title: Rituximab (chimeric anti-CD20 monoclonal antibody) inhibits the constitutive nuclear factor-{kappa}B signaling pathway in non-Hodgkin's lymphoma B-cell lines: role in sensitization to chemotherapeutic drug-induced apoptosis.
abstract: The chimeric anti-CD20 antibody rituximab (Rituxan, IDEC-C2B8) 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-x(L) expression. We hypothesized that the mechanism by which rituximab down-regulates Bcl-x(L) may be, in part, due to inhibition of constitutive nuclear factor-kappaB (NF-kappaB) activity that regulates Bcl-x(L) expression. This hypothesis was tested in CD20(+) drug-resistant Ramos (Bcl-2(-)/Bcl-x(L)(+)) and Daudi (Bcl-2(+)/Bcl-x(L)(+)) cell lines. Rituximab decreased the phosphorylation of NF-kappaB-inducing kinase, IkappaB kinase, and IkappaB-alpha, diminished IKK kinase activity, and decreased NF-kappaB 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-kappaB signaling pathway concomitant with Bcl-x(L) and Bfl-1/A1 down-regulation. The role of NF-kappaB in the regulation of Bcl-x(L) transcription was shown using promoter reporter assays in which deletion of the two-tandem NF-kappaB binding sites in the upstream promoter region significantly reduced the luciferase activity. This was further corroborated by using IkappaB superrepressor cells and by NF-kappaB-specific inhibitors. The direct role of Bcl-x(L) in drug resistance was assessed by using Bcl-x(L)-overexpressing cells, which exhibited higher drug resistance that was partially reversed by rituximab. Rituximab-mediated inhibition of the NF-kappaB signaling pathway and chemosensitization was corroborated by the use of specific inhibitors. These findings reveal a novel pathway mediated by rituximab through Raf-1 kinase inhibitor protein induction that negatively regulates the constitutive NF-kappaB pathway and chemosensitization of the NHL B-cells.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA 90095, USA.
authors.forename: Ali R
authors.initials: AR
authors.lastname: Jazirehi
authors.forename: Sara
authors.initials: S
authors.lastname: Huerta-Yepez
authors.forename: Genhong
authors.initials: G
authors.lastname: Cheng
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bonavida
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000633:toxicity*; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects*; D001402:B-Lymphocytes / Q000187:drug effects / Q000473:pathology; D002051:Burkitt Lymphoma / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000069283:Rituximab; D015398:Signal Transduction / Q000187:drug effects*; D051020:bcl-X Protein
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D000069283:Rituximab; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases