Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We ...investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1(-/-) mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.
Melanoma is a severe metastatic skin cancer with poor prognosis and no effective treatment. Therefore, novel therapeutic approaches using nanotechnology have been proposed to improve therapeutic ...effectiveness. Lipid-core nanocapsules (LNCs), prepared with poly(ε-caprolactone), capric/caprylic triglyceride, and sorbitan monostearate and stabilized by polysorbate 80, are efficient as drug delivery systems. Here, we investigated the effects of acetyleugenol-loaded LNC (AcE-LNC) on human SK-Mel-28 melanoma cells and its therapeutic efficacies on melanoma induced by B16F10 in C57B6 mice. LNC and AcE-LNC had z-average diameters and zeta potential close to 210 nm and -10.0 mV, respectively. CytoViva(®) microscopy images showed that LNC and AcE-LNC penetrated into SK-Mel-28 cells, and remained in the cytoplasm. AcE-LNC in vitro treatment (18-90×10(9) particles/mL; 1 hour) induced late apoptosis and necrosis; LNC and AcE-LNC (3-18×10(9) particles/mL; 48 hours) treatments reduced cell proliferation and delayed the cell cycle. Elevated levels of nitric oxide were found in supernatant of LNC and AcE-LNC, which were not dependent on nitric oxide synthase expressions. Daily intraperitoneal or oral treatment (days 3-10 after tumor injection) with LNC or AcE-LNC (1×10(12) particles/day), but not with AcE (50 mg/kg/day, same dose as AcE-LNC), reduced the volume of the tumor; nevertheless, intraperitoneal treatment caused toxicity. Oral LNC treatment was more efficient than AcE-LNC treatment. Moreover, oral treatment with nonencapsulated capric/caprylic triglyceride did not inhibit tumor development, implying that nanocapsule supramolecular structure is important to the therapeutic effects. Together, data herein presented highlight the relevance of the supramolecular structure of LNCs to toxicity on SK-Mel-28 cells and to the therapeutic efficacy on melanoma development in mice, conferring novel therapeutic mechanisms to LNC further than a drug delivery system.
We report a method to obtain biaryl dipeptide tyrosine via Suzuki–Miyaura and alkynyl dipeptide tyrosine by Sonogashira cross-coupling reactions. Analysis of the biological action of biaryl dipeptide ...tyrosine 4d compound showed its ability to impair the metabolism and proliferation of SK-Mel-28 human melanoma lineage cells, independently of mitochondrial membrane depolarization, apoptosis and necrosis. Moreover, 4d compound did not cause toxicity to human umbilical vein endothelial cells (HUVEC), suggesting its toxic specificity to cancer cells.
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•Synthesis of tyrosine dipeptides.•Toxicity of a dipeptide tyrosine derivative demonstrated in a melanoma cell lineage.•Biaryl dipeptide tyrosine compounds have selective cytotoxic potential.
Metastatic melanoma is an aggressive cancer with increasing incidence and limited therapies in advanced stages. Systemic neutrophilia or abundant neutrophils in the tumor contribute toward its worst ...prognosis, and the interplay of cancer and the immune system has been shown in tumor development and metastasis. We recently showed the in vivo efficacy of poly(ε-caprolactone) lipid-core nanocapsule (LNC) or LNC loaded with acetyleugenol (AcE-LNC) to treat B16F10-induced melanoma in mice. In this study, we investigated whether LNC or AcE-LNC toxicity could involve modifications on crosstalk of melanoma cells and neutrophils. Therefore, melanoma cells (B16F10) were pretreated with vehicle, LNC, AcE or AcE-LNC for 24 h, washed and, further, cocultured for 18 h with peritoneal neutrophils obtained from C57Bl/6 mice. Melanoma cells were able to internalize the LNC or AcE-LNC after 2 h of incubation. LNC or AcE-LNC pretreatments did not cause melanoma cells death, but led melanoma cells to be more susceptible to death in serum deprivation or hypoxia or in the presence of neutrophils. Interestingly, the production of reactive oxygen species (ROS), which causes cell death, was increased by neutrophils in the presence of LNC- and AcE-LNC-pretreated melanoma cells. LNC or AcE-LNC treatments reduced the concentration of transforming growth factor-β (TGF-β) in the supernatant of melanoma cells, a known factor secreted by cancer cells to induce pro-tumoral actions of neutrophils in the tumor microenvironment. In addition, we found reduced levels of pro-tumoral chemical mediators VEGF, arginase-1, interleukin-10 (IL-10) and matrix metalloproteinase-9 (MMP-9) in the supernatant of LNC or AcE-LNC-pretreated melanoma cells and cocultured with neutrophils. Overall, our data show that the uptake of LNC or AcE-LNC by melanoma cells affects intracellular mechanisms leading to more susceptibility to death and also signals higher neutrophil antitumoral activity.
TSPO (Translocator 18 KDa; tryptophan-rich sensory protein oxygen sensor) is a constitutive outer mitochondrial membrane protein overexpressed in inflammatory cells during local or systemic ...processes. Despite its expression is characterized, role of TSPO in inflammation remains elusive. For this study, we investigated the role of TSPO ligands on neutrophil functions elicited by two different inflammatory pathways. Peritoneal neutrophils were isolated from male Balb-C mice, treated with TSPO ligand diazepam, Ro5-4864 or PK11195 (1,100 or 1000 nM; 2 h) and further stimulated with lipopolysaccharide from
(LPS), a binding for Toll-Like Receptor-4 (TLR4), or leukotriene B4 (LTB4), a G-protein coupled receptor (GPCR) ligand. LPS treatment did not lead to overexpression of TSPO on neutrophils, and pre-treatment with any TSPO ligand did not alter cytokine expression, adhesion molecule expression, or the production of reactive oxygen and nitrogen species caused by LPS stimulation. Conversely, all TSPO ligands impaired LTB4's actions, as visualized by reductions in L-selectin shedding, β2 integrin overexpression, neutrophil chemotaxis, and actin filament assembly. TSPO ligands showed distinct intracellular effects on LTB4-induced neutrophil locomotion, with diazepam enhancing cofilin but not modifying Arp2/3 expression, and Ro5-4864 and PK11195 reducing both cofilin and Arp2/3 expression. Taken together, our data exclude a direct role of TSPO ligands in TLR4-elicited pathways, and indicate that TSPO activation inhibits GPCR inflammatory pathways in neutrophils, with a relevant role in neutrophil influx into inflammatory sites.
Local treatment with LYSO-7 impairs leukocyte-endothelial interactions evoked by fMLP stimulation in mesentery postcapillary venules of male Wistar rats. In vitro LYSO-7 treatment reduces membrane ...and gene expression of adhesion molecules CD62 L and CD18, impairs NFκB nuclear translocation and blocks the elevation of intracellular calcium induced by fMLP activation in neutrophils. In vitro LYSO-7 treatment also induces expression of PPARγ and β/δ by neutrophils.
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•Local treatment with LYSO-7 reduced the leukocyte-endothelial interactions.•LYSO-7 treatment reduced nuclear translocation of NF-κB evoked by fMLP activation.•LYSO-7 treatment induced expression of PPARγ and PPARβδ by neutrophils.•LYSO-7 inhibited neutrophil chemotaxis and reduced levels of intracellular calcium.•LYSO-7 inhibited protein and gene expression of adhesion molecule expression.
Neutrophils are the first line of defence during inflammatory processes; nevertheless, exacerbated influx and actions of neutrophils in terms of uncontrolled inflammation are harmful to the host. Hence, neutrophil activity is the target of drugs seeking to address undesired inflammation. Here, we investigated the mechanisms of action of a ligand of the three isoforms of peroxisome proliferator-activated receptors (PPAR; (5Z)-5-(5-bromo-1H-indole-3-yl)methylene-3-(4-chlorobenzyl)-thiazolidine-2,4-dione), dubbed LYSO-7, on neutrophils activated by N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), an agonist of G-protein coupled receptors (GPCRs) that binds to membrane-formylated peptide and activates intracellular inflammation pathways. Neutrophils were collected from the peritoneal cavity of male Wistar rats four hours after oyster glycogen injection. Afterwards, the neutrophils were incubated with saline or LYSO-7 (1 or 10 μM, 30 min), washed and stimulated with fMLP (10−7 μM, 1 h). LYSO-7 treatment inhibited gene and protein expression of adhesion molecules, CD62 L and CD18, abolished adhesion of neutrophils to endothelial cells, impaired chemotaxis, blocked the enhancement of intracellular calcium levels, induced the expression of PPARγ as well as PPARβδ and reduced nuclear translocation of nuclear factor κB (NF-κB). Moreover, topical application of LYSO-7 (10 mM) prior to local application of fMLP (10−7 μM) diminished the in vivo leukocyte-endothelial interactions in the mesentery microcirculation of rats. Together, our data highlight the effectiveness of anti-inflammatory actions of LYSO-7 on neutrophils activated by GPCRs, depending, at least in part, on impaired of NF-κB activation and induction of PPAR expression.
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•Synthesis of N-triazolyl maleimides.•N-Triazolyl maleimide, associated or not with paclitaxel, was evaluated on metabolism and on death mechanisms.•N-triazolyl maleimides may be ...useful co-adjuvant molecules to cause toxicity to melanoma cells.
Novel N-triazolyl maleimide derivatives were synthesized by azide–alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.
A small library of compounds was prepared by a combination of toluene dioxygenase (TDO)-catalyzed enzymatic dihydroxylation and copper(I)-catalyzed Hüisgen cycloaddition. Some compounds were obtained ...by coupling an alkyne and a conduritol derivative, while more complex structures were obtained by a double Hüisgen reaction of a dialkyne and two molecules of the cyclitol. The compounds were fully characterized and subjected to preliminary biological screening.
PCB126 is a dioxin-like polychlorinated biphenyl (PCB) environmental pollutant with a significant impact on human health, as it bioaccumulates and causes severe toxicity. PCB126-induced immune ...toxicity has been described, although the mechanisms have not been fully elucidated. In this study, an in vivo protocol of PCB126 intoxication into male Wistar rats by intranasal route was used, which has not yet been described. The intoxication was characterised by PCB126 accumulation in the lungs and liver, and enhanced aryl hydrocarbon receptor expression in the liver, lungs, kidneys, and adipose tissues. Moreover, an innate immune deficiency was characterised by impairment of adhesion receptors on blood leukocytes and by reduced blood neutrophil locomotion and oxidative burst activation elicited by ex vivo G protein-coupled receptor (GPCR) activation. Specificity of PCB126 actions on the GPCR pathway was shown by normal burst oxidative activation evoked by Toll-like receptor 4 and protein kinase C direct activation. Moreover, in vivo PCB180 intoxication did not alter adhesion receptors on blood leukocytes either blood neutrophil locomotion, and only partially reduced the GPCR-induced burst oxidative activation on neutrophils. Therefore, a novel mechanism of in vivo PCB126 toxicity is described which impairs a pivotal inflammatory pathway to the host defence against infections.
Skin graft successful depends on reduction of local inflammation evoked by the surgical lesion and efficient neovascularization to nutrition the graft. It has been shown that N-terminal portion of ...the Annexin A1 protein (AnxA1) with its anti-inflammatory properties induces epithelial mucosa repair and presents potential therapeutic approaches. The role of AnxA1 on wound healing has not been explored and we investigated in this study the effect of the peptide Ac2–26 (N-terminal AnxA1 peptide Ac2–26; AnxA1
2–26
) on heterologous skin scaffolds transplantation in BALB/c mice, focusing on inflammation and angiogenesis. Treatment with AnxA1
2–26
, once a day, from day 3–60 after scaffold implantation improved the take of the implant, induced vessels formation, enhanced gene and protein levels of the vascular growth factor-A (VEGF-A) and fibroblast influx into allograft tissue. It also decreased pro- while increasing anti-inflammatory cytokines. The pro-angiogenic activity of AnxA1
2–26
was corroborated by topical application of AnxA1
2–26
on the subcutaneous tissue of mice. Moreover, treatment of human umbilical endothelial cells (HUVECs) with AnxA1
2–26
improved proliferation, shortened cycle, increased migration and actin polymerization similarly to those evoked by VEGF-A. The peptide treatment instead only potentiated the tube formation induced by VEGF-A. Collectively, our data showed that AnxA1
2–26
treatment favors the tissue regeneration after skin grafting by avoiding exacerbated inflammation and improving the angiogenesis process.
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