title: Norcantharidin facilitates LPS-mediated immune responses by up-regulation of AKT/NF-κB signaling in macrophages.
abstract: Norcantharidin (NCTD), a demethylated analog of cantharidin, is a common used clinical drug to inhibit proliferation and metastasis of cancer cells. But the role of NCTD in modulating immune responses remains unknown. Here, we investigated the function and mechanism of NCTD in regulation of TLR4 associated immune response in macrophages. We evaluated the influence of NCTD on host defense against invaded pathogens by acute peritonitis mouse model, ELISA, Q-PCR, nitrite quantification, phagocytosis assay and gelatin zymography assay. Our data showed that the survival and the serum concentrations of IL-6 and TNF-α were all enhanced by NCTD significantly in peritonitis mouse model. Accordingly, LPS-induced cytokine, nitric oxide and MMP-9 production as well as the phagocytosis of bacteria were all up-regulated by NCTD in a dose dependent manner in both RAW264.7 cells and bone marrow-derived macrophages (BMMs). Then we further analyzed TLR4 associated signaling pathway by Western blot, Immunofluorescence and EMSA in the presence or absence of LPS. The phosphorylation of AKT and p65 at serine 536 but not serine 468 was enhanced obviously by NCTD in a dose dependent manner, whereas the degradation of IκBα was little effected. Consequently, the nuclear translocation and DNA binding ability of NF-κB was also increased by NCTD obviously in RAW264.7 cells. Our results demonstrated that NCTD could facilitate LPS-mediated immune response through promoting the phosphorylation of AKT/p65 and transcriptional activity of NF-κB, thus reprofiling the traditional anti-tumor drug NCTD as a novel immune regulator in promoting host defense against bacterial infection.
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
authors.forename: Qufei
authors.initials: Q
authors.lastname: Zhao
authors.forename: Yu
authors.initials: Y
authors.lastname: Qian
authors.forename: Ruimei
authors.initials: R
authors.lastname: Li
authors.forename: Binghe
authors.initials: B
authors.lastname: Tan
authors.forename: Honghui
authors.initials: H
authors.lastname: Han
authors.forename: Mingyao
authors.initials: M
authors.lastname: Liu
authors.forename: Min
authors.initials: M
authors.lastname: Qian
authors.forename: Bing
authors.initials: B
authors.lastname: Du
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D015850:Interleukin-6 / Q000097:blood / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism*; D010538:Peritonitis / Q000097:blood / Q000276:immunology / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects / Q000276:immunology; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000276:immunology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000097:blood / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; C069741:norcantharidin; D051057:Proto-Oncogene Proteins c-akt