Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1123-1124
CORRESPONDENCE
Induction of Apoptosis by Salicylates in B-Cell Chronic
Lymphocytic Leukemia
 |
LETTER |
To the Editor:
We have read with great interest the report by Bellosillo et
al1 demonstrating the induction of apoptosis in leukemic
cells by aspirin and salicylate. This finding could have important
implications for the understanding of the biology of chronic
lymphocytic leukemia (CLL) as well as for the design on new therapeutic
approaches for this disease. While the characterization of the
phenomenon is clear, including the involvement of caspases in the
effector phase, some points may deserve additional comments.
First, normal mononuclear cells (PBMC) were shown to be less sensitive
than frozen CLL B cells to apoptosis induction by salicylates. We
wonder whether the storage could have influenced the apoptosis resistance of the leukemic cells. In addition, if all experiments with
leukemic cells were done at 5 × 106 cells/mL, the high
cell density could have influenced (at least partly) their
susceptibility to apoptosis. Some additional data could have provided
additional insight about the relevance and extent of the susceptibility
reported by the authors, including the bcl-2 expression in the leukemic
cells and the study of the effect of salicylate on cells from patients
with lower cell counts.
A second point refers to aspirin and salicylate concentration required
to induce apoptosis, which are quite high (IC50
5.9 ± 1.13 mmol/L, and 6.96 ± 1.13 mmol/L, respectively), as
stated by Bellosillo et al. In the same reference they used (the
Goodman and Gilman textbook of pharmacology), the lower value for toxic concentration of salicylate is 200 µg/mL,2 making quite
improbable the use of these levels of aspirin or salicylate in the
clinical setting. These concentrations are above those required for
cyclooxigenase (COX) inhibition and the authors clearly demonstrate
that COX-independent mechanism(s) must be involved, although the sample
of other COX inhibitors used is too small to exclude all
nonsteroidal anti-inflammatory drugs (NSAIDs) of sharing similar
results. As stated below, some other targets of aspirin and salicyte,
besides COX, are also affected by several NSAIDs.
Third, some other molecular targets of salicylates have been identified
in the 100-year-long record of aspirin research, some of which may be
involved in the triggering or regulation of apoptosis. For instance,
nitric oxide is a mediator involved both in triggering or inhibition of
apoptosis, depending on the cell and mechanisms under evaluation,
although it is most frequently an inhibitor, through its effect on
caspases (see ref 3 and references therein for recent review).
Inducible nitric oxide synthase (iNOS) is inhibited by both aspirin and
salicylate at concentration near the values reported by Bellosillo et
al (IC50, 3 mmol/L and 20 mmol/L, respectively). The
effects are exerted at the level of translational/posttranslational
modification and directly on the catalytic activity of
iNOS.4
A potentially significant target is the mitochondrion, which plays a
central role in most models of apoptosis.5 Since the 1970s,
aspirin has been known to uncouple the respiratory chain6; it also inhibits enzymes such as NADH oxidase and cytochrome c oxidase.7 This effect associates with lipid peroxidation
and damage, probably by apoptosis.
Recently, the mitochondrial protein cytochrome c has deserved
substantial attention as a key component of the apoptosis machinery in
several models. The release of cytochrome c from the mitochondria as a
consequence of changes in their membranes has been shown to activate
procaspase 9 and other caspases downstream. The most characterized
change is the formation of a channel that includes several proteins of
the bcl-2 family (bcl-Xs, bax) and participates in the
regulation of Ca++, pH, or electric properties of the
matrix.8 Though controversial, this process is accompanied
by an important change in the permeability of the mitochondrial
membrane, referred to as permeability transition. Salicylate induces
mitochondrial permeability transition.9 This effect has
been involved in the pathophysiology of Reye's syndrome induced by
aspirin.10 Interestingly, several other NSAIDs also induce
changes in the permeability of mitochondrial membranes.11
In summary, the interesting report by Bellosillo et al deals with a
very central point in cell and cancer biology, and the effect of
salicylates they describe may have a significant impact on the way we
consider these drugs in therapy. However, we suggest that the
exploration of additional targets, in particular related to the effect
of salicylates on mitochondria, deserves further attention.
M. Teresa Guereño
Roberto A. Diez
Laboratorio
de Inmunofarmacología
Facultad de Medicina, UBA
Buenos
Aires, Argentina
| |
REFERENCES |
1.
Bellosillo B, Piqué M, Barragán M, Castaño E, Villamor N, Colomer D, Montserrat E, Pons G, Gil J:
Aspirin and salicylate induce apoptosis and activation of caspases in B-cell chronic lymphocytic leukemia cells.
Blood
92:1406, 1998[Abstract/Free Full Text]
2.
Insel PA:
Analgesis-antipyretic and anti-inflammatory agents and drugs employed in the treatment of gout, in
Hartman JG,
Limberd LE,
Molinoff PB,
Ruddon RW,
Goodman-Gilman A
(eds):
Goodman & Gilman's The Pharmacological Basis of Therapeutics. New York, NY, MacGraw-Hill, 1996, p 617.
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Proteases to die for.
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12:1551, 1998[Free Full Text]
4.
Amin AR, Vyas P, Attur M, Leszczynska-Piziak J, Patel IR, Weissmann G, Abramson SB:
The mode of action of aspirin-like drugs: Effect on inducible nitric oxide synthase.
Proc Natl Acad Sci USA
92:7926, 1995[Abstract/Free Full Text]
5.
Kroemer G, Dallaporta B, Resche-Rigon M:
The mitochondrial death/life regulator in apoptosis and necrosis.
Annu Rev Physiol
60:619, 1998[Medline]
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6.
Pachman LM, Esterly NB, Peterson RD:
The effect of salicylate on the metabolism of normal and stimulated human lymphocytes in vitro.
J Clin Invest
50:226, 1971
7.
Jorgensen TG:
Ulcer formation and histochemical changes in rat-stomach mucosa induced by acetylsalicylic acid.
Acta Pathol Microbiol Scand [A]
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8.
Zammami N, Brenner C, Marzo I, Susin SA, Kroemer G:
Subcellular and submitochondrial mode of action of the bcl-2-like oncoproteins.
Oncogene
16:2265, 1998[Medline]
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9.
Trost LC, Lemasters JJ:
The mitochondrial permeability transition: A new pathophysiological mechanism for Reye's syndrome and toxic liver injury.
J Pharmacol Exp Ther
278:1000, 1996[Abstract/Free Full Text]
10.
Trost LC, Lemasters JJ:
Role of the mitochondrial permeability transition in salicylate toxicity to cultured rat hepatocytes: Implications for the pathogenesis of Reye's syndrome.
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11.
Uyemura SA, Santos AC, Mingatto FE, Jordani MC, Curti C:
Diclofenac sodium and mefenamic acid: Potent inducers of the membrane permeability transition in renal cortex mitochondria.
Arch Biochem Biophys
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Response
We thank Drs Guereño and Díez for their interest in our
study1 and we appreciate the opportunity to respond to
their thoughtful comments. For our experiments we used frozen normal peripheral blood mononuclear cells (PBMC). In this regard they raise
the possibility that a "selection" effect may take place as a
consequence of freezing. We have observed that a fraction of cells die
during freezing (1% to 10% of B-CLL cells and 10% to 15% of PBMC),
but we believe that this fraction is too low to explain the difference
in sensitivity of PBMC and B-CLL cells to aspirin by a
"selection" effect. Furthermore, B and T lymphocytes from normal
donors and T lymphocytes from B-CLL patients are more resistant than
B-CLL cells to aspirin-induced apoptosis, as determined by analysis of
phosphatidylserine exposure by flow cytometry, where percentages are
referred to the total number of cells analyzed. Moreover, cell density
was 5 × 106 cells/mL in cell viability assayed by the
MTT method, whereas for analysis of apoptosis cells were
incubated at densities of 2 to 4 × 106 cells/mL and
sensitivities to aspirin similar to those obtained by the MTT assay
were found for each patient. Therefore, we consider that cell density
does not influence susceptibility to aspirin-induced apoptosis.
Regarding the mechanism of aspirin-induced apoptosis, aspirin did not
modify Bcl-2 protein level (unpublished results, July 1997). As discussed in our study, inhibition of
cyclooxygenase (COX) is not sufficient to induce apoptosis in B-CLL
cells. We proposed several COX-independent mechanisms that may be
involved in aspirin-induced apoptosis in B-CLL cells (inhibition of
NF
B, inhibition of AP-1, activation of p38), but we agree that
additional mechanisms should also be considered. In this respect,
the mechanisms proposed by Drs Guereño and Díez
(induction of mitochondrial permeability transition and inhibition of
nitric oxide synthase) deserve investigation. Interestingly, it has
recently been described that B-CLL cells spontaneously express a
functional inducible nitric oxide synthase, which has an anti-apoptotic
role.2 Experiments are in progress to study the mechanisms
involved in the apoptotic action of aspirin and salicylate in B-CLL
cells.
Beatriz Bellosillo
Joan Gil
Department de
Ciències Fisiològiques II
Campus de Bellvitge
Universitat de Barcelona
Barcelona, Spain
| |
REFERENCES |
1.
Bellosillo B, Piqué M, Barragán M, Castaño E, Villamor N, Colomer D, Montserrat E, Pons G, Gil J:
Aspirin and salicylate induce apoptosis and activation of caspases in B-cell chronic lymphocytic leukemia cells.
Blood
92:1406, 1998
2.
Zhao H, Dugas N, Mathiot C, Delmer A, Dugas B, Sigaux F, Kolb JP:
B-cell chronic lymphocytic leukemia cells express a functional inducible nitric oxide synthase displaying anti-apoptotic activity.
Blood
92:1031, 1998[Abstract/Free Full Text]
