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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1406-1414
Aspirin and Salicylate Induce Apoptosis and Activation of Caspases in
B-Cell Chronic Lymphocytic Leukemia Cells
By
Beatriz Bellosillo,
Maria Piqué,
Montserrat Barragán,
Esther Castaño,
Neus Villamor,
Dolors Colomer,
Emilio Montserrat,
Gabriel Pons, and
Joan Gil
From the Departament de Ciències Fisiològiques II, Campus
de Bellvitge, Universitat de Barcelona, L'Hospitalet, Barcelona,
Spain; and the Unitat d'Hematopatologia and Servei d'Hematologia,
Institut d'Investigacions Biomèdiques August Pi i Sunyer
(IDIBAPS), Hospital Clínic, Barcelona, Spain.
 |
ABSTRACT |
We analyzed the effect of aspirin, salicylate, and other
nonsteroidal antiinflammatory drugs (NSAIDs) on the viability of B-chronic lymphocytic leukemia (B-CLL) cells. Aspirin induced a
decrease in cell viability in a dose- and time-dependent manner. The
mean IC50 for cells from 5 patients was 5.9 ± 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In some cases, 2.5 mmol/L aspirin produced
an important cytotoxic effect after 4 days of incubation. No effect was
observed with other NSAIDs, at concentrations that inhibit
cyclooxygenase, such as ketorolac (10 µmol/mL), NS-398 (100 µmol/mL), or indomethacin (20 µmol/mL), thus suggesting the involvement of cyclooxygenase-independent mechanisms in aspirin-induced cytotoxicity. Salicylate also produced dose-dependent cytotoxic effects
on B-CLL cells and the mean IC50 for cells from 5 patients was 6.96 ± 1.13 mmol/L (range, 5 to 7.8 mmol/L). Both aspirin and
salicylate induced DNA fragmentation and the proteolytic cleavage of
poly(ADP(adenosine 5 -diphosphate)-ribose) polymerase (PARP), demonstrating that both compounds induce apoptosis of B-CLL cells. Finally, inhibition of caspases by Z-VAD.fmk blocked proteolytic cleavage of PARP, DNA fragmentation, and cytotoxicity induced by
aspirin. Mononuclear cells from normal donors showed a lower sensitivity than cells from B-CLL patients to aspirin as determined by
analysis of cell viability. B and T lymphocytes from normal donors and
T lymphocytes from CLL patients are more resistant to aspirin-induced
apoptosis, as determined by analysis of phosphatidylserine exposure.
These results indicate that aspirin and salicylate induce apoptosis of
B-CLL cells by activation of caspases and that this activation involves
cyclooxygenase-independent mechanisms.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
B-CELL CHRONIC lymphocytic leukemia
(B-CLL) is characterized by the accumulation of monoclonal
CD5+ B lymphocytes.1,2 Most circulating cells
appear to be arrested at the G0 phase of the cell cycle and
it has been suggested that the clonal excess of B cells results from
decreased apoptosis rather than increased proliferation.3
Apoptosis is the physiological process whereby most cells, including B
lymphocytes, are eliminated, which leads to homeostasis.4 The evidence obtained in recent years indicates that many cancer chemotherapy agents induce apoptosis of tumor cells.5
Glucocorticoids and other chemotherapeutic agents, such as
chlorambucil, 2-chloro-2-deoxyadenosine, fludarabine,
camptothecin, and mitoxantrone,6-11 induce apoptosis in
B-CLL lymphocytes, suggesting that apoptosis is one of the mechanisms of their therapeutic action.
Cysteine-proteases of the CED-3/ICE family, now named caspases, play an
important role in apoptosis.12-14 Some of these proteases can cleave and inactivate poly(ADP-ribose) polymerase (PARP), and this
cleavage can be used as a marker of activation of caspases and
apoptosis. We recently reported that drug-induced apoptosis of B-CLL
cells involves the activation of caspases.15
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to induce
apoptosis of v-src-transformed chicken embryo fibroblasts and
different human cell lines.16-20 The mechanism responsible for this apoptotic effect of NSAIDs is not clear. Aspirin and other
NSAIDs directly target cyclooxygenase (COX),21 a key enzyme in the production of prostaglandins, prostacyclins, and
thromboxanes.22 There are 2 COX isoenzymes, COX-1 and
COX-2, which differ in their expression regulation and tissue
distribution23 and could be selectively inhibited. Although
COX is the only molecular target known of most NSAIDs, both
COX-dependent and COX-independent mechanisms in the apoptotic action of
NSAIDs have been reported.17,24,25
In the present study, we have analyzed the effect of aspirin,
salicylate, and other NSAIDs on the viability of B-CLL cells and
whether these drugs induce apoptosis of these leukemic cells.
| |
MATERIALS AND METHODS |
Patients.
Seven patients (1 man and 6 women) with B-CLL who had not received
treatment, with a median age of 75 years (range, 63 to 78 years), were
studied. B-CLL was diagnosed according to standard clinical and
laboratory criteria. The median peripheral blood leukocytosis was 85 × 109 leukocytes/L (range, 34 to 194 × 109). Leukemic cells were phenotyped for cell surface
markers by flow cytometry and were positive in all cases for CD5 and
CD19. According to Binet's classification,26 at the time
of inclusion, 5 patients were at stage A, 1 patient was at stage B, and
1 patient was at stage C.
Isolation of B-CLL cells.
Mononuclear cells from peripheral blood samples were isolated by
centrifugation on a Ficoll/Hypaque (Seromed, Berlin, Germany) gradient
and cryopreserved in liquid nitrogen in the presence of 10% dimethyl
sulfoxide (DMSO).
Reagents.
Aspirin (acetylsalicylic acid), sodium salicylate, indomethacin,
3,(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), and
propidium iodide (PI) were obtained from Sigma Chemicals Co (St Louis,
MO). N-benzyloxycarbonyl-Val-Ala-Asp-fluromethyl ketone (Z-VAD.fmk) was
obtained from Enzyme Systems Products (Dublin, CA). Ketorolac was
kindly provided by Almirall Laboratories (Barcelona, Spain).
N-(2-ciclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398) was
obtained from BIOMOL Research Laboratories, Inc (Plymouth Meeting, PA).
Stock solutions of aspirin and indomethacin in absolute ethanol, NS-398
in DMSO, and sodium salicylate and ketorolac in double-distilled water
were freshly prepared for each experiment. The final concentrations of
ethanol and DMSO did not affect cell viability (data not shown).
Anti-PARP polyclonal antibody (Vi.5) raised against the recombinant
human PARP overproduced in Sf9/baculovirus was kindly provided by Dr
Gilbert de Murcia (Strasbourg, France).
Cell culture.
B-CLL lymphocytes were cultured immediately after the thawing of the
cells at a concentration of 5 × 106 cells/mL in RPMI
1640 culture medium (GIBCO-BRL, Paisley, UK) supplemented with 10%
heat-inactivated fetal calf serum (FCS), 2 mmol/L glutamine, and 0.04 mg/mL gentamicin at 37°C in a humidified atmosphere containing 5%
carbon dioxide.27 Factors were added at the beginning of
the culture.
Cell viability assay.
Cell viability was determined by the MTT assay.28 B
lymphocytes (5 × 105 cells/well) were incubated in
96-well plates in the absence or in the presence of factors in a final
volume of 100 µL. After 48 hours, 10 µL of MTT (5 mg/mL in
phosphate-buffered saline) was added to each well for an additional 6 hours. The blue MTT formazan precipitated was dissolved in 100 µL of
isopropanol:1 mol/L HCl (24:1) and the absorbance values at 550 nm were
determined on a multiwell plate reader.
Western blot analysis of PARP cleavage.
Cells were lysed with Laemmli sample buffer29 and protein
extracts were electrophoresed on 10% polyacrylamide gel and
transferred to Immobilon-P (Millipore, Bedford, MA) membranes. After
blocking for 1 hour with 5% dried skimmed milk in TBST (50 mmol/L Tris HCl, pH 8.0, 150 mmol/L NaCl, 0.5% Tween-20), the filters were incubated with Vi.5 PARP antibody diluted 1:1,000 in 5% dried skimmed
milk in TBST, which recognizes both the native enzyme (116 kD) and the
cleavage product (~85 kD).15 Antibody binding was
detected using a secondary antibody (swine antirabbit Ig; DAKO,
Glostrup, Denmark) conjugated to horseradish peroxidase diluted 1:500
in 5% dried skimmed milk in TBST and an enhanced chemiluminescence
(ECL) detection kit (Amersham, Buckinghamshire, UK). As an internal
control of total cellular protein levels, blots were also probed with
 -tubulin antibody (Oncogene Science, Inc, Cambridge, MA).
Analysis of DNA fragmentation.
Analysis of DNA fragmentation by agarose gel electrophoresis was
performed as previously described.15 Five million cells were pelleted and lysed for 20 minutes at 4°C in 10 mmol/L
Tris-HCl, pH 7.4, 0.2% Triton X-100, 1 mmol/L EDTA. After
centrifugation at 14,000g for 15 minutes, the supernatant was
treated with 0.2 mg/mL of proteinase K in a buffer containing 150 mmol/L NaCl, 10 mmol/L Tris HCl, pH 8.0, 40 mmol/L EDTA, and 1% sodium
dodecyl sulfate (SDS) for 6 hours at 37°C. DNA was
extracted twice with buffered phenol and precipitated with 140 mmol/L
NaCl and 2 vol of ethanol at  20°C overnight. DNA
precipitates were washed twice in 70% ethanol, dissolved in
double-distilled water, and treated for 1 hour at 37°C with RNase
(Boehringer Mannheim, Mannheim, Germany). Finally, DNA preparations
were electrophoresed in 1% agarose gels. Gels were stained with
ethidium bromide and visualized under UV light.
PI DNA staining.
Quantification of apoptosis by PI staining and fluorescence-activated
cell sorting (FACS) analysis was performed as described previously.30 Briefly, cells were harvested and fixed in
70% ethanol. Cells were centrifuged, washed in phosphate-buffered saline (PBS), and resuspended in 0.5 mL PBS containing PI (5 µg/mL) and RNase (100 µg/mL). Tubes were incubated for 30 minutes at 37°C and placed at 4°C in the dark overnight before flow
cytometry analysis to identify the sub-G0 peak
corresponding to apoptosis.
Analysis of apoptosis by annexin binding.
Exposure of phosphatidylserine was quantified by surface annexin V
staining as described previously.31 One
million cells were incubated during 24 hours with the indicated
factors. After that time, cells were washed with PBS and incubated with
phycoerythrin-conjugated anti-CD19 (DAKO) and tri-color-conjugated
anti-CD2 (Caltag Laboratories, Burlingame, CA) for 15 minutes in the
dark. Cells were then washed, resuspended in 200 µL of binding buffer
(10 mmol/L HEPES, pH 7.4 , 2.5 mmol/L CaCl2, 140 mmol/L
NaCl), and incubated with 0.5 µg/mL of ApoAlert Enhanced Annexin
V-FITC (Clontech Laboratories Inc, Palo Alto, CA) for 5 to 15 minutes
in the dark, before analysis by flow cytometry (FACScan;
Becton Dickinson, Mountain View, CA). Samples were
acquired using Lysis-II software and data were analyzed with the
Paint-a-gate Pro software (Becton Dickinson). To analyze a sufficient
number of cells, a live-gate in side scatter (SSC) versus CD19 or SSC
versus CD2 was drawn and at least 5,000 CD19+ cells or
CD2+ cells were acquired.
Statistical analysis.
Levels of significance between samples were determined using the
t-test for nonpaired samples and the analysis of variance (ANOVA), Fisher's protected least significant difference (PLSD). To
compare the effect of aspirin on normal PBMCs and B-CLL cells, we
performed a multiple analysis of variance (MANOVA), Hottelling T2.
| |
RESULTS |
Cytotoxic effect of aspirin on B-CLL lymphocytes.
First, the effect of aspirin on the viability of B-CLL lymphocytes was
studied. Cells from 5 patients were incubated for 48 hours with several
doses of aspirin, ranging from 1 to 10 mmol/L, and cell viability was
determined by the MTT assay. As seen in Fig 1A, a dose-dependent
decrease in cell viability could be observed in all the cases studied,
although cell sensitivity to aspirin differed from one patient to
another. The mean IC50 of these dose-response studies was
5.9 ± 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In 3 patients
(patients no. 1, 4, and 5), an effect was detected with 2.5 mmol/L
aspirin.
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| Fig 1.
Cytotoxic effect of aspirin on B-CLL cells. (A)
Dose-response of the cytotoxic effect of aspirin on B-CLL cells. Cells
from 5 patients were incubated for 48 hours with various concentrations of aspirin that ranged from 1 to 10 mmol/L. (B) Time course of aspirin-induced cytotoxicity. Cells from patients no. 1 and 2 were
incubated with 2.5, 5, and 10 mmol/L aspirin (ASA) for the times
indicated. Cell viability was determined by the MTT assay as described
in the Materials and Methods and is expressed as a percentage of the
viability of control cells at the beginning of the culture. Data are
shown as the mean value ± SD of 3 independent experiments.
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The cytotoxicity of aspirin on B-CLL lymphocytes was further studied
with time-course assays that were performed with cells from patients
no. 1 and 2. Cells were incubated without or with aspirin (2.5, 5, or
10 mmol/L) for different periods of time, as indicated in Fig 1B. A
significant decrease in cell viability was observed in both patients
after only 24 hours of incubation with 10 mmol/L aspirin (P < .003). A dose of 5 mmol/L aspirin produced a significant decrease in
cell viability of lymphocytes from patient no. 1 after 24 hours of
incubation (P = .006) that increased with time of incubation.
No significant effect was seen with this dose in cells from patient no.
2 after 24 hours (P = .29), and longer periods of incubation
were needed to achieve an important cytotoxic effect. Incubation for 48 hours with 2.5 mmol/L aspirin induced cytotoxicity in cells from
patient no. 1, but the viability of cells from patient no. 2 was not
significantly decreased after 96 hours of incubation.
COX inhibition is not sufficient to induce apoptosis of B-CLL cells.
Because aspirin targets the enzyme COX, we tested whether other
inhibitors of this enzyme could produce cytotoxic effects on B-CLL
lymphocytes. An inhibitor of both COX-1 and COX-2 (indomethacin), a
specific inhibitor of COX-1 (ketorolac), and a specific inhibitor of
COX-2 (NS-398) were tested. Neither COX-1 nor COX-2 inhibitors produced
any effect on cell viability with doses up to 10 µmol/L ketorolac, 20 µmol/L indomethacin, and 100 µmol/L NS-398, which have been
previously described to inhibit COX32-36
(Fig 2). Furthermore, we used ketorolac and
NS-398 together, and no effect was observed either. All these assays
were performed with cells from 5 patients and in all cases there were
no effects. However, at the concentrations used in these experiments,
these NSAIDs inhibited the production of prostaglandin E2
(PGE2) by B-CLL cells, thus demonstrating that
COX was being inhibited (data not shown). Taken together, these results
indicate that inhibition of COX is not sufficient to induce the
decrease in cell viability produced by aspirin on B-CLL lymphocytes.
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| Fig 2.
Effect of COX inhibitors on the viability of B-CLL
lymphocytes. Cells were incubated for 48 hours with 5 mmol/L aspirin,
10 µmol/L ketorolac, 100 µmol/L NS-398, or 20 µmol/L
indomethacin. Cell viability was determined by the MTT assay as
described in the Materials and Methods and is expressed as a percentage
of the viability of control cells at the beginning of the culture. Data
are shown as the mean value ± SD of 3 independent experiments. Statistical significance of differences between control cells and cells
treated with COX inhibitors was assayed by ANOVA, Fisher's PLSD.
**P < .0001.
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Cytotoxic effect of salicylate on B-CLL lymphocytes.
To test if salicylate, a structure-related compound of aspirin,
produced any effect on cell viability, dose-response studies were
performed with 1 to 20 mmol/L sodium salicylate. As seen in
Fig 3, this substance produced cytotoxic
effects on B-CLL cells when concentrations of 5 to 10 mmol/L were used.
This effect was observed in cells from 5 patients studied. The mean
IC50 for these 5 patients was 6.96 ± 1.13 mmol/L
(range, 5 to 7.8 mmol/L).
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| Fig 3.
Dose-response of the cytotoxic effect of salicylate on
B-CLL cells. Cells from 5 patients were incubated for 48 hours with various concentrations of sodium salicylate as indicated. Cell viability was determined by the MTT assay as described in the Materials
and Methods and is expressed as a percentage of the viability of
control cells at the beginning of the culture. Data are shown as the
mean value ± SD of 3 independent experiments.
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Aspirin and salicylate induce apoptosis and activation of caspases in
B-CLL lymphocytes.
To determine whether this cytotoxic effect was due to induction of
apoptosis, we analyzed whether incubation with aspirin or salicylate
induced DNA fragmentation and proteolytic cleavage of PARP, a hallmark
of apoptosis in most cells, including B-CLL cells.15 Cells
from patients no. 1 and 2 were incubated with various doses of aspirin
as described previously, ranging from 1 to 10 mmol/L. As seen in
Fig 4, both DNA fragmentation (Fig 4A) and
PARP cleavage (Fig 4B) were dose-dependent and a clear effect could be
observed with 2.5 to 5 mmol/L aspirin. These doses corresponded to the
doses that induced a cytotoxic effect as previously determined by the
MTT assay. Similar results were obtained with cells from patients no. 3 and 4. The apparent decrease in the total amount of PARP in the Western
blot analysis of patient no. 1 at the doses of 5 and 7.5 mmol/L is due to a decrease in the total amount of loaded protein, as
demonstrated by immunoblotting of -tubulin.
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| Fig 4.
Induction of apoptosis by aspirin on B-CLL cells. (A)
Effect of aspirin on DNA fragmentation in B-CLL cells. Cells from
patients no. 1 and 2 were incubated for 24 hours with 1 to 10 mmol/L
aspirin (ASA). DNA was extracted and subjected to agarose gel
electrophoresis as described in the Materials and Methods. (B) Effect
of aspirin on PARP cleavage. Cells from patients no. 1 and 2 were
incubated for 48 hours with 1 to 10 mmol/L aspirin (ASA). PARP cleavage was analyzed on protein extracts from these cells by Western blot as
described in the Materials and Methods. The position of the native PARP
(116 kD) and the proteolytic fragment (85 kD) is indicated. As an
internal control, blots were also probed with -tubulin antibody. (C)
Effect of aspirin on DNA content in a representative CLL patient. Cells
from patient no. 1 were incubated for 48 hours with 1 to 10 mmol/L
aspirin (ASA). DNA content was quantified by PI staining and flow
cytometry analysis as described in the Materials and Methods. (D)
Quantification of aspirin-induced apoptosis in cells from 4 patients by
PI staining and FACS analysis after 48 hours of incubation with 1 to 10 mmol/L aspirin.
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Furthermore, we corroborated these results measuring apoptosis by PI
staining and FACS analysis. As shown in Fig 4C and D, aspirin induced
in a dose-dependent manner an increase in the percentage of subdiploid
cells in 4 patients studied.
As seen in Fig 5, salicylate also induced
DNA fragmentation and cleavage of PARP when cells were incubated with
the doses that produced cytotoxic effects. Similar results to that
shown in Fig 5 were obtained with cells from 2 additional patients
(results not shown). These results demonstrate that the cytotoxicity of both aspirin and salicylate is due to apoptosis induction.
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| Fig 5.
Induction of apoptosis by salicylate on B-CLL
cells. (A) Effect of salicylate on DNA fragmentation in B-CLL cells.
Cells from patients no. 3 and 4 were incubated for 24 hours with 5 and
10 mmol/L sodium salicylate (NaSal). DNA was extracted and subjected to
agarose gel electrophoresis as described in the Materials and Methods.
(B) Effect of salicylate on PARP cleavage. Cells from patients no. 3 and 4 were incubated for 48 hours with 5 and 10 mmol/L sodium
salicylate (NaSal). PARP cleavage was analyzed on protein extracts from
these cells by Western blot as described in the Materials and Methods.
The position of the native PARP (116 kD) and the proteolytic
fragment (85 kD) is indicated.
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To demonstrate the involvement of activation of caspases in the
apoptotic effect of aspirin, we studied whether the caspase inhibitor
Z-VAD.fmk prevented aspirin-induced apoptosis. We incubated B-CLL cells
with aspirin in the presence or absence of 200 µmol/L Z-VAD.fmk, a
concentration that inhibits apoptosis of B-CLL cells.15 This concentration of Z-VAD.fmk had no cytotoxic effects (results not
shown) and prevented the cytotoxic effect of both 5 and 10 mmol/L
aspirin in the 3 cases studied, 2 of which are shown in Fig 6. Furthermore, Z-VAD.fmk
completely blocked aspirin-induced DNA fragmentation
(Fig 7A) and PARP cleavage (Fig 7B).
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| Fig 6.
Effect of the caspase inhibitor Z-VAD.fmk on the
viability of B-CLL cells. Cells from patients no. 2 and 6 were seeded
in 96-microwell plates and incubated for 48 hours either alone or in
the presence of aspirin (ASA; 5 or 10 mmol/L) or aspirin with 200 µmol/L Z-VAD.fmk. Z-VAD.fmk was added 1 hour before aspirin administration. Cytotoxicity was analyzed by the MTT method as described in the Materials and Methods and is expressed as a percentage of the viability of control cells at the beginning of the culture. Data
are shown as the mean value ± SD of 3 independent experiments. Statistical significance of differences between treatment with aspirin
alone or in combination with Z-VAD.fmk was assayed ANOVA (Fisher's
PLSD). *P < .03; **P < .0001.
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| Fig 7.
Effect of the caspase inhibitor Z-VAD.fmk on
aspirin-induced apoptosis in B-CLL cells. (A) Effect of Z-VAD.fmk on
aspirin-induced DNA fragmentation. B lymphocytes from 2 B-CLL patients
were incubated for 24 hours with 10 mmol/L aspirin (ASA) in the
presence or absence of 200 µmol/L Z-VAD.fmk. DNA was extracted and
subjected to agarose gel electrophoresis as described in the Materials
and Methods. (B) Effect of Z-VAD.fmk on aspirin-induced PARP cleavage.
Cells from patients no. 2 and 6 were incubated for 24 hours with 10 mmol/L aspirin (ASA) in the presence or absence of 200 µmol/L Z-VAD.fmk. Cells were lysed and analyzed by Western blot as described in the Materials and Methods. Z-VAD.fmk was added 1 hour before aspirin
administration.
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Aspirin effect on normal peripheral blood mononuclear cells (PBMCs).
Finally, we studied the cytotoxic effects of aspirin in normal PBMCs.
Incubation of PBMCs with aspirin produced no significant cytotoxic
effect (P > .12) with doses up to 7.5 mmol/L
(Fig 8A). The mean aspirin IC50
value for normal cells was 15.8 ± 6.2 mmol/L (range, 9.8 to 26.3 mmol/L). The comparison between the dose-responses of the cytotoxic
effect of aspirin on B-CLL cells and normal PBMCs indicates that normal
PBMCs are more resistant to aspirin (P = .003). A similar
dose-dependency was found when DNA fragmentation and PARP cleavage were
analyzed. The concentration of aspirin or salicylate necessary to
induce detectable DNA fragmentation and PARP cleavage was 7.5 mmol/L
(results not shown).
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| Fig 8.
Effect of aspirin on normal PBMCs and B cells. (A)
Dose-response of the cytotoxic effect of aspirin on normal PBMCs. Cells from 6 normal donors were incubated with various concentrations of
aspirin as indicated for 48 hours. Data are presented as the means ± SD of 3 experiments each for all 6 normal donors and compared with the
means of the 5 B-CLL patients represented in Fig 1A. Cell viability was
determined by the MTT assay as described in the Materials and Methods
and is expressed as a percentage of the viability of control cells at
the beginning of the culture. (B) Comparison between the induction of
apoptosis in B cells and T cells from B-CLL patients and normal donors.
Cells were incubated with 1 to 10 mmol/L aspirin for 24 hours and
phosphatidylserine exposure was measured by binding of annexin V to
CD19+ or CD2+ cells as described in the
Materials and Methods. Statistical significance was determined using
the t-test for nonpaired samples. *P < .05;
**P < .01; ***P < .001.
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Because most normal PBMCs are T lymphocytes, it was interesting to
distinguish the effects of aspirin on B and T cells. To specifically
analyze the induction of apoptosis in normal B cells by aspirin, we
quantified phosphatidylserine exposure in response to aspirin in
CD19+ cells. As seen in Fig 8B, aspirin did not induce a
significant increase in the apoptosis of normal B cells with doses up
to 2.5 mmol/L (P > .13); in contrast, aspirin induced
apoptosis of B-CLL cells at 2.5 mmol/L, indicating that B cells from
normal donors have a lower degree of sensitivity to aspirin than that
of B-CLL cells. Furthermore, T cells from both normal donors and B-CLL patients appeared to be more resistant to aspirin-induced apoptosis than B-CLL cells (Fig 8B).
| |
DISCUSSION |
The results shown in this report demonstrate that aspirin and its
metabolite salicylate induce apoptosis of B-CLL cells. This is the
first report showing apoptotic action of NSAIDs in human primary tumor
cells.
NSAIDs have been shown to induce apoptosis of human colon carcinoma and
in vitro transformed colon adenoma cell lines.17-20 Furthermore, different NSAIDs cause apoptosis in v-src-transformed chicken embryo fibroblasts16 and sulindac sulfide induces
apoptosis of promyelocytic leukemia cell line HL-60.18 It
has been reported that aspirin, in contrast to other NSAIDs, does not
induce apoptosis in either HT-29 colon carcinoma cells or
v-src-transformed chicken fibroblasts.16,20 The
discrepancy between these results and our results could be explained by
the fact that the doses used in previous studies were too low (150 µmol/L and 1.5 mmol/L). Very recently, it has been reported that
aspirin induces apoptosis of HT-29 cells at concentrations higher that
1 mmol/L.37
The mechanisms by which NSAIDs induce apoptosis are not clear. In
agreement with the hypothesis that the apoptotic action of NSAIDs is
mediated by the inhibition of COX, overexpression of COX-2 in rat
epithelial intestinal cells inhibits butyrate-induced apoptosis and
this inhibition was reversed by the NSAID sulindac sulfide.24 However, the sulfone metabolite of sulindac,
which does not inhibit cyclooxygenases,38 also induces
apoptosis of HT-29 cells,17 and treatment with different
prostaglandins failed to reverse the effects of NSAIDs on
apoptosis.25 Considering the results herein reported, two
different arguments indicate the involvement of
cyclooxygenase-independent mechanisms in the apoptotic action of
aspirin and salicylate on B-CLL cells. (1) Neither COX-1 nor COX-2
inhibitors induced cytotoxicity in B-CLL cells. (2) Although aspirin is
a much more potent inhibitor of COX than salicylate, both drugs show
similar potency as inducers of apoptosis. Taken together, these results
suggest the existence of COX-dependent and COX-independent mechanisms
in the apoptotic action of NSAIDs. Interestingly, we recently
demonstrated that aspirin inhibits DNA synthesis in Swiss 3T3
fibroblasts by both COX-dependent and COX-independent mechanisms,
depending on the dose used.39
COX-independent mechanisms have been proposed to explain the inhibition
of the transcription factor NF- B by aspirin and
salicylate.40-43 It is noteworthy that inhibition of
NF-B causes apoptosis of B lymphocytes44,45 and that
NF-B has an essential role in preventing tumor necrosis factor-
(TNF-)- and cancer therapy-induced apoptosis.46-49
Another COX-independent mechanism induced by aspirin and salicylate is
the inhibition of AP-1 activity, which may involve the elevation of
H+ concentration.50 In addition, it was very
recently reported that salicylate induces apoptosis of fibroblasts via
p38 mitogen-activated protein kinase,51 and this kinase has
been involved in mIgM-induced apoptosis of human B
lymphocytes.52 The results presented in this report
demonstrate that aspirin-induced apoptosis of B-CLL cells involves the
activation of caspases, as previously described for glucocorticoid-,
fludarabine-, and mitoxantrone-induced apoptosis.11,15 The
elucidation of the mechanisms involved in the apoptotic action of
aspirin and salicylate in B-CLL cells needs further investigation.
The finding that salicylate also induces apoptosis of B-CLL cells is
important, because aspirin is substantially hydrolyzed to salicylate on
first pass through portal circulation. Consequently, plasma
concentrations of salicylate rapidly exceed plasma concentrations of
aspirin after oral administration of aspirin, and the half-life of
salicylate in the plasma is considerably longer than that of aspirin.53 Although we only observe effects of aspirin and
salicylate on apoptosis of B-CLL cells at relatively high
concentrations (1.5 to 10 mmol/L), the lower range of such
concentrations is achieved and even maintained in the treatment of
chronic inflammatory states such as rheumatoid arthritis, in which the
therapeutic plasma concentration is 150 to 300 mg/L (0.94 to 1.88 mmol/L).54-57 Unfortunately, not all patients are able to
tolerate the adequate dosage to maintain these plasma levels for a long
time.57 On the other hand, normal peripheral blood
mononuclear cells are more resistant to aspirin than B-CLL cells, thus
suggesting that clinical use of aspirin at concentrations that induce
apoptosis of B-CLL cells could have only minor effects on normal
lymphocytes in certain patients.
Several epidemiological studies have demonstrated an association
between the long-term consumption of aspirin and a reduced risk of
colon cancer.58-61 Although no association with reduced risk of other types of cancer has been clearly
demonstrated,61 the incidence of B-CLL in cohorts of
individuals taking aspirin on a regular basis has not been analyzed.
This issue, as well as whether there is a potential role for aspirin in
B-CLL management, deserves investigation. Finally, the elucidation of
the mechanisms underlying the induction of the apoptosis of B-CLL cells
by aspirin and salicylate would provide important information to
understand the biology of B-CLL and to design new strategies for its
therapy.
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted April 17, 1998.
B.B., M.P., M.B., and E.C. are recipients of a research fellowship from
Fundación de la Asociación Española Contra el
Cáncer, CIRIT, Fundació August Pi i Sunyer, and Universitat
de Barcelona, respectively. This work was supported by "Fondo de
Investigaciones Sanitarias de la Seguridad Social" (FIS 95/0873), by
Química Farmacéutica Bayer S.A. (Division Consumer Care),
and by the "Generalitat de Catalunya" (1995 SGR 00427).
Address reprint requests to Joan Gil, PhD, Departament de
Ciències Fisiològiques II, Campus de Bellvitge, Universitat
de Barcelona, Pavelló de Govern, 4a planta, 08907 L'Hospitalet, Spain; e-mail: joangil{at}bellvitge.bvg.ub.es.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are indebted to Dr G. de Murcia for the kind gift of PARP
antibodies. We thank Drs V. Jiménez and J. Ros for the
determinations of PGE2 levels. We thank Drs R. Bartrons, C. Pastor, and P. Feliz for their help and suggestions and R. Rycroft for
language assistance.
| |
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Abstract
Introduction
Abstract