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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 307-313
NEOPLASIA
Oxidative stress interferes with cancer chemotherapy: inhibition
of lymphoma cell apoptosis and phagocytosis
Emily Shacter,
Joy A. Williams,
Roger M. Hinson,
Sema Sentürker, and
Yang-ja Lee
From the Laboratory of Immunology, Division of Therapeutic Proteins,
Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD.
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Abstract |
Many antineoplastic drugs kill tumor cells by inducing apoptosis.
This highly controlled mechanism of cell death is thought to be
physiologically advantageous because apoptotic cells are removed by
phagocytosis before they lose their permeability barrier, thus
preventing induction of an inflammatory response to the dying cells. In
contrast, necrotic cells lyse and release their contents into the
extracellular space, thus inducing inflammation. In this report, we
examine the effects of oxidative stress on chemotherapy-induced cell
killing. We find that H2O2 inhibits the ability
of 4 different chemotherapy drugs (VP-16, doxorubicin, cisplatin, and
AraC) to induce apoptosis in human Burkitt lymphoma cells.
H2O2 shifts the form of cell death from
apoptosis to pyknosis/necrosis, which occurs after a significant delay
compared with chemotherapy-induced apoptosis. It can also lower the
degree of cell killing by these drugs. These effects of
H2O2 can be prevented by the antioxidant agents
Desferal, Tempol, and dimethylsulfoxide. Phagocytosis by monocyte-derived macrophages of VP-16-treated lymphoma cells is also
inhibited by H2O2. Cells killed with
H2O2 (with or without VP-16) do ultimately
undergo phagocytosis, but this occurs only after they have lost their
permeability barrier. Thus, membrane-intact apoptotic cells are
recognized and phagocytosed by monocyte-derived macrophages, but
membrane-intact pyknotic/necrotic cells are not. The results
suggest that chemotherapy-induced apoptosis and phagocytosis of cancer
cells may be enhanced by including certain antioxidant agents in the
treatment protocol.
(Blood. 2000;96:307-313)
© 2000 by The American Society of Hematology.
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Introduction |
Chemotherapy is one of the mainstays of medical
intervention for cancer. Many antineoplastic drugs kill tumor cells by
inducing a form of cell death called apoptosis,1 which is
characterized by unique biochemical and morphologic
features.2 Most notably, cells dying by apoptosis fragment
into small, subcellular, membrane-bound "apoptotic
bodies."3 In contrast, cells that die by necrosis swell
and then lyse, releasing their contents into the extracellular space.
It is thought that death by apoptosis is physiologically advantageous
because apoptotic cells are cleared by phagocytosis and subsequent
intracellular degradation.4,5 In this manner, apoptotic
cells are removed without causing damage to the surrounding tissue. In
contrast, necrotic cells are thought to promote an inflammatory
response caused by the leakage of intracellular proteins and nucleic
acids. Agents that interfere with the ability of an antineoplastic
agent to induce apoptosis may limit drug efficacy and induce a
tissue-damaging inflammatory response.
This work examines the possible influence of oxidants on the effects of
cancer chemotherapy. Oxidants such as superoxide, hydrogen peroxide
(H2O2), and hydroxyl radical are generated by activated phagocytes (eg, neutrophils and macrophages) as part of the
inflammatory response.6 Under normal circumstances, oxidants serve a protective function by killing invading bacteria and
tumor cells.7 However, they can have detrimental side
effects as well, causing tissue damage and contributing to the
development or progression of numerous different diseases including
cancer.8 Solid tumors are often infiltrated by inflammatory
phagocytes,9 which can generate oxidative stress
within the tumor tissue. In support of this possibility, cancer
patients are reported to have higher levels both of generalized
oxidative stress10-12 and of oxidative damage within tumor
tissues as compared with normal tissues.13-15
In previous studies with Burkitt lymphoma cells, we found that
relatively low concentrations of H2O2 (50-100 µmol/L) inhibited the induction of apoptosis by the chemotherapy drug
VP-16 (etoposide) and the calcium ionophore A23187.16 Cell
death still occurred, but it was by a nonapoptotic mechanism. Thus,
nuclear condensation and fragmentation of the cells into apoptotic
bodies were prevented, as were many of the biochemical steps that are
known to mediate apoptosis, such as oligonucleosomal degradation of the
DNA and caspase activation. The goal of the present work was to
determine whether oxidants such as H2O2
interfere with the actions of cancer chemotherapeutic agents in
general. Using Burkitt lymphoma cells as a model, we found that
H2O2 inhibited the induction of apoptosis by 4 different antineoplastic agents. The presence of
H2O2 could also reduce the overall level of
cell killing by 3 of these drugs. Because of its inhibition of
apoptosis, H2O2 also inhibited the phagocytosis
of VP-16-treated lymphoma cells by human monocyte-derived macrophages. Induction of apoptosis could be restored by
coadministration of selected antioxidants which did not, by
themselves, induce apoptosis. The potential ramifications of these
findings for cancer chemotherapy are discussed.
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Materials and methods |
Cells and treatments
The Burkitt lymphoma cell lines JLP 119, ST-486, and BL-41 were
provided by Kishor Bhatia (National Cancer Institute, National Institutes of Health, Bethesda, MD). Cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L L-glutamine, and 50 µmol/L  -mercaptoethanol
("medium") at 37°C in 5% CO2 in air.
Exponentially growing cells were harvested by centrifugation and
resuspended in fresh medium to achieve a culture density of
5 × 105 cells/mL. H2O2 was
added 30 minutes after chemotherapy drugs. This time point was chosen
to minimize the possibility of any direct interactions between
H2O2 and the chemotherapeutic agents. In
previous studies with VP-16, we found that H2O2
does not have any direct chemical effect on VP-16,16 but
similar experiments were not carried out for all of the other drugs
used in the present studies. To avoid any possible interactions, we
allow the drug to enter the cells before adding the
H2O2. The time of addition of
H2O2 is not critical as long as it is added before the onset of apoptosis; it inhibits the induction of apoptosis equally well if added 30 minutes before or after adding the
chemotherapy drugs. When antioxidants were tested, they were added 30 to 60 minutes before the chemotherapy drugs. Cell incubations were for 4 to 22 hours, as indicated in the text.
Quantification of cell death using Hoechst/propidium iodide nuclear
staining and fluorescence microscopy
Cells were stained with Hoechst 33342 and propidium iodide
(PI) and were visualized using fluorescence microscopy, as
described previously.17 A minimum of 200 cells were counted
and classified as follows: (1) live cells (normal nuclei: blue
chromatin with organized structure); (2) membrane-intact apoptotic
cells (bright blue chromatin that is highly condensed, marginated, or
fragmented); (3) membrane-permeable apoptotic cells (bright red
chromatin, highly condensed, or fragmented); (4) necrotic cells (red,
enlarged nuclei with smooth normal structure); and (5)
pyknotic/necrotic cells (bright red, slightly condensed nuclei
sometimes divided into 2-3 spheres). All experiments were repeated at
least 3 times.
Phagocytosis assay
A fluorescence microscope-based assay was developed for quantifying
phagocytosis of lymphoma cells by macrophages. Monocyte-derived macrophages were prepared by incubating normal human monocytes (prepared by elutriation) for 7 to 14 days in RPMI
containing 10% FCS, penicillin/streptomycin, and recombinant human
macrophage colony-stimulating factor (rhM-CSF; 100 ng/mL).
Monocytes were seeded at a starting density of
2 × 105 cells/well in 24-well plates. Burkitt
lymphoma cells were stained with the stable, cell-permeable green
fluorescent dye CFDA [5-(and-6)-carboxyfluorescein diacetate,
succinimidyl ester] (0.16 mg/mL stock in dimethylsulfoxide [DMSO])
by suspending 8 × 105 cells in 1 mL
phosphate-buffered saline (PBS) in a test tube and adding 0.75 µL
CFDA. The dye becomes modified by esterases once it enters cells so
that it gains fluorescence, binds to intracellular proteins, and
becomes trapped inside the cell. Cells were incubated for 20 minutes at
37°C, mixed with one-half volume of medium, centrifuged at 4°C,
and resuspended to a cell density of 5 × 105
cells/mL in medium. Treatments with H2O2 and
chemotherapy drugs were performed as described earlier. The intensity
of green staining of the target cells did not diminish over the course
of any of the incubations. At the end of the incubations, cells were
added at a 2:1 ratio to the macrophages, and the cells were allowed to
coincubate for 1 hour at 37°C. Nonphagocytosed lymphoma cells were
removed by thorough washing with medium. The macrophages were stained
with phycoerythrin (PE)-labeled anti-CD14 antibody (1:15 dilution in
buffer containing 1% albumin and azide) at 4°C for 45 minutes.
Cells were detached from the wells by adding 1% lidocaine/0.5% serum
in PBS and incubating for 10 minutes at 37°C. The wells were mixed
vigorously to remove all cells (verified by phase contrast microscopy),
which were then centrifuged and resuspended in PBS. Cells were analyzed
by fluorescence microscopy using a 2-color (FITC/TRITC)
fluorescence cube ( ex 450-490 nm, em
520-650 nm). The percent phagocytosis is the percentage of total
macrophages (red/orange cells, at least 200 per measurement) containing
internalized Burkitt lymphoma cells (green spheres). Macrophages
containing multiple green cells were counted only once. The presence of
free (nonphagocytosed) Burkitt lymphoma cells was rare ( 1 per
measurement) and was not quantified.
Measurement of H2O2 production by Burkitt
lymphoma cells
To determine whether the Burkitt lymphoma cells used in these
experiments produce their own H2O2, we
suspended JLP 119 or ST-486 cells (5 × 105/mL) in
PBS/1 mg/mL glucose ± 1 mmol/L azide to inhibit
endogenous catalase activity. At various time points (10 minutes to 2 hours), samples were removed and centrifuged, and cell-free
supernatants were assayed for the presence of
H2O2 by the phenol red assay, performed as
described previously.17 Because
H2O2 can diffuse freely in and out of cells,
the concentration of H2O2 outside of the cells
should reflect the intracellular concentration as well.
Reagents
VP-16, doxorubicin, AraC (cytosine
-D-arabinofuranoside), cisplatin
(cis-Platinum(II)diammine dichloride), Desferal (deferoxamine mesylate), Tempol (4-hydroxy-Tempo), 1,10-o-phenanthroline,
ascorbic acid, ebselen
(2-phenyl-1,2-benzisoselenazol-3[2H]-one), DMSO, Hoechst
33342, and PI were purchased from Sigma Chemical, Inc. (St. Louis, MO).
CFDA (catalog no. 1157) was from Molecular Probes, Inc. (Eugene, OR).
PE-labeled anti-CD14 antibody was from PharMingen (San Diego,
CA). rhM-CSF was kindly provided by Valerie Calvert (Food
and Drug Administration/Center for Biologics Evaluation and Research
[CBER]).
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Results |
VP-16 kills Burkitt lymphoma cells almost entirely by inducing
apoptosis, and this mode of cell death is inhibited by the presence of
H2O2.16 Cells treated either with
H2O2 alone or with VP-16 in the presence of
H2O2 are mostly nonapoptotic, showing cytoplasmic swelling, mild pyknosis of the nucleus, and little or no
DNA fragmentation, caspase activation, or annexin-V
binding.16 Because these cells resemble necrotic cells
except for the pyknosis of the nucleus, they are referred to as
pyknotic/necrotic. Using cell morphology as the criterion for assessing
the mode of cell death, we quantified cell death by fluorescence
microscopy after staining the cells with the nuclear dyes Hoechst 33342 and PI. This method allowed us to distinguish apoptotic from
nonapoptotic cells and also revealed whether the cells had lost their
plasma-membrane permeability barrier (see "Materials and
methods"). Examples of the different cell death morphologies
identified with this method are shown in Figure
1.
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| Fig 1.
Morphologies of live and dead Burkitt lymphoma cells as
determined by fluorescence microscopy.
JLP 119 cells were treated with 50 µmol/L
H2O2. Cells were harvested after 22 hours,
stained with Hoechst 33342 and PI, and examined by fluorescence
microscopy as described in "Materials and methods."
Characteristic live and dead cell morphologies are identified by the
arrows as follows: double arrow, live cell; long thin arrow, necrotic
cell; long fat arrows, pyknotic/necrotic cells; short white arrow,
membrane-permeable apoptotic cells; short open arrow, membrane-intact
apoptotic cell.
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The results in Figure 2 show the effects of
H2O2 on the induction of apoptosis by 4 different antineoplastic drugs with diverse mechanisms of action: VP-16
(1.7 µmol/L), doxorubicin (0.2 µmol/L), cisplatin (50 µmol/L),
and AraC (2 µmol/L). H2O2 was added 30 minutes after each drug addition. As shown, all of these drugs killed
the lymphoma cells by inducing apoptosis. The presence of increasing
concentrations of H2O2 progressively inhibited
the occurrence of apoptosis and converted the mode of cell death to pyknosis/necrosis. In addition, decreased overall cell killing by
VP-16, doxorubicin, and cisplatin occurred in the presence of
H2O2. For the representative experiments shown
in Figure 2, the fraction of cells appearing alive and healthy after
VP-16 treatment increased from 16% ± 4% (mean ± SD,
n = 3) in the absence of H2O2 to
43% ± 11% in the presence of 50 µmol/L
H2O2. With doxorubicin, the respective values
were 18% ± 4% without H2O2 and
36% ± 5% with 50 µmol/L H2O2
(n = 4). For cisplatin, values were 4% ± 1% and
24% ± 5% without and with H2O2,
respectively (n = 4). Because the level of cell killing by 75 µmol/L H2O2 alone exceeded the level of
killing by AraC, cells treated with 75 µmol/L
H2O2 and AraC together incurred a level of cell
death that was comparable to the higher level induced by
H2O2 alone. Control experiments with VP-16
showed that H2O2 had no direct chemical effect
on the drug.16
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| Fig 2.
Induction of cell death by chemotherapy drugs in the
presence and absence of H2O2.
JLP 119 cells were treated with either VP-16, doxorubicin, cisplatin,
or AraC followed 30 minutes later by the addition of the indicated
concentrations of H2O2. After 22 hours of
incubation, cells were harvested and analyzed by fluorescence
microscopy, as described in "Materials and methods." The fraction
of total cells representing each form of cell death is shown. For ease
of interpretation, the numbers of blue (membrane-intact) and red
(PI-permeable) apoptotic cells were added together ( ), as were the
numbers of pyknotic/necrotic and necrotic cells ( ). The number of
typical necrotic cells generally represented less than 10% of the
number of pyknotic/necrotic cells. Each experiment was repeated at
least 3 times. Error bars show the SD for the total number of dead
cells.
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Further experiments were carried out to determine whether the decreased
cell killing seen after incubation with a chemotherapy drug plus
H2O2 can translate into increased long-term
survival of tumor cells. For these studies, JLP 119 cells were treated with 1.7 µmol/L VP-16 in the presence or absence of 50 µmol/L H2O2, and the cells were allowed to incubate
for 12 hours. Live cells were then counted by trypan blue exclusion,
diluted in fresh growth medium, and plated at 3 cells/well in 96-well
plates (288 wells per treatment). After 3 weeks, the number of wells
containing growing cells was counted. The cell densities in each
positive well were equivalent, suggesting that each growth was derived from a single cell (clone). In 2 independent experiments (Table 1), we found that 1.5- to 2-fold more cells
survived VP-16 treatment when H2O2 was also
present, consistent with the results obtained at 22 hours.
In the experiments shown in Figure 2, we used concentrations of
chemotherapy drugs (eg, 1.7 µmol/L VP-16) that killed 80% of the
cells over the course of 22 hours of incubation. With increased drug
concentrations (eg, 8.5 µmol/L VP-16), apoptosis occurs much more
rapidly, such that 60% to 70% of JLP 119 cells are killed within 4 hours.16 Under these conditions, the presence of 75 µmol/L H2O2 completely inhibited induction of
apoptosis (Figure 3). However, these cells
will ultimately die from H2O2 toxicity over the
course of an overnight incubation, and most of this delayed cell death
is pyknotic/necrotic (eg, as seen in Figure 2). Identical results have
been obtained with 2 additional Burkitt lymphoma cell lines, ST-486 and
BL-41 (see following text).
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| Fig 3.
Rapid inhibition of VP-16-induced apoptosis by
H2O2.
JLP 119 cells were treated for 4 hours with 8.5 µmol/L VP-16 in the
presence or absence of 75 µmol/L H2O2, added
30 minutes after the VP-16. Cell death was assayed by fluorescence
microscopy, as described in "Materials and methods."
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A series of agents known to counteract the actions of reactive oxygen
species were tested for their ability to prevent
H2O2 from inhibiting chemotherapy-induced
apoptosis. Three compounds were found to restore the induction of
apoptosis by antineoplastic drugs: the iron chelator Desferal
(desferrioxamine), the nitroxide spin trap Tempol, and the nonspecific
radical scavenger DMSO. The results of experiments carried out using
Desferal with VP-16, doxorubicin, and cisplatin are shown in Figure
4. Cells were pretreated for 1 hour with
Desferal to provide time for uptake of the compound into the cells. At
concentrations of 5 or 10 µmol/L, the chelator lowered the overall
level of cell killing induced by 50 µmol/L H2O2
alone. The cell death that remained was primarily apoptotic instead of pyknotic/necrotic. When administered together with chemotherapy drugs, Desferal inhibited the antiapoptotic effects of
H2O2, and the cells died by apoptosis instead
of pyknosis/necrosis. Similar results were observed in experiments
using Tempol and 75 µmol/L H2O2 (Figure
5) and DMSO (data not shown). Like
Desferal, Tempol also reduced the overall level of cell killing induced by 50 µmol/L H2O2 alone from 70% to 30%,
converting the mode of cell death from pyknosis/necrosis to apoptosis.
Neither Desferal nor Tempol had a significant effect on the level or
type of cell death induced by the chemotherapy drugs alone (data not
shown).
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| Fig 4.
Effect of Desferal on cell killing induced by
chemotherapy drugs in the presence and absence of
H2O2.
After a 1-hour preincubation with Desferal (10 µmol/L), cells were
treated for 22 hours with 1.7 µmol/L VP-16, 0.2 µmol/L doxorubicin,
or 50 µmol/L cisplatin in the presence or absence of 50 µmol/L
H2O2 (added 30 minutes after the chemotherapy
drugs) and analyzed as described in the legend to Figure 2.
Pyknotic/necrotic cells are indicated by , and apoptotic cells are
indicated by .
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| Fig 5.
Effect of Tempol on cell killing induced by chemotherapy
drugs in the presence and absence of H2O2.
Cells were treated for 18 hours with 1.7 µmol/L VP-16 or 50 µmol/L
cisplatin in the presence or absence of 75 µmol/L
H2O2 and 0.5 mmol/L Tempol and analyzed as
described in the legend to Figure 2. The order of addition of reagents
was Tempol first, followed by chemotherapy drug, followed by
H2O2 (30-minute delay). Pyknotic/necrotic cells
are indicated by , and apoptopic cells are indicated by .
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Four other antioxidant compounds that were tested failed to inhibit the
effects of H2O2 on the induction of apoptosis
by VP-16 (0.85-1.7 µmol/L) or doxorubicin (0.2 µmol/L) during
overnight incubations. These were the divalent metal cation chelator
1,10-o-phenanthroline (1-4 µmol/L), which may have failed to
inhibit the effects of H2O2 because of its
inherent cytotoxicity to the cells at concentrations greater than 2 µmol/L; vitamin C (10-500 µmol/L), which was toxic at
concentrations of 100 µmol/L or greater; ebselen (10 µmol/L, an
inhibitor of lipid peroxidation), which was also toxic to the cells;
and trolox (a vitamin E analog), tested at 10 to 500 µmol/L.
One of the main physiologic advantages of apoptotic death compared with
necrotic death derives from how the cells are cleared from tissues. It
is believed that apoptotic cells are taken up and removed from tissues
by phagocytosis, either by professional phagocytes (eg, tissue
macrophages) or by other neighboring cells.18 We sought to
determine whether oxidative stress interferes with phagocytosis of
dying tumor cells, given that it inhibits the induction of apoptosis. A
fluorescence microscopy-based in vitro assay was developed for
measuring phagocytosis of apoptotic Burkitt lymphoma cells by human
monocyte-derived macrophages. A photograph of a representative result
from this assay is shown in Figure 6.
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| Fig 6.
Fluorescence microscopy assay for phagocytosis of Burkitt
lymphoma cells by human monocyte-derived macrophages.
The phagocytosis assay was performed as described in Materials and
methods. The photograph shows 3 macrophages (red-orange cells), which
have engulfed VP-16-treated ST-486 (green) cells.
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Phagocytosis of cells treated with VP-16 in the presence or absence of
H2O2 was tested using 3 different Burkitt
lymphoma cell lines: JLP 119, ST-486, and BL-41. Cells were treated
with VP-16 at concentrations that induce morphologic apoptosis within 4 hours of treatment. H2O2 was used at 75 µmol/L, which is sufficient to convert most of the VP-16-induced
phagocytosis to pyknosis/necrosis. As shown in Figure
7, all 3 cell lines underwent
significantly increased phagocytosis after VP-16-induced
apoptosis as compared with control cells. Note that the
apoptotic cells underwent phagocytosis even though the membranes were
still intact (impermeable to PI). H2O2
inhibited apoptosis and subsequent phagocytosis of all 3 cell lines.
The inhibition of phagocytosis was not due to a direct effect of
H2O2 on the macrophages because there is no
residual H2O2 present at the time when the
lymphoma cells are added to the macrophages; the
H2O2 is removed by cellular peroxidases and other consumption mechanisms within 2 hours after addition to the tumor
cells.17
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| Fig 7.
Effect of H2O2 on
chemotherapy-induced apoptosis and phagocytosis of Burkitt lymphoma
cells.
The Burkitt lymphoma cell lines JLP 119, BL-41, and ST-486 were treated
for 4 hours with VP-16 (8.5, 340, or 85 µmol/L,
respectively) in the presence or absence of 75 µmol/L
H2O2 and then subjected to phagocytosis by
human monocyte-derived macrophages, as described in "Materials and
methods." The fractions of cells dying by apoptosis,
pyknosis/necrosis, or typical necrosis are shown in the top row of
graphs. Percent phagocytosis for these cell populations is shown
in the lower row of graphs. The results represent the mean ± range or SD for 2 to 3 separate
experiments for JLP 119 and ST-486 cells. The data for BL-41 represent
1 experiment.
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Cells killed with H2O2 (with or without VP-16)
do ultimately undergo phagocytosis, but this occurs only after they
have lost their permeability barrier. As shown in Figure
8A, JLP 119 cells treated with 75 µmol/L
H2O2 remained mostly impermeable to PI during
the first 8 hours of incubation. After this time, PI permeability increased sharply, rising at a steady rate thereafter. However, a shift
in the rate of phagocytosis does not occur until 13 hours after adding
H2O2. A similar result was obtained when BL-41
cells were treated with H2O2; increased
phagocytosis followed the loss of membrane integrity (Figure 8B).
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| Fig 8.
Time course of lymphoma cell permeabilization and
phagocytosis following H2O2 treatment.
JLP 119 cells (A) or BL-41 cells (B) were treated with 75 or 250 µmol/L H2O2, respectively, and allowed to
incubate for the times indicated in the figure. Cells were then
harvested and incubated with monocyte-derived macrophages for 1 hour.
Percent phagocytosis (open circles) was analyzed as described in
"Materials and methods." Cell death was quantified by Hoechst/PI
staining. The total percentage of cells that had become permeable to PI
is shown (closed circles). Under these conditions, at least 82% of the
PI-permeable cells at each time point were pyknotic/necrotic. The
results for JLP 119 are averaged from 2 separate experiments, and those
for BL-41 are from a single experiment.
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Discussion |
These results demonstrate that H2O2
interferes with the ability of antineoplastic drugs to kill tumor
cells. This oxidant shifts the form of cell death from apoptosis to
pyknosis/necrosis, which occurs after a significant delay compared with
chemotherapy-induced apoptosis. H2O2 also
lowers the degree of cell killing by antineoplastic agents. This
finding provides a mechanism whereby cancer cells may escape killing
from chemotherapy agents. Tumors are often infiltrated with
inflammatory phagocytes, which can generate large levels of reactive
oxygen species within the tumor tissue. Tumor cells themselves have
also been found to generate oxidants,19 although the
Burkitt lymphoma cells used in these studies did not generate
significant levels of H2O217 (data
not shown). Cancer patients are reported to have higher levels of
generalized oxidative stress.10-15 Thus, some tumor tissues
may contain significant levels of reactive oxygen species.
H2O2 is pivotal among the array of oxidants
generated in vivo because it is produced by virtually every form of
oxidative stress (eg, by activated phagocytes, reperfusion of ischemic
tissues, ionizing radiation) and because it is required for the
generation of secondary reactive oxygen species (eg, the highly
reactive hydroxyl radical and hypochlorous acid). Our findings suggest
that if this H2O2 is present at the time of
administration of cancer chemotherapy, some neoplastic cells that would
otherwise be killed may survive drug treatment.
An important consequence of the effect of H2O2
on cell death was the delay of phagocytosis of the tumor cells by human
monocyte-derived macrophages. Cells treated with VP-16 undergo the
morphologic changes associated with apoptosis before losing the
membrane permeability barrier.16 These early apoptotic
cells are capable of being recognized and phagocytosed by
monocyte-derived macrophages within 4 hours of drug treatment. If the
cells are treated with VP-16 in the presence of
H2O2, this phagocytosis is lost even though the
cells have been lethally damaged; ie, they will go on to die by
pyknosis/necrosis over the course of an overnight incubation. Thus,
membrane-intact apoptotic cells are recognized and phagocytosed by
monocyte-derived macrophages, but membrane-intact pyknotic/necrotic cells are not. The finding that pyknotic/necrotic cells become phagocytosed after they have lost their membrane permeability barrier
may derive from macrophage recognition of intracellular or inner
membrane markers, which are exposed only upon cell lysis. To our
knowledge, the extent and time course of phagocytosis of nonapoptotic
cells have not been documented previously. Our results show that
pyknotic/necrotic cells are taken up only after they have undergone
drastic changes, unlike apoptotic cells, which are recognized early in
the death process. One potential physiologic ramification of this
finding is that cells killed in vivo in the presence of
H2O2 (with or without chemotherapy drugs) are
not phagocytosed until after they have begun to leak their contents into the extracellular space, thus allowing an inflammatory response to
ensue.18 This may induce a cycle of chronic inflammation and further interference with chemotherapy action.
If oxidants have the same effect on drug-induced apoptosis in vivo as
they do in vitro, then the overall effectiveness of chemotherapy might
be improved by coadministering antioxidants with the chemotherapeutic
agents. Support for this concept first came from a report by Chinery et
al,20 who found that administration of vitamin E and
pyrrolidinedithiocarbamate (PDTC) together with the chemotherapy agents
5-fluorouracil and doxorubicin enhanced the tumor-reduction capacity of
the drugs. In this model for colon cancer treatment, the proposed
mechanism of action for the antioxidants was significantly different
from that shown here because vitamin E and PDTC alone also induced
apoptosis in the colon tumor cells when tested in vitro. Under our
experimental conditions, the antioxidants that were effective in
preventing H2O2 from inhibiting
apoptosis Desferal, Tempol, and DMSOdid not by themselves induce any
cell death. In addition, we did not find inhibition of the effects of
H2O2 by 2 lipid-active compounds, trolox (a
vitamin E analog) and ebselen (which inhibits lipid peroxidation
reactions). Vitamin C was similarly ineffective, possibly because of
its inherent toxicity to these cells. Although Desferal and Tempol have
very different activity profiles in biologic systems, they share the
ability to prevent redox reactions of iron in the presence of
H2O2; Desferal is a potent iron
chelator21 and Tempol has the ability to keep iron in an
oxidized state, thus preventing catalysis of the Haber-Weiss reaction
with H2O2.22 Our finding that both
Desferal and Tempol prevented the effects of
H2O2 implies that free radicals mediate the
effects of H2O2. These results differ from but
are complimentary to those of Chinery et al,20 suggesting
that there may be different mechanisms whereby antioxidants can improve
the efficacy of chemotherapy. Additional animal-based studies to verify
the applicability of these findings in vivo are merited.
In the studies presented here, 4 different drugs were examined. With
all drugs, the mode of cell death was diverted away from apoptosis to
pyknosis/necrosis in the presence of 50 to 75 µmol/L H2O2. The drugs were VP-16, doxorubicin,
cisplatin, and AraC, which represent 4 different classes of
chemotherapeutic agents: a topoisomerase II inhibitor
(VP-1623), an anthracycline (doxorubicin24), a
bulky adduct (cisplatin25), and a metabolic inhibitor
(AraC26). Thus, H2O2 acts
independently of the mechanism of action of the drug; ie, it acts by
preventing downstream events initiated by the drugs and does not block
a specific action of any of the drugs. This is consistent with our
previous finding16 that H2O2 acts by depleting the cells of adenosine triphosphate (ATP), which is
required for apoptosis to proceed.27,28 In the absence of ATP, numerous markers of VP-16-induced apoptosis are blocked, including activation of caspases, oligonucleosomal DNA fragmentation, and exposure of phosphatidylserine on the cell surface.16
Other researchers have suggested that ATP must be maintained above a critical threshold level of approximately 25% to 50% of control steady-state levels for apoptosis to occur.29,30 Of note,
we have found that both Tempol and Desferal inhibit the
H2O2 (75 µmol/L)-induced depletion of
intracellular ATP, such that ATP levels are maintained at 60% or more
of control levels in the presence of these compounds (data not shown).
Desferal and Tempol act by inhibiting the induction of DNA
single-strand breaks by H2O2,31,32
thus preventing activation of poly(ADP)-ribosylation and the subsequent
depletion of ATP.33 The protection of ATP levels can
explain why Tempol and Desferal overcome the
antiapoptotic effects of H2O2 and may also
clarify why the residual cell death induced by
H2O2 alone in the presence of these compounds
is primarily apoptotic; that is, H2O2 by itself
has the capacity to induce apoptosis in Burkitt lymphoma cells, but
this apoptosis is prevented because of the irreversible drop in ATP
induced by concentrations of H2O2 greater than
50 µmol/L.16 By reducing the drop in ATP levels, Desferal
and Tempol allow apoptosis to occur.
| |
Acknowledgments |
We are grateful to Valerie Calvert (Food and Drug
Administration/Center for Biologics Evaluation and
Research) for supplying human monocytes. We also thank
Boon Chock and Giovanna Tosato for careful reading of the manuscript
and for many useful suggestions.
| |
Footnotes |
Submitted August 4, 1999; accepted March 3, 2000.
Supported by a grant from The Food and Drug Administration
Office of Women's Health.
Reprints: Emily Shacter, Bldg 29A, Rm 2A-11, 29 Lincoln Dr,
Bethesda, MD 20892; e-mail: shacter{at}cber.fda.gov.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction