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Chronic Lymphocytic Leukemia and B Cells

Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia

Abstract

The effects of the hyperforin (HF), a natural phloroglucinol purified from Hypericum perforatum, were investigated ex vivo on leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). HF was found to promote apoptosis of B-CLL cells, as shown by time- and dose-dependent stimulation of phosphatidylserine externalization and DNA fragmentation, by disruption of the mitochondrial transmembrane potential, caspase-3 activation and cleavage of the caspase substrate PARP-1. Moreover, HF-induced downregulation of Bcl-2 and Mcl-1, two antiapoptotic proteins that control mitochondrial permeability. HF also downregulated two proteins which are overexpressed by B-CLL patients' cells, the cell cycle inhibitor p27kip1 through caspase-dependent cleavage into a p23 form, and the nitric oxid (NO) synthase of type 2 (inducible NO synthase). This latter was accompanied by reduction in the production of NO known to be antiapoptotic in B-CLL cells. Preventing effects of the general caspase inhibitor z-VAD-fmk indicated that HF-promoted apoptosis of B-CLL cells was mostly caspase dependent. Furthermore, normal B lymphocytes purified from healthy donors appeared less sensitive to HF-induced apoptosis than B-CLL cells. These results indicate that HF may be of interest in the development of new therapies for B-CLL based on the induction of apoptosis and combination with cell cycle-dependent antitumor drugs.

Introduction

Hyperforin (HF) is a natural phloroglucinol extracted from Hypericum perforatum (St John's wort). The latter has been utilized in traditional medicine and contains other biologically active constituents, including naphthodianthrones such as hypericin and various flavonoids.1 Extracts of St John's wort alleviate symptoms of mild depression2 by inhibiting the neuronal reuptake of neurotransmitters including serotonin, noradrenaline, dopamine, glutamate and GABA.3 HF seems to be the main active ingredient and inhibits the synaptosomal uptake of 5-HT by elevating free intracellular [Na+]i concentration4 through the activation of nonselective cation channels.5 Furthermore, HF exerts antibacterial activity against multiresistant Staphylococcus aureus and Gram-positive bacteria.6 HF displays antioxidant properties, impairs the production of superoxide from activated neutrophils7 and inhibits various cytochrome P450 isoforms,8 a chronic exposure leading to inhibition of CYP3A4 activity in vivo.9 HF acts as a dual inhibitor of 5-lipooxygenase and of cyclooxygenase 1 (COX-1), hence its interest in the treatment of inflammatory and allergic diseases connected to eicosanoids.10 HF inhibits the proliferation of peripheral blood mononuclear cells (PBMC) without displaying toxic effects; moreover, incubation of endothelial cells with HF suppressed the proliferation of alloreactive T cells.11

Recently, it was reported that extracts of St John's wort containing HF induce growth inhibition and apoptosis of human and rat malignant cells in vitro as well as in vivo.12, 13 Similar effects, associated with activation of caspases, were also shown with the myeloid leukemia cell lines K562 and U937.14 However, it is not known whether HF could exert activity in B-cell chronic lymphocytic leukemia (B-CLL), a malignancy which results from a defect of apoptosis rather than an impaired control of lymphoproliferation. Actually, the leukemic cells from B-CLL are characterized by resistance to apoptotic signals, and most B-CLL cells are arrested in the G0 phase of the cell cycle (for reviews, see Bannerji and Byrd15 and Kolb et al.16).

We therefore investigated the effects of HF on B-CLL patients' cells ex vivo. Membrane, mitochondrial and nuclear events of apoptosis were studied as well as the expression of several proteins known to be involved in the control of apoptosis and cell cycle. Our results show the pro-apoptotic capacities of HF in B-CLL and its interest in new strategies of combined therapy for this leukemia.

Materials and methods

Patients, cells and cell culture

Blood samples from B-CLL patients were obtained from the Hematology Department of Hôtel-Dieu hospital (Paris, France) after written informed consent, in accordance with the rules and tenets of the revised Helsinki protocol. Diagnosis was established according to standard clinical and international CLL workshop criteria, including lymphocyte morphology and co-expression of CD5, CD20 and CD23 antigens. A total of 41 patients (23 men and 18 women) with a mean age of 67.8±10.4 years (range 33–91 years) were selected and the time since diagnosis varied between 0 (newly diagnosed patients) and 10 years. The patients were randomly chosen for each type of experiment inasmuch as CD38 and ZAP-70 expression, cytogenetics and mutational VH status were only available for a fraction of them, thus hampering a risk-group analysis. The leukemic B cells were isolated with a purity greater than 96%, as previously described.17 Except when indicated, all the experiments were performed with freshly purified B-CLL leukemic cells. Blood samples from healthy donors were obtained from the Institut Français du Sang as residues from platelet preparations. The healthy blood donors were under 60 years old, according to the French legislation, and therefore they could not be exactly age matched with the B-CLL patients. PBMC were prepared as described elsewhere17 and normal B lymphocytes were purified by positive selection on anti-CD19-coated magnetic beads according to the specifications of the manufacturer (Dynal, Oslo, Norway). Their purity usually ranged from 92 to 95%, as estimated by labeling with an anti-CD20-phycoerythrin antibody, and monocyte contamination never exceeded 2%.18 All cell cultures were performed at 37°C in an humidified atmosphere containing 5% CO2, either in RPMI-1640 medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% FCS or in Quantum 007 medium, both from PAA Laboratories (Pasching, Austria) at seeding densities of 2 × 106 cells/ml. The use of these two culture medium resulted in no detectable differences in cell survival and apoptotic responses.

Reagents

HF (MW=538) was prepared and analyzed according to the method described by JD Fourneron and Y Naït-Si (Effect of eluent pH on HPLC/UV analysis of HF, Phytochem Analysis, in press). Two different batches were used and 1 mg/ml stock solutions were made in ethanol. Flavopiridol was provided by Aventis Pharmaceuticals (Bridgewater, NJ, USA). A 10 mM stock solution was prepared in DMSO, aliquoted and kept at −20°C. The broad specificity caspase inhibitor z-VAD-fmk was obtained from Biomol (Plymouth Meeting, PA, USA). Except when otherwise stated, FITC-conjugated mAbs were purchased from Becton Dickinson (Mountain View, CA, USA). The slow-releasing nitric oxide (NO) donor NOC-18 or DETA-NO (half-life of 5 h at physiological pH) of the NONOate family, which mimics in part the effect of NO released by an inducible NO synthase (iNOS), was purchased from Alexis Biochemicals (Q-BIOgene, Illkirch, France). The other reagents and chemicals were from Sigma (St Louis, MO, USA).

Annexin V-binding assay

Phosphatidylserine (PS) externalization, a membrane marker of cells undergoing apoptosis, was quantified by specific binding of FITC-conjugated annexin V (Bender Medsystems, Vienna, Austria), with or without simultaneous labeling with propidium iodide (PI), according to a modification of the technique described by Koopman et al.19 The percentages of annexin V-FITC-positive and PI-negative cells were determined by cytometry on an EPICS Altra flow cytometer (Beckman Coulter).

DNA fragmentation assay

Apoptosis was also quantified by DNA fragmentation as evaluated by detection of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) in cell lysates from aliquots of 20 000 cells using an ELISA with anti-histone and anti-DNA fragment mAbs (Cell Death Detection ELISAPLUS, Roche Diagnostics, Indianapolis, IN, USA) as previously described.20

Mitochondrial membrane permeability

The Mit-E-Ψ mitochondrial permeability detection kit (Biomol) was used to monitor the variations of the mitochondrial transmembrane potential, Δψm, according to the manufacturer's specifications.21 Briefly, washed cells were incubated at 2 × 106 cells/ml in assay buffer containing the fluorescent probe JC-1 for 15 min at 37°C. After washing, cells were distributed in triplicates in microtitration plate and fluorescence was recorded in a microplate reader cytofluorometer (Wallac Victor-2, Perkin Elmer, Norwalk, MT, USA) with simultaneous detection of green and red fluorescence (respectively: excitation wavelength 485 nm, emission wavelength 525 nm, and excitation wavelength 485 nm, emission wavelength 595 nm). The dissipation of ΔΨm is characterized by a significant shift of the red to the green fluorescence and therefore by a reduction of the red/green fluorescence ratio.

Caspase activity assay

Aliquots of cell lysates (10–20 μg) were incubated with the fluorogenic caspase-3 substrate DEVD-aminocoumarin, DEVD-AMC (caspase-3 cellular assay, Biomol). The release of AMC was recorded every 10 min during 2 h in a Victor-2 microplate reader thermostated at 37°C (excitation wavelength: 380 nm; emission wavelength: 460 nm).19 The specificity of the reaction was assessed by the addition of unlabeled Ac-DEVD-CHO caspase-3 inhibitor (Biomol). Caspase-8 activity was determined similarly using the specific tetrapeptide IETD-pNA (Biomol) as a substrate.

Western blot analysis

The expression of several proteins, poly(ADP-ribose) polymerase-1 (PARP-1), Bcl-2, Mcl-1, iNOS and p27kip1 was studied by Western blotting, as detailed previously.21 Briefly, equal amounts of the cell lysates (generally 30–50 μg of proteins in SDS-reducing buffer) were electrophoresed by SDS-PAGE (either 12% for p27kip1, Bcl-2, Mcl-1 and β-actin or 8% for iNOS and PARP or a gradient gel for simultaneous analysis). The polyclonal rabbit antibodies against iNOS (N-20), p27kip1 (N-20) recognizing both the p27 and its cleavage product p23, and anti-Mcl-1 (S-19) were from Santa Cruz Biotechnology, CA, USA. The monoclonal anti-Bcl-2 antibody was from Biosource (Clinisciences, Montrouge, France). The monoclonal anti-PARP-1 (C2-10) was from Alexis Biochemicals (QBiogene, Illkirch, France). The immunoblotted proteins were revealed with HRP-goat anti-rabbit or anti-mouse antibodies (DakoCytomation, Glostrup, Denmark) and a system of chemiluminescence (Western lightning chemiluminescence reagent plus; Perkin-Elmer, Boston, MA, USA). Finally, the membranes were also hybridized with a mouse anti-β actin mAb (clone C4; ICN, Costa Mesa, CA, USA) for protein content monitoring and standardization. Films were analyzed with a digital imager (Vilber Lourmat, Marne-la-Vallée, France) system and the NIH Image 1.44b11 software was used for the quantification of the intensity of the bands.

Endogenous NO production

The production of NO was measured by in situ binding of the NO-specific fluorescent probe DAF-2 DA (Calbiochem, La Jolla, CA, USA), as described previously.22

Statistical analysis

Cells were cultured in duplicates or triplicates and each experiment repeated two to five times. Statistical analysis was performed using the Statview software; the unpaired two tail t-test was used for the comparison of test and control groups.

Results

HF promotes apoptosis of B-CLL patients' cells

The effects of HF on membrane and nuclear events of apoptosis were investigated in B-CLL cells by studying PS externalization (binding of annexin V-FITC) and DNA fragmentation (release of cytoplasmic nucleosomes). Treatment with HF induced a dose-dependent stimulation of both annexin V binding (Figure 1) and nucleosome fragmentation as compared to untreated cells (Table 1). Nucleosome fragmentation increased significantly after 24 h of treatment with concentrations of HF0.5 μg/ml, then slightly decreased by 48 h. Maximum nucleosome release was observed with a concentration of 5 μg/ml HF (about 9 μ M). As reported previously, 1 μ M flavopiridol induced roughly similar enrichments in nucleosomes (246±60) for six patients tested.21 This observation was confirmed when the effects of both agents were compared directly on thawed leukemic B cells from three patients, inasmuch as the enrichment in nucleosomes obtained with 5 μg/ml HF (167±20) and 1 μ M flavopiridol (173±11) were very close (Table 2). HF was thus used at this concentration of 5 μg/ml (chosen as optimal to trigger apoptosis) in most of the following experiments. Purified normal B lymphocytes were, in contrast, much less sensitive to the pro-apoptotic effect of HF. For the three donors tested, the mean percentages of nucleosome enrichment in B lymphocytes treated with 1 and 5 μg/ml HF were, respectively, of 112 and 132% after 1 day of treatment, and of 105 and 128% after 2 days as compared to control values of untreated cells.

Figure 1
figure1

Hyperforin stimulates PS externalization on B-CLL patients' cells. Leukemic cells from four B-CLL patients were cultured for 48 h in the absence (control) or in the presence of three different concentrations of HF. Phosphatidylserine (PS) externalization was evaluated by binding of annexin V-FITC using flow cytometry, and the percentages of annexin-positive cells were determined by Kolmogorov–Smirnov analysis after subtraction of the values of the control cells. NT: not tested.

Table 1 Hyperforin stimulates DNA fragmentation in B-CLL patients' cells
Table 2 Hyperforin-induced DNA fragmentation is caspase dependent

HF-induced apoptosis involves the caspase pathway

Exposure to HF resulted in a dose-dependent stimulation in the capacity of B-CLL cell lysates to cleave DEVD-AMC, a fluorogenic substrate of caspase-3, that was totally abolished with the caspase-3 inhibitor DEVD-CHO (Figure 2a). This indicated that HF was able to induce caspase-3 activity. In contrast, HF had little effect on caspase-8 activity, yet a small stimulation of its activity following treatment with HF was observed (Figure 2b). Furthermore, HF-induced nucleosome fragmentation was nearly completely prevented by the presence of the general caspase inhibitor z-VAD-fmk (Table 2), indicating that apoptosis of B-CLL cells triggered by HF was mostly caspase dependent. Investigating the effect of HF on the expression of PARP-1, a caspase substrate, showed clearly a strong cleavage of the 116 kDa PARP molecule into its 85 kDa fragment. This cleavage was totally prevented in the presence of z-VAD-fmk, demonstrating that it was caspase-dependent, as expected (Figure 3a). It is interesting to note that 5 μg/ml HF induced the same nearly complete cleavage as that observed with 1 μ M flavopiridol (Figure 3c), a plant-derived compound displaying a remarkable capacity to induce B-CLL cells to apoptosis in vitro (see Kolb et al.16 and Billard et al.21).

Figure 2
figure2

Effect of hyperforin on the activity of caspase-3 and -8 in B-CLL cells. B-CLL leukemic cells were treated in culture for 20 h with 1 or 5 μg/ml HF (HF 1 and HF 5, respectively) or left untreated (control). Cells were then lysed and 20 μg of proteins were incubated with the fluorogenic (DEVD-AMC) or chromogenic (IETD-pNA) substrates of caspase-3 and -8, respectively, in the presence or absence of their specific inhibitors (Ac-DEVD-CHO for caspase-3 and Ac-IETD-CHO for caspase-8). The release of fluorescent AMC (a) or chromogenic pNA (b) was recorded every 10 min during 2 h. The results are representative of data obtained with three other patients. Identical observations were obtained with two different batches of purified HF.

Figure 3
figure3

Hyperforin elicits the cleavage of PARP-1, p27kip1, Bcl-2 and Mcl-1 and inhibits iNOS expression. B-CLL patients' cells were treated in culture for 20 h with 1 or 5 μg/ml HF, or with 1 μ M flavopiridol (F) or left untreated (control, C) in the presence or absence of z-VAD-fmk (40 μ M). The expression of the various proteins was analyzed by Western blot from cell lysates containing equal amount of proteins (see Materials and methods). The analysis of β-actin content (as a control of protein loading) was performed in parallel. (a) iNOS downregulation, cleavage of PARP, p27 and of Bcl-2 and prevention by z-VAD-fmk. The data are representative of at least three different patients that gave comparable results. (b) Cleavage of Mcl-1 (and Bcl-2) after treatment with 5 μg/ml HF. Data are representative of two different patients that gave comparable results. (bd) Comparison of the effects of 5 μg/ml HF with those of 1 μ M flavopiridol (F).

HF induces the cleavage of Bcl-2 and Mcl-1 and a dissipation of the mitochondrial transmembrane potential ΔΨm. Western blot analysis of the expression of Bcl-2 and Mcl-1, two antiapoptotic proteins,23 evidenced that both proteins were cleaved upon treatment with HF (Figure 3a and b). These cleavages were caspase dependent inasmuch as they were prevented by z-VAD-fmk, as exemplified for Bcl-2 (Figure 3a). The observation that Bcl-2 expression was reduced by HF was confirmed by measuring Bcl-2 content in cell lysates by ELISA (not shown). The effect of 5 μg/ml HF on Bcl-2 cleavage was similar to that of 1 μ M flavopiridol, while it was slightly less strong for Mcl-1 cleavage (Figure 3b). The cleavages of Bcl-2 and Mcl-1 suggested the contribution of mitochondrial alterations during apoptosis promotion by HF. Actually, exposure to HF resulted in a dissipation of the ΔΨm, as measured by the decrease in the ratio of red/green fluorescence of the JC-1 probe (Figure 4). Therefore HF elicited mitochondrial alterations typical of the ‘intrinsic’ apoptotic pathway in B-CLL cells.

Figure 4
figure4

Hyperforin triggers a dissipation of the mitochondrial transmembrane potential ΔΨm. B-CLL patient's cells were cultured for 18 h in the presence of 1, 5 or 10 μg/ml HF (HF 1, HF 5 and HF 10, respectively) or in its absence (control). Then, cells were incubated for 15 min at 37°C with the fluorescent probe JC-1, and subsequently washed and distributed in triplicates in the wells of a microtitration plate. Green and red fluorescences were immediatly measured as detailed in Materials and methods. The dissipation of ΔΨm is characterized by a significant shift of the red to the green fluorescence and therefore by a reduction of the red/green fluorescence ratio. The data are representative of three different patients that gave comparable results.

HF inhibits iNOS expression and NO production in B-CLL cells

The iNOS is known to be constitutively overexpressed by B-CLL cells but not by normal B lymphocytes.17, 24 Western blot experiments showed clearly that treatment of B-CLL cells with HF induced a dose-dependent reduction of iNOS expression and that this downregulation correlated with PARP-1 cleavage, taken as a marker of caspase-dependent apoptosis (Figure 3a). Note also that the inhibition of iNOS expression seen with 5 μg/ml HF was almost as strong as the effect of 1 μ M flavopiridol (Figure 3c) which was reported recently.21 Moreover, iNOS downregulation by HF was accompanied by a marked reduction of NO production by B-CLL cells, as detected with the fluorescent probe DAF-2 DA (Figure 5a). For the five different patients tested, the reductions in the rate of NO release after treatment with 5 μg/ml HF ranged from 52 to 97% as compared to untreated controls. As shown in Figure 5b, normal B lymphocytes released much less NO (more than four times less, as estimated from the value at t=0) and this production was also somewhat inhibited with 5 μg/ml HF.

Figure 5
figure5

Effect of hyperforin on NO production by B lymphocytes from B-CLL patients and normal individuals. Purified B lymphocytes from B-CLL patient (a) or normal blood donor (b) were treated for 20 h with 1 or 5 μg/ml HF or left untreated (Control). Cells were then incubated for 1 h at 37°C with the fluorescent probe DAF-2 DA and the cell fluorescence was recorded every 10 min during 2 h, as described in Materials and methods. The data are representative of four patients that gave comparable results.

Interestingly, HF-driven apoptosis was partially reverted by the addition of low concentrations of DETA-NO, a slow releasing chemical NO donor (Figure 6); at higher concentrations, DETA-NO exhibited pro-apoptotic effect, as already observed21 and in accordance with its well-known biphasic effect on apoptosis.

Figure 6
figure6

Reversal of hyperforin-induced apoptosis by low concentrations of DETA-NO. Leukemic cells from a representative B-CLL patient were cultured for 20 h as before in medium alone (control) or with HF (2 μg/ml), in the presence or absence of varying concentrations of DETA-NO/NOC-18. DNA fragmentation was evaluated by the release of cytoplasmic nucleosomes (see Materials and methods). Results are expressed as the percentages of nucleosome enrichment as compared to control cells taken as 100%.

HF elicits the cleavage of p27kip1

The cdk inhibitor p27kip1 is another protein which is overexpressed by B-CLL cells.25 It was previously shown to be downregulated during apoptosis of B cells through caspase-dependent cleavage into a p23 form.26 It appeared that HF treatment elicited a dose-dependent cleavage of p27kip1 into the p23 fragment and that this effect was totally prevented in the presence of z-VAD-fmk, as expected (Figure 3a). The extent of p27 cleavage depended on each patient, appearing in some cases lower than the effect of flavopiridol, used as reference (Figure 3a and d).

Discussion

The present work shows for the first time that HF, a natural phloroglucinol, displays ex vivo pro-apoptotic activities in B-CLL patients' cells. Indeed, HF elicited PS externalization, DNA fragmentation, caspase-3 activation, PARP-1 cleavage, dissipation of the transmembrane mitochondrial potential ΔΨm and cleavage-induced downregulation of two antiapoptotic proteins, Mcl-1 and Bcl-2 in the leukemic B cells. These data seem of interest because the accumulation of leukemic cells in B-CLL results mostly from defective apoptotic processes.

Our results showing that DNA fragmentation and several effects of HF described here, including PARP cleavage, are inhibited by the general caspase inhibitor z-VAD-fmk and that caspase-3 activity is stimulated highlight the role of the caspase pathway in HF-mediated apoptosis of B-CLL cells. These results are in agreement with previous reports on other types of tumor cells,13, 14 except that only a slight caspase-8 activation was found here. HF is not known to bind to a receptor displaying a death domain and, with the exception of the D(1) dopamine receptor, it does not interact with biogenic amine receptors and transporters.27

Other data provide evidence that the mitochondria-mediated pathway is also involved. First, the mitochondrial potential ΔΨm is dissipated, as observed in MT-450 cells.13 In addition, the antiapoptotic proteins Bcl-2 and Mcl-1, that control the permeability of the mitochondrial membrane and the release of apoptogenic molecules, are somewhat downregulated upon HF treatment. Bcl-2 and Mcl-1 play important roles in the resistance of B-CLL cells to various chemotherapeutic agents.23

Furthermore, HF also downregulates two other proteins, iNOS and p27kip1, which are overexpressed in B-CLL cells.17, 25 The former, iNOS, is responsible for the production of NO that displays antiapoptotic properties in B-CLL cells17, 24 and is a component of their resistance to cell death.28 We showed recently that several plant-derived compounds, such as flavopiridol,21 polyphenols (including resveratrol) and their acetate derivatives as well as a synthetic aminoflavone, induced both apoptosis of B-CLL patients' cells and downregulation of iNOS expression and NO production.22, 20, 29 HF thus represents another example of a natural compound capable of promoting apoptosis and inhibiting the NO pathway in B-CLL cells. This inhibition of the NO pathway could be either a cause or a consequence of apoptosis. In favor of the former possibility, our experiments indicate that low concentrations of a NO donor can partially revert HF-elicited apoptosis, as previously found for flavopiridol.21 This would suggest that HF-induced suppression of NO release is a molecular switch contributing to subsequent apoptosis, perhaps through a release of the blocking effect of NO on the active site of caspases,30 as previously suggested and discussed.21, 31 Whatever, our results suggest the existence of a complex interplay between NO and caspases that may finely tune apoptosis induction by various compounds such as HF.

The other protein overexpressed in B-CLL being downregulated by HF, p27kip1, is an inhibitor of the cell cycle known to be cleaved by caspases during apoptosis of B cells.26 An inverse relation exists between p27kip1 levels and the susceptibility of B-CLL cells to fludarabine in vitro.25 These data suggest that p27kip1 may be involved in the cell cycle arrest and in the resistance of patients to chemotherapy.21 Thus, cleavage-induced downregulation of p27kip1 by HF could permit the cell cycle to resume and a better action of cell cycle-dependent chemotherapeutic agents. From our data, HF appears of promising interest in the development of new strategies for the therapy of B-CLL, through not only its in vitro pro-apoptotic properties, but also its potential capacity to improve antitumor chemotherapy.

Finally, HF displays potent antitumor activities in different cancer models. Its poor solubility and stability restrain its potential clinical use. However, HF derivatives retaining the antitumor properties of the parental compound but with improved pharmacological activity were recently synthesized, such as aristoforin.32 In addition, the stable dicyclohexylammonium salt of HF was found to trigger apoptosis in both murine and human tumor cells and to reduce in vivo the number of metastasis in mice grafted with the C-26 and B16-LU8 tumors.33 The sodium salt is also water soluble and improves the stability of HF. HF derivatives might therefore represent a new class of therapeutic molecules for B-CLL patients.

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Acknowledgements

We thank Mrs S Pasco-Dubrulle for her skillful assistance and the clinicians of the Department of Hematology at the Hôtel-Dieu hospital. This work was supported by INSERM, Canceropôle Ile-de-France and by a grant from ARC (no. 3322).

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Correspondence to J-P Kolb.

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Quiney, C., Billard, C., Faussat, A. et al. Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia. Leukemia 20, 491–497 (2006). https://doi.org/10.1038/sj.leu.2404098

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Keywords

  • hyperforin
  • B-CLL
  • apoptosis
  • nitric oxide
  • p27kip1

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