Heat-inactivation inhibited the protease catalytic site, but managed the apparent molecular weight, as observed in SDS-PAGE gels, maintaining also the acknowledgement by the rabbit arazyme-specific polyclonal antibody in western blotting assay (data not shown). increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing main and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease conversation with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a encouraging novel option for metastatic melanoma treatment. (gingipains) have immunoregulatory properties, acting as virulence factors in periodontitis. Arg- and Lys-gingipains reduced CD14 expression causing macrophage hyporesponsiveness,8 suppressed inflammatory responses by human gingival fibroblasts,9 activated platelets, and cleaved the chemokine RANTES.10 Interestingly, the adhesin, but not the catalytic subunit of HMN-214 gingipains mediated the strong upregulation of proinflammatory cytokines by macrophages.8 The surface-associated subtilisin-like protease (SspA) of spider,13 had a strong antitumor effect in a murine metastatic melanoma B16F10-Nex2 model, mediated by the cleavage of tumor cell surface CD44 and the induction of arazyme-specific antibodies that cross-react with tumor matrix metalloprotease 8 (MMP-8).14 In HMN-214 this study, we show that, in addition to its proteolytic-dependent activity, arazyme has a secondary, non-proteolytic antitumor effect dependent on an intact immune system. The anti-melanoma immune response induced by heat-inactivated arazyme was dependent on IFN, and CD8+ T lymphocytes were identified as the main effector cells for tumor rejection. Both macrophages and dendritic cells (DCs) were activated by arazyme, triggering TLR4-MyD88-TRIF- and MAPK-dependent signaling pathways. Materials and methods Animals Inbred male C57BL/6 (WT), BALB/c, culture medium supernatant, obtained from InsectBiotech, Korea, was purified as previously explained. 13 Purified arazyme was completely inactivated by incubating at 50C for 30?min.13 Arazyme treatment in murine models C57Bl/6, test when two groups were compared. Comparisons of three or more groups were performed HMN-214 using the one-way or two-way ANOVA test, followed by Dunnett’s or Tukey’s multiple comparisons, as explained in the Physique legends. In all studies, a value 0.05 was considered statistically significant. Results Anti-metastatic effect of active and heat-inactivated arazyme Recently, we showed that treatment of melanoma-bearing C57Bl/6 mice with active arazyme, a bacterial metalloprotease secreted by arazyme-specific antibodies were cytotoxic to tumor cells, an effect increased by match, and passive transfer to tumor-bearing mice partially inhibited melanoma lung metastasis.14 In order to verify whether the proteolytic activity of the enzyme was responsible for the antitumor effect, arazyme was heat-inactivated at 56C,13 and used in the same treatment protocol. Melanoma-bearing C57Bl/6 male mice inoculated intraperitoneally with active or inactive arazyme on alternate days for 2?weeks showed a significant reduction in the number of metastatic lung nodules compared to PBS-treated mice (Figs.?1A and B). Active arazyme was slightly more efficient than the heat-inactivated protease, suggesting that this direct effect of arazyme on tumor cells is usually, at least partially, important for the inhibition of metastasis. However, the metalloprotease can also control melanoma cell growth by other means 0.0001, analyzed by one-way ANOVA with HMN-214 Tukey’s multiple comparisons. An intact immune system and IFN are necessary for the antitumor effect of active and inactive arazyme To evaluate the role of the immune system in the inhibition of metastasis by active or inactive arazyme, immunodeficient NSG mice were inoculated with B16F10-Nex2 cells and treated with the protease. NSG mice carry the SCID mutation and a deletion of the IL-2 receptor, and thus lack functional T and B lymphocytes and NK cells, and have deficient cytokine signaling. These mice have been widely used as hosts for both normal and malignant human cells.18,19 Neither active nor inactive arazyme inhibited metastasis in immunodeficient NSG mice, as both the treated and untreated animals experienced the same Rabbit polyclonal to ITPK1 quantity of lung nodules as the C57Bl/6 (WT) control mice (Fig.?1B). This result demonstrates the fundamental importance of an intact immune response for the full protease-mediated antitumor effect. IFN and nitric oxide (NO) play unique functions in the intricate relationship between the immune system and tumor development.20 To.
Heat-inactivation inhibited the protease catalytic site, but managed the apparent molecular weight, as observed in SDS-PAGE gels, maintaining also the acknowledgement by the rabbit arazyme-specific polyclonal antibody in western blotting assay (data not shown)