Christian Billard

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells.

Background The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. Methodology and Results HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser473) and Akt1 substrate Bad (at Ser136) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Significance Our data provide new evidence that HFs pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.

Stimulation of iNOS expression and apoptosis resistance in B-cell chronic lymphocytic leukemia (B-CLL) cells through engagement of Toll-like receptor 7 (TLR-7) and NF-κB activation

B-CLL cells are characterized by in vivo resistance to apoptosis due, in part, to the presence of an inducible nitric oxide synthase, iNOS, as the NO released plays anti-apoptotic role, notably by inhibiting caspases. The mechanisms leading to spontaneous expression of iNOS in these cells are presently unknown. The restricted use of some V(H) sub-groups and the sequences of the monoclonal immunoglobulins of the B-cell receptor expressed by the leukemia cells suggested that the latter have encountered specific auto-antigens and/or microbial derived antigens. Their binding to the BCR provides an activation signal resulting in enhanced survival, hence could be involved in the aetiology of the disease. At the interface of innate and cognate immunity, Toll-like receptors, TLR, recognize PAMPs (pathogen-associated molecular patterns) expressed by various bacteria and virus as well as some self-antigens. We thus hypothesized that TLR were involved in the early steps of B-CLL oncogenesis, notably apoptosis resistance through the induction of iNOS expression and the production of NO. Our results show that B-CLL cells express TLR-7 and TLR-9. Incubation of B-CLL cells with TLR-7 agonists effectively resulted in an increased resistance to apoptosis that was reverted with the NOS inhibitor L-NMMA. This resistance was associated with enhanced iNOS expression (protein and mRNA) and NO release, stimulation of NF-kappaB activation, phosphorylation of I kappaB alpha, all these events being suppressed with wedelolactone or Bay 11-7085, two inhibitors of I kappaB alpha phosphorylation. Our present data thus suggest that TLR-7 signaling stimulates apoptosis resistance, notably through an NF-kappaB-dependent activation of the NO pathway.

Apoptotic effects on B-cell chronic lymphocytic leukemia (B-CLL) cells of heterocyclic compounds isolated from Guttiferaes

A series of 10 heterocyclic compounds purified from Allanblackia were tested on two B cell lines, ESKOL and EHEB, and on cells from B-CLL patients. Several molecules inhibited the proliferation of both cell lines and promoted apoptosis of B-CLL cells through different mechanisms, some of them elicited a dissipation of the mitochondrial transmembrane potential, other triggered caspase-3 activation and cleavage of the inducible nitric oxide synthase. Blood mononuclear cells and B-lymphocytes from healthy donors appeared less sensitive than B-CLL cells. These results indicate that these molecules may be of interest in the development of new therapies for B-CLL.

Types I and II interferons upregulate the costimulatory CD80 molecule in monocytes via interferon regulatory factor-1.

CD80/B7.1 expressed on monocytes plays a prominent role in the activation of T cell-mediated immunity and its level is reduced in monocytes from cancer patients. Type I (alpha/beta) and type II (gamma) IFNs are widely administered as adjuvant therapy. We show here that both classes of IFNs upregulate CD80 mRNA and protein in primary monocytes ex vivo. The stimulatory action of IFN-alpha/beta on CD80 is accompanied by the activation of both interferon regulatory factors IRF-1 and IRF-7, whereas IFN-gamma stimulating effect is associated only with IRF-1 induction. IFNs concomitantly upregulate the transcription of CD40 costimulatory molecule whose activation is known to require IRF-1. In monocytic U937 cells, IRF-1 is activated by IFN-gamma but not by IFN-alpha/beta, whereas it is the reverse for IRF-7; in the latter cells, only IFN-gamma is capable of stimulating CD80 transcription emphasizing the essential role of IRF-1. Moreover, siRNA against IRF-1 prevents IFN-gamma-mediated CD80 activation. In AML cells, IFNs upregulate CD40, CD80 and IRF-1 in the FAB-M4/M5 subtypes but not in the less differentiated M1/M2 subtypes. Monitoring the expression of CD80 on AML cells and its modulation by IFNs could help to predict the patients more susceptible to benefit from therapeutic strategies aimed at eliciting specific T cell responses to leukemia-associated antigens.

Therapeutic activity of two xanthones in a xenograft murine model of human chronic lymphocytic leukemia.

BackgroundWe previously reported that allanxanthone C and macluraxanthone, two xanthones purified from Guttiferae trees, display in vitro antiproliferative and proapoptotic activities in leukemic cells from chronic lymphocytic leukemia (CLL) and leukemia B cell lines.ResultsHere, we investigated the in vivo therapeutic effects of the two xanthones in a xenograft murine model of human CLL, developed by engrafting CD5-transfected chronic leukemia B cells into SCID mice. Treatment of the animals with five daily injections of either allanxanthone C or macluraxanthone resulted in a significant prolongation of their survival as compared to control animals injected with the solvent alone (p = 0.0006 and p = 0.0141, respectively). The same treatment of mice which were not xenografted induced no mortality.ConclusionThese data show for the first time the in vivo antileukemic activities of two plant-derived xanthones, and confirm their potential interest for CLL therapy.