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NLRP3 inflammasome [NLR (nucleotide-binding domain, leucine-rich repeat containing protein) Pyrin-domain-containing 3 ] functions as an innate sensor of several PAMPs and DAMPs (pathogen- and damage-associated molecular patterns). It has been also reported as a transcription factor related to Th2 pattern, although its role in the adaptive immunity has been controversial, mainly because the studies were performed using gene deletion approaches. In the present study, we have investigated the NLRP3 gain-of-function in the context of encephalomyelitis autoimmune disease (EAE), considered to be a Th1- and Th17-mediated disease. We took advantage of an animal model with NLRP3 gain-of-function exclusively to T CD4+ lymphocytes (CD4CreNLRP3fl/fl). These mice presented reduced clinical score, accompanied by less infiltrating T CD4+ cells expressing both IFN-γ and IL-17 at the central nervous system (CNS) during the peak of the disease. However, besides NLRP3 gain-of-function in lymphocytes, these mice lack NLRP3 expression in non-T CD4+ cells. Therefore, in order to circumvent this deficiency, we transferred naive CD4+ T cells from WT, NLRP3−/− or CD4CreNLRP3fl/fl into Rag-1−/− mice and immunized them with MOG35–55. Likewise, the animals repopulated with CD4CreNLRP3fl/fl T CD4+ cells presented reduced clinical score and decreased IFN-γ production at the peak of the disease. Additionally, primary effector CD4+ T cells derived from these mice presented reduced glycolytic profile, a metabolic profile compatible with Th2 cells. Finally, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under a Th2-related cytokine milieu cocktail exhibited in vitro an increased IL-4 and IL-13 production. Conversely, naive CD4+ T cells from CD4CreNLRP3fl/fl mice under Th1 differentiation produced less IFN-γ and T-bet. Altogether, our data evidence that the NLRP3 gain-of-function promotes a Th2-related response, a pathway that could be better explored in the treatment of multiple sclerosis.

To report a case of a child with strabismus and delayed discovery of a metallic intraocular foreign body with good recovery of visual acuity and stereopsis with 36-month follow-up. A 4-year-old girl was evaluated due to exotropia of right eye initiated 9 months before with progressive worsening. Visual acuity was 1.00 logMAR (20/200) in the right eye and 0.00 logMAR (20/20) in the left eye. Anterior segment evidenced a small paracentral corneal leukoma, posterior synechia and mild lens opacity in the temporal quadrant only in the right eye. Fundus examination in the right eye identified the presence of an intraocular foreign body, with appearance of metallic components surrounded by retinal pigmented endothelial cells atrophy. Full-field electroretinography (ERG) showed reduced amplitudes and delayed implicit times for both rods and cones in the affected eye. All tests were normal in the fellow eye. Pars plana vitrectomy was promptly performed in the right eye, followed by phacoemulsification with intraocular lens implantation 4 months later due to worsening of the lens opacification. The full-field ERG was repeated after the surgical procedures. The ERG showed mild worsening of all responses in the right eye. After 36 months of follow-up, visual acuity was 0.20 logMAR (20/32) with improvement of the ocular misalignment and with 60 s of arc stereopsis with ERG responses unchanged. In this young girl perforating ocular trauma with metallic material was lately diagnosed with strabismus as a sign of alert. Prompt surgical intervention and proper management were essential to provide reasonable visual function including some degree of stereopsis, even though retinal dysfunction characterized by ERG was persistent.

Protease roles in cancer progression have been demonstrated and their inhibitors display antitumor effects. Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, and tellurium compounds were described as potent cysteine protease inhibitors and also assayed in several animal models. In this work, the two enantiomeric forms of 1-[Butyl(dichloro)-λ4-tellanyl]-2-[1S-methoxyethyl]benzene (organotelluranes RF-13R and RF-13S) were evaluated as inhibitors of cathepsins B and L, showing significant enantiodiscrimination. We observed their cytotoxic effects on a murine melanoma model, effectively inhibiting tumor progression in vivo. The enantiomers were able to inhibit melanoma cell viability, migration and invasion in vitro. Besides, RF-13S and RF-13R were able to inhibit endothelial cell angiogenesis using a tube formation assay in vitro, in a stereodependent manner. These organotelluranes affected cell morphology, showing disassembling of the actin cytoskeleton. These results suggest organotelluranes as potential antitumor agents, acting directly on tumor cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis, showing low toxicity and high efficiency. Taken together our results suggest that this class of compounds should be further studied to reveal their potential as antitumoral agents.

There is a strong correlation between obesity and cancer. Here, we investigated the influence of IL-6 and gut microbiota of obese mice in melanoma development. We first evaluated B16F10 melanoma growth in preclinical models for obesity: mice deficient for leptin

Purpose To describe retinal pigment epithelium (RPE) disease detected by fundus autofluorescence (FAF) imaging in eyes with idiopathic choroidal neovascularization (ICNV). Methods A retrospective review of patients seen during a 14-month period with the diagnosis of ICNV was performed to identify patients with RPE disease, defined as hypo or hyperautofluorescent lesions on FAF. The presence of ICNV was confirmed by clinical history, ophthalmoscopic examination, fluorescein angiography (FA), and spectral domain-optical coherence tomography (SD-OCT). The clinical diagnosis of an underlying inflammatory condition was based on the FAF appearance of multiple punched-out hyper or hypoautofluorescent spots in the retinal fundus. Results The mean age was 27 years (range, 21-33 years). Best-corrected visual acuity ranged from 20/25 to 20/200 with a median visual acuity of 20/80. Ten eyes of 8 patients presented RPE abnormalities on FAF. Of the 10 study eyes, ICNV was observed in 8 eyes. ICNV appeared as a type 2 neovascular membrane at the macular area on FA, and SD-OCT revealed neurosensory detachment in all study eyes. FAF demonstrated abnormalities of the RPE that were not appreciated on clinical examination or by other imaging modalities. Conclusions FAF may reveal an underlying inflammatory condition in patients diagnosed as ICNV, modifying the diagnosis and management.

Background Anti-angiogenic drugs remain the mainstay therapy for several vascular retinal pathologies. The repurposing of approved anti-angiogenic drugs for use in ophthalmology can increase therapeutic options and reduce costs. The purpose of this study was to investigate the ocular safety profile of intravitreal (IVT) ramucirumab, an approved anti-vascular endothelial growth factor molecule for systemic treatment, using cell culture and animal models. Methods The cytotoxicity of ramucirumab at different concentrations was evaluated in human retinal pigment epithelial cells (ARPE-19) using the MTT assay. In addition, 250 or 500 µg of ramucirumab or vehicle was injected in the eye of 16 chinchilla rabbits. The eyes were evaluated by ophthalmoscopy, electroretinography, spectral-domain optical coherence tomography (SD-OCT) and by light and transmission electron microscopy. Results Electroretinography or SD-OCT did not detect functional or morphological alterations at 24 h or one week after injection. Light and transmission electron microscopy confirmed the absence of major signs of toxicity, although we found a statistically significant reduction in ganglion cell number between the controls and the eyes that received 500 µg of ramucirumab after 7 days. Compared to lower concentrations, 500 µg of ramucirumab caused reduction in cell viability and changes in morphology in ARPE-19 cells. Compared to the baseline, ocular and serum osmolarity showed no difference after IVT injection at all timepoints. Conclusion In conclusion, IVT injection of ramucirumab in rabbits is safe and does not cause functional damage to the retina. At the lower dose tested in vivo (250 µg), the morphology and ultrastructural anatomy were normal at 24 h and 1 week after the injection. However, the 500 µg dose can cause a decrease in ganglion cell number seven days after the injection.

The antitumor effect of metformin has been demonstrated in several types of cancer; however, the mechanisms involved are incompletely understood. In this study, we showed that metformin acts directly on melanoma cells as well as on the tumor microenvironment, particularly in the context of the immune response. In vitro, metformin induces a complex interplay between apoptosis and autophagy in melanoma cells. The anti-metastatic activity of metformin in vivo was assessed in several mouse models challenged with B16F10 cells. Metformin's activity was, in part, immune system-dependent, whereas its antitumor properties were abrogated in immunodeficient (NSG) mice. Metformin treatment increased the number of lung CD8-effector-memory T and CD4+Foxp3+IL-10+ T cells in B16F10-transplanted mice. It also decreased the levels of Gr-1+CD11b+ and RORγ+ IL17+CD4+ cells in B16F10-injected mice and the anti-metastatic effect was impaired in RAG-1-/- mice challenged with B16F10 cells, suggesting an important role for T cells in the protection induced by metformin. Finally, metformin in combination with the clinical metabolic agents rapamycin and sitagliptin showed a higher antitumor effect. The metformin/sitagliptin combination was effective in a BRAFV600E/PTEN tamoxifen-inducible murine melanoma model. Taken together, these results suggest that metformin has a pronounced effect on melanoma cells, including the induction of a strong protective immune response in the tumor microenvironment, leading to tumor growth control, and the combination with other metabolic agents may increase this effect.

Background: Inflammation is a key feature of aldosterone-induced vascular damage and dysfunction, but molecular mechanisms by which aldosterone triggers inflammation remain unclear. The NLRP3 inflammasome is a pivotal immune sensor that recognizes endogenous danger signals triggering sterile inflammation. Methods: We analyzed vascular function and inflammatory profile of wild-type (WT), NLRP3 knockout ( NLRP3 −/− ), caspase-1 knockout ( Casp-1 −/− ), and interleukin-1 receptor knockout ( IL-1R −/− ) mice treated with vehicle or aldosterone (600 µg·kg −1 ·d −1 for 14 days through osmotic mini-pump) while receiving 1% saline to drink. Results: Here, we show that NLRP3 inflammasome plays a central role in aldosterone-induced vascular dysfunction. Long-term infusion of aldosterone in mice resulted in elevation of plasma interleukin-1β levels and vascular abnormalities. Mice lacking the IL-1R or the inflammasome components NLRP3 and caspase-1 were protected from aldosterone-induced vascular damage. In vitro, aldosterone stimulated NLRP3-dependent interleukin-1β secretion by bone marrow–derived macrophages by activating nuclear factor-κB signaling and reactive oxygen species generation. Moreover, chimeric mice reconstituted with NLRP3-deficient hematopoietic cells showed that NLRP3 in immune cells mediates aldosterone-induced vascular damage. In addition, aldosterone increased the expression of NLRP3, active caspase-1, and mature interleukin-1β in human peripheral blood mononuclear cells. Hypertensive patients with hyperaldosteronism or normal levels of aldosterone exhibited increased activity of NLRP3 inflammasome, suggesting that the effect of hyperaldosteronism on the inflammasome may be mediated through high blood pressure. Conclusions: Together, these data demonstrate that NLRP3 inflammasome, through activation of IL-1R, is critically involved in the deleterious vascular effects of aldosterone, placing NLRP3 as a potential target for therapeutic interventions in conditions with high aldosterone levels.

Background Functional and anatomical evaluation of patients with ischemic diabetic macular edema after monthly injections of Bevacizumab. Methods Five eyes from five patients with diabetic macular edema associated with macular ischemia in fluorescein angiography (FA), received 6 monthly intravitreal injections of Bevacizumab. All subjects underwent SD-OCT, FA, OCT angiography (OCTA) and microperimetry at baseline and after 6 months follow-up. Primary outcome measures were improvement of best corrected visual acuity (BCVA), microperimetry and assessment of macular perfusion (foveal avascular zone size and capillary loss). Results Five patients completed the follow-up. BCVA improved from 20/180 to 20/74 (p = 0.01) and macular sensitivity improved from 11.66 to 16.26 dB (p

BackgroundFor the purpose of studying functional human dendritic cells (DCs) in a humanized mouse model that mimics the human immune system (HIS), a model referred to as HIS mice was established.MethodsHuman immune system mice were made by engrafting NOD/SCID/IL2Rgammanull (NSG) mice with human hematopoietic stem cells (HSCs) following the transduction of genes encoding human cytokines and human leukocyte antigen (HLA)-A2.1 by adeno-associated virus serotype 9 (AAV9) vectors.ResultsOur results indicate that human DC subsets, such as CD141+CD11c+ and CD1c+CD11c+ myeloid DCs, distribute throughout several organs in HIS mice including blood, bone marrow, spleen, and draining lymph nodes. The CD141+CD11c+ and CD1c+CD11c+ human DCs isolated from HIS mice immunized with adenoviruses expressing malaria/human immunodeficiency virus (HIV) epitopes were able to induce the proliferation of malaria/HIV epitopes-specific human CD8+ T cells in vitro. Upregulation of CD1c was also observed in human CD141+ DCs 1 day after immunization with the adenovirus-based vaccines.ConclusionsEstablishment of such a humanized mouse model that mounts functional human DCs enables preclinical assessment of the immunogenicity of human vaccines in vivo.

Background Swept-source optical coherence tomography (SS-OCT) has a higher scanning rate and longer wavelength in comparison with spectral-domain OCT (SD-OCT), allowing an improved imaging of retinal vascular plexuses and choriocapillaris. The present two patients diagnosed with multiple evanescent white dot syndrome (MEWDS) underwent fundus autofluorescence (FAF), en-face SS-OCT, and SS-OCT angiography (OCTA) imaging, and its features were described and correlated. Results The clinical and imaging findings of both cases were consistent with the diagnosis of MEWDS. Color fundus photograph revealed subtle deep retinal white spots in the posterior pole and around the optic disk. FAF showed several hyperautofluorescent lesions corresponding topographically to the subtle deep retinal white lesions observed on color fundus photographs. Cross-sectional SS-OCT showed disruption of the ellipsoid zone (EZ) within the macular area in all study patients. En-face SS-OCT at the level of the outer retina showed lower reflectivity correspondent to the diffuse attenuation due to the EZ disruption on cross-sectional OCT. SS-OCTA demonstrated flow preservation within the retinal vasculature and choriocapillaris. Conclusions SS-OCT imaging allows a better visualization of the choriocapillaris, and its normal appearance in MEWDS may suggest that the outer retina and photoreceptors represent the primary site of inflammation.

Stat3 as a transcription factor regulating gene expression in lymphocytes during the immune response is well known. However, since the pioneering studies discovering the presence of Stat3 in mitochondria and its role in regulating mitochondrial metabolism, only a few studies have investigated this non-conventional function of Stat3 in lymphocytes. From this perspective, we review what is known about Stat3 as a transcription factor and what is known and unknown about mitochondrial Stat3 (mitoStat3) in lymphocytes. We also provide a framework to consider how some of the functions previously assigned to Stat3 as regulator of gene transcription could be mediated by mitoStat3 in lymphocytes. The goal of this review is to stimulate interest for future studies investigating mitoStat3 in the immune response that could lead to the generation of alternative pharmacological inhibitors of mitoStat3 for the treatment of chronic inflammatory diseases.

PURPOSE To compare the efficacy of different concentrations of topical lidocaine gel with standard subconjunctival anesthesia. METHODS This was a prospective randomized controlled pilot study with 3 different groups. Group SC received subconjunctival lidocaine and proparacaine drops as needed during surgery. Group L2 received 2% lidocaine gel and group L5 received 5% lidocaine gel. Both lidocaine gel groups could receive another gel dose as needed during the procedure. The primary outcome was pain, which was experienced during and after surgery. The assessment was done using a 10-point linear analog scale at predetermined stages of surgery (1-first incision, 2-pterygium body excision, 3-conjunctival suturing, 4-immediate postoperative period after patching, and 5-assessment by a trained nurse in the absence of a surgeon after operation). RESULTS We enrolled 45 consecutive patients who underwent primary pterygium surgery. Statistical significance between groups was found in mean pain at administration (2.2 vs. 0.4 vs. 0.0 in SC, L2, and L5 groups, respectively, with P < 0.05). The number of reapplications of anesthetic drops did not differ between the groups. We found a correlation between reapplication of the anesthetic and pain at administration and duration of the procedure. CONCLUSIONS We demonstrated that the use of lidocaine gel at concentrations of 2% and 5% in pterygium surgery was as effective as subconjunctival injection of lidocaine in reducing intraoperative and postoperative pain, producing significantly less pain during initial administration of the anesthetic.

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.

Silencing of SOCS1 protein with shRNAi lentivirus (shR-SOCS1) led to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS1 silencing inhibited cell migration and invasion as well as in vitro growth by cell cycle arrest at S phase with increased cell size and nuclei. Down-regulation of SOCS1 decreased the expression of epidermal growth factor receptor, Ins-Rα, and fibroblast growth factor receptors. The present work aimed at analyzing the SOCS1 cell signaling and expression of proteins relevant to tumor development. An RNA microarray analysis of B16F10-Nex2 melanoma cells with SOCS1 silenced by shRNAi-SOCS1 was undertaken in comparison with cells transduced with the empty vector. Among 609 differentially expressed genes, c-Kit, Met and EphA3 cytokine/tyrosine-kinase (TK) receptors were down regulated. A significant decrease in the expression of TK receptors, the phosphorylation of mediators of ERK1/2 and p38 pathways and STAT3 (S727) were observed. Subcutaneous immunization with shR-SOCS1-transduced viable tumor cells rendered protection against melanoma in a syngeneic model, with decreased expression of PD-L1 and of matrix metallo-proteinases (MMPs) and CD-10 in those cells. The present work shows the role of SOCS1 in murine melanoma development and the potential of SOCS1-silenced tumor cells in raising an effective anti-melanoma immune response.

Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.

In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8(+) T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8(+) T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8(+) T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44(+)CD62L(-)NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation.

Malignant melanoma is one the most aggressive types of cancer and its incidence has gradually increased in the last years, accounting for about 75% of skin cancer deaths. This poor prognosis results from the tumor resistance to conventional drugs mainly by deregulation of apoptotic pathways. The aim of this work was to investigate the cell death mechanism induced by α-pinene and its therapeutic application. Our results demonstrated that α-pinene was able to induce apoptosis evidenced by early disruption of the mitochondrial potential, production of reactive oxygen species, increase in caspase-3 activity, heterochromatin aggregation, DNA fragmentation and exposure of phosphatidyl serine on the cell surface. Most importantly, this molecule was very effective in the treatment of experimental metastatic melanoma reducing the number of lung tumor nodules. This is the first report on the apoptotic and antimetastatic activity of isolated α-pinene.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Melanoma is the most aggressive form of skin cancer, and its incidence has increased dramatically over the years. the murine B16F10 melanoma in syngeneic C57Bl/6 mice has been used as a highly aggressive model to investigate tumor development. Presently, we demonstrate in the B16F10-Nex2 subclone that silencing of SOCS-1, a negative regulator of Jak/Stat pathway, leads to reversal of the tumorigenic phenotype and inhibition of melanoma cell metastasis. SOCS-1 silencing with short hairpin RNA affected tumor growth and cell cycle regulation with arrest at the S phase with large-sized nuclei, reduced cell motility, and decreased melanoma cell invasion through Matrigel. A clonogenic assay showed that SOCS-1 acted as a modulator of resistance to anoikis. in addition, down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor ( mainly the phosphorylated-R), Ins-R alpha, and fibroblast growth factor receptor. in vivo, silencing of SOCS-1 inhibited subcutaneous tumor growth and metastatic development in the lungs. Because SOCS-1 is expressed in most melanoma cell lines and bears a relation with tumor invasion, thickness, and stage of disease, the present results on the effects of SOCS-1 silencing in melanoma suggest that this regulating protein can be a target of cancer therapy. Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Unidade Oncol Expt, BR-04023062 São Paulo, Brazil Univ São Paulo, Dept Pharmacol, São Paulo, Brazil Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Unidade Oncol Expt, BR-04023062 São Paulo, Brazil FAPESP: 06/50634-2 Web of Science

This paper presents the results of simulation analysis executed by the TPN – Tanque de Provas Numerico (Numerical Offshore Tank), about the POLVO FPSO (Floating, Production, Storage Offloading) offloading operation, located at Campos basin in the state of Rio de Janeiro, Brazil. Moreover, it is shown the viability to use numerical codes, as TPN simulator, as auxiliary tools instead of carry out expensive and time spending procedures like reduced model tests.

Made available in DSpace on 2015-03-18T15:56:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: Pyrostegia venusta (Ker. Gawl.) Miers (Bignoniacea) is a medicinal plant from the Brazilian Cerrado used to treat leucoderma and common diseases of the respiratory system. Objective: To investigate the antitumor activity of P.venusta extracts against melanoma. Materials and Methods: The cytotoxic activity and tumor induced cell death of heptane extract (HE) from P. venusta flowers was evaluated against murine melanoma B16F10-Nex2 cells in vitro and in a syngeneic model in vivo. Results: We found that HE induced apoptosis in melanoma cells by disruption of the mitochondrial membrane potential, induction of reactive oxygen species and late apoptosis evidenced by plasma membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation, exposure of phosphatidylserine on the cell surface and activation of caspase-2,-3,-8,-9. HE was also protective against singeneyc subcutaneous melanoma HE compounds were also able to induce cell cycle arrest at G2/M phases on tumor cells. On fractionation of HE in silica gel we isolated a cytotoxic fraction that contained a mixture of saturated hydrocarbons identified by (1) H NMR and GC-MS analyses. Predominant species were octacosane (C 28 H 58 -36%) and triacontane (C 30 H 62 -13%), which individually showed significant cytotoxic activity against murine melanoma B16F10-Nex2 cells in vitro and a very promising antitumor protection against subcutaneous melanoma in vivo. Conclusion: The results suggest that the components of the heptane extract, mainly octasane and triacontane, which showed antitumor properties in experimental melanoma upon regional administration, might also be therapeutic in human cancer, such as in the mostly epidermal and slowly invasive melanomas, such as acral lentiginous melanoma, as an adjuvant treatment to surgical excision. Fed Univ Sao Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, Div Cell Biol, BR-04023062 Sao Paulo, Brazil Fed Univ Sao Paulo UNIFESP, Expt Oncol Unit UNONEX, BR-04023062 Sao Paulo, Brazil Univ Estadual Paulista UNESP, Phytochem Lab, Dept Biol Sci, Assis, SP, Brazil Fed Univ Sao Paulo UNIFESP, Inst Environm Chem & Pharmaceut Sci, BR-04023062 Sao Paulo, Brazil Univ Estadual Paulista UNESP, Phytochem Lab, Dept Biol Sci, Assis, SP, Brazil

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished.

Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8(+) T lymphocytes were the main effector antitumor population, although B and CD4(+) T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells 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 primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction 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 promising novel alternative for metastatic melanoma treatment.

Knowledge on the regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now recognized that multiple signaling pathways that affect motility, growth control, invasion, metabolism, cytokine release and the ability to escape the immune response, regulate melanoma development. Silencing of SOCS-1 protein, a negative regulator of Jak/Stat pathway, leads to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS-1 silencing with shRNAi lentivirus (shR-SOCS-1) inhibits in vitro growth by cell cycle regulation with S phase arrest and increased size of cells and nuclei. It also inhibited tumor cell migration and invasion. Down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptors. Based on the results of SOCS-1 silencing, the present work aims at analyzing the expression of proteins relevant to tumor development, searching for signaling pathways related with SOCS-1. A protein microarray analysis of murine melanoma cells silenced for SOCS-1 by shRNAi-SOCS-1 lentivirus transduction was undertaken using as controls, cells transduced with the empty vector. Among 614 differentially expressed genes, c-kit, met and EphA3 cytokine/tyrosine-kinase (TK) receptors were downregulated. A significant decrease in the expression of TK receptors as observed by Western blotting of B16 shR-SOCS-1 extracts showed that SOCS-1 positively regulates the MAPK signaling pathways in these cells. A decrease in the phosphorylation of mediators of ERK1/2 and p38 pathways and of STAT3 (S727) was observed. These results demonstrate that SOCS-1 plays an important role in tumor development and progression up-regulating a number of growth factor receptors in murine melanoma cells. Interestingly, p-CREB (S133) and total AP-2 alpha were up-regulated in the SOCS-1-deficient B16F10-Nex2 cells. In vivo experiments using a syngeneic model and a prophylactic protocol showed that the subcutaneous immunization with shR-SOCS-1-transduced viable tumor cells, rendered protection against either subcutaneously grafted or metastatic murine melanoma cells, with significant reduction in tumor growth and lung metastatic nodules. Such immunization increased the expression of CD11c, CD80, CD86 and MHC-II in splenic dendritic cells, as compared to DCs from a group that received only B16F10-Nex2 cells. Of notice was the decreased expression of PD-L1 in SOCS-1-silenced B16F10-Nex2 cells (B16shR-SOCS-1). Such molecular phenotype may have a role in the immune response and protective effect of subcutaneous immunization with these cells, rather than with wild-type B16F10-Nex2 cells. By overexpressing PD-L1, WT-melanoma cells negatively regulates T-cell proliferation in the tumor environment. Down regulation of PD-L1 is compatible with inactive ERK. The present results show the important role of SOCS-1 in murine melanoma development and the potential of SOCS-1-silenced tumor cells in raising an effective anti-tumor immune response, inducing protection even against a metastatic melanoma challenge. Funded by FAPESP 2010/51423-0 and 2012/17473-6. Citation Format: Rodrigo Berzaghi, Esq., Vera SC Maia, II, Felipe V. Pereira, III, Filipe Manegatti De Melo, Luiz R. Travassos, Luiz R. Travassos. The role of SOCS-1 in stimulating melanoma development through tyrosine-kinase receptors and MAPK pathways and prevents antitumour immunity by PD-L1 expression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A146.

Background Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S (-) C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. Methods B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Results Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. Conclusions The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.

Study background: Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, may translocate to the cell surface and be secreted. They play a role in tumor angiogenesis. Cystatins are natural cysteine protease inhibitors that can inhibit tumor development, growth and metastasis. In the present work we evaluated the potential therapeutic use of sugarcane cystatin CaneCPI-4 as an anticancer drug. Methods: Viability, migration, invasion and anchorage-independent growth were investigated in B16F10-Nex2 melanoma and HUVEC cells in the presence of CaneCPI-4. The in vivo effect of CanceCPI-4 on tumor development was assessed using a murine model. Angiogenesis in vitro was evaluated using HUVEC cells plated on Matrigel. Immunohistochemical analysis of CD34 expression in primary melanoma was also carried out. Results: Sugarcane cystatin CaneCPI-4 was not cytotoxic to melanoma or HUVEC cells growing in vitro, but efficiently inhibited melanoma cell development in vivo. CaneCPI-4 inhibited melanoma and endothelial cell migration and tumor invasion in vitro. Using a Matrigel angiogenesis assay, CaneCPI-4 at 1 mM was able to completely abolished endothelial cell sprouting in vitro. Angiogenesis inhibition was confirmed in vivo by immunohistochemistry. Conclusions: Sugarcane cystatin CaneCPI-4 inhibits melanoma development in vivo by angiogenesis disruption and inhibition of melanoma invasion, migration and anchorage-independent growth.

Knowledge on the regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now recognized that multiple signaling pathways that affect motility, growth control, invasion, metabolism, cytokine release and the ability to escape the immune response, regulate melanoma development. Silencing of SOCS-1 protein, a negative regulator of Jak/Stat pathway, leads to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS-1 silencing with shRNAi inhibits in vitro growth by cell cycle regulation with S phase arrest and increased size of cells and nuclei. It also inhibited tumor cell migration and invasion. Down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor (mainly the phosphorylated-R), Ins-Rα, and fibroblast growth factor receptors. Based on the results of SOCS-1 silencing, the present work aims at analyzing the expression of proteins relevant to tumor development, searching for signaling pathways related with SOCS-1. A protein microarray analysis of murine melanoma cells silenced for SOCS-1 by shRNAi SOCS-1 lentivirus transduction was undertaken using as controls, cells transduced with the empty vector. Among the 614 differentially expressed genes some cytokine/growth factor receptors were down regulated, e.g. c-kit, met and EphA3. Western blotting of B16 shR-SOCS-1 cell extracts showed a significant decrease in the expression of these receptors, indicating that SOCS-1 positively regulates the MAPK pathways in these cells. We analyzed the main MAPK pathways that were up regulated in melanoma and observed a decrease in the phosphorylation of mediators of ERK1/2 and p38 pathways and of STAT3 (S727). These results demonstrate that SOCS-1 can play an important role in tumor development and progression up regulating a number of growth factor receptors in murine melanoma cells. Interestingly, p-CREB (S133) was up regulated in the SOCS-1-deficient B16F10-Nex2 cells. In vivo experiments showed that using a prophylactic protocol, the subcutaneous immunization of syngeneic mice with B16 shR-SOCS-1 viable cells, rendered a protective effect against subcutaneously grafted and metastatic murine melanoma, with significant reduction in tumor growth and metastates in the lungs. A flow cytometry assay showed decreased expression of PD-L1 at the surface of B16 shR-SOCS-1 cells. This might explain the protective effect of subcutaneous immunization with these cells, rather than with wild-type B16F10-Nex2 cells that overexpress PD-L1, which negatively regulates T-cell immune response. Down regulation of PD-L1 is compatible with inactive ERK. The present results show the important role of SOCS-1 in murine melanoma stimulating cell growth and migration, allowing for the invasive phenotype. Citation Format: Rodrigo Berzaghi, Felipe V. Pereira, Vera S.C. Maia, Jorge B. Scutti, Luiz R. Travassos. The role of SOCS-1 in stimulating melanoma development through growth factor receptors and MAPK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5296. doi:10.1158/1538-7445.AM2014-5296

The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

Malignant melanoma is the most aggressive and deadly type of skin cancer. The Wilms tumor protein 1 (WT1) is a transcription factor that has been associated with melanoma oncogenesis and proliferation, hence could be a target of tumor control procedures. A lysine-arginine rich peptide (WT1 pTj) based on WT1 C-terminal zinc finger domain was synthesized and tested for anti-proliferative effects on human and murine melanoma cell lines (A2058 and B16F10-Nex2, respectively), including inhibition of cell growth and colony formation, cell cycle arrest and induction of cell senescence. Internalization of WT1 pTj into A2058 cells was established by confocal microscopy, using biotinylated peptide and staining by streptoavidin-FITC. An effect on p53 activity was measured by a luciferase reporter assay. Competitive ELISA assay was performed to verify whether the peptide interferes with p53-WT1 interaction. In vivo, athymic nude mice and C57Bl/6 were inoculated with A2058 and B16F10-Nex2 cells, respectively, and treated for 5 consecutive days with C-amidated WT1 pTj. Here, we show that WT1 pTj decreased, in a dose and time dependent manner, the number of melanoma viable cells. The anti-proliferative activity of WT1 pTj was linked to an irreversible G1 cell cycle arrest and induction of senescence as suggested by positive staining for beta-galactosidase. Treatment with WT1 pTj reduced the total number of soft-agar colonies by A2058 melanoma cells. The peptide entered the cell within 15 minutes and was diffusely distributed throughout the cell, including the nucleus. Moreover, WT1 pTj enhanced p53 activity and competed with WT1 protein for binding to p53. In vivo, WT1 pTj reduced the number of lung nodules in the B16F10-Nex2 syngeneic model of metastatic melanoma and prolonged survival of mice in the A2058 subcutaneous xenograft model. In conclusion, WT1 pTj peptide entered cancer cells and promoted antitumor activity in vitro and in vivo. The data support the hypothesis that WT1 pTj may prevent important interactions between WT1 protein and its partners via a zinc finger motif, inhibiting melanoma development. Therefore, WT1 pTj is a promising agent against WT1-expressing malignancies (e.g. MCF-7 but not SK-BR-3 breast cancer or normal human fibroblasts; and also HL60 acute myeloid leukemia). WT1 pTj may act also as a trojan carrier peptide for potential drug delivery. Supported by FAPESP grant 2010/51423-0 and the Brazilian National Research Council (CNPq) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2867. doi:1538-7445.AM2012-2867