Molecules from animal venoms are promising candidates for the development of new drugs. previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (pnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG −/− mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18 F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. tumors from pnV-treated animals were smaller and did not uptake detectable amounts of 18 F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro-and antiinflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from pnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of pnV on tumor development in vivo, considering the complex neoplastic microenvironment. The
Chloroquine (CQ) is a weak base that was originally used to treat malaria infection. 1 However, recent findings have pinpointed the modulatory effect of CQ in models of chronic inflammation and viral infections. The antiviral effect of CQ and its derivative hydroxychloroquine has attracted great attention due to the recent Sars-CoV-2 virus outbreak. 2,3 As an immunomodulatory agent, CQ reduces the severity of experimental autoimmune encephalomyelitis (EAE), likely by inducing regulatory T cells and tolerogenic dendritic cells (DCs). 4,5 EAE and multiple sclerosis (MS) are demyelinating disorders of the CNS in which interleukin (IL)-17-producing helper T (Th17) cells play a major role in disease severity. To date, there has been no report of the effect of CQ on T cell differentiation; thus, we aimed to investigate whether CQ interferes with Th17 cell differentiation.We isolated CD4 + T cells from the spleens of wild-type (WT) C57BL/6 mice and activated the cells with agonistic anti-CD3/ CD28 antibodies under Th17 polarizing conditions in the presence or absence of CQ. We found that cultures with CQ had a significant decrease in IL-17 production ( Fig. 1a). Moreover, CQ at low concentrations was toxic to Th17 cells but not to Th1 cells or DCs (Fig. 1b). Indeed, CQ augmented Th1 cell differentiation (Fig. 1b). These results show that Th17 cells are more susceptible to CQ toxicity than Th1 cells and DCs. We then investigated whether CQ also impacts Tc17 cell differentiation and found that IL-17 production by CD8 + T cells was also reduced by CQ treatment (Fig. 1c). Because T-bet and STAT1 are major suppressors of Th17 cell differentiation, 6,7 we aimed to investigate T-bet and STAT1 expression in CQ-treated cells. Cultures of CQ-treated Th17 cells with the least toxic dose of CQ had a significant increase in T-bet but not STAT1 expression ( Fig. 1d). This result is interesting because we recently showed that CQ-induced tolerogenic DCs rely on STAT1 expression to maintain their suppressive function. 8 Collectively, our current results indicate that, in T cells, CQ suppresses Th17 cell differentiation by stimulating T-bet rather than STAT1 expression.Finally, we investigated whether EAE suppression in CQtreated mice is associated with an increase in T-bet expression in Th17 cells. We immunized WT mice with MOG 35-55 peptide in complete Freund's adjuvant to induce EAE and monitored the mice daily for disease development. CQ treatment started once the mice showed the first signs of EAE (approximately day 10 p.i.) and continued for 7 consecutive days. Similar to what we previously published, 4 CQ-treated mice displayed significantly less severe EAE than PBS-treated mice (Fig. 1e). Analysis of CNS cells at day 21 p.i. revealed that CQ-treated mice had a significant decrease in IL-17-producing Th17 cells and no effect on IFN-γ-producing Th1 cells compared with cells from PBStreated mice (Fig. 1f). Further analyses showed that STAT1 expression was unchanged, while T-bet expression was increased in Th17 cells in the CNS of CQ...
Background Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells, also evaluating RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) involvement. Methods Human (NG97) GB cells were treated with twelve subfractions (SFs—obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound healing and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Values were analyzed by the GraphPad Prism software package and the level of significance was determinate using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. Results Two (SF1 and SF11) of twelve SFs, decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase β2. Conclusion All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway.
Immunomodulation has been considered an important approach in the treatment of malignant tumors. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumor size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated twenty-three subfractions named Low Weight (LW-1 to LW-12) and High Weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumor cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.
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