Tumor gene signatures that correlate with release of extracellular vesicles shape the immune landscape in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) evade immune responses through multiple resistance mechanisms. Extracellular vesicles (EVs) released by the tumor and interacting with immune cells induce immune dysfunction and contribute to tumor progression. This study evaluates the clinical relevance and impact on anti-tumor immune responses of gene signatures expressed in HNSCC and associated with EV production/release. Expression levels of two recently described gene sets were determined in The Cancer Genome Atlas Head and Neck Cancer cohort (n = 522) and validated in the GSE65858 dataset (n = 250) as well as a recently published single-cell RNA sequencing dataset (n = 18). Clustering into HPV(+) and HPV(-) patients was performed in all cohorts for further analysis. Potential associations between gene expression levels, immune cell infiltration, and patient overall survival were analyzed using GEPIA2, TISIDB, TIMER, and the UCSC Xena browser. Compared to normal control tissues, vesiculation-related genes were upregulated in HNSCC cells. Elevated gene expression levels positively correlated (P < 0.01) with increased abundance of CD4(+) T cells, macrophages, neutrophils, and dendritic cells infiltrating tumor tissues but were negatively associated (P < 0.01) with the presence of B cells and CD8(+) T cells in the tumor. Expression levels of immunosuppressive factors NT5E and TGFB1 correlated with the vesiculation-related genes and might explain the alterations of the anti-tumor immune response. Enhanced expression levels of vesiculation-related genes in tumor tissues associates with the immunosuppressive tumor milieu and the reduced infiltration of B cells and CD8(+) T cells into the tumor.

Metastatic Melanoma: A Preclinical Model Standardization and Development of a Chitosan-Coated Nanoemulsion Containing Temozolomide to Treat Brain Metastasis.

Melanoma is the most aggressive type of skin cancer. Brain metastasis is the worst scenario in metastatic melanoma and the treatment options for these patients are limited. Temozolomide (TMZ) is a chemotherapy agent used to treat primary central nervous system tumors. Our objective was to develop chitosan-coated nanoemulsion containing temozolomide (CNE-TMZ) for nasal route administration to melanoma brain metastasis treatment. A preclinical model of metastatic brain melanoma was standardized, and the efficiency of the developed formulation was further determined in vitro and in vivo. The nanoemulsion was done by spontaneous emulsification method and the formulation was characterized by size, pH, polydispersity index, and zeta potential. Culture assessments to determine cell viability were done in the A375 human melanoma cell line. To determine the safety of formulation, healthy C57/BL6 mice were treated with a nanoemulsion without TMZ. The model in vivo used B16-F10 cells implanted by stereotaxic surgery in C57/BL6 mice brains. The results demonstrate that the preclinical model used showed to be useful to analyze the efficiency of new candidate drugs to treat melanoma brain metastasis. The chitosan-coated nanoemulsions with TMZ showed the expected physicochemical characteristics and demonstrated safety and efficacy, reducing around 70% the tumor size compared to control mice, and presenting a tendency in mitotic index reduction, becoming an interesting approach to treat melanoma brain metastasis.

Effects of different maternal diets on adipose tissue inflammation and liver tissue oxidative stress in dams and their female offspring.

Pregnancy and lactation are important stages of fetal development. Therefore, this study investigated how different maternal diets offered during gestation and lactation periods affect adipose tissue inflammation and liver tissue oxidative stress of dams and their female offspring. Female BALB/c albino mice (60 days old) were randomized into three groups receiving a standard (CONT), hypercaloric (HD), or restricted (RD) diet during the pregnancy. After birth, female offspring weaned at 21 days were divided into two groups that received a standard or restricted diet (CONT/CONT, CONT/RD, RD/CONT, RD/RD, HD/CONT, and HD/RD) until 100 days old. Histological, oxidative parameters and inflammatory infiltrate of dams' and offspring's liver and adipose tissue were evaluated. HD dams presented non-alcoholic steatohepatitis (NASH) diagnosis and an increase in tumor necrosis factor-alpha (TNF-α) concentrations when compared to the RD and CONT dams, indicating a pro-inflammatory state. High concentrations of malondialdehyde (MDA) formation and catalase (CAT) activity in HD when compared to the CONT in the liver. SOD activity decreased in RD mice compared to CONT, and the SOD/CAT ratio was decreased in the RD and HD in comparison to the CONT. The maternal diet leads to an increase in SOD in RD/RD compared to HD/RD. RD-fed dams showed an increase in inflammatory infiltrates compared to CONT, evidencing changes caused by a restrictive diet. In the HD/CONT offspring, we verified an increase in inflammatory infiltrates in relation to the offspring fed a standard diet. In conclusion, HD, and RD, during pregnancy and lactation, altered the liver and adipose tissues of mothers. Furthermore, the maternal diet negatively impacts the offspring's adipose tissue but does not cause liver damage in these animals in adult life.

ENTPD1 (CD39) and NT5E (CD73) expression in human glioblastoma: an in silico analysis.

Glioblastoma (GB) is the most common primary brain tumor in adults and carries a dismal prognosis, despite the best available treatment. The 2021 WHO Classification of CNS tumors incorporated molecular profiling to better define the characteristics and prognosis of tumor types and subtypes. These recent advances in diagnosis have not yet resulted in breakthrough therapies capable of shifting the treatment paradigm. NT5E/CD73 is a cell surface enzyme that participates in a complex purinergic pathway in synergy with ENTPD1/CD39 producing extracellular adenosine (ADO) from ATP. ADO promotes tumor progression by inducing immunosuppression, stimulating adhesion, invasion, and angiogenesis. In this study, we performed an in silico analysis of 156 human glioblastoma samples in an unexplored public database to investigate the transcriptional levels of NT5E and ENTPD1. The analysis revealed a significant increase in transcription levels of the genes under study in GB samples versus non-tumor brain tissue samples, in concordance with previous studies. High transcriptional levels of NT5E or ENTPD1 were independently related to a decrease in overall survival (p = 5.4e-04; 1.1e-05), irrespective of the IDH mutation status. NT5E transcriptional levels were significantly higher in GB IDH wild-type patients compared to GB IDH-mutant; however, ENTPD1 levels showed no significant difference, p ≤ 0.001. This in silico study indicates the need for a deeper understanding of the purinergic pathway relation to GB development, also inspiring future population studies that could explore ENTPD1 and NT5E not only as prognostic markers but also as potential therapeutic targets.

Ectonucleotidases in Inflammation, Immunity, and Cancer.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes that hydrolyze nucleotides such as ATP, UTP, ADP, and UDP to monophosphates derivates such as AMP and UMP. The NTPDase family consists of eight enzymes, of which NTPDases 1, 2, 3, and 8 are expressed on cell membranes thereby hydrolyzing extracellular nucleotides. Cell membrane NTPDases are expressed in all tissues, in which they regulate essential physiological tissue functions such as development, blood flow, hormone secretion, and neurotransmitter release. They do so by modulating nucleotide-mediated purinergic signaling through P2 purinergic receptors. NTPDases 1, 2, 3, and 8 also play a key role during infection, inflammation, injury, and cancer. Under these conditions, NTPDases can contribute and control the pathophysiology of infectious, inflammatory diseases and cancer. In this review, we discuss the role of NTPDases, focusing on the less understood NTPDases 2-8, in regulating inflammation and immunity during infectious, inflammatory diseases, and cancer.

Neuroinflammatory responses following zinc or branched-chain amino acids supplementation in obese rats.

The excessive production of pro-inflammatory mediators, characteristic of obesity, leads to neuroinflammation. Zinc (Zn) and the branched-chain amino acids (BCAA) are supplements known for their immunomodulatory properties. Our goal was to evaluate if Zn or BCAA supplementation can affect long-term recognition memory and neuroinflammatory parameters of obese rats after a high-fat diet (HFD). Three-month-old Wistar rats were divided into six groups: Standard diet (SD) + vehicle; SD + Zn; SD + BCAA; High-fat diet (HFD) + vehicle; HFD + Zn; and HFD + BCAA. Diets were administrated for 19 weeks, Zn (1,2 mg/kg/day) or BCAA (750 mg/kg/day) supplementation was conducted in the last 4 weeks. Long-term recognition memory was evaluated by the novel object recognition test. IL-1ß immunoreactivity in the cortex and hippocampus, and IL-6 levels in the cortex tissue were assessed. Astrogliosis were evaluated through GFAP + cell count and morphological analysis (Sholl Method). Zn supplementation improved object recognition memory in HFD-fed rats, which was not observed following BCAA supplementation. The levels of IL-6 in the cerebral cortex were higher after HFD, which was not diminished after neither supplementation. Obesity also led to increased IL-1ß immunoreactivity in the cerebral cortex and hippocampus, which was reduced by Zn. BCAA supplementation also diminished IL-1ß immunoreactivity, but only in the hippocampus. We also showed that astrocyte reactivity caused by HFD is area-dependent, being the cerebral cortex more susceptible to the diet. Even though BCAA and Zn can affect IL-1ß immunoreactivity and astrocyte morphology, only Zn improved memory. Future studies are needed to clarify the pathways by which Zn improves cognition in obesity.

Neutrophils: fast and furious-the nucleotide pathway.

Nucleotide signaling is a key element of the neutrophil activation pathway. Neutrophil recruitment and migration to injured tissues is guided by purinergic receptor sensitization, mostly induced by extracellular adenosine triphosphate (ATP) and its hydrolysis product, adenosine (ADO), which is primarily produced by the CD39-CD73 axis located at the neutrophil cell surface. In inflammation unrelated to cancer, neutrophil activation via purinergic signaling aims to eliminate antigens and promote an immune response with minimal damage to healthy tissues; however, an antagonistic response may be expected in tumors. Indeed, alterations in purinergic signaling favor the accumulation of extracellular ATP and ADO in the microenvironment of solid tumors, which promote tumor progression by inducing cell proliferation, angiogenesis, and escape from immune surveillance. Since neutrophils and their N1/N2 polarization spectrum are being considered new components of cancer-related inflammation, the participation of purinergic signaling in pro-tumor activities of neutrophils should also be considered. However, there is a lack of studies investigating purinergic signaling in human neutrophil polarization and in tumor-associated neutrophils. In this review, we discussed the human neutrophil response elicited by nucleotides in inflammation and extrapolated its behavior in the context of cancer. Understanding these mechanisms in cancerous conditions may help to identify new biological targets and therapeutic strategies, particularly regarding tumors that are refractory to traditional chemo- and immunotherapy.

Extracellular ectonucleotidases are differentially regulated in murine tissues and human polymorphonuclear leukocytes during sepsis and inflammation.

Sepsis is life-threatening organ dysfunction caused by a dysregulated inflammatory and immune response to infection. Sepsis involves the combination of exaggerated inflammation and immune suppression. During systemic infection and sepsis, the liver works as a lymphoid organ with key functions in regulating the immune response. Extracellular nucleotides are considered damage-associated molecular patterns and are involved in the control of inflammation. Their levels are finely tuned by the membrane-associated ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family. Although previous studies have addressed the role of NTPDase1 (CD39), the role of the other extracellular NTPDases, NTPDase2, -3, and -8, in sepsis is unclear. In the present studies we identified NTPDase8 as a top downregulated gene in the liver of mice submitted to cecal ligation-induced sepsis. Immunohistochemical analysis confirmed the decrease of NTPDase8 expression at the protein level. In vitro mechanistic studies using HepG2 hepatoma cells demonstrated that IL-6 but not TNF, IL-1ß, bacteria, or lipopolysaccharide are able to suppress NTPDase8 gene expression. NTPDase8, as well as NTPDase2 and NTPDase3 mRNA was downregulated, whereas NTPDase1 (CD39) mRNA was upregulated in polymorphonuclear leukocytes from both inflamed and septic patients compared to healthy controls. Although the host's inflammatory response of polymicrobial septic NTPDase8 deficient mice was no different from that of wild-type mice, IL-6 levels in NTPDase8 deficient mice were higher than IL-6 levels in wild-type mice with pneumonia. Altogether, the present data indicate that extracellular NTPDases are differentially regulated during sepsis.

Xanthan gum-based hydrogel containing nanocapsules for cutaneous diphenyl diselenide delivery in melanoma therapy.

The aim of this study was to further evaluate the antitumoral effect of (PhSe)2-loaded polymeric nanocapsules (NC (PhSe)2) against a resistant melanoma cell line (SK-Mel-103) and develop a xanthan gum-based hydrogel intending the NC (PhSe)2 cutaneous application. For the in vitro evaluation, cells were incubated with free (PhSe)2 or NC (PhSe)2 (0.7-200 µM) and after 48 h the MTT assay, propidium iodide uptake (necrosis marker) and nitrite levels were assessed. The hydrogels were developed by thickening of the NC (PhSe)2 suspension or (PhSe)2 solution with xanthan gum and characterized in terms of average diameter, polydispersity index, pH, drug content, spreadability, rheological profiles and in vitro permeation in human skin. The results showed that NC (PhSe)2 provided a superior antitumoral effect in comparison to free (PhSe)2 (IC50 value of 47.43 µM and 65.05 µM, respectively) and increased the nitrite content. Both compound forms induced propidium iodide uptake, suggesting a necrosis-related pathway could be involved in the cytotoxic action of (PhSe)2. All hydrogels showed pH values around 7, drug content close to the theoretical values (5 mg/g) and mean diameter in the nanometric range. Besides, formulations were classified as non-Newtonian flow with pseudoplastic behavior and suitable spreadability factor. Skin permeation studies revealed that the compound content was higher for the nano-based hydrogel in the dermis layer, demonstrating its superior permeation, achieved by the compound encapsulation. It is the first report on an adequate formulation development for cutaneous application of NC (PhSe)2 that could be used as an adjuvant treatment in melanoma therapy.

Glioprotective Effect of Chitosan-Coated Rosmarinic Acid Nanoemulsions Against Lipopolysaccharide-Induced Inflammation and Oxidative Stress in Rat Astrocyte Primary Cultures.

Rosmarinic acid (RA) is a natural polyphenolic compound with a well-documented neuroprotective effect mainly associated with its anti-inflammatory and antioxidant activities. Recently, our research group developed and optimized chitosan-coated RA nanoemulsions (RA CNE) intended to be used for nasal delivery as a new potential neuroprotective therapy. In this sense, the present study aimed to evaluate the protective and/or therapeutic potential of RA CNE in inflammation/oxidative stress induced by LPS (1 µg mL-1) in rat astrocyte primary cultures. In summary, pre-treatment with RA CNE before exposure to LPS (protective protocol) reduced significantly the LPS-induced alterations in astrocyte cell viability, proliferation, and cell death by necrosis, which was not observed in therapeutic protocol. RA CNE protective protocol also enhanced anti-oxidative status by ~ 50% by decreasing oxygen reactive species production and nitric oxide levels and preventing total thiol content decrease. Finally, our results demonstrate the protective effect of RA CNE in migratory activation and GFAP expression of reactive astrocytes. Overall, our findings indicate for the first time the RA CNE glioprotective potential, associated with an increase in cell viability and proliferation, a preventive effect on cellular death by necrosis, migratory ability and hypertrophic reactive astrocytes, and the reparation of astrocyte redox state.

Characterization of macrophage phenotype, redox, and purinergic response upon chronic treatment with methionine and methionine sulfoxide in mice.

Hypermethioninemia is a disorder characterized by high plasma levels of methionine (Met) and its metabolites such as methionine sulfoxide (MetO). Studies have reported associated inflammatory complications, but the mechanisms involved in the pathophysiology of hypermethioninemia are still uncertain. The present study aims to evaluate the effect of chronic administration of Met and/or MetO on phenotypic characteristics of macrophages, in addition to oxidative stress, purinergic system, and inflammatory mediators in macrophages. In this study, Swiss male mice were subcutaneously injected with Met and MetO at concentrations of 0.35-1.2 g/kg body weight and 0.09-0.3 g/kg body weight, respectively, from the 10th-38th day post-birth, while the control group was treated with saline solution. The results revealed that Met and/or MetO induce an M1/classical activation phenotype associated with increased levels of tumor necrosis factor alpha and nitrite, and reduced arginase activity. It was also found that Met and/or MetO alter the activity of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, as well as the levels of thiol and reactive oxygen species in macrophages. The chronic administration of Met and/or MetO also promotes alteration in the hydrolysis of ATP and ADP, as indicated by the increased activity of ectonucleotidases. These results demonstrate that chronic administration of Met and/or MetO promotes activated pro-inflammatory profile by inducing M1/classical macrophage polarization. Thus, the changes in redox status and purinergic system upon chronic Met and/or MetO exposure may contribute towards better understanding of the alterations consistent with hypermethioninemic patients.

Tannic acid elicits selective antitumoral activity in vitro and inhibits cancer cell growth in a preclinical model of glioblastoma multiforme.

Glioblastoma is a devastating tumor affecting the central nervous system with infiltrative capacity, high proliferation rate and chemoresistance. Therefore, it is urgent to find new therapeutic alternatives that improve this prognosis. Herein, we focused on tannic acid (TA) a polyphenol with antioxidant and antiproliferative activities. In this work, the antitumor and antioxidant effects of TA on rat (C6) glioblastoma cells and their cytotoxicity relative to primary astrocyte cultures were evaluated in vitro. Cells were exposed to TA of 6.25 to 75 µM for 24, 48 and/or 72 h. In addition, colony formation, migration and cell adhesion were analyzed and flow cytometry was used to analyze cell death and cell cycle. Next, the action of TA was evaluated in a preclinical glioblastoma model performed on Wistar rats. In this protocol, the animals were treated with a dose of 50 mg/kg/day TA for 15 days. Our results demonstrated that TA induced in vitro selective antiglioma activity, not demonstrating cytotoxicity in astrocyte culture. It induced cell death by apoptosis and cell cycle arrest, reducing formation and size of colonies, cell migration/adhesion and showing to be a potential antioxidant. Interestingly, the antiglioma effect was also observed in vivo, as TA decreased tumor volume by 55%, accompanied by an increase in the area of intratumoral necrosis and infiltration of lymphocytes without causing systemic damage. To the best of our knowledge, this is the first study to report TA activity in a GBM preclinical model. Thus, this natural compound is promising as a treatment for glioblastoma.

Purinergic Signaling in Glioma Progression.

Among the pathological alterations that give tumor cells invasive potential, purinergic signaling is emerging as an important component. Studies performed in in vitro, in vivo and ex vivo glioma models indicate that alterations in the purinergic signaling are involved in the progression of these tumors. Gliomas have low expression of all E-NTPDases, when compared to astrocytes in culture. Nucleotides induce glioma proliferation and ATP, although potentially neurotoxic, does not evoke cytotoxic action on the majority of glioma cells in culture. The importance of extracellular ATP for glioma pathobiology was confirmed by the reduction in glioma tumor size by apyrase, which degrades extracellular ATP to AMP, and the striking increase in tumor size by over-expression of an ecto-enzyme that degrades ATP to ADP, suggesting the effect of extracellular ATP on the tumor growth depends on the nucleotide produced by its degradation. The participation of purinergic receptors on glioma progression, particularly P2X7, is involved in the resistance to ATP-induced cell death. Although more studies are necessary, the purinergic signaling, including ectonucleotidases and receptors, may be considered as future target for glioma pharmacological or gene therapy.

Arginase-1+ Exosomes from Reprogrammed Macrophages Promote Glioblastoma Progression.

Interactions between tumor cells and tumor-associated macrophages (TAMs) are critical for glioblastoma progression. The TAMs represent up to 30% of the glioblastoma mass. The role of TAMs in tumor progression and in the mechanisms underlying tumor growth remain unclear. Using an in vitro model resembling the crosstalk between macrophages and glioblastoma cells, we show that glioblastoma-derived exosomes (GBex) reprogram M1 (mediate pro-inflammatory function) and M2 (mediate anti-inflammatory function) macrophages, converting M1 into TAMs and augmenting pro-tumor functions of M2 macrophages. In turn, these GBex-reprogrammed TAMs, produce exosomes decorated by immunosuppressive and tumor-growth promoting proteins. TAM-derived exosomes disseminate these proteins in the tumor microenvironment (TME) promoting tumor cell migration and proliferation. Mechanisms underlying the promotion of glioblastoma growth involved Arginase-1+ exosomes produced by the reprogrammed TAMs. A selective Arginase-1 inhibitor, nor-NOHA reversed growth-promoting effects of Arginase-1 carried by TAM-derived exosomes. The data suggest that GBex-reprogrammed Arginase-1+ TAMs emerge as a major source of exosomes promoting tumor growth and as a potential therapeutic target in glioblastoma.

Development and characterization of CD73-siRNA-loaded nanoemulsion: effect on C6 glioma cells and primary astrocytes.

Introduction: Glioblastoma (GB) is the most common malignant brain tumor and is characterized by high invasiveness, poor prognosis, and limited therapeutic options. Silencing gene expression, through the use of small interfering RNA (siRNA), has been proposed as an alternative to conventional cancer therapy. Here, we evaluated the potential of CD73 as a new therapeutic target, since it is overexpressed in solid tumors and has emerged as a promising target to control GB progression.Methods: A cationic nanoemulsion (NE) as an intravenous siRNA-CD73 delivery system was developed and its effect on C6 glioma cell viability was determined.Results: The nanostructured system was effective in complexing oligonucleotides for delivery to target cells. In addition, we observed that the NE-siRNA-CD73 complex was effective in reducing CD73 protein levels and AMPase activity, which were related to decreased C6 glioma cell viability.Conclusions: These findings indicate the potential of siRNA-CD73-loaded cationic NE as a therapeutic alternative for glioma treatment.

Design of Pegylated-Nanocapsules to Diphenyl Diselenide Administration: In Vitro Evidence of Hemocompatible and Selective Antiglioma Formulation.

Diphenyl diselenide [(PhSe)2] is a pleiotropic pharmacological agent, but it has low aqueous solubility. The nanoencapsulation of (PhSe)2 allowed the preparation of an aqueous formulation as well as potentiated its in vitro antitumor effect and the effectiveness in a preclinical model of glioblastoma when administered by the intragastric route. Thus, aiming at maximizing the therapeutic potential of (PhSe)2, the present study designed a pegylated-formulation intending to intravenous administration of the (PhSe)2 as a new approach for glioma therapy. The poly(Ɛ-caprolactone) nanocapsules containing (PhSe)2 were physically coated with polyethyleneglycol (PEG) using the preformed polymer interfacial deposition technique and evaluated through physicochemical, morphological, spectroscopic, and thermal characteristics. Hemocompatibility was determined by the in vitro hemolysis test and cytotoxicity assays were performed in astrocytes and glioma C6 cells (10-100 µM). The pegylated-nanocapsules had an average diameter of 218 ± 25 nm, polydispersity index of 0.164 ± 0.046, zeta potential of - 8.1 ± 1.6 mV, pH 6.0 ± 0.09, (PhSe)2 content of 102.00 ± 3.57%, and encapsulation efficiency around 98%. Besides, the (PhSe)2 pegylated-nanocapsules were spherical, presented absence of chemical interaction among the constituents, and showed higher thermal stability than the non-encapsulated materials. PEG-coated nanocapsules did not cause hemolytic effect while formulations without PEG induced a hemolysis rate above 10%. Moreover, pegylated-nanocapsules had superior in vitro antiglioma effect in comparison to free compound (IC50: 24.10 µM and 74.83 µM, respectively). Therefore, the (PhSe)2-loaded pegylated-nanocapsule suspensions can be considered a hemocompatible formulation for the glioma treatment by the intravenous route.

2-(2-Methoxyphenyl)-3-((Piperidin-1-yl)ethyl)thiazolidin-4-One-Loaded Polymeric Nanocapsules: In Vitro Antiglioma Activity and In Vivo Toxicity Evaluation.

Among gliomas types, glioblastoma is considered the most malignant and the worst form of primary brain tumor. It is characterized by high infiltration rate and great angiogenic capacity. The presence of an inflammatory microenvironment contributes to chemo/radioresistance, resulting in poor prognosis for patients. Recent data show that thiazolidinones have a wide range of pharmacological properties, including anti-inflammatory and antiglioma activities. Nanocapsules of biodegradable polymers become an alternative to cancer treatment since they provide targeted drug delivery and could overcome blood-brain barrier. Therefore, here we investigated the in vitro antiglioma activity and the potential in vivo toxicity of 2- (2-methoxyphenyl) -3- ((piperidin-1-yl) ethyl) thiazolidin-4-one-loaded polymeric nanocapsules (4L-N). Nanocapsules were prepared and characterized in terms of particle size, polydispersity index, zeta potential, pH, molecule content and encapsulation efficiency. Treatment with 4L-N selectively decreased human U138MG and rat C6 cell lines viability and proliferation, being even more efficient than the free-form molecule (4L). In addition, 4L-N did not promote toxicity to primary astrocytes. We further demonstrated that the treatment with sub-therapeutic dose of 4L-N did not alter weight, neither resulted in mortality, toxicity or peripheral damage to Wistar rats. Finally, 4L as well as 4L-N did not alter makers of oxidative damage, such as TBARS levels and total sulfhydryl content, and did not change antioxidant enzymes SOD and CAT activity in liver and brain of treated rats. Taken together, these data indicate that the nanoencapsulation of 4L has potentiated its antiglioma effect and does not cause in vivo toxicity.

HPLC-UV method for temozolomide determination in complex biological matrices: Application for in vitro, ex vivo and in vivo studies.

A high-performance liquid chromatography method for temozolomide (TMZ) determination in complex biological matrices was developed and validated for application in in vitro, ex vivo and in vivo studies of new nanotechnology-based systems for TMZ nasal delivery. The method was able to quantify TMZ in nanoemulsions, following cellular uptake, in the porcine nasal mucosa and in mouse plasma and brain. Analyses were performed on a C18 column at 35°C, under UV detection at 330 nm. The mobile phase was methanol-acetic acid 0.5% (30:70, v/v), eluted at an isocratic flow rate of 1.1 mL/min. The method was found to be specific, precise, accurate, robust and linear (0.05 to 5 µg/mL) for TMZ determination in all matrices. No interference of TMZ degradation products was found under various stress conditions such as acidic, alkaline, oxidative, light and thermal exposure, demonstrating stability. The method was applied for the quantification of TMZ in different matrices, i.e. the efficiency of nanoemulsions in vitro in increasing TMZ cellular uptake, ex vivo TMZ permeation and retention in the porcine nasal mucosa tissue, and for in vivo TMZ quantification in mouse brain following intranasal nanoemulsion administration compared with free TMZ.

Inhibition of the Adenosinergic Pathway in Cancer Rejuvenates Innate and Adaptive Immunity.

The adenosine pathway plays a key role in modulating immune responses in physiological and pathological conditions. Physiologically, anti-inflammatory effects of adenosine balance pro-inflammatory adenosine 5'-triphosphate (ATP), protecting tissues from damage caused by activated immune cells. Pathologically, increased adenosine monophosphatase (AMPase) activity in tumors leads to increased adenosine production, generating a deeply immunosuppressed microenvironment and promoting cancer progression. Adenosine emerges as a promising target for cancer therapy. It mediates protumor activities by inducing tumor cell proliferation, angiogenesis, chemoresistance, and migration/invasion by tumor cells. It also inhibits the functions of immune cells, promoting the formation of a tumor-permissive immune microenvironment and favoriting tumor escape from the host immune system. Pharmacologic inhibitors, siRNA or antibodies specific for the components of the adenosine pathway, or antagonists of adenosine receptors have shown efficacy in pre-clinical studies in various in vitro and in vivo tumor models and are entering the clinical arena. Inhibition of the adenosine pathway alone or in combination with classic immunotherapies offers a potentially effective therapeutic strategy in cancer.

Incorporation of 3,3'-Diindolylmethane into Nanocapsules Improves Its Photostability, Radical Scavenging Capacity, and Cytotoxicity Against Glioma Cells.

3,3'-Diindolylmethane (DIM) is a phytochemical that presents health benefits (antitumor, antioxidant, and anti-inflammatory effects). However, it is water insoluble and thermo- and photolabile, restraining its pharmaceutical applications. As a strategy to overcome such limitations, this study aimed the development and characterization of DIM-loaded nanocapsules (NCs) prepared with different compositions as well as the in vitro assessment of scavenging activity and cytotoxicity. The formulations were obtained using the interfacial deposition of preformed polymer method and were composed by Eudragit® RS100 or ethylcellulose as polymeric wall and primula or apricot oil as the core. All the formulations had adequate physicochemical characteristics: nanometric size (around 190 nm), low polydispersity index (< 0.2), pH value at acid range, high values of zeta potential, drug content, and encapsulation efficiency (~ 100%). Besides, nanoencapsulation protected DIM against UVC-induced degradation and increased the scavenging activity assessed by the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and 1-1-diphenyl-2-picrylhydrazyl methods. The developed DIM-loaded nanocapsules were further evaluated regarding the in vitro release profile and cytotoxicity against a human glioblastoma cell line (U87 cells). The results demonstrated that the nanoencapsulation promoted a sustained release of the bioactive compound (in the range of 58-78% after 84 h) in comparison to its free form (86% after 12 h), as well as provided a superior cytotoxic effect against the U87 cells in the highest concentrations. Therefore, our results suggest that nanoencapsulation could be a promising approach to overcome the DIM physicochemical limitations and potentialize its biological properties.