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 p27 kip1 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.

Noxa upregulation is associated with apoptosis of chronic lymphocytic leukemia cells induced by hyperforin but not flavopiridol

“…The patient-dependent effects on Mcl-1, Bcl-2 and p27 were in accordance with previous data. 4,8 Similar results were observed with four other patients (9)(10)(11)(12). Indeed, in all 12 patients, the ratio of Noxa/actin band intensities increased after hyperforin treatment as compared to untreated controls (Figure 2a).…”

“…Indeed, only marginal effects of the phloroglucinol on Mcl-1 or Bcl-2 were detected here; in addition, Mcl-1 cleavage seems rather a secondary mechanism inasmuch as it was previously found to be caspase-dependent. 4 Our observation that flavopiridol did not stimulate Noxa expression indicates that the flavonoid does not act through the same mechanism as hyperforin or other apoptosis inducers in CLL cells. The strong inhibition of Mcl-1 expression by flavopiridol, as a result of either protein degradation or transcriptional inhibition, may be sufficient to allow a sustained Bak activity, together with convergent downregulation of other antiapoptotic proteins.…”

“…Expression of Mcl-1 and Bcl-2 was also analyzed, as well as two proteins, which are constitutively overexpressed in CLL cells and downregulated upon flavopiridol and hyperforin-triggered apoptosis: the inducible NO synthase that produces the antiapoptotic NO, and p27kip1 that is cleaved by caspases into a p23 fragment. 4,8 Figure 1 shows that Noxa was expressed at low levels by CLL cells from eight patients (1)(2)(3)(4)(5)(6)(7)(8). In all cases, hyperforin treatment resulted in a strong increase of Noxa expression and this effect was accompanied with stimulation of PARP cleavage (and p27 cleavage in some cases), as compared to untreated controls.…”

“…The strong inhibition of Mcl-1 expression by flavopiridol, as a result of either protein degradation or transcriptional inhibition, may be sufficient to allow a sustained Bak activity, together with convergent downregulation of other antiapoptotic proteins. 4,8 Moreover, the effects of certain apoptosis inducers could involve proapoptotic Bcl-2 familly members distinct from Noxa and Bak, such as Bak or the BH3-only Puma and Bim. 11 Finally, our results favor the concept that hyperforin and agents having the capacity to induce or mimic Noxa represent a novel attractive therapeutic strategy for CLL, as recently suggested.…”

“…These experimental conditions were previously demonstrated to allow optimal apoptosis induction (notably DNA fragmentation, dissipation of the mitochondrial transmembrane potential and caspase-3 activation). 4,8 Noxa expression was analyzed by western blotting from equal amounts of cell lysates (30-50 mg of proteins in SDS-reducing buffer), as described previously. 8 Two mAb against Noxa were used: IMG-249 from Imgenex (Clinisciences, Montrouge, France) and clone 6D619 from the US Biological (Euromedex, Mundolsheim, France).…”