Fusion Protein Ompc-CD154 Inhibits Proliferation and Induces Apoptosis in B-NHL Cell Lines By a Mechanism That Involves MAPK P38 and TRAF2 Activation

CD40 is a member of the TNF super family, which is expressed mainly in B cells, dendritic cells and macrophages, and the interaction with its ligand CD154 is essential for the development of the immune response. The activation or selective inhibition of this pathway is part of various therapeutic strategies for the treatment of diseases with inflammatory or immune origin, as well as various types of tumors. The fusion protein OmpC-CD154 expresses 10 amino acids of CD154 (Ser149-Trp140) and is capable of binding to CD40, inducing apoptosis and inhibiting proliferation in NHL cell lines. In this study we demonstrate that the fusion protein OmpC-CD154 induced apoptosis and inhibition of proliferation in several B-NHL cell lines. In addition, we analyzed the mechanisms involved in the induction of apoptosis and the inhibition of proliferation. Our results by Western blot demonstrate that OmpC-CD154 induced degradation of Bcl-6 by a MAPK p38 activation dependent mechanism. Pre-treatment of Ramos and Raji cell lines with a specific chemical p38 inhibitor reverted the effect of OmpC-CD154 in proliferation. Our results showed that the fusion protein can also inhibit the constitutive activation of both the canonical and non-canonical NF-kB pathways, and this inhibition was associated with the activation of the TRAFs pathway, as it promotes TRAF2 phosphorylation, and the decreasing of the expression of TRAF3. All these effects were greater than those observed with recombinant human CD154 on B-NHL cell lines. The present findings establish that a CD154 peptide fused with a protein exhibits significant cytotoxicity on B-NHL cells. Current studies are evaluating fusion proteins with CD154 peptide that are not antigenic in vivo and that have significant anti-tumor effects, both direct and on the host immune response, with minimal toxicity.