Rituximab (Chimeric Anti-CD20 Monoclonal Antibody) Inhibits the Constitutive Nuclear Factor-KB Signaling Pathway in Non-Hodgkin’s Lymphoma B-Cell Lines: Role in Sensitization to Chemotherapeutic Drug-induced Apoptosis

The chimeric anti-CD20 antibody rituximab (Rituxan, IDECC2B8) is widely used in the clinical treatment of patients with non-Hodgkin’s lymphoma (NHL). Rituximab sensitizes NHL B-cell lines to drug-induced apoptosis via down-regulation of Bcl-xL expression. We hypothesized that the mechanism by which rituximab down-regulates Bcl-xL may be, in part, due to inhibition of constitutive nuclear factor-KB (NF-KB) activity that regulates Bcl-xL expression. This hypothesis was tested in CD20 drug-resistant Ramos (Bcl-2 /Bcl-xL ) and Daudi (Bcl-2/Bcl-xL ) cell lines. Rituximab decreased the phosphorylation of NF-KB-inducing kinase, IKB kinase, and IKB-a, diminished IKK kinase activity, and decreased NF-KB DNA binding activity. These events occurred with similar kinetics and were observed 3 to 6 hours post-rituximab treatment. Rituximab significantly up-regulated Raf-1 kinase inhibitor protein expression, thus interrupting the NF-KB signaling pathway concomitant with Bcl-xL and Bfl-1/A1 down-regulation. The role of NF-KB in the regulation of Bcl-xL transcription was shown using promoter reporter assays in which deletion of the two-tandem NF-KB binding sites in the upstream promoter region significantly reduced the luciferase activity. This was further corroborated by using IKB superrepressor cells and by NF-KB–specific inhibitors. The direct role of Bcl-xL in drug resistance was assessed by using BclxL–overexpressing cells, which exhibited higher drug resistance that was partially reversed by rituximab. Rituximab-mediated inhibition of the NF-KB signaling pathway and chemosensitization was corroborated by the use of specific inhibitors. These findings reveal a novel pathwaymediated by rituximab through Raf-1 kinase inhibitor protein induction that negatively regulates the constitutive NF-KB pathway and chemosensitization of the NHL B-cells. (Cancer Res 2005; 65(1): 264–76)