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Introduction
Acute myelogenous leukemia is a serious and often lethal disease. There is a need for new, effective, and relatively
non-toxic therapeutic modalities. Recent research has focused on targeted the disruption of proliferation signal transduction and
the utility of 2-methoxyestradiol (2-ME) in the treatment of leukemia. 2-ME is a physiological metabolite of the endogenous
estrogen estradiol-17β which fails to bind the estrogen
receptor[1]. This compound is currently used in clinical trials as a
single agent or in combination with other anticancer agents, for treatment of several types of human cancer, including breast
cancer, prostate cancer, and multiple
myeloma[2,3]. 2-ME also shows antileukemic activity
in vitro with therapeutic
selectivity[4].
2-ME is another inhibitor of microtubule dynamic
causing mitotic arrest followed by cell cycle
G2/M-phase arrest[5]. It was also found to have an anti-angiogenesis effect in
tumors through its direct cytotoxicity on endothelial
cells[6,7]. It has also been reported that 2-ME induces apoptosis in
various cell types. In addition, recent studies have shown
that 2-ME inhibits the transcription of the superoxide
dismutase (SOD) enzymes, which protect cells from damage
induced by superoxide radicals, and the inhibition of SOD
activity results in apoptosis of human leukemia
cells[8,9]. Because 2-ME is relatively non-toxic to normal
tissues[9], an important implication of these findings is that such agents
might play a useful role in the therapy of leukemia.
Two major apoptotic pathways are known to date. The
intrinsic pathway is initiated by the mitochondria, while the
extrinsic pathway is initiated by cell surface
receptors[10]. Mitochondria-mediated apoptosis occurs in response to a
wide range of stimuli. Mitochondria are the major
generators of ATP by oxidative phosphorylation. Reactive oxygen
species (ROS) are generated in and around mitochondria,
and they are buffered by antioxidants. ROS may not only
arise from endogenous sources, but also from exogenous
sources. Our previous studies suggested that manumycin
increases ROS levels and results in
apoptosis[11]. Although many studies have shown that 2-ME has an effect in various
tumor cells, including leukemia cells, currently the role of
mitochondrial functional changes in the response of
leukemia cells to 2-ME has not yet been explored.
We hypothesized that 2-ME would induce functional
changes in the mitochondria in association with ROS
generation in the course of apoptosis induction in leukemia cells.
To test this hypothesis, we evaluated the effect of 2-ME on
mitochondrial membrane potential, ROS, and apoptosis in
leukemia cell lines. Our results indicated the requirement of
ROS generation in apoptosis induced by 2-ME in leukemia
cells.
Materials and methods
Cell culture Human leukemia cell lines HL-60 and U937
from American Type Culture Collection (Manassas, VA, USA)
were maintained in suspension culture with RPMI-1640
medium supplemented with heat-treated fetal bovine serum
(10%) at 37 oC in a humidified atmosphere with 5%
CO2. All experiments were done in exponentially growing cultures.
Chemicals and reagents Manumycin,
N-acetyl-L-cysteine (NAC), 2-ME,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT), carbonyl cyanide 4-phenylhydrazone
(FCCP), and DMSO were purchased from Sigma Chemical Co
(St Louis, MO, USA). All tissue culture media were
purchased from Life Technologies (Gaithersburg, MD, USA).
ApoAlert Nitric Oxide/Annexin V Dual Sensor Kits were
purchased from BD Biosciences Clontech Laboratories (Palo
Alto, CA, USA).
5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylben-zimidazolylcarbocyanine iodide (JC-1)and dihydroethidium
(DHE) were purchased from Molecular Probes (Eugene, OR,
USA) and prepared separately in DMSO. The stock
solutions were then further diluted in tissue culture medium to
the desired concentrations. 2-ME and manumycin were first
dissolved in DMSO (tissue-culture grade). The stock
solution was diluted in tissue culture medium at appropriate
concentrations such that the final concentration of DMSO in
culture medium would not exceed 0.1%
(v/v).
Assessment of mitochondrial membrane potential
Measurement of mitochondrial membrane potential (Dym) was
performed with the JC-1 stain, a lipophilic cation fluorescent
dye that accumulates in the mitochondria, showing green
fluorescence at a low mitochondrial membrane potential and
forming red fluorescent J-aggregates at higher membrane
potential. Drop in mitochondrial membrane potential is
indicated by a decrease in the ratio of the red signal to the green
signal. HL-60 and U937 cells were cultured in 96-well tissue
culture plates with black walls and clear bottoms. After
attached to the vessels overnight, the cells were treated with
experimental treatments, 10 µmol/L FCCP (positive control
for depolarization), or 0.1% DMSO (negative control) for
various durations. The cells were then incubated with 5
μg/mL JC-1 at 37 °C for 15 min in dark as previously described. The
cells were washed twice with phosphate buffered solution
(PBS), and the plates were immediate read using a
fluorescent plate reader (Spectrafluor Plus, Tecan, Zurich,
Switzer-land) with the excitation and emission wavelengths set at
540 and 595 nm, respectively for red fluorescence, and 485
and 535 nm, respectively for green fluorescence. For each
sample, the results were calculated as follows after the
fluorescence values had been corrected for the background:
red fluorescence of sample-average of red fluorescence of
blanks)/(green fluorescence of sample-green
fluorescence-average of green fluorescence of blanks. Data from 3
independent experiments were analyzed using one-way ANOVA
with a post-hoc comparison between the groups.
Flow cytometry assay Flow cytometry assays were
performed at our Flow Cytometry Core Facility (Becton
Dickinson, Mountain View, CA, USA ). Phycoerythrin
(PE)-conjugated Annexin V was used to detect cells undergoing
apoptosis. In brief, the cells were collected by
centrifugation (200×g for 5 min at 4
oC) after the experimental treatments and resuspended in Annexin V binding buffer with
Annexin V-PE staining for 15 min at room temperature. After
dilution, the cells were analyzed with flow cytometry for PE
fluorescence (excitation wavelength, 488 nm; emission
wavelength, 578 nm). To measure changes of intracellular
nitric oxide (NO) level, a proprietary membrane-permeable
NO sensor dye was used according to the manufacturer's
instructions. Briefly, the cells were pre-incubated with the
NO sensor dye for 30 min prior to experimental treatments in
the continued presence of the dye. The cells were pelleted
by centrifugation, rinsed, and analyzed with flow cytometry
for green fluorescence (excitation wavelength, 488 nm;
emission wavelength, 515 nm). For dual NO/Annexin V
simultaneous flow cytometry analysis, the ApoAlert Nitric
Oxide/Annexin V Dual Sensor Kit was used.
Measurement of intracellular superoxide anion
The cells were cultured in 96-well plates. HL-60 and U937 cells
were treated with 2 µmol/L 2-ME for various durations, and
then the cells were incubated with 5 µmol/L DHE as
previously described. In brief, after experimental treatments, the
cells were incubated with 5 µmol/L DHE in culture medium
for 10 min at 37 °C in dark. The cells were rinsed twice with
PBS, and then ethidium-DNA fluorescence was immediately
measured with a fluorescent plate reader (Spectrafluor Plus,
Tecan, Switzerland) with an excitation wavelength at 430 nm
and an emission wavelength at 590 nm. The average
ethidium-DNA fluorescence of each group with the 95% confidence
intervals was plotted over time. Three independent
experiments were performed
Cytotoxicity assays Cell growth inhibition or the relative
number of viable cells was determined by MTT assay as
previously described. In brief, HL-60 or U937 cells were
seeded onto 96-well plates at the initial
density of 5000 cells/well. After incubation with various
concentrations of 2-ME, NAC, and a combination of both, respectively for 48 h, 20 µL
MTT reagent (3 mg/mL) was added to each well and
incubated at 37 oC for 4 h. The
cells were then centrifuged
(700×g for 15 min), and the medium was
removed. The cell pellets were dissolved in 200 µL
DMSO. Absorbance was measured at a wavelength of 590 nm with reference at wavelength of
635 nm. All of the experiments were performed with the
absorbance values within the linear range of this colorimetric
assay. Percentage viability was defined as 100% times the
ratio of absorbance above the background in the sample to
the average absorbance above the background in the
control (DMSO-treated) samples. The
background absorbance was measured in the wells with the cells that had been
killed by exposure to 70% ethanol for 10 min.
Statistical analysis Comparison in experiments was
performed using one-way ANOVA with Tukey's test or Student's
t-test to assess the statistical significance of differences
between the groups. Differences with P<0.05 were
considered significant.
Results
2-ME decreased viability and induced apoptosis of
leukemia cells 2-ME decreased the viability in the leukemia
cell lines (Figure 1A). Modest degrees of cytotoxicity were
noted at 1 µmol/L 2-ME (48 h), which reached near maximal
levels of cytotoxicity at concentrations more than 2 µmol/L.
Meanwhile, the time course of 2-ME-mediated apoptosis in
HL-60 and U937 cells was analyzed (Figure 2B).
Apoptosis-induced by 2 µmol/L 2-ME treatment became apparent after
12 h, and apoptosis was enhanced while the drug exposure
time was elongated.
Effects of 2-ME on mitochondrial membrane
potential It is well established that apoptosis induced by some agents is
associated with the perturbation of mitochondrial functions.
Therefore, we investigated changes in mitochondrial
function in response to 2-ME treatment. Using the fluorescent
dye JC-1, we assessed relative changes in mitochondrial
membrane potential following drug treatments in leukemia
cell lines. In both HL-60 and U937 cells, treatment with 2
µmol/L 2-ME induced mitochondrial membrane
hyperpolarization in the early phase of treatment followed by
depolari-zation. The positive control for depolarization was achieved
by treating the cells with FCCP (Figure 2).
2-ME induced ROS generation in leukemia cells
2-ME induced NO generation in HL-60 and U937 cells
2-ME 2 µmol/L induced NO generation as demonstrated by
the significant increase (P<0.05, ANOVA, post-hoc Tukey's
test) in the percentage of cells stained by NO sensor dye in
2-ME-treated cells compared with the DMSO sham-treated
control cells (Figure 3A)
2-ME induced generation of superoxide anion Another major ROS is the superoxide anion. To determine
whether 2-ME increased superoxide anion in leukemia cell
lines, we measured superoxide anion by detecting the
oxidation of DHE. HL-60 and U937 cells were exposed to 2
µmol/L 2-ME for 2, 4, or 6 h. Three independent experiments were
performed for each cell line. 2-ME significantly increased
superoxide anion levels relative to levels in untreated
control cells at all 3 time points (Figure 3B).
2-ME induced apoptosis was associated with the
generation of ROS in leukemia cells 2-ME induced NO generation
before the cells underwent apoptosis. A dual-fluorescence
flow cytometry analysis was used to determine the
relationship between NO and apoptosis. The cells were incubated
with 2 µmol/L 2-ME for various times and then double-stained
with NO sensor dye and Annexin V. A substantial amount of
NO accumulation was observed after 1 h of 2-ME treatment,
as evidenced by a shift of the cell population from the
lower-left quadrant, rightward along the x-axis. However, no
detectable drug-induced apoptosis was observed at the same
time (1 h), as evidenced by the lack of change in Annexin V
labeling. In the continuous presence of 2-ME, NO remained
elevated up to 6 h, whereas a significant number of Annexin
V-positive cells did not appear until 6 h, and cell apoptosis
was further increased at 6 h (Figure 4A). Similar results were
obtained with U937 cells (data not shown). These results
suggested that treatment with 2-ME in both cell lines would
induce NO generation before the cells underwent apoptosis.
NAC blocked 2-ME-induced ROS generation NAC is a
commonly used quencher of ROS. The ability of NAC to
block increases in superoxide anion and NO has previously
been documented. Co-culture with 5 mmol/L NAC for 6 h
effectively blocked 2-ME-induced NO and superoxide anion
generation in HL-60 and U937 cells (Figure 4B, 4C). These
findings indicate that we could use NAC to demonstrate
whether ROS is essential for 2-ME-induced apoptosis.
NAC blocked 2-ME-induced apoptosis HL-60 and U937
cells were incubated with 2 µmol/L 2-ME for 6 h, and
apoptosis was detected by Annexin V-PE. 2-ME significantly
(P<0.05, ANOVA, post-hoc Tukey's test) increased the
percentage of cells stained by Annexin V-PE compared with
binding in the corresponding DMSO sham-treated control. At 5
mmol/L, NAC did not have effect on apoptosis, but the
presence of NAC almost completely suppressed 2-ME-induced
apoptosis, as evidenced by a near-complete reversal of the
percentage of Annexin V-positive cells (Figure 4D). Taken
together, these findings suggest that an increase in ROS is
required for 2-ME-induced apoptosis in leukemia cells.
NAC protects leukemia cells from 2-ME
cytotoxicity To further investigate whether NAC could protect leukemia cells
from 2-ME cytotoxicity, we measured the relative viability of
cells treated for 48 h with control medium: 2 µmol/L 2-ME, 5
mmol/L NAC, or a combination of both, respectively. The
results of 3 independent experiments were plotted.
Concurrent treatment with NAC significantly prevented the decrease
in viability with 2-ME treatment (P<0.05; Figure 4E). These
results suggest that the effect of 2-ME on viability was mostly
mediated by ROS.
Manumycin enhanced 2-ME-induced NO generation and
apoptosis We previously found that manumycin induced
apoptosis of leukemia cells via the generation of NO. We
also found 2-ME induced apoptosis of leukemia cells through
the functional change of mitochondria and the increase in
NO and superoxide anion. Therefore, we combined
manumy-cin with 2-ME to evaluate the enhancement of apoptosis.
Manumycin significantly increased the percentage of cells
stained with the NO sensor dye only and the percentage of
cells stained positive by both Annexin V-PE and the NO
sensor dye in HL-60 cells. Compared with 2-ME alone, the
combination of manumycin and 2-ME further increased the
percentage of cells stained with the NO sensor dye only and the
percentage of cells stained by both Annexin V-PE and the
NO sensor dye (one-way ANOVA, post-hoc between-group
comparisons, Tukey's test, P<0.05; Figure 5). A similar
result was obtained for the U937 cells (data not shown).
Discussion
We demonstrated here that 2-ME induced apoptosis in
leukemia cells through the generation of ROS. This
interpretation was based on the increase in the 2-ME-treated
leukemia cells and on the significant protection against apoptosis
exerted by NAC, a quencher of ROS. Our results
corroborated and extended the findings of Huang
et al on the induction of oxygen free radicals, mitochondria damage, and
apoptosis in chronic lymphocytic
leukemia[4_9].
It is clear that changes of mitochondrial membrane
potential are associated with
apoptosis[12_14]. Both hyperpolarization and depolarization have previously been
observed in association with mitochondrial cytochrome c
release[15]. However, mitochondrial depolarization in
association with apoptosis appears to be more common.
Resveratrol induces apoptosis with mitochondrial
depolarization in acute lymphocytic leukemia
cells[16]. Oxidative damage and mitochondrial depolarization induced by imexon
highly correlates with imexon-induced apoptosis in several
myeloma cell lines and an acute promyelocytic leukemia cell
line[17]. The mitochondrial permeability transition (MPT) pore
opening is expected to affect mitochondrial membrane
potential. Vieira et al proposed a 2-step model for MPT in
which hyperpolarization was associated with step 1 and
non-specific pore opening and depolarization was associated with
step 2. We found that 2-ME induced hyperpolarization at an
early stage and subsequent depolarization in Hl-60 and U937
cells. It is conceivable that the duration of step 1 is very
brief in HL-60 and U937 cells. The increase in superoxide
also appears to correlate with mitochondrial membrane
hyperpolarization induced by 2-ME.
Mitochondria are the rich source of ROS, which are toxic
byproducts of aerobic cells and play an important role in cell
proliferation, aging, and cancer development. An excessive
amount of ROS can lead to cell death by apoptosis or by
necrosis. The ability of ROS to damage cellular components
and cause cell death suggests the possibility to explore this
chemical for killing cancer cells[18]. The importance of
chemotherapy-induced change in redox status to signal
apoptosis and regulate the apoptosis effector is emerging.
Although the role of ROS in apoptosis induction has been
described for a diverse collection of xenobiotics, this report
provides experimental evidence for the role of ROS in
apoptosis induced by 2-ME. The mechanisms of action of
2-ME may involve the inhibition of Akt and the activation of
Jun N-terminal kinase (JNK), the inhibition of SOD,
anti-angiogenetic effects, and interruption microtubule
assembl-ing. However, the question as to which one induces ROS
generation is unclear. Future work to bridge this gap in
knowledge will be important because this knowledge will
provide the theoretical basis for the rational design of
synergistic combination chemotherapy regimens, including 2-ME.
Superoxide and NO are the major ROS in living cells, and
superoxide is the first species to be generated by the
auto-oxidation of the mitochondria redox groups. ROS, including
NO and superoxide anion generated by 2-ME, serves as
stimuli to induce apoptosis in leukemia cells. NO in
particular can be both pro- and anti-apoptotic depending on
circumstances. We studied the production of NO and
superoxide anion in HL-60 and U937 cells treated with 2-ME.
Our results provided evidence of apoptosis induced by
2-ME. NAC blocked apoptosis induced by 2-ME indicated
that ROS was a requirement in the apoptotic signaling
cascade.
Farnesyltransferase inhibitors (FTIs) are a group of new
chemotherapeutic agents being studied in clinical trials for
both solid tumors and hematological malignancies.
Manumycin is an FTI that mimics the farnesyl
pyrophosphate group[21]. FTI have demonstrated preclinical activity
in a variety of experimental models, and we previously
demonstrated that manumycin induced ROS generation, which
mediated DNA damage and induced apoptosis of anaplastic
thyroid cancer (ATC) cells and leukemia cell lines. Oxidative
damage has been suggested to be the key mechanism by
which manumycin induced apoptosis. On the basis of the
ability of manumycin to induce the generation of ROS in
several experiment models, it is reasonable to expect that its
combination with 2-ME could enhance apoptosis. The
results of the present study demonstrated that the
combination of manumycin with 2-ME enhanced the generation of
NO and resulted in enhanced apoptosis induction in
leukemia cells. This result from the in
vitro assay suggested that it was possible to combine 2-ME with other ROS-generating
agents to enhance the therapeutic effect on leukemia cells.
In conclusion, 2-ME induced oxidative stress and
mitochondrial damage, which then caused apoptosis in leukemia
cells. The mechanism of apoptosis induction by 2-ME
involved the generation of ROS. The ROS-generating agent
manumycin significantly enhanced apoptosis induced by
2-ME. These results may help to elucidate the mechanisms
underlying efficacious combinations of 2-ME with other
drugs and provide a new strategy to enhance therapeutic
activity. Future studies will further investigate this possibility.
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