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[630]Sildenafil (Viagra®) Induces Apoptosis of B-CLL

Cells.

Helene Merle-Beral, Véronique Matéo, Sylvie Baudet,

Rubio, Fernandez, Jacques-Louis

Binet, Marika Sarfati. Hematology, Pitié-Salpêtrière,

Paris, France; Immunology and Inflammation, Institut

Pasteur, Paris, France; Pharmacy, Pitié-Salpêtrière,

Paris, France

We and others have previously shown that theophylline,

an inhibitor of type 4 cyclic adenosine monophosphate

(cAMP) phosphodiesterase (PDE4), induced apoptosis in

chronic lymphocytic leukemia (CLL) cells. Because the

PDE target of theophylline remains not clearly

elucidated, we examined the ability of other

isoform-specific PDE inhibitors to elicit apoptosis in

CLL cells.

In this respect, we tested the in vitro effect of

three PDE5 inhibitors, including sildenafil (Viagra®),

zaprinast and the chemical compound methoxyquinazoline

(MQZ) and compared the results to those obtained with

theophylline. Determination of CLL cell death was

performed by double staining with FITC-labeled annexin

V and propidium iodide (PI) and by analysis of

mitochondrial transmembrane potential (DYm) using

DiOC6.

Results showed that sildenafil (50 mg/ml)

significantly induced apoptosis in the leukemic cells

after 24 hr of culture. The mean cell death values (±

SD) in the presence or absence of sildenafil are 29.6%

± 13.7 vs 11.7% ± 7.3 for CLL-PBMC (n=14) (p< 0.0001)

and 51.5% ± 9.2 vs 35.5% ± 13.4 for purified B-CLL

cells (n=6) (p<0.0008).

We also demonstrated that sildenafil induced apoptosis

in a dose-dependent manner [0.5-100 mg/ml] in B-CLL

but not normal B cells isolated from control donors.

The percentage of annexin V positive cells increased

from 15 to 40% in leukemic cells and remained stable

in normal B cells. Various PDE inhibitors resulted in

differential induction of apoptosis in CLL cells with

mean percentage values (± SEM) of 33.4% ± 3.5 with

sildenafil (50 mg/ml) (n=18), 16.6% ± 2.2 with MQZ (10

mg/ml) (n=24), 9.1% ± 1.7 with zaprinast (10 mg/ml)

(n=7), and 38.1% ± 3.3 with theophylline (60 mg/ml)

(n=18) compared to 7.6 % ± 1.2 in medium alone (p<

0.0001).

Sildenafil-induced apoptosis was caspase-dependent as

demonstrated by a complete inhibition of cell death in

presence of the caspase inhibitor z-VAD.fmk and an

increase in caspase 3 activity (CPP32) in drug-treated

cultures.

Finally, IL-4 and to a lesser extent SDF1-a protected

B-CLL cells from sildenafil-induced apoptosis.

We conclude that sildenafil (Viagra®) induces

apoptosis of B-CLL cells in vitro and thus might be

considered in the treatment of CLL patients. Further

in vivo investigations should be warranted.

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