling upstream of the mitochondria, thus blocking the recruitment of mitochondrial-derived
amplification factors, such as cytochrome c We also provide evidence
that RSV has a potent effect on the intracellular redox status, a critical determinant of the efficacy of the death signal (9, 42, 43) In that respect, it has been shown previously that maintaining a slightly elevated intracellular O2⫺promotes cellular proliferation (14, 44) and inhibits apoptotic signaling (18, 42) A pro-oxidant intracellular mi-lieu is an invariable finding in cancer cells and has been shown to endow cancer cells with a survival advantage over their normal counterparts (15) Because a decrease in intracellular O2⫺and cyto-solic pH is critical for efficient death execution, our results suggest that low concentrations of RSV could enable cancer cells to evade death signaling by creating a pro-oxidant intracellular milieu and inhibiting cytosolic acidification
RSV Induces Increase in O 2ⴚvia NADPH Oxidase Activation.
Considering the earlier reported ability of RSV to inhibit mitochon-drial complex III-induced reactive oxygen species production, our paradoxical findings provide evidence for a pro-oxidant effect of RSV
at concentrations that do not trigger apoptosis (45) Such pro-oxidant activity of polyphenolics, such as RSV, has been reported recently in different systems (46, 47) In one model, reactive oxygen species generation at concentrations of RSV that triggered cell death in human cancer cells was proposed to be responsible for its cytotoxic activity (47) In addition, our results implicate the membrane NADPH oxidase complex as a potential source of O2⫺on incubation with low doses of RSV Inhibition of the NADPH oxidase complex not only restored death signaling but also resulted in reverting the negative effect of RSV on drug-induced intracellular H2O2production This fits in well with our hypothesis that a balance between intracellular O2⫺ and
H2O2could be a critical factor in the response of cells to apoptotic triggers, with a tilt toward the former favoring survival and a pre-dominance of the latter facilitating death execution (10) A careful
look at the data shown in Fig 5B reveals that, similar to RSV alone,
exposure of cells to the anticancer drug C2 also results in a slight elevation in intracellular O2⫺ However, it is important to note that C2-induced O2⫺ is accompanied by a surge in intracellular H2O2, which is not observed with low concentrations of RSV In addition, preincubation with RSV completely blocks C2-induced H2O2 produc-tion and maintains a significantly elevated intracellular O2⫺level By
Fig 4 Resveratrol (RSV) inhibits apoptosis triggered by a novel anticancer drug, C2.
A, HL60 cells (1⫻ 10 6 /ml) were incubated simultaneously with RSV (8 M ) and C2 (50
g/ml) for 18 h (RSV/C2) or preincubated for 2 h with RSV (8 M ) before the addition
of C2 (RSV⫹ C2) Cell viability was determined by MTT assay B, activity of caspase-3
and -9 was determined by fluorimetric assays Poly(ADP ribose) polymerase (PARP)
cleavage and (C) cytosolic cytochrome c were detected by Western blot analyses.
Trang 6inhibiting the decrease in intracellular pH It is intriguing as to how an
increase in intracellular O2 ⫺blocks mitochondrial-derived H2O2 One
possibility could be inhibition of upstream caspase activation, as has
been shown previously, or other proapoptotic factors, such as
death-promoting Bcl-2 family members, required for the engagement of the
mitochondrial pathway (48) Impeding these upstream pathways could
account for the inhibition of drug-induced mitochondrial H2O2
pro-duction and downstream cytochrome c release observed in cells
preincubated with RSV This in turn fails to amplify downstream
caspase cascade (caspase-9 and -3) and leads to a substantial decrease
in the sensitivity of cells to apoptotic stimuli that require mitochon-drial amplification factors Taken together, these results indicate that the increase in intracellular O2⫺on exposure of leukemia cells to RSV was mediated through the activity of NADPH oxidase complex and linked directly or indirectly to the apoptosis-inhibitory activity of RSV In addition, data presented here not only implicate mitochon-drial H2O2production as a critical effector mechanism during drug-induced apoptosis but also demonstrate the ability of an increase in intracellular O2⫺to prevent H2O2production and thereby impede the recruitment of the mitochondrial death pathway
Fig 5 Resveratrol (RSV) creates a pro-oxidant
intracellular milieu and inhibits C2-mediated
de-crease in cytosolic pH and cell death A, HL60
(1 ⫻ 10 6 ) cells were treated with 50 g/ml of C2
for 4 h with or without previous incubation with 8
M RSV, and intracellular H2O2was determined.
B, 2⫻ 10 6 cells (in 2 ml) were treated with RSV (8
M ) for 4 h or 50 g/ml of C2 for 4 h with or
without 2 h of previous incubation with RSV (8
M ), and intracellular O2⫺was determined C, cells
were treated as in (A), and cytosolic pH was
as-sessed with 2
⬘,7⬘-bis(2-carboxyethyl)-5,6-carboxy-fluorescein D, HL60 cells (1⫻ 10 6 /ml) were
pre-incubated with DPI (1.25 M ) or with DPI (1.25
M ) ⫹ RSV (8 M ) for 4 h before the addition of
50 g/ml C2 for 18 h Cell survival was assessed
by the MTT assay.
Fig 6 The inhibitory effect of resveratrol (RSV)
on vincristine-induced apoptosis can be neutralized
by inhibition of the NADPH oxidase complex A,
HL60 cells (1⫻ 10 6 cell/ml) were preincubated
with RSV (2, 4, or 8 M ) for 2 h before treatment
with 1.25 g/ml of vincristine for 18 h Cell
viability was determined by the MTT assay B,
caspase-9 and -3 activity was determined by
fluo-rimetric assays C, HL60 cells (1⫻ 10 6 /ml) were
preincubated with DPI (1.25 M or 2.5 M ) ⫹ RSV
(8 M ) for 4 h before the addition of 1.25 g/ml of
vincristine for 18 h Cell survival was assessed by the
MTT assay D, CEM leukemia cells (1⫻ 10 6 /ml)
were cotransfected with pCMV- -galactosidase
(-gal) and pIRES-RacN17 and exposed to 1.25 g/ml
of vincristine for 18 h with or without previous
incu-bation for 2 h with 8 M RSV Cell survival was
assessed by the -gal assay.
1457
Trang 7Potential Implications These findings could be potentially
im-portant in light of the recent interest in the biological activity of
flavonoids or flavonoid-like molecules, such as RSV, for their
possi-ble use in combination chemotherapy regimens Although in vitro
exposure of tumor cells to RSV at relatively high concentrations
results in apoptotic cell death, because of the low bioavailability of
RSV, plasma levels as high as 50 –100Mmay not be physiologically
attainable (49, 50) Our data suggest death-inhibitory and/or
prosur-vival activity of RSV in leukemia cells at doses that may be relevant
physiologically (49, 50) Thus, the use of RSV in combination with
drugs such as C2, vincristine, or daunorubicin could be a dangerous
mixture because the slight pro-oxidant effect may provide tumor cells
with not only a survival advantage but also impede death signals This
could present an ideal environment for the propagation and
prolifer-ation of tumor cells
ACKNOWLEDGMENTS
We thank Kartini Bte Iskander and M Ali Shazib for technical assistance
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Fig 7 Resveratrol (RSV) inhibits
daunorubicin-induced apoptosis, and preincubation with DPI
re-verts the effect of RSV on drug-induced H 2 O 2
production A, 1⫻ 10 6 cell/ml were incubated with
RSV (8 M ) for 2 h, followed by 18 h of exposure
to 0.2 g/ml of daunorubicin Cell viability was
determined by the MTT assay B, caspase-9 and -3
activity was determined by fluorimetric assays C,
HL60 (1 ⫻ 10 6 ) cells were preincubated with 1.25
MDPI before the addition of RSV (DPI/RSV),
followed by treatment with C2 (50 g/ml),
dauno-rubicin (0.2 g/ml), or vincristine (1.25 g/ml) for
4 h Intracellular H 2 O 2 was determined by flow
cytometry.
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