RESUMEN
Clinical treatment of B-cell chronic lymphocytic leukemia (B-CLL) is limited by the progressive drug resistance and nonselectivity of most drugs towards malignant cells. Depsipeptides are present in certain bacteria and display potent antitumor activity. We have studied the effect of the novel cyclodepsipeptide AT514 (serratamolide) from Serratia marcescens on B-CLL cell viability. AT514 induced apoptosis of B-CLL cells from the 21 patients studied, as confirmed by Annexin-V binding and nuclei condensation, with an average IC50 of 13 microM. AT514 was effective in those B-CLL cases resistant to fludarabine, but had no effect on normal PBL. AT514 preferentially activated the intrinsic apoptotic pathway, as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9 and -3, but not of caspase-8. Importantly, AT514 interfered with phosphatidylinositol-3 kinase and protein kinase C survival signals since it increased the apoptotic effect of LY294002 and Bisl inhibitors, and induced Akt dephosphorylation at Ser 473. AT514 also decreased NF-kappaB activity by dramatically reducing the levels of p65 in B-CLL. This was confirmed on functional assays using NF-kappaB-luc-transfected Raji cells and transgenic mice. Our results establish that AT514 induces apoptosis of primary B-CLL cells and could be useful for clinical treatment of this malignancy.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , FN-kappa B/metabolismo , Péptidos Cíclicos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serratia marcescens/química , Animales , Caspasa 3 , Caspasa 9 , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Depsipéptidos/farmacología , Humanos , Técnicas In Vitro , Leucemia de Células B/tratamiento farmacológico , Luciferasas/genética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Transgénicos , Mitocondrias/fisiología , FN-kappa B/genética , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transfección , Proteína X Asociada a bcl-2RESUMEN
Apoptosis is a regulated event crucial to the development and proliferation of normal and malignant B cells. We have studied the role of signals delivered via alpha4 integrin on apoptosis triggered by three different pathways on these cells. For apoptosis induced by serum deprivation, culturing B cells on the recombinant fibronectin fragment H89, a known ligand for alpha4beta1 integrin, resulted in statistically significant (P < 0.005) higher viability values (68%, 65% and 67%) for Ramos, Nalm-6 and EHEB cells, respectively, than culturing cells on poly lysine (42%, 42% and 48%). An antialpha4 MoAb reverted the protecting effect, thus confirming that it was due specifically to alpha4 engagement. Similarly, cells cultured on FN-III4-5, a recently identified fibronectin region which binds activated alpha4 integrin, also showed statistically significant higher viability than poly lysine cultures. Alpha4 engagement however, did not prevent apoptosis induced on Ramos cells via surface IgM. Adhesion of IM-9 cells, a myeloma cell line carrying functional Fas receptors, to the H89 fragment neither increased cell viability upon triggering apoptosis via Fas when compared to poly lysine. These results indicate that alpha4 signalling may overcome B cell apoptosis induced by the lack of growth factors but does not seem to affect the IgM or Fas apoptotic pathways, thus suggesting different intracellular mechanisms for these processes.