Extracellular ATP/P2X7 receptor, a regulatory axis of migration in ovarian carcinoma-derived cells.

ATP is actively maintained at high concentrations in cancerous tissues, where it promotes a malignant phenotype through P2 receptors. In this study, we first evaluated the effect of extracellular ATP depletion with apyrase in SKOV-3, a cell line derived from metastatic ovarian carcinoma. We observed a decrease in cell migration and an increase in transepithelial electrical resistance and cell markers, suggesting a role in maintaining a mesenchymal phenotype. To identify the P2 receptor that mediated the effects of ATP, we compared the transcript levels of some P2 receptors and found that P2RX7 is three-fold higher in SKOV-3 cells than in a healthy cell line, namely HOSE6-3 (from human ovarian surface epithelium). Through bioinformatic analysis, we identified a higher expression of the P2RX7 transcript in metastatic tissues than in primary tumors; thus, P2X7 seems to be a promising effector for the malignant phenotype. Subsequently, we demonstrated the presence and functionality of the P2X7 receptor in SKOV-3 cells and showed through pharmacological approaches that its activity promotes cell migration and contributes to maintaining a mesenchymal phenotype. P2X7 activation using BzATP increased cell migration and abolished E-cadherin expression. On the other hand, a series of P2X7 receptor antagonists (A438079, BBG and OxATP) decreased cell migration. We used a CRISPR-based knock-out system directed to P2RX7. According to the results of our wound-healing assay, SKOV3-P2X7KO cells lacked receptor-mediated calcium mobilization and decreased migration. Altogether, these data let us propose that P2X7 receptor is a regulator for cancer cell migration and thus a potential drug target.

Cytotoxic and Antitumoral Effects of Methanolic Extracts of Avocado Fruit Mesocarp in Colorectal Cancer Cell Line HT29.

Colorectal cancer is a widespread neoplasia with high ratios of chemoresistance. Phytochemicals in plant-based extracts could be useful to treat colorectal cancer, and/or reduce chemoresistance. Methanolic extract of avocado mesocarp (MEAM) has demonstrated antitumoral properties, depending on the fruit ripening stage (RS). The aim of this study was to analyze the effects of methanolic extracts of "Hass" avocado fruit at different RS on cytotoxicity, antioxidative, anti-inflammatory, anti-invasive, cell cycle, and epithelial-mesenchymal transition inhibition in colorectal adenocarcinoma cell line HT29. The MEAM showed an increasing concentration of total phenolic compounds as the RS progressed, which was correlated with antioxidant capacity measured by the Ferric Reducing Antioxidant Power assay but not with the 2.2-diphenyl-1-picrylhydrazyl assay. The specific phenolic compounds of MEAM were determined by high-performance liquid chromatography, and it was found that concentrations of epicatechin decreased while concentrations of chlorogenic acid increased as the RS progressed. The HT29 cell line was treated with MEAM for 48 h, and all MEAM had a cytotoxic effect, reported by MTT assay, nevertheless, the strongest effect was associated with the presence of chlorogenic acid. MEAM induced apoptosis and cell cycle arrest in phase G0/G1, reported by flow cytometry. Moreover, MEAM inhibited cell migration evidenced by the wound healing assay. On the other hand, MEAM significantly reduced expression of mRNA of tumor necrosis factor-alpha and cyclooxygenase 2. These effects comprise important inhibition of some hallmarks of cancer. This, in turn, may provide interesting guidelines for developing antitumoral intervention agents.

Neuroprotectin D1 Attenuates Blast Overpressure Induced Reactive Microglial Cells in the Cochlea.

OBJECTIVE/HYPOTHESIS: We examined a neuroinflammatory response associated with glial activation in the cochlea exposed to blast overpressure and evaluated the potential therapeutic efficacy of specialized pro-resolving mediators such as neuroprotectin D1, NPD1; (10R, 17S-dihydroxy-4Z, 7Z, 11E, 13E, 15Z, 19Z-docosahexaenoic acid) in a rodent blast-induced auditory injury model. STUDY DESIGN: Animal Research. METHODS: A compressed-air driven shock tube was used to expose anesthetized adult male Long-Evan rats to shock waves simulating an open-field blast exposure. Approximately 30 minutes after blast exposure, rats were treated with NPD1 (100 ng/kg body wt.) or vehicle delivered intravenously via tail vein injection. Rats were then euthanized 48 hours after blast exposure. Unexposed rats were included as controls. Tissue sections containing both middle and inner ear were prepared with hematoxylin-eosin staining to elucidate histopathological changes associated with blast exposure. Cochlear tissues were evaluated for relative expression of ionized calcium-binding adaptor 1 (Iba1), as an indicator of microglial activation by immunohistochemistry and western blot analyses. RESULTS: Our animal model resulted in an acute injury mechanism manifested by damage to the tympanic membrane, hemorrhage, infiltration of inflammatory cells, and increased expression of Iba1 protein. Moreover, therapeutic intervention with NPD1 significantly reduced Iba1 expression in the cochlea, suggesting a reduction of a neuroinflammatory response caused by blast overpressure. CONCLUSIONS: Blast overpressure resulted in an increased expression of proteins involved in gliosis within the auditory system, which were reduced by NPD1. Treatment of NPD1 suggests an effective strategy to reduce or halt auditory microglial cell activation due to primary blast exposure. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2018-E2025, 2021.

Altered expression of aquaporin 1 and aquaporin 5 in the cornea after primary blast exposure.

Purpose: Our study aimed to determine whether the altered expression of biomarkers linked to corneal injuries, such as the edema-regulating proteins aquaporin-1 and aquaporin-5 (AQP1 and AQP5), occurred following primary blast exposure. Methods: Adult male Dutch Belted rabbits were anesthetized and exposed to blast waves with peak overpressures of 142.5-164.1 kPa (20.4-23.4 psi). These exposure groups experienced peak blast overpressure-specific impulses (impulse per unit surface area) of 199.6-228.5 kPa-ms. Unexposed rabbits were included as controls. The animals were euthanized at 48 h post-exposure. Corneas obtained from the euthanized blast-exposed and control rabbits were processed for quantitative PCR and western blot to quantify mRNA and the protein expression of AQP1 and AQP5. Immunohistochemical analysis was conducted to determine the cellular localization of AQP1 and AQP5. Results: Corneal thickness increased up to 18% with the peak blast overpressure-specific impulses of 199.6-228.5 kPa-ms at 48 h after blast exposure. mRNA levels of AQP1 and AQP5 increased in the whole cornea lysates of blast-exposed rabbits relative to those of the controls. Western blot analyses of whole cornea lysates revealed that the expression levels of AQP1 and AQP5 were approximately 2- and 1.5-fold higher, respectively, in blast-exposed rabbits compared to controls. The extent of AQP1 immunostaining (AQP1-IS) increased in the epithelial cell layer after blast exposure. The AQP5-IS pattern changed from a mixed membrane and cytoplasmic expression in the controls to predominantly cytoplasmic expression in the basally located cornea epithelial cells of blast-exposed rabbits. Conclusions: Primary blast exposure resulted in edema-related changes in the cornea manifested by the altered expression of the edema-regulating proteins AQP1 and AQP5 with blast overpressure-specific impulses. These findings support potential acute corneal injury mechanisms in which the altered regulation of water permeability is caused by primary blast exposure.

Sjögren's syndrome-like ocular surface disease in thrombospondin-1 deficient mice.

Thrombospondin-1 (TSP-1) is a major activator of latent transforming growth factor-beta in vitro as well as in vivo. Mice deficient in TSP-1, despite appearing normal at birth, develop a chronic form of ocular surface disease that is marked by increased apoptosis and deterioration in the lacrimal gland, associated dysfunction, and development of inflammatory infiltrates that result in abnormal tears. The increase in CD4(+) T cells in the inflammatory infiltrates of the lacrimal gland, and the presence of anti-Sjögren's syndrome antigen A and anti-Sjögren's syndrome antigen B antibodies in the serum resemble autoimmune Sjögren's syndrome. These mice develop an ocular surface disorder dry eye that includes disruption of the corneal epithelial layer, corneal edema, and a significant decline in conjuctival goblet cells. Externally, several mice develop dry crusty eyes that eventually close. The inflammatory CD4(+) T cells detected in the lacrimal gland, as well as those in the periphery of older TSP-1 null mice, secrete interleukin-17A, a cytokine associated with chronic inflammatory diseases. Antigen-presenting cells, derived from TSP-1 null, but not from wild-type mice, activate T cells to promote the Th17 response. Together, these results indicate that TSP-1 deficiency results in a spontaneous form of chronic dry eye and aberrant histopathology associated with Sjögren's syndrome.

Age-dependent alterations in mouse exorbital lacrimal gland structure, innervation and secretory response.

Several studies investigated the effect of aging on rat and human lacrimal gland physiology. However, in most of these studies, only two age groups were investigated. Furthermore, those studies did not correlate the age-related histological changes that occur in the lacrimal gland to the functional changes (nerve activity and protein secretion) that might occur with aging. Thus, the purpose of the present study was to investigate the effect of aging on lacrimal gland structure, innervation and function using BALB/c mice at different ages. Exorbital lacrimal glands were removed from 3, 8, 12, 24, and 32-month-old, male BALB/c mice, fixed, embedded and processed for histology and immunohistochemistry. Sections were stained with hematoxylin and eosin to determine morphological changes and lymphocytic infiltration; giemsa to identify mast cells; and Kinyoun's carbol fucsin solution to indicate lipofuscin-like inclusions. Parasympathetic and sympathetic nerves were identified by immunofluorescence techniques. To measure acetylcholine release and protein secretion, lacrimal gland pieces were incubated in Krebs Ringer buffer containing 5 mM KCl (control), 75 mM KCl (depolarizing buffer which activates nerves), carbachol (a cholinergic agonist, 10(-4) M), or phenylephrine (an alpha1-adrenergic agonist, 10(-4) M) for 20 min. The media were collected and analysed for acetylcholine and peroxidase using a spectrofluorometric assay. KCl-, carbachol- and phenylephrine-stimulated peroxidase secretion decreased in lacrimal glands from 8, 12, and 24-month-old mice when compared to 3-month-old animals. Both the density and distribution of parasympathetic and sympathetic nerves surrounding the acini decreased with increasing age. Acetylcholine release from lacrimal gland nerves decreased in 24-month-old mice compared to 3- and 12-month-old animals. Similarly, progressive morphological changes, including increased numbers of lipofuscin-like inclusions, mast cells and lymphocytic infiltration occurred in an age-dependent manner. We conclude that structural alterations of mouse lacrimal gland, including increased accumulation of lipofuscin-like inclusions, chronic inflammation and functional alterations including decreased acetylcholine release and protein secretion occurred with aging.