JBC Papers in Press.

Published on March 14, 2007 as Manuscript M610616200

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M610616200

CYANIDIN-3-RUTINOSIDE, A NATURAL POLYPHENOL ANTIOXIDANT,

SELECTIVELY KILLS LEUKEMIC CELLS BY INDUCTION OF OXIDATIVE
STRESS
Rentian Feng1, Hong-Min Ni1, Shiow Y. Wang2, Irina L. Tourkova 1, Michael R. Shulin1, Hisashi
Harada3 and Xiao-Ming Yin1

From the 1Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA,
2
The Fruit Laboratory, Beltsville Agricultural Research Center, U.S. Department of Agriculture,
Beltsville, MD,
3
Departmetn of Internal Medicine, Virginia Commonwealth University, Richmond, VA

Running title: Polyphenol-induced oxidative stress in leukemic cells

Address corresponding to: Dr. Xiao-Ming Yin, Department of Pathology, University of Pittsburgh
School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261, Phone: 412-648-8436; Fax: 412-684-
9564; E-mail: xmyin@pitt.edu

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Anthocyanins are a group of naturally
occurring phenolic compounds widely available
in fruits and vegetables of human diets. They
have broad biological activities including anti-
mutagenesis and anti-carcinogenesis, which are
generally attributed to their antioxidant
activities. We studied the effects and the
mechanisms of the most common type of
anthocyanins, cyanidin-3-rutinoside, in several
leukemia and lymphoma cell lines. We found
that cyanidin-3-rutinoside extracted and
purified from black raspberry cultivar Jewel
induced apoptosis in HL-60 cells in a dose- and
time-dependent manner. Paradoxically, this
compound induced the accumulation of
peroxides, which are involved in the induction
of apoptosis in HL-60 cells. In addition,
cyanidin-3-rutinoside treatment resulted in
reactive oxygen species (ROS)-dependent
activation of p38 MAPK and JNK, which
contributed to cell death by activating the
mitochondria pathway mediated by Bim.
Down-regulation of Bim or over-expression of
Bcl-2 or Bcl-xL considerably blocked apoptosis.
Notably, cyanidin-3-rutinoside treatment did
not lead to increased ROS accumulation in
normal human peripheral blood mononuclear
cells and had no cytotoxic effects on these cells.
These results indicate that cyanidin-3-
rutinoside have the promising potential to be
used in leukemia therapy with the advantages
of being wildly available and being selective
against tumors.
Natural products derived from plants have
recently received much attention as potential
chemopreventive and chemotherapeutic agents.
Among them great attention has been given to
natural products with established antioxidant
activities and less toxicity in normal cells, such as
tea polyphenols and resveratrol (1-3). These
substances appear very promising for cancer
prevention and treatment in pre-clinical models
and clinical trials (1-4).
Perhaps the most common type of plant
polyphenols is flavonoids, which provide much of
the flavor and color to fruits and vegetables. More
than 5,000 different flavonoids have been
described (5). There are six major subclasses of
flavonoids (5): anthocyanins, flavones (e.g.,
apigenin), flavonols (e.g, quercetin), flavanones
(e.g., naringenin), catechins of flavanols (e.g.,
epicatechin) and isoflavones (e.g., genistein).
Anthocyanins probably deserve the most attention,
as the daily uptake of anthocyanins in human diet
is remarkable, estimated to be 180 to 215 mg/day
in the United States (6), which is much higher than
the total intake estimated for other flavonoids (23
mg/day).
Anthocyanins are the glycosylated form of
anthocyanidins, which are polyhydroxyl and
polymethoxy derivatives of 2-

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
phenylbenzopyrylium or flavylium salts (7).
Structurally, there are more than a dozen different
anthocyanidins in the plants but cyanidin is the
most frequently found naturally occurring
anthocyanidin. Cyanidin is widely available in
human diet through crops, beans, fruits, vegetables
and red wine (7), and cyanidin-3-rutinoside (C-3-
R)1 is a common glycosylated form (8).
Anthocyanins demonstrate strong anti-oxidant
activities in a variety of in vitro assays (7-10).
These natural compounds can react with reactive
oxygen species (ROS) and thus interrupt the
propagation of new free radical species. The
double bonds present in the phenolic ring, the
hydroxyl side chains and even the glycosylation
contribute to the scavenging activity.
Anthocyanins, particularly cyanidin glycosides,
have been found to possess a broad spectrum of
biological activities, including the scavenging
effects on activated carcinogens and mutagens and
the effects on cell cycle regulation (7,8,11-13)
Previous work indicated that cyanidin-rich berry
or pomegranate extracts, or purified cyanidins
possess remarkable chemopreventive activities in
cell culture and animal models (11,14-17)
Furthermore, cyanidin glycosides purified from
berry extracts possess pro-apoptotic properties in
human cancer cells including leukemia cells
(13,18-20). These studies suggest that the anti-
tumor activities of cyanidin glycosides could be
related to their ability to induce apoptosis and that
these products may have potential as agents for
cancer treatment as well. All these activities have
been thought to be related to the antioxidant
properties of the cyanidins (8,21-23). However,
this notion has not been carefully examined,
particularly for the apoptotic activity.
Furthermore, the molecular mechanisms and
signaling pathways initiated by cyanidin
glycosides in cell death are also poorly
understood.
In the present study, we have found that
cyanidin-3-rutinoside paradoxically increases the
level of peroxides in the human leukemia cells,
which is responsible for its apoptotic effects.
Cyanidin-3-rutinoside subsequently activates the
p38MAP kinase and the pro-death Bcl-2 family
proteins, leading to mitochondrial release of
apoptogenic factors and cell death. In contrast,
cyanidin-3-rutinoside caused little ROS generation
in the normal human peripheral blood
mononuclear cells (PBMC) and had very low
toxicity toward these cells. Our study thus
indicates that anti-oxidative natural products, such
as cyanidin 3-rutinoside, could exhibit pro-oxidant
activities selectively in the leukemic cells, which
could be exploited for the development of anti-
tumor agents with a low toxicity toward normal
cells.
Experimental Procedures
Reagents. Cyanidin-3-rutinoside (C-3-R)
(>99% purity) was purified from black raspberry
cultivar Jewel extract as previously described (10).
Purified C-3-R was stored at -80°C and the stock
solution (150 mM) was freshly prepared before
use by dissolving the powder in DMSO.
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The following primary or secondary
antibodies were used: anti-Bak (NT) (Upstate
Biotechnology, Lake Placid, NY); anti-Bak (Ab-1)
(Oncogene, Cambridge, MA); anti-Bax (N-20)
(Santa Cruz Biotechnology, Santa Cruz, CA); anti-
Bax (clone 6A7), anti-Bcl-2, anti-JNK (clone 666),
anti-cytochrome c and anti-Smac (all from BD
Biosciences, San Jose, CA); anti-Bcl-xL, anti-Bim,
anti-phosphorylated JNK and p38MAP kinase (all
from Cell Signaling, Beverly, MA); anti-β-actin
(Sigma, St. Louis, MO); anti-COX IV subunit II
(Invitrogen Molecular Probes, Carlsbad, CA); and
HRP-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA).
Cell line and cell culture. The human
leukemia and lymphoma cell lines HL-60
(myeloblastic), MOLT-4 (lymphoblastic) and
Daudi (lymphoblastic) were obtained from
American Type Culture Collection (Manassas,
VA). Human leukemia cells HL-60/neo, HL-
60/Bcl-2, HL-60/Bcl-xL were derived from the
laboratory of Dr. Kapil N. Bhalla (Medical
College of Georgia) and cultured as previously
described (24). The T cell acute lymphoblastic
leukemia cell line, CCRF-CEM and its subline
stably expressing shRNA against Bim were
cultured as previously described (25). Human
PBMC were prepared from healthy donors
(Pittsburgh Blood Bank) using Ficoll-Hypaque
centrifugation. The leukemia/lymphoma cell lines
and PBMC were cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum (FBS).
Analysis of cell death. Apoptotic and
necrotic cell death were determined as previously
2
described with modifications (26). Briefly, cells
were double-stained with 10 µmol/L bis-
benzimide Hoechst 33258 and propidium iodide
(PI, 1µg/ml) (Invitrogen, Molecular Probes,
Carlsbad, CA) for 30 minutes and analyzed by
fluorescence microscopy. Approximately 300-500
cells per condition were randomly selected and
assessed. Hoechst 33258 positive cells with
apoptotic (condensed and/or fragmented) nuclei
were considered as apoptotic cells regardless
whether they are PI positive or not, whereas PI-
stained cells without apoptotic nuclear changes
were considered as necrotic cells.
Caspase activities were measured using 15 µg
(caspase-3) or 20µg (caspase-8 and -9) of proteins
and 20 µM of fluorescent substrates (Ac-DEVD-
AFC, AC-IETD-AFC, and Ac-LEHD-AFC for
caspase-3, -8, and -9, respectively)(Biomol,
Plymouth Meeting, PA)(26). The fluorescence
signals were detected by a fluorometer (Tecan
GENios, Durham, NC) at excitation and emission
wavelengths of 400 nm and 510 nm, respectively.
Cytochrome c release and Bax translocation to
the mitochondria were analyzed by immunoblot
assay using the cytosolic and membrane fractions
prepared from treated cells. Subcellular
fractionation was conducted using limited plasma
membrane permeabilization with 0.05% digitonin
as previously described (27).
Measurement of ROS. For the in vitro study,
superoxide radicals were quantified
spectrophotometrically in a xanthine/xanthine-
oxidase system as previously described (28).. The
final results were expressed as percent inhibition
of superoxide radical production in the presence of
an antioxidant (C-3-R, ascorbic acid or Trolox).
The assay for hydrogen peroxide (H2O2) levels
was carried out following procedures previously
described (29). The final results were expressed
as percent inhibition of H2O2 levels in Ti(IV)-
H2O2 complex system in the presence of
antioxidants.
For determination of the cellular ROS level,
two different methods were used. First, at various
times (15 minutes to 4 hours) after C-3-R
treatment, HL-60 cells were incubated for another
15 minutes with either 2 µM of dihydroethidium
bromide or 2 µM of 2’,7’-
dichlorodihydrofluorescein diacetate (H2DCFDA).
These ROS-dependent fluorescent probes were
converted to ethidium or 2’,7’-dichlorofluorescein
(DCF) in the presence of superoxide radicals or
H2O2, respectively, which can be detected by flow
cytometry (26). To measure the intracellular
accumulation of C-3-R-induced H2O2 production,
cells were simultaneously treated with C-3-R and
H2DCFDA for 1.5-3 hours and then harvested for
flow cytometry analysis. In another set of
experiments, intracellular H2O2 levels were
determined by a method based on horseradish
peroxidase-dependent oxidation of phenol red,
which is assessed by the increased absorbance at
610 nm (30).
Immunofluorescence and immunoblot
analysis. Immunofluorescence staining was
carried out as previously described (26). Treated
cells were harvested and pelleted to glass slides by
Cytospin® (Thermo Shandon Inc., Pittsburgh,
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PA). Cells were fixed in 4%
paraformaldehyde/PBS (pH 7.4) for 1 hour at 4°C
and permeabilized in 0.5% Triton X-100 for
another 1 hour. Following the incubation of the
primary antibodies, the molecules of interest were
detected using Alexa Fluor-conjugated secondary
antibodies and fluorescence microscopy.
For the immunoblot assay, treated cells were
lysed with RIPA buffer supplemented with 1 µM
EDTA and a cocktail of protease inhibitors.
Proteins in the lysates were quantified and
separated by SDS- PAGE, transferred to a PVDF
membrane and probed with relevant antibodies.
Results
C-3-R selectively induces apoptosis in
human leukemic cells. Treatment of the human
leukemia cell line HL-60 with C-3-R induced a
significant amount of apoptosis in a time and dose-
dependent manner (Figure 1A) based on the
morphology of the nucleus. About 50% of cells
became apoptotic in 18 hours when 50 µM of C-3-
R was applied and virtually all cells were
apoptotic in the presence of 120 µM or more of C-
3-R. There was very little necrosis as measured by
propidium iodide (PI) staining. Consistently, a
significant amount of caspase-3 and caspase-9
activities could be detected in a dose-dependent
manner, which peaked around 16 hours (Figure
1B-C). The detected activity was reduced
thereafter, likely due to the increased cell death.
Consequently, C-3-R induced apoptosis and
caspase activation in HL-60 cells could be
3
suppressed by the effector caspase inhibitor,
DEVD-CHO (data not shown), and the pan-
caspase inhibitor, z-VAD-FMK (Figure 1D). The
mitochondria pathway would be an important
mechanism contributing to C-3-R induced
apoptosis, as we found that cytochrome c (Figure
1E) and Smac (see below) was released upon C-3-
R treatment.
C-3-R also induced apoptosis in other human
leukemia/lymphoma cell lines including MOLT-4,
Daudi and CCRF-CEM (Figure 1F). It had little
toxicity against the normal human PBMC as
measured by nuclear staining with Hoechst 33258
and by the MTT assay (Figure 2A-B). This
probably is not surprising, as C-3-R is a common
constituent in human diets (5,6). This property is
similar to Resveratrol, a natural product widely
studied for its chemopreventive benefits (4). C-3-
R had some toxicity against proliferating PBMC
stimulated with PHA, based on Hoechst staining
(Figure 2A), although this was not as obvious in
the MTT assay. This toxicity, however, was much
lower than that in the proliferating HL-60 cells
(Figure 1A). We had also observed a similar level
of toxicity of Resveratrol in the proliferating
PBMC using the Hoechst assay (data not shown).
These observations suggest that proliferating
PBMC could be in general more susceptible to
these chemicals.
C-3-R selectively alters the intracellular
level of peroxides in HL-60 cells. Structurally,
C-3-R possesses the phenolic rings typical of the
anthocyanidins (Figure 3A), suggesting that it has
the similar anti-oxidant activity as the other
members of the family. Indeed, when using the
standard in vitro assays, we found that C-3-R
demonstrated concentration-dependent inhibition
of H2O2 production in the Ti (IV)-H2O2 complex
system (Figure 3B). C-3-R could also effectively
inhibit superoxide and peroxyl radicals (ROO.)
production (data not shown). This activity might
be related to its chemo-preventive activity in the
normal cells (8,21,22). Consistently, we have
found that treatment of normal human PBMC with
C-3-R led to reduced intracellular peroxide levels
in the time frame of 15 minutes to 4 hours based
on the use of the cell permeable nonfluorescent
H2DCFDA, which is oxidized to generate the
fluorescent product, DCF, by the peroxides
(Figure 2C). In addition, the increased reduction
of MTT in the C-3-R treated cells could also
suggest an overall less oxidative environment in
these cells (Figure 2B).
In contrast, when the leukemic HL-60 cells
were treated with C-3-R, there was an increased
level of H2O2 accumulated in the cells. In the first
assay, we added H2DCFDA only for the last 15
minutes of the culture at each given time point to
detect the intracellular level of H2O2 at that
particular moment. We found that while similar to
that in the normal PBMC, C-3-R treatment in HL-
60 cells led to the reduction of intracellular
peroxides in the first 15 minutes of the treatment.
The peroxide level began to reverse in 30 minutes
and was significantly above the control level by 1
hour of culture (Figure 3C). The accumulated
level of peroxide was gradually reduced thereafter
to the control level by 4 hours after treatment. To
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examine the overall impact of C-3-R treatment on
the intracellular accumulation of H2O2, we added
the H2DCFDA at the beginning of the culture
through the first 1.5 and 3 hours. This could allow
the level of DCF accumulated as the production of
H2O2 was increased. The result indicated that
despite the initial reduction of H2O2 in the first 15
minutes after treatment, the net level of H2O2 was
increased during the first 3 hours, which was
peaked at the first 1.5 hours (Figure 3D). We
confirmed this result using an enzyme-based assay
that measures the concentration of H2O2 based on
the horseradish peroxidase-mediated oxidation of
phenol red (30) (Figure 3E). In contrast to the
observed change in H2O2 levels, there were no
significant changes in the intracellular superoxide
(O2-) levels using dihydroethidium staining (data
not shown). These results suggested that C-3-R
led to a bi-phasic change in the intracellular redox
status and the net impact is the increase of the
intracellular H2O2 level.
C-3-R-induced apoptosis could be mediated
by ROS, p38MAP kinase and JNK. Based on
the above observations, it was possible that C-3-R-
induced apoptosis could be related to its activity in
enhancing cellular peroxide level. To investigate
this possibility, we first determined whether a pre-
incubation of a non-toxic low dose of H2O2 would
inhibit C-3-R-induced apoptosis. Such treatment
had been well known to induce an adaptive
response from the cells, which leads to cellular
resistance to the subsequent challenge of a larger
dose of H2O2 due to the induction of anti-oxidative
gene expression (31). Indeed, this pre-treatment
4
with 0.5 to 4 µM of H2O2 for a period of 3 hours
dramatically suppressed C-3-R induced apoptosis
in HL-60 cells (Figure 4A). Consistently, C-3-R-
induced apoptosis, caspase activity and
mitochondrial release of apoptogenic factors could
be also suppressed by the general anti-oxidant, N-
acetyl-cysteine (NAC) or the H2O2 scavenger,
catalase (Figure 4B-C). These observations
suggest that C-3-R-induced oxidative stress can
activate the mitochondria apoptosis pathway in
HL-60 cells.
To explore the signaling events upstream the
mitochondria, we examined the re-dox sensitive
stress kinase pathways, which are involved in
apoptosis (32). In untreated HL-60 cells, neither
p38MAP kinase nor JNK was phosphorylated
(Figure 4D). Upon C-3-R treatment, there was a
rapid increase in the phosphorylation of both
p38MAP kinase and JNK, correlating with the
increase of intracellular peroxides levels (Figure
3C-E). Indeed, treatment of cells with the anti-
oxidant, NAC, and the H2O2 scavenger, catalase,
suppressed the activation of p38MAP kinase and
JNK at the early time point (Figure 4E).
While JNK activation seemed to be relatively
transient, p38MAP kinase phosphorylation was
sustained through the time course. However, both
p38MAP kinase inhibitor, SB203580, and JNK
inhibitor, SP600125, could significantly inhibit
mitochondrial release of cytochrome c and Smac,
caspase activation and apoptosis induced by C-3-R
(Figure 4B-C), indicating that both stress kinases
were involved in C-3-R-activated mitochondria
apoptosis pathway following C-3-R-induced
oxidative stress.
Activation of the mitochondria pathway by
C-3-R. To understand how C-3-R-mediated
oxidative stress and stress kinase activated the
mitochondria apoptosis pathway, we examined
activation of the pro-death Bcl-2 family proteins,
which are perhaps the most important molecules
promoting this pathway (33). The multi-domain
pro-death molecules, Bax and Bak, are responsible
for the mitochondrial release of apoptogenic
factors. When activated, they become
oligomerized, which can be detected by
conformation-sensitive antibodies. Indeed, the
mitochondria-localized Bak could be activated by
C-3-R treatment as shown by the immunostaining
with a conformation-sensitive antibody (Ab-1)
(Figure 5A). Immunostaining with a
conformation-sensitive anti-Bax antibody (clone
6A7) could also detect Bax in a distinctive
punctated pattern following C-3-R treatment,
suggesting that the normally cytosol-located Bax
was translocated to the mitochondria and was in an
activated status (Figure 5A). Fractionation
studies confirmed Bax translocation upon C-3-R
treatment (Figure 5B). Interestingly, catalase,
SB203580 or SP600125 had much stronger effects
on Bax conformational change (Figure 5C) than
on Bax translocation (Figure 5B). These data
suggest that ROS and stress kinases mainly
mediate the second step of Bax activation, the
oligomerization.
This activation step of Bax and Bak
(oligomerizaiton and conformation change) was
most likely caused by the BH3-only Bcl-2 family
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proteins (33). Among the possible BH3-only
molecules that could be activated under the current
experimental system, we focused on Bim as it had
been reported to be activated by stress kinases,
mostly via direct phosphorylation of protein (34-
37). We found that while the general level of
BimEL did not change significantly following C-3-
R treatment, the migration patterns of BimEL was
altered notably (Figure 5D). There were about
three species of BimEL that could be detected in
the non-treated HL-60 cells, which represent
different phosphorylation status of BimEL as
previously shown (34-36,38). Treatment of C-3-R
rapidly induced the increase of the slowest
migrating species, which remained for at least
several hours (Figure 5D). When the inhibitors of
the stress kinases were added to the culture, this
up-shift was significantly suppressed (Figure 5E).
These results thus indicate that increased BimEL
phosphorylation following C-3-R treatment was
correlated with the activation of p38MAP kinase
and JNK, which could contribute to its pro-
apoptotic activity as shown in earlier studies (34-
37).
Since suppression of Bim expression using the
RNAi-based approach in HL60 cells was not
successful, we used another leukemic cell line,
CCRF-CEM, for which a subline stably expressing
a shRNA against Bim had been established and
characterized (25). This cell line was susceptible
to C-3-R-mediated apoptosis in a dose-dependent
manner (Figure 1F), which could be further
suppressed by p38MAP kinase inhibitor,
SB203580 and the anti-oxidant, NAC and catalase,
5
to the same extent as observed in HL-60 cells
(Figure 5F), suggesting that a conserved pathway
is activated. Bim expression was considerably
reduced in the CCRF-CEM subline stably
expressing a shRNA against Bim and indeed this
subline was significantly resistant to C-3-R-
indcued apoptosis, compared to the subline
expressing the shRNA vector only (Figure 5F).
Furthermore, no further reduction of apoptosis was
seen in the Bim knocked-down cells upon
treatment of SB203580, NAC or catalase,
supporting the notion that Bim is the downstream
target of ROS and p38 MAP kinase.
Conversely, HL-60 cells that over-expressed
the anti-death Bcl-2 or Bcl-xL (Figure 6A) were
significantly resistant to C-3-R induced apoptosis
and caspase activation at all tested doses (Figure
6B-C). These results indicate that C-3-R-induced
HL-60 apoptosis is mainly mediated by the
mitochondria pathway, which is regulated by the
Bcl-2 family proteins.
Discussion
Anthocyanins, such as C-3-R, have broad
biological effects in anti-mutagenesis and anti-
carcinogenesis and have been hailed as useful
natural agents for chemoprevention (8,18,21,22).
These activities are generally attributed to their
anti-oxidant properties, as determined in a variety
of in vitro assays. While we have confirmed that
C-3-R, purified from black raspberries, does
possess the anti-oxidant activity in these in vitro
assays, we find that it actually causes ROS
accumulation in the leukemic cells and that the
increased oxidative stress is responsible for its
pro-apoptotic activity.
It is not yet clear as to how C-3-R promotes
oxidative stress in the leukemic cells. We have
observed the increase of peroxides, but not
superoxides, in the treated cells. The surge of
ROS can be cumulative and is sufficient to
activate the downstream apoptotic events. It is
likely that treatment with C-3-R could inactivate
the glutathione anti-oxidant system, which is
involved in scavenging of the peroxides. This
assumption is supported by the protective effects
of NAC observed in this study, which could regain
the glutathione levels in C-3-R-treated cells.
C-3-R, on the other hand, did not induce
increased levels of peroxide in the normal PBMC,
where the intracellular H2O2 levels actually
remained lower following the treatment,
suggesting that C-3-R could have a radical-
scavenging activity in these cells. It is not quite
clear as to why C-3-R has different impact on the
redox status in the transformed leukemic cells
versus the normal PBMC. But this could be
related to the notion that transformed cells and
normal cells may have different resting levels of
ROS and anti-oxidant capacity (39,40).
It may be interesting to note that several other
natural products, such as β-phenylethyl
isothiocyanate (PEITC), a natural compound
found in consumable cruiferous vegetables (41),
which are known to be anti-oxidants in the in vitro
assays, demonstrate pro-oxidant activities in the in
vivo studies which are responsible for their
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apoptotic effects in tumor cells. Notably, PEITC
also shows a relatively higher selection toward the
tumor cells for its killing effects and ROS
generation (41). These observations indicate that
the dual effects on the redox status could be a
shared feature in some biologically active natural
products that are being evaluated for their
chemoprevention and/or chemotherapeutic
activities.
The intracellular redox environment could be
determined by the relative rate of production and
removal of ROS. Changes in ROS level or the
intracellular redox environment can significantly
modulate physiologic and pathologic processes. It
has been generally postulated that tumor cells
produce more ROS than normal cells, which is
connected with increased proliferation and
tumorigenesis (39,40). Oxygen radicals such as
superoxide and hydrogen peroxide can act as
signaling molecules directly. Alternatively, a
change in redox environment can affect signaling
cascades and redox sensitive transcription factors
involved in proliferation, survival, and hormonal
response.
However, excessive ROS production can lead
to cellular damage and induce cell death (40).
Because of the pro-oxidant status of the cancer
cells, they seem to be more susceptible than
normal cells to treatment with agents that cause
oxidative stress, as epitomized by the study using
PEITC (41). Our findings that C-3-R, also a
natural product commonly found in the fruits and
vegetables, selectively induces the accumulation
of peroxides in the leukemic cell line, HL-60, but
6
not in the normal PBMC, point to the same
direction and suggest that this approach is valid in
seeking new generation of therapeutic agents with
lower side effects, considering that many
anticancer drugs currently used in the clinic have
strong cytotoxicity toward normal cells.
Not all tumor cells may be subjected to such a
selective killing. While we have observed that
several leukemia/lymphoma cell lines (HL-60,
MOLT4, Daudi and CCRF-CEM) are sensitive to
C-3-R, we have also found that a lung
adenocarcinoma cell line A549 and a
hepatocellular carcinoma cell line SMMC-7721
are resistant to the treatment of C-3-R (data not
shown). It is possible that the redox status of these
cells is different from that of leukemic cells like
HL-60. Alternatively C-3-R may not be
sufficiently potent in changing the re-dox balance
to allow the development of oxidative stress in
some cancer cells. These hypotheses could be
tested in future experiments.
ROS can cause cell death via a variety of
mechanisms, among which the activation of stress
kinases plays an important role (32). Our studies
indicate the activation of p38MAP kinase and JNK
by C-3-R in a ROS-dependent manner in the HL-
60 and CCRF-CEM cells. The exact mechanisms
by which ROS activate the MAPK pathways are
not fully clear in many cases and could depend on
the types of cells and the stimulus. ROS could
activate the upstream ASK1 (32), or inactivate the
MAP kinase phosphatase (42), leading to
sustained phosphorylation and activation of
p38MAP kinase or JNK. Future work should be
directed to explore these possibilities.
Both ROS and JNK/p38MAP kinase-initiated
apoptosis have been closely linked to the intrinsic
pathway characterized by the mitochondrial
release of apoptogenic factors, such as cytochrome
c and Smac (32). Mitochondrial activation is most
critically regulated by the Bcl-2 family proteins.
While the anti-death molecules, such as Bcl-2 and
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Bcl-xL, inhibit mitochondria activation, the pro-
death molecules promote it (33). Our findings
indicate that C-3-R induced p38MAP kinase and
JNK activation promotes the activation of BH3-
only molecule, BimEL, and Bax and Bak
conformation change. Earlier studies showed that
Bim could be sequestered in the cytosol by its
binding with the dynein light chain-1, and is
released from the microtubules following
phosphorylation in response to apoptotic stimuli
(35,38). It binds and antagonizes the anti-death
Bcl-2 or Bcl-xL, thus activating Bax or Bak (43).
BimEL could be phosphorylated at Ser65 by JNK
(36) or p38MAP kinase (37) in response to growth
factor deprivation, UV stimulation or sodium
arsenate, agents that all can readily induce
oxidative stress. p38MAP kinase could also
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transcriptionally up-regulate Bim in leukemic cells
following glucocorticoids treatment (25). Our
finding that C-3-R could promote p38MAP kinase
and JNK-mediated BimEL phosphorylation is
consistent with these studies. Furthermore,
inhibiting Bim expression led to the suppression of
C-3-R-induced apoptosis to the same extent as the
p38MAP kinase inhibitor and the anti-oxidant,
supporting the role of Bim in C-3-R-induced
apoptosis. It has to be pointed, however, that this
pathway is likely not the only pro-death
mechanism activated by C-3-R as suppressing
ROS, stress kinases or Bim could block C-3-R-
induced apoptosis significantly, but not completely.
In conclusion, the selective toxicity of C-3-R
in the leukemic cells is contributed significantly
by its paradoxical pro-oxidant effects in the cancer
cells, which activate the stress kinases and
subsequently the pro-death Bcl-2 family proteins
and the mitochondria apoptotic pathway. This
property of C-3-R could be further explored for
the development of anti-tumor agents with a
higher therapeutic index.
7
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Footnotes

* We would like to thank Dr. Kapil N. Bhalla (Medical College of Georgia) for the HL-60/neo, HL-
60/Bcl-2 and HL-60/Bcl-xL cell lines, and Drs. Wen-Xing Ding and Wentao Gao for advice in certain
experimental procedures. Ms. Lu Feng (Mt. Lebanon Senior High School) partially took part in the
experiment as a summer student. X.-M. Yin is in part supported by NIH grants (CA83817, CA111456
and NS045252).

1
The abbreviation used are: C-3-R: Cyanidin-3-rutinoside; CAT: catalase; DCF: 2’,7’-
dichlorofluorescein; H2DCFDA: 2’,7’-dichlorodihydrofluorescein diacetate; NAC: N-acetyl-cysteine;
PBMC: peripheral blood mononuclear cells; PEITC: β-phenylethyl isothiocyanate; PI: propidium iodide.
STS: staurosporine.

Figures Legends

Figure 1. C-3-R induces apoptosis in HL-60 cells in a dose- and time-dependent way. (A-C). HL-60
cells were incubated with C-3-R at difference dose for 18 hours (A, B) or at 60 µM for indicated hours
(A, C). Cell death was analyzed by Hoechst 33258 and PI staining. Apoptotic and necrotic cell death
were defined in the Experimental Procedure Section. Caspase-3 and caspase-9 activities were measured
using 20 µM of Ac-DEVD-AFC or Ac-LEHD-AFC, respectively (B-C). (D) HL-60 cells were exposed
to C-3-R (60 µM) for 18h in the absence or presence of the pan-caspase inhibitor z-VAD (50 µM).
Percentage of apoptotic cells (upper panel) or caspase-3 activity (lower panel) was measured as above.
Data (mean±SD) represent at least three independent experiments. (E) HL-60 cells were treated with 80

9
µM of C-3-R for the indicated times. Both total cell lysates and cytosolic fractions were prepared and
subjected to immunoblot analysis using antibodies against cytochrome c and β-actin. (F). MOLT-4,
Daudi and CCRF-CEM cells were treated with C-3-R for 18h at the indicated concentrations and then
stained with Hoechst 33258. Cells with the typical apoptotic nuclear changes were quantified (mean ±
SD).

Figure 2. Normal human peripheral blood mononuclear cells are resistant to C-3-R-induced
apoptosis and redox stress. (A-B). Normal resting human PBMC or PMBC stimulated with PHA (10
µg/ml) for 48 hours were incubated with the designated chemicals for 18 hours. C-3-R was used at 80
µM and STS (staurosporine) was used at 1.0 µM or as indicated. Percentage of apoptotic cells was
determined using Hoechst 33258 staining (A). Alternatively, cells were subjected to MTT assay (B).
(C). Normal resting PBMC were incubated with C-3-R (80 µM) or vehicle control for the designated time
point. H2DCFDA (2 µM) was added 15 minutes before the harvest and cells were then analyzed by flow
cytometry. Light line: control untreated samples; Dark line: C-3-R-treated samples.

Figure 3. C-3-R treatment perturbs ROS production in HL-60 cells. (A) The polyphenol structure of
C-3-R. (B) C-3-R suppressed hydrogen peroxide formation in the in vitro H2O2-Ti (IV) system.

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Titanium reagent was added to 200 µl of C-3-R of various concentrations in the presence of NH4OH and
H2O2. The absorbance of the supernatant from the Ti- H2O2 complex was measured at 410 nm and the
inhibition of H2O2 production by C-3-R was determined against blank control. (C). HL-60 cells were
treated with or without C-3-R for the indicated time. H2DCFDA (2 µM) was added in the last 15 minutes
of the culture before cells were harvested for flow cytometry analysis. Light line: control untreated
samples; Dark line: C-3-R treated samples. (D) HL-60 cells were treated with C-3-R (80 µM) for 0, 1.5
or 3 hours and then harvested for flow cytometry analysis. H2DCFDA was added at the beginning of the
culture. (E) HL-60 cells were incubated with C-3-R at the designated dose for 1 hour. The intracellular
H2O2 levels were determined by the red-phenol method as described in Materials and Methods.

Figure 4. C-3-R-induced apoptosis and mitochondria activation can be mediated by ROS via p38
MAPK and JNK. (A). HL-60 cells were pretreated with H2O2 for 3 hours and then cultured in fresh
medium without H2O2, but treated with C-3-R or vehicle control for 18 hours. Apoptotic cells were
determined by Hoechst 33258 staining. (B) HL-60 cells were pretreated with N-acetyl-cysteine (NAC, 5
mM), catalase (CAT, 500 U/ml), SB203580 (SB, 10 µM) or SP600125 (SP, 1 µM) for 1 hour, followed
by incubation with C-3-R (80 µM) for another 18 hours. Apoptosis was determined by Hoechst 332598
staining (upper panel) and capase-3 activities in the total cell lysates were measured using Ac-DEVD-
AFC as the substrate (lower panel). (C) HL-60 cells were pretreated with various reagents as in B,
followed by incubation with C-3-R (80 µM) for another 8 hours. Subcellular fractionation was performed
and the cytosolic fraction was subjected to immunoblot analysis with the indicated antibodies. (D). HL-
60 cells were treated with 80 µM of C-3-R for various times and subjected to immunoblot analysis using
antibodies against total and phosphorylated p38MAP kinase or JNK. (E). HL-60 cells were pretreated
with vehicle control, NAC (5 mM) or CAT (500 U/ml) for 1 hour, followed by incubation with C-3-R (80
µM) for another 2 hours. Immunoblot analysis was carried out as in E.

Figure 5. C-3-R activates the pro-death Bcl-2 family proteins via ROS, p38MAP kinase and JNK.
(A) HL-60 cells were treated with C-3-R (80 µM) for 8 hours and then immunostained with an anti-Bax
(6A7) or anti-Bak (Ab-1) to detect conformationally changed Bax or Bak. (B-C) HL-60 cells were pre-
treated with catalase (CAT, 500 U/ml), SB203580 (SB, 10 µM), or SP600125 (SP, 2 µM) for 1 hour,
followed by incubation with C-3-R (80 µM) for another 8 hours. The mitochondria-enriched heavy
membrane fraction was analyzed by immunoblot assay with the anti-Bax (NT) and anti-COX IV subunit
II antibodies (B). The latter serves as the loading control for the mitochondria fraction. Alternatively,
percentage of HL-60 cells showing positive 6A7 staining in the punctated pattern was quantified (C). (D)

10
HL-60 cells were treated with C-3-R (80 µM) for the designated time point and subjected to immunoblot
analysis using an anti-Bim antibody. (E) HL-60 cells were treated as in B. Total cell lysates were
prepared and analyzed by immunoblot using the anti-Bim and anti-β-actin antibodies. (F). CCRF-CEM
cells expressing the shRNA-vector or expressing the specific shRNA against Bim were treated with
vehicle control (white column), C-3-R (80 µM) in the absence (black column) or presence of SB203580
(10 µM, gray column), NAC (5 mM, horizontal line column) or CAT (500 U/ml, vertical line column) for
24 hours. Percentages of apoptotic cells were determined following Hoechst staining. The insert shows
the expression of BimEL in the two cell lines used.

Figure 6. Bcl-2 or Bcl-xL can protect HL-60 cells from apoptosis induced by C-3-R. (A) HL-60 cell
lines stably expressing the mammalian expression vector (neo) or the human Bcl-xL or Bcl-2 were
analyzed by immunoblot assay for Bcl-xL (left panel) or Bcl-2 (right panel). (B-C) HL-60 stable cell lines
expressing the vector, Bcl-xL or Bcl-2 were treated with C-3-R (0-80 µM) for 18 hours. The percentage of
cells with apoptotic nuclei as determined by Hoechst 33258 staining were quantified (B). Alternatively,
cell lysates were prepared and analyzed for the activities of caspase-3 (C, upper panel) and -9 activities
(C, lower panel).

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11
 Figure 1

A B C
100 Apoptosis
40,000 40,000
Necrosis
Cell Death (%)

80

Caspase-3 Activity
Caspase-3 Activity
60 30,000 30,000

40
20,000 20,000
20
10,000 10,000
0
0 10 20 40 60 80 100 120 160
0 0
C-3-R (µM)
15,000 15000
100
Caspase-9 Activity
Apoptosis

Caspase-9 Activity
Cell Death (%)

80
Necrosis 10,000 10000
60

40 5,000 5000

20

0 0 0

0 8 16 24 48 0 20 40 60 80 0 8 16 24 32
Time (Hr) C-3-R (µM) Time (Hrs)

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 Figure 1

D E
80
0 6 8 16 Time (Hrs)

60
Apoptotic Cells %)

Cytosol Cytochrome c

β-Actin
40
Total Cytochrome c

20
F
0 70
30,000
60

Apoptotic Cells (%)

Caspase-3 Activity

50
20,000
40
30
10,000 20
10
0 0
Control C-3-R C-3-R C-3-R (µM): 0 20 40 80 0 20 40 80 0 20 40 80
+ z-VAD
MOLT-4 Daudi CCRF-CEM

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 C Figure 2
A 15 min
100 C-3-R Control
Apoptotic Cells (%)
80

60
30 min
40
C-3-R Control

20

0
0 40 80 40 80 1.0 0 80 1.0 (µM)
C-3-R Resveratrol STS C-3-R STS 1 hr
- + PHA C-3-R Control
B
0.6
0.5
2 hr
0.4 C-3-R Control
OD 570 (nm)

0.3
0.2
0.1
4 hr
0 C-3-R Control
Control C-3R STS Control C-3-R STS
- + PHA

DCF Fluorescence Intensity

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 C 15 min Figure 3
C-3-R Control

A D

Control
30 min
Control

Events
C-3-R
3 Hrs 1.5 Hrs

Rutinoside 1 hr
Control
C-3-R
DCF Fluorescence Intensity
B E
Inhibition of H2O2 Formation (%)

30
25 3
2 hr
20

H2O2 (µM)
Control
C-3-R 2
15
10
1
5
0 4 hr 0
Control
10 20 40 60 80 100 0 20 40 80 160
C-3-R (µM) C-3-R C-3-R (µM)

DCF Fluorescence Intensity

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A 100
Figure 4

Apoptotic Cells (%)

80
C
- + C-3-R
60
- - SB SP CAT NAC
40 Cytochrome c

20 Smac

0 β-Actin
H2O2 (µM): 0 0 0.5 1 2 4
C-3-R: - +
B 100
D
0 0.5 1 2 4 8 16 Time (Hr)
Apoptotic Cells (%)

80 Phospho-p38

Total p38
60
Phospho-JNKs
40
Total JNKs
20

0
50,000
E
- + C-3-R
Caspase-3 Activity

40,000
- - NAC CAT
30,000 Phospho-p38

Total p38
20,000
Phospho-JNKs
10,000
Total JNKs
0
C-3-R: - +
- - SP SB NAC CAT

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 Figure 5
A Control C-3-R
B
- + C-3-R
- - CAT SB SP
Bax (6A7)

Bax

COX-IV

D
Bak (ab-1)

0 1 2 4 8 (Hrs.)

BimEL

β-actin

C
30 E
- + C-3-R
6A7+ Cells (%)

20 - - SB SP

BimEL
10

β-actin
0
- - SP SB CAT
C-3-R: - +

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F Figure 5

80
Control
70 shRNA
C-3-R
Vector Bim
C-3-R+SB
60 BimEL

Apoptotic Cells (%)

C-3-R+NAC
C-3-R+CAT
50

40

30

20

10

0
shRNA-Vector shRNA-Bim

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 Figure 6

A C 30,000

Cell Line: Neo Bcl-xL Neo Bcl-2 25,000

Relative Caspase-3 Activity

20,000

Bcl-xL Bcl-2 15,000

B 10,000

5,000

80
0
Apoptotic Cells (%)

8,000
60
7,000

Relative Caspase-9 Activity

6,000
40
5,000

20 4,000
3,000
0 2,000
C-3-R (µM): 0 20 40 80 0 20 40 80 0 20 40 80
Cell lines: HL-60-Neo HL-60-Bcl-XL HL-60-Bcl-2 1,000
0
C-3-R (µM): 0 40 80 0 40 80 0 40 80
Cell lines: HL-60-Neo HL-60-Bcl-xL HL-60-Bcl-2

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 Cyanidin-3-rutinoside, a natural polyphenol antioxidant, selectively kills leukemic
cells by induction of oxidative stress
Rentian Feng, Hong-Min Ni, Shiow Y. Wang, Irina L. Tourkova, Michael R. Shulin,
Hisashi Harada and Xiao-Ming Yin
J. Biol. Chem. published online March 14, 2007

Access the most updated version of this article at doi: 10.1074/jbc.M610616200

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