Cancer Letters 215 (2004) 43–52
www.elsevier.com/locate/canlet
Nitric oxide is synthesized in acute leukemia cells after exposure to
phenolic antioxidants and initially protects against mitochondrial
membrane depolarization
Christian Kellner, Susan J. Zunino*
Chair of Genetics, Friedrich-Alexander University of Erlangen-Nürnberg, D91058 Erlangen, Germany
Received 4 February 2004; received in revised form 8 June 2004; accepted 11 June 2004
Abstract
We investigated the early events involved in loss of mitochondrial membrane potential (DJmt) leading to apoptosis in cells
derived from patients with acute lymphocytic leukemia after exposure to phenolic antioxidants. Using the nitric oxide binding
dye diaminofluorescein-FM diacetate, we found that intracellular nitric oxide (NO) levels increased significantly within 4 h
after exposure to the antioxidants curcumin, carnosol, and quercetin. Inhibition of nitric oxide synthetase (NOS) activity with
mercaptoethylguanidine increased the percentage of leukemia cells with depolarized mitochondria membranes after antioxidant
treatment. These data suggest that NO production in the leukemia-derived cells may be a protective response to maintain DJmt
after antioxidant exposure and inhibition of NOS increases the disruption of mitochondrial homeostasis induced by the
antioxidants.
q 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Leukemia; Phenolic antioxidants; Apoptosis; Mitochondria; Nitric oxide
1. Introduction
Plant-derived phenolic antioxidants have received a
great deal of attention in recent years for their anticancer activities. Curcumin, found in the spice turmeric,
carnosol, found in the herb rosemary, and the flavonoid
quercetin, found in many fruits and vegetables have
* Corresponding author. Address: USDA Western Human Nutrition Research Center and Department of Nutrition, 3202A Meyer
Hall, One Shields Ave., University of California, Davis, CA 95616,
USA. Tel.: C1-530-752-5156; fax: C1-530-752-5271.
E-mail address: [email protected] (S.J. Zunino).
displayed anti-carcinogenic properties in vitro and in
vivo. Both curcumin and carnosol have been effective
in suppressing tumor initiation by benzo[a]pyrene and
7,12-dimethylbenz[a]anthracene and inhibiting tumor
promotion induced by phorbol ester [1–4]. Carnosol
has also been shown to inhibit breast cancer in vivo
and the growth of leukemia-derived cells in vitro [3,5].
Curcumin has shown effective suppression of stomach,
colon, and breast cancers, whereas quercetin inhibited
growth of cervical cancer, melanoma, and intestinal
tumors in mouse models [6–10]. Both curcumin and
quercetin have been investigated in phase I clinical
trials to evaluate toxicity and pharmacokinetic
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2004.06.046
44
C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
properties and potential efficacy as anti-cancer agents
[11,12].
We have previously shown that phenolic antioxidants induce depolarization of mitochondrial membranes, which then leads to activation of apoptosis in
leukemia-derived cell lines, including acute lymphocytic leukemia (ALL) lines with chromosomal translocation t(4;11) [5,13]. The t(4;11) ALLs are a
high-risk subgroup of leukemia found in 60% of
infants with ALL and these cells are particularly
resistant to conventional chemotherapy, necessitating
the evaluation of alternative treatment strategies [14].
Little is known about the early events induced by
antioxidant chemicals that lead to the loss of DJmt. In
the current study, changes initiated by the phenolic
antioxidants curcumin, carnosol, and quercetin that
may be responsible for the disruption of mitochondrial
homeostasis were analyzed. Using cell lines derived
from patients with t(4;11) pro-B ALL, as well as pre-B
and T-ALL, we discovered that synthesis of the free
radical nitric oxide (NO) was stimulated within 4 h
after exposure to the antioxidants. Interestingly,
inhibition of nitric oxide synthetase (NOS) activity
increased the depolarization of mitochondrial membranes observed after antioxidant treatment,
suggesting increased NO production may be part of a
protective mechanism against mitochondrial depolarization in these cells. Although exposure of the acute
leukemia-derived cells to phenolic antioxidants does
eventually induce loss of mitochondrial homeostasis
and apoptosis, these data suggest that NO production
may play an important early role in the resistance of the
leukemia cells to environmental insult.
2. Materials and methods
2.1. Cell culture and antioxidant treatments
SEM and RS4;11 lines were established from
patients diagnosed with pro-B ALL containing the
chromosomal translocation t(4;11)(q21;q23) [15,16].
The presence of the t(4;11) translocation has been
confirmed in these cell lines by polymerase chain
reaction (PCR) as previously described [17]. The two
pro-B ALL lines, the pre-B leukemia line REH, and
the T-cell ALL line CEM were maintained at 37 8C,
5% CO2 in RPMI medium (Invitrogen, Karlsruhe,
Germany) supplemented with 10% fetal bovine
serum, 100 IU/ml penicillin, 100 mg/ml streptomycin,
50 mg/ml gentamycin, 1 mM sodium pyruvate, and
2 mM L-glutamine (Invitrogen).
Curcumin and quercetin were purchased from
Calbiochem (Schwalbach, Germany) and carnosol
was purchased from Alexis Corp. (Gruenberg,
Germany). Quercetin and carnosol were dissolved in
dimethylsulfoxide (DMSO, Sigma Chemical Co.,
Deisenhofen, Germany) and curcumin was dissolved
in ethanol before use. Cells were split to a density of
0.5!106/ml for treatment with antioxidants. Equivalent amounts of diluent were always added to control
cell populations in each experiment (designated
untreated cells).
2.2. Analysis of mitochondrial membrane potential
Fluorescence analysis was performed on a FACSCalibur fluorescence-activated cell sorter (FACS)
using CELLQuest software (Becton-Dickinson, BD,
Mountain View, CA, USA). JC-1 dye (5,5 0 ,6,6 0 -tetratetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide, Molecular Probes, Leiden, The
Netherlands) was used to measure changes in
mitochondrial membrane potential as previously
described [5,13]. All analyses of whole cells were
performed using appropriate scatter gates to exclude
cellular debris and aggregated cells. Ten thousand
events were collected for each sample.
2.3. Analysis of nitric oxide synthesis
To measure changes in nitric oxide levels, the cells
were incubated with the cell permeable dye DAF-FM
DA (4-amino-5-methylamino-2 0 , 7 0 -difluorofluorescein diacetate, Molecular Probes) for 30 min at
37 8C and at a final concentration of 1 mM prior to
the addition of antioxidants. Cells were then exposed
to the antioxidants for the indicated time, washed 1x
with PBS (Ca2C/Mg2C-free, Invitrogen), and analyzed by FACS. For experiments requiring inhibition
of NOS activity, the cells were pre-incubated for
30 min at 37 8C with both mercaptoethylguanidine
(MEG, Calbiochem) and DAF-FM DA before
addition of the antioxidants. The final concentrations
of MEG were 1 mM for curcumin treatments and
0.5 mM for carnosol and quercetin treatments.
C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
2.4. Cell fractionation and western blot analysis
For analysis of protein expression levels of NOS
isoforms after antioxidant treatment, protein lysates
were prepared by vortexing 1!108 cells per ml of
lysis buffer containing 1% Triton X-100 (Sigma),
50 mM Tris–Cl (pH 8), 150 mM NaCl, and the
following proteinase inhibitors (Roche Diagnostics,
Mannheim, Germany): aprotinin, leupeptin, chymostatin, and EDTA. Protein concentrations were determined using the Bio-Rad DC protein assay (Munich,
Germany). Equal amounts of protein were electrophoresed on 6% SDS-polyacrylamide gels under
reducing conditions and electroblotted on to PVDF
membrane (Amersham Pharmacia). Blocking and
staining of membranes were carried out using 1!
PBS containing 2% nonfat dry milk and 0.1% Tween
20 (Sigma). Rabbit polyclonal antibodies against
neuronal NOS (nNOS, K-20), inducible NOS
(iNOS, N-20), and endothelial cell NOS (ecNOS,
N-20) and also blocking peptides were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA,
USA) and used according to the manufacturer’s
recommendations. Positive controls for each NOS
isoform were purchased from BD-Transduction Labs
(Heidelberg, Germany): rat cerebellum lysate (nNOS
control), LPS/IFN-g-treated mouse macrophage
lysate (iNOS control), and endothelial cell lysate
(ecNOS control). HRP-conjugated goat anti-rabbit
IgG (BD-Transduction Labs, Heidelberg, Germany)
was used as secondary antibody at a dilution of
1:1000. The ECL Western blotting system (Amersham Pharmacia) was used for visualization of
immunoreactive bands at manufacturer suggested
concentrations.
Crude membrane and cytosolic fractions were
generated from the leukemia cells using the ApoAlert
Cell Fractionation Kit (BD-Clonetech, Heidelberg,
Germany). Cells were dounced in the fractionation
buffer using mini-dounce glassware (Carl Roth) and
passed through a 25 gauge syringe needle. Fractions
were then obtained following the manufacturer’s
recommendations. Protein concentrations were determined as described above and 10 mg of protein from
each fraction were electrophoresed on 6 and 15%
SDS-PAGE gels. Blots from the 6% gels were stained
with the anti-iNOS antibody and blots from the 15%
gels were stained with anti-COX4 provided in
45
the ApoAlert kit to estimate the purity of the
membrane and cytosolic fractions.
2.5. Statistical analysis
All statistical analyses were performed with
GraphPad software (GraphPad Software, Inc., San
Diego, CA, USA) and data were displayed as
arithmetic meansGSE. P values were obtained
using two-tailed paired t-tests with a confidence
interval of 95% for evaluation of the significance of
differences between treatment groups.
3. Results
3.1. Depolarization of mitochondrial membranes
is an early response to antioxidant treatment
in ALL-derived cell lines
We have previously shown that significant
depolarization of mitochondrial membranes leading
to apoptosis was induced after 24 h or longer
treatment with the antioxidant carnosol in several
ALL lines [5]. However, the early events involved
in the disruption of mitochondrial homeostasis by
phenolic antioxidants in these leukemia cells has
not been elucidated. To determine the time course
of action for carnosol and also the phenolics
curcumin and quercetin, early changes in DJmt
were monitored using the mitochondria-selective
dye JC-1 [5,13,18]. The pro-B t(4;11) lines SEM,
RS4;11, the pre-B ALL line REH, and the T-ALL
line CEM were treated with 10 mM curcumin, 6 mg/
ml (18 mM) carnosol, or 2.5 mM quercetin and then
stained with JC-1. By 4 h after antioxidant treatment, a clear increase in the percentage of cells
that emitted only green fluorescence was observed,
representing cells with depolarized mitochondrial
membranes (Fig. 1, lower right quadrants). All
three of these antioxidants were able to kill the
ALL lines after extended exposure (data not
shown). These data indicated that curcumin,
carnosol, and quercetin act early in the leukemiaderived cells by a common mechanism involving
the disruption of mitochondrial membrane potential
(DJmt).
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C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
Fig. 1. Curcumin, carnosol, and quercetin induce loss of DJmt in the leukemia-derived cell lines. The following cells were untreated or treated
with 10 mM curcumin, 6 mg/ml (18 mM) carnosol, or 2.5 mM quercetin for 4 h at 37 8C and then stained with the mitochondria-selective dye JC1: (A) SEM cells, (B) RS4;11 cells, (C) REH cells, and (D) CEM cells. Cells with normal polarized mitochondrial membranes emit greenorange fluorescence (top right quadrant). The number in the bottom right quadrant of each dot plot represents the percentage of cells that emit
only green fluorescence due to depolarized mitochondrial membranes. Dot plot profiles for DMSO and ethanol treated cells (Untreated) were
similar. The data are representative of at least three separate experiments.
3.2. Nitric oxide is produced in the ALL-derived cells
in response to antioxidant treatment
Nitric oxide has been shown to play a key role in
regulating the electron transport chain due to its
ability to reversibly inhibit cytochrome c oxidase, part
of the terminal electron acceptor complex IV [19,20].
However, overproduction of NO has been linked to
prolonged inhibition of electron transport resulting in
increased electron leakage and generation of reactive
oxygen and nitrogen species, which can lead to
disruption of DJmt [21]. Due to the pivotal role of NO
C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
in maintaining mitochondrial homeostasis, we hypothesized that treatment of the leukemia-derived cells with
the phenolic antioxidants induced changes in the
levels of NO in the leukemia-derived cells, resulting
in loss of DJmt. To determine if NO levels were
altered after treatment with curcumin, carnosol, and
quercetin, the cells were incubated with the cellpermeable, NO-binding dye DAF-FM diacetate
(DAF-FM DA). DAF-FM DA is essentially nonfluorescent until it binds to NO and forms a strongly
fluorescent benzotriazole derivative [22]. By 4 h after
antioxidant treatment, an increase in the fluorescent
signal was observed, indicating that NO was
synthesized in response to the treatment with
all three antioxidants (Fig. 2, black histogram).
The untreated leukemia cells labeled with DAF-FM
DA (gray) also showed detectable fluorescent
signals compared to the unlabeled controls (white),
indicating that NO was constitutively present in
detectable levels in all four leukemia-derived lines.
47
The increases in fluorescence of the DAF-FM DA
induced by antioxidant treatment were statistically
significant for all cell lines and treatment groups
when compared to the untreated, labeled cells (P!
0.05). The strongest increase in NO synthesis was
observed in the populations of cells treated with
curcumin, which ranged from approximately 3- to 5fold increases compared to untreated cells.
It has been reported that the diaminofluorescein
(DAF) moiety can bind to Ca2C as well as NO [23].
To ensure that the fluorescent signals observed in the
FACS analyses represented binding of DAF-FM to
NO, the cells were co-incubated with mercaptoethylguanidine (MEG), an inhibitor of NOS [24], plus
DAF-FM DA for 30 min, and subsequently treated
with antioxidants. Indeed, MEG was able to reduce
the fluorescent signals of the DAF-FM DA dye in both
the untreated cells and the antioxidant-treated cells
(Fig. 3). These data indicate that DAF-FM DA was
binding to NO. Furthermore, these results confirm that
Fig. 2. Nitric oxide (NO) levels are increased in the ALL lines in response to antioxidant treatment. The leukemia cells were incubated with
1 mM of the NO-binding dye DAF-FM DA and then left untreated or treated with the antioxidants for 4 h at 37 8C. One group of cells was left
untreated by both DAF-FM DA and the antioxidants and used as the baseline fluorescence control (white histogram). The treatments were as
follows: (A) 10 mM curcumin, (B) 6 mg/ml carnosol, (C) 2.5 mM quercetin. After 4 h, cells were washed and analyzed by FACS. Untreated cells
pre-incubated with the DAF-FM DA are represented by the gray histograms, which were similar for both DMSO and ethanol treatments.
Antioxidant-treated cells pre-incubated with the DAF-FM DA are represented by the black histograms. The data is representative of at least
three separate experiments.
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C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
Fig. 3. DAF-FM DA fluorescence is inhibitable by the NOS
inhibitor mercaptoethylguanidine (MEG). Cells were pre-incubated
with MEG for 30 min at 37 8C in the presence or absence of DAFFM DA: (A) SEM cells, (B) RS4;11 cells, (C) REH cells, and (D)
CEM cells. Cells treated with curcumin were pre-incubated with
1 mM MEG, whereas cells treated with carnosol and quercetin were
pre-incubated with 0.5 mM MEG. One group of cells was left
untreated by DAF-FM DA and served as the baseline fluorescence
control (white bar). Antioxidant treatments were: 10 mM curcumin,
6 mg/ml carnosol, and 2.5 mM quercetin. Cells were pre-incubated
with DAF-FM DA and left untreated (diagonal lined bar) or treated
with both DAF-FM DA and antioxidant (black bar). Cells were
washed after 4 h antioxidant treatment and analyzed by FACS. The
data represent the arithmetic meanGSE of three separate
experiments. MFI, mean fluorescence intensity.
untreated leukemia cells have detectable levels of NO
already present and that in response to antioxidant
treatment, the leukemia cells increased their production of NO above basal levels.
3.3. Inhibition of NOS enhances mitochondrial
membrane depolarization induced by antioxidant
treatment
We had hypothesized that overproduction of NO
may be partly responsible for the early depolarization
of mitochondria membranes in these leukemiaderived cells after antioxidant exposure. Consequently, if NO production was inhibited, the cells
should maintain proper mitochondrial membrane
integrity. To test this hypothesis, the cells were preincubated with MEG, exposed to the antioxidants for
4 h, and then stained with JC-1. Unexpectedly,
inhibition of NOS by MEG enhanced rather than
suppressed the depolarization of mitochondrial membranes that was induced by treatment with curcumin,
quercetin, and carnosol in all four cell lines (Fig. 4).
After 4 h of antioxidant treatment, there was a
statistically significant increase in the percentage of
cells with depolarized mitochondrial membranes that
were treated with MEG plus antioxidants compared to
cells treated with the antioxidants alone for all cell
lines (P!0.05). In these experiments, REH and CEM
cells were treated with only 1 mM of quercetin due to
the high degree of depolarization observed with
2.5 mM quercetin (Fig. 1). These data suggested that
increased production of nitric oxide actually may be
an early protective mechanism against disruption of
DJmt after exposure of these leukemia-derived cells
to the phenolic antioxidants.
3.4. Differential expression of iNOS in the ALL lines
Several isoforms of NOS have been identified
including neuronal NOS (nNOS, type 1, bNOS),
inducible NOS (iNOS, type 2), and endothelial cell
NOS (ecNOS, type 3) [25] Western blot analysis was
used to identify the NOS isoform present in the ALL
lines. The nNOS and ecNOS proteins were not
observed in the ALL lines. However, the anti-iNOS
antibody detected two distinct molecular species in all
four lines (Fig. 5A). The band of the doublet with the
slower electrophoretic mobility was estimated to have
an apparent molecular weight of approximately
150 kD, while the faster migrating protein corresponded to an approximate molecular weight of
145 kD. The protein detected in the positive control
lysate from murine macrophages was approximately
135 kD. Detection of both proteins in the doublet
could be inhibited by blocking peptide (data not
shown), suggesting that both bands were antigenically
similar to iNOS. Carnosol treatment repeatedly
produced the appearance of a smaller anti-iNOS
C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
49
Fig. 4. Inhibition of NOS activity enhances loss of DJmt in the leukemia cells after antioxidant exposure. The leukemia cells were pre-incubated
for 30 min at 37 8C with 1 mM MEG prior to curcumin treatment (A), or 0.5 mM MEG prior to carnosol (B) and quercetin (C) treatment.
Antioxidant treatments were generally 10 mM curcumin, 6 mg/ml carnosol, and 2.5 mM quercetin, except for REH and CEM which were treated
with only 1 mM quercetin (*) due to the high degree of depolarization observed at the 2.5 mM concentration. After 4 h antioxidant treatment, the
cells were stained with JC-1 and analyzed by FACS. The percentage of cells with depolarized mitochondrial membranes are displayed for
untreated cells (white bars) and antioxidant-treated cells (black bars). The data represent the arithmetic meanGSE of three separate
experiments.
reactive fragment in REH and CEM cells with an
apparent moleular weight of approximately 120 kD.
To determine the location of the two anti-iNOS
reactive proteins, cells were fractionated into crude
membrane (containing mitochondria) and cytosolic
fractions and analyzed by Western blot (Fig. 5B). To
estimate the efficiency of the fractionation procedure,
the fractions were also analyzed by Western blot with
an antibody against COX4, that is imbedded in the
mitochondrial membrane. The 145 kD protein was
found predominantly in the cytosolic fractions of each
cell line. The cytosolic fractions did not contain
detectable COX4 protein, indicating that the cytosolic
fractions were reasonably free of contamination by
mitochondria (Fig. 5B). The 150 kD protein was
primarily present in the membrane fraction of SEM
and RS4;11 cells. Both anti-iNOS reactive proteins
were faintly detectable in the mitochondria/membrane
fractions of REH and CEM. However, the 145 kD
protein was also more strongly detected in the
cytosolic fractions of these latter two lines. These
data suggest that the two isoforms reacting with the
anti-iNOS antibody may have a distinct subcellular
localization in the ALL-derived cell lines: the larger
protein was found predominantly in the membrane
fractions, whereas the smaller protein was present
mostly in the cytosol.
4. Discussion
Evidence has accumulated that supports a role for
NO in the maintenance of DJmt, due to the ability of
NO to reversibly and temporarily inhibit cytochrome
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C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
Fig. 5. (A) The ALL lines constitutively express anti-iNOS reactive
isoforms that are not further induced by antioxidant treatment.
Protein lysates were prepared from untreated leukemia cells (U) and
cells treated for 4 h with curcumin (Cu), carnosol (Ca), and
quercetin (Q). Approximately 30 mg of total protein from each
lysate were electrophoresed on 6% SDS-PAGE gels and transferred
to PVDF membranes for Western blot analysis. Protein lysate
prepared from mouse macrophages (Mac) was used as a positive
control for iNOS protein. Arrows indicate the two iNOS proteins
present in the leukemia cells. (B) Cells were fractionated into
membrane/mitochondrial fractions, M, and cytosolic fractions, C.
Approximately 10 mg of total protein from each fraction was
electrophoresed on either 6% SDS-PAGE gels for detection of
iNOS isoforms or 15% SDS-PAGE gels for detection of COX4, a
subunit of cytochrome c oxidase. Protein was transferred to PVDF
membranes for Western blot analysis. Protein lysate prepared from
mouse macrophages (Mac) was used as a positive control for iNOS
protein (Cont.). Endothelial cell lysate was used in the control lane
for COX4 detection.
c oxidase [19,20]. Because of the reversible nature of
inhibition, NO appears to play a critical role in
controlling O2 consumption, ATP production, and
maintenance of DJmt [21]. Our data indicated that
one of the early responses of acute lymphocytic
leukemia-derived cells to treatment with curcumin,
carnosol, and quercetin was to increase production of
the free radical NO. Therefore, these phenolic
compounds appear to be inducing a pro-oxidant
response in the leukemia-derived cells. Inhibition of
NOS activity and reduction of NO levels by MEG
increased the percentage of cells with depolarized
mitochondrial membranes, suggesting that endogenous synthesis of NO in response to the phenolic
compounds was initially a protective response from
these cells. The increase in NO may be an attempt to
arrest the electron transport chain and, therefore,
maintain DJmt during an environmental insult.
The current study indicated that treatment of the
acute leukemia-derived cells with the phenolic
compounds eventually resulted in the loss of DJmt
by 4 h post-exposure (Fig. 1). Recently, Beltran et al.
[26] showed that exposure of the acute leukemia
line Jurkat to an exogenous NO donor also initiated
a protective response against serum-deprivation and
staurosporine treatment, but an eventual collapse of
DJmt was observed after the exposure to NO
exceeded 5 h. Collapse of DJmt was proposed to be
due to the accumulation of oxidative damage and/or
exhaustion of ATP, since one of the protective actions
of NO in these experiments was the reversal of the
ATP synthase pump [26]. Overproduction of NO has
been linked to the increased generation of superoxide
anion and other reactive oxygen species and also
reactive nitrogen species, such as peroxynitrite, which
can lead to disruption of DJmt [21]. Peroxynitrite,
formed through the interaction of superoxide anion
and NO, can irreversibly inhibit the electron transport
complexes I, II, and III [21], inducing a further
collapse of DJmt. Therefore, the induction of NO
synthesis in these leukemia-derived cells after
exposure to the curcumin, carnosol, and quercetin,
although initially protective, may indirectly be
responsible for the subsequent disruption of mitochondrial homeostasis and cell death.
Nitric oxide is a free radical synthesized from
L-arginine and oxygen by several isoforms of NOS
including nNOS, iNOS, ecNOS, and, more recently,
a mitochondrial form (mtNOS) [25,27]. The nNOS and
ecNOS isoforms are constitutively expressed in a variety
of tissues. The iNOS isoform is normally transcriptionally regulated by a variety of inflammatory mediators, as
well as pathophysiological conditions, such as hypoxia/
ischemia (reviewed in Refs. [28 and 29]). Expression of
iNOS protein has been observed in brain, breast, lung,
and colon tumors and low output of NO may enhance the
blood flow to the tumor tissue. Constitutive expression
of a functional iNOS and elevated production of NO
C. Kellner, S.J. Zunino / Cancer Letters 215 (2004) 43–52
have also been observed in B-cell chronic lymphocytic
leukemia, and appears to play a survival role in these
types of cells [28,30]. We observed two constitutively
expressed proteins that reacted with the anti-iNOS
antibody. The 145 kD protein was located predominantly in the cytosol, whereas, the 150 kD protein was
found predominantly in the mitochondria/membrane
fraction, and the expression of both protein bands
remained constant after antioxidant treatment. Although
it is currently unclear whether the NO generated in
response to the antioxidants originates in the cytosol or
the mitochondria, these data suggest that an increase in
the activation status of an existing iNOS-like isoform
rather than de novo synthesis was responsible for the
increase in NO synthesis.
Although most studies of these phenolic compounds have measured serum concentrations after
ingestion to determine bioavailability, limited data is
present concerning the levels and clearance of these
substances in the blood after intravenous injection. In
phase I clinical trials, up to 8 g of curcumin were
administered orally with peak serum concentrations
reaching approximately 1.77 mM after ingestion [11].
No apparent toxicity by curcumin was observed in
patients consuming 8 g of curcumin per day over a 3
month period. Quercetin was administered via bolus
intravenous infusions in phase I trials to determine
pharmacokinetics and toxicity [12]. Infusions were
administered at several concentrations during these
experiments, with some renal toxicity identified at the
highest dose of 1700 mg/m2 (approximately 1–2 mM
total blood volume). The concentration of quercetin
(2.5 mM) used in our studies is significantly less than
that used in the phase I trials. Currently, there is no
information available on the pharmacokinetics of
carnosol. Further experiments utilizing animal models
will need to be performed to analyze the efficacy of
these phenolic compounds against the t(4;11) and other
ALLs at the concentrations utilized in our studies.
The t(4;11) ALL cell lines SEM and RS4;11 represent
a high-risk subgroup of infant ALLs that are generally
refractory to conventional chemotherapies after relapse
resulting in a poor prognosis for survival [14]. In the
present study, we have utilized these t(4;11) and other
ALL-derived lines to understand how several potent
phenolic antioxidant compounds may disrupt DJmt
leading to cell death. Curcumin, carnosol, and quercetin
have distinct chemical structures, but appeared to act by
51
a common mechanism to induce NO synthesis. Of
interest is the initial protective effect that increased NO
appeared to give these leukemia cells, since inhibiting
NOS significantly increased the loss of DJmt. These
latter results suggest that NOS may be a potential
chemotherapeutic target for the treatment of ALL, since
inhibiting NOS may render the ALL cells more sensitive
to agents that disrupt DJmt. Therefore, the combination
of phenolic antioxidant compounds, such as curcumin,
carnosol, or quercetin in conjunction with NOS inhibitors
may be particularly valuable as a novel strategy for
treating the high-risk t(4;11) subgroup of ALLs.
Acknowledgements
Supported by the Wilhelm Sander-Stiftung,
Germany, Grant #2001.048.1. We thank Dr George
H. Fey, Dr Darshan Kelley, and Dr Carl Keen for
helpful discussions and critical review of this manuscript. We also thank Katrin Sapala for excellent
technical assistance.
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