Tumor Tissue Oxidative Stress Changes and Na, K-ATPase Evaluation in Head and Neck Squamous Cell Carcinoma.

Changes in metabolism are mechanisms that are largely implicated in the development, progression, and metastasis of head and neck squamous cell carcinoma (HNSCC) and also in resistance to different anticancer therapies. Identification of biomarkers for differentiation between cancerous and normal epithelium, treatment design and prognosis remain a vital issue in the field of head and neck cancer. The present study analyzed the main biochemical changes that occur in HNSCC tumors by through mechanisms involving oxidative stress. The release of substances reactive to thiobarbituric acid was significantly lower in HNSCC tumor tissue as compared to healthy tissue. The assays related to the lipid profile assays showed changes in membrane biophysics of tumor cells due to an increase in total phospholipids and total cholesterol, as well as an increased activity and expression of the α1 subunit of Na, K-ATPase, which is fundamental in the process of carcinogenesis. The modulation of the antioxidant system was also affected, with a decrease in the catalytic activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as a reduction of glutathione (GSH) content and an increase in H2O2 content. A reduction in catalase (CAT) activity was observed. The data presented here are in accordance with important findings described by us in a previous study, involving the same individuals, but with a focus on the damage generated in red blood cells, resulting from tumor installation. Therefore, it was possible to conclude that the biochemical alterations found in HNSCC cells are fundamental for transformation and maintenance of the tumor cell and once it is installed, it is also capable of generating injuries in the patients' red blood cells. Our data demonstrate that this could be a promising biomarker for HNSCC.

Chemical composition, anti-inflammatory and antinociceptive effects of the butanolic fraction of Annona nutans (Annonaceae) leaves.

The species Annona nutans (R. E. Fries) is a plant found in Bolivia, Paraguay, Argentina and the Brazilian cerrado. Considering the anti-inflammatory and antinociceptive activities of the hydrometanolic fraction (FHMeOH) of A. nutans leaves previously reported, the present study aimed to evaluate in vivo anti-inflammatory and antinociceptive activities of a subfraction obtained from FHMeOH, the butanolic fraction (FBuOHf). Intraperitoneal (i.p.) treatment with FBuOHf (50 and 100 mg · kg-1) inhibited paw edema induced by carrageenan. Moreover, FBuOHf (100 mg · kg-1, i.p.) also suppressed polymorphonuclear (PMN) leukocyte migration in the footpad. Regarding the antinociceptive activity, FBuOHf (50, 100, and 200 mg · kg-1, i.p.) inhibited acetic acid-induced abdominal writhing. In the formalin test, this fraction (200 mg · kg-1, i.p.) reduced licking time only in the inflammatory phase. The FBuOHf contents flavonoids and cinnamic acid derivatives, such as quercetin-3-O-galactoside, quercetin-3-O-glucoside, isorhamnetin-3-O-galactoside, quercetin-3-O-ß-D-apio-furanosyl-(1→2)-galactopyranoside and chlorogenic acid, identified and quantified by LC-MS. The FBuOHf possesses anti-inflammatory and peripheral antinociceptive activities.

Anti-inflammatory and antinociceptive activities of a phenylpropanoid-enriched fraction of Duguetia furfuracea.

A previous study reported the in vivo anti-inflammatory and antinociceptive activities of essential oil of the underground stem bark of Duguetia furfuracea, termed EODf. This study aimed to obtain a phenylpropanoid-enriched fraction from the D. furfuracea (EFDf) essential oil and to investigate its anti-inflammatory and antinociceptive effects. The chemical composition of the EFDf was determined by gas chromatography-mass spectrometry (GC-MS). The in vivo anti-inflammatory activity was evaluated with a lipopolysaccharide (LPS)-induced paw oedema model. The effects of the EFDf on the polymorphonuclear leukocyte recruitment and the inducible nitric oxide synthase (iNOS) expression were evaluated in mice footpads. Moreover, the in vivo antinociceptive effect was assayed using the formalin test and the LPS-induced thermal hyperalgesia model. In the EFDf, 8 major compounds were identified, with α-asarone (36.4%) and 2,4,5-trimethoxystyrene (27.8%) the main constituents. A higher concentration of phenylpropanoid derivatives was found in the EFDf, 64.2% compared to the EODf (38%). The oral (p.o.) treatment with the EFDf at a dose of 3 mg/kg significantly attenuated the paw oedema, polymorphonuclear leukocyte migration, iNOS expression, and tumour necrosis factor alpha (TNF-α) production. The EFDf (10 and 30 mg/kg) also inhibited both phases of the formalin test and caused a significant increase in the reaction time in the LPS-induced thermal hyperalgesia model. Finally, EFDf-treated animals did not show any alteration of motor coordination. The results suggest that the enrichment of 2,4,5-trimethoxystyrene and α-asarone enhances the anti-inflammatory activity of the EFDf compared to the EODf. In contrast, the antinociception promoted by the EFDf was similar to the EODf and was mediated via activation of adenosinergic and opioidergic receptors.

Evaluation of the Erythrocyte Membrane in Head and Neck Cancer Patients.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous and complex disease, both from a clinical and molecular point of view. The prolonged use of alcohol and tobacco, along with the release of tumor secretions can modulate blood cells, such as erythrocytes. Here, this study was conducted with 24 patients diagnosed with HNSCC and an equal number of healthy individuals are matched by age and gender. The levels of lipid peroxidation were measured using the individual plasma, while for lipid concentrations, identification and quantification Na, K-ATPase activity and osmotic fragility, the red blood cell concentrate were used. The release of TBARS was significantly higher in patients with HNSCC. The lipid profile assays demonstrated a rearrangement of the erythrocyte membrane due to a decrease in total phospholipids and phosphatidylethanolamine followed by an increase in total cholesterol and phosphatidylcholine. Na, K-ATPase activity also increased. Erythrocytes were more fragile in patients with HNSCC than in health individuals. Therefore, the membrane of erythrocytes were rearranged and Na, K-ATPase function altered in the HNSCC patients. Our findings suggests that the alcohol, tobacco and tumor secretion modulate in a specific manner that the erythrocytes membranes of these patients making this system a potential tool for HNSCC biomarker of tumor progression.

Anti-inflammatory and central and peripheral anti-nociceptive activities of α-asarone through the inhibition of TNF-α production, leukocyte recruitment and iNOS expression, and participation of the adenosinergic and opioidergic systems.

Alpha-asarone has been found to possess many pharmacological activities, which can improve cognitive function and exert anti-oxidant, anxiolytic, anti-epileptic and protective effects against endothelial cell injury. The anti-inflammatory activity of α-asarone was evaluated using lipopolysaccharide (LPS)-induced paw oedema. Moreover, leukocyte migration, inducible nitric oxide synthase (iNOS) expression and tumour necrosis factor-alpha (TNF-α) levels were quantified in footpads. Formalin and LPS-induced thermal hyperalgesia models were generated using adenosinergic, opioidergic, serotonergic and muscarinic receptor antagonists. The effects on motor coordination were evaluated by means of the rota-rod test. Oral treatment (p.o.) with α-asarone (3 mg/kg) significantly inhibited paw oedema by 62.12 and 72.22%, 2 and 4 h post LPS injection, respectively. Alpha-asarone (3 mg/kg, p.o.) attenuated the inflammatory infiltrate 1, 3 and 6 h after LPS injection. Furthermore, α-asarone (3 mg/kg, p.o.) suppressed iNOS expression and TNF-α production, 6 and 1 h after inflammatory stimulus, respectively. Alpha-asarone (3, 10 and 30 mg/kg, p.o.) inhibited both phases of formalin-induced licking. In the hot-plate test, α-asarone (10 and 30 mg/kg, p.o.) increased the latency to response 3 and 5 h post LPS stimulus. Caffeine and naloxone abolished the central anti-nociceptive effect of α-asarone (neurogenic phase of formalin and hot plate tests), suggesting the participation of the adenosinergic and opioidergic systems. Furthermore, naloxone reversed the peripheral activity of α-asarone (inflammatory phase of formalin test), indicating the possible involvement of the opioidergic pathway. In the rota-rod test, α-asarone did not change motor coordination. These findings suggest that α-asarone has anti-inflammatory, peripheral and central anti-nociceptive effects and could represent a promising agent for future research.

Estuarine fish assemblages present a species-specific difference in the multixenobiotics resistance activity.

We investigated the activity of the multixenobiotic resistance (MXR) phenotype, a biological defense system in aquatic organisms, in the fish assemblages of two tropical estuaries with different degrees of environmental impacts, the Paraiba River and Mamanguape River Estuaries. The aim of this work was to compare the activity of the MXR phenotype of different fishes to test the hypothesis that each species has an inherent activity level and to use this activity as a bioindicator of aquatic contamination. We assessed the MXR activity of the gills, using rhodamine B (RB) accumulation assay. The results demonstrated a species-specific difference in the MXR activity of fishes caught in the same estuarine zone. Also, the pelagic species Eucinostomus melanopterus, Eucinostomus argenteus, and Lutjanus jocu had higher RB accumulation, while the demersal species Sphoeroides testudineus and Sphoeroides greeleyi had the lowest RB accumulation, suggesting that the ecological characteristic of fish in the water column exerts an influence on MXR activity. Besides, we demonstrated the potential of using the gill MXR activity of the key estuarine species, the Brazilian silversides Atherinella brasiliensis, as a tool for biomonitoring estuaries.

WIN55,212-2 induces caspase-independent apoptosis on human glioblastoma cells by regulating HSP70, p53 and Cathepsin D.

Despite the standard approaches to treat the highly aggressive and invasive glioblastoma (GBM), it remains incurable. In this sense, cannabinoids highlight as a promising tool, because this tumor overexpresses CB1 and/or CB2 receptors and being, therefore, can be susceptible to cannabinoids treatment. Thus, this work investigated the action of the cannabinoid agonist WIN55-212-2 on GBM cell lines and non-malignant cell lines, in vitro and in vivo. WIN was selectively cytotoxic to GBM cells. These presented blebbing and nuclear alterations in addition to cell shrinkage and chromatin condensation. WIN also significantly inhibited the migration of GAMG and U251 cells. Finally, the data also showed that the antitumor effects of WIN are exerted, at least to some extent, by the expression of p53 and increased cathepsin D in addition to the decreased expression of HSP70.This data can indicate caspase-independent cell death mechanism. In addition, WIN decreased tumoral perimeter as well as caused a reduction the blood vessels in this area, without causing lysis, hemorrhage or blood clotting. So, the findings herein presented reinforce the usefulness of cannabinoids as a candidate for further evaluation in treatment in glioblastoma treatment.

Reduced CD8+ T cells infiltration can be associated to a malignant transformation in potentially malignant oral epithelial lesions.

OBJECTIVE: The aim of this study was to evaluate the immunohistochemical expressions of PD1, CD4+, and CD8+ in premalignant lesions (OPML) that were transformed into oral squamous cell carcinoma OSCC (OPML-OSCC), in OSCC and also in premalignant lesions that were not transformed into OSCC (OPML-NOSSC). MATERIALS AND METHODS: Retrospective analyses were performed in order to verify the demographic characteristics of the patients. CD4, CD8, and PD1 IMH studies were carried out on OPML and OSCC samples from 11 patients with OPML-OSCC and OPML, together with samples from 14 patients with OPML-NOSCC. The differences between OPML-OSCC and OPML-NOSCC were analyzed. RESULTS: Non-homogenous leukoplakia, together with the related oral subsite, and the lack of an exposure to tobacco, were all associated with malignant transformations. There were no statistical differences in the PD1 expression and the CD4+ cells in OPML-OSCC and OPML-NOSCC. A significant increment in the CD8+ cells was noted in the OPML that evolved into carcinomas when compared with OPML-NOSCC (p = 0.05), whereas there were higher CD8+ cells levels in the carcinomas when compared with the OPML that evolved into carcinomas (p = 0.027). CONCLUSIONS: CD8+ cells infiltrate more in OPML-NOSCC than in OPML-OSCC. Carcinoma is more infiltrated by CD8+ cells than its associated OPML. CLINICAL RELEVANCE: Understanding immunological factors associated with malignant transformation of oral premalignant lesions can open a new way to treat this disease.

Are the Statins promising antifungal agents against invasive candidiasis?

The medical importance of intra-abdominal candidiasis (IAC) contrasts with the limited number of pharmacological treatment options available and the increasing rate of resistance to antifungal drugs. Thus, the repositioning of compounds in clinical use can contribute to the broadening of treatment possibilities for this infection. Statins, a class of drugs used to reduce cardiovascular event risk, have shown antiparasitic, antibacterial, and antiviral activities; however, their antifungal effects remain poorly studied. In this context, the present study aimed to elucidate the antifungal potential of six statins in vitro, as well as to evaluate the therapeutic use of fluvastatin in a mouse model of IAC. The biological effects of statins were evaluated against Candida spp., through the determination of the minimum inhibitory concentration (MIC). For the statins that showed activity, the fungicidal concentration, toxicity/selectivity, synergism with azoles and polyenes, phenotypic effects, and activity against virulence factors were also determined. Atorvastatin, rosuvastatin and fluvastatin were highly active, especially against C. albicans (MIC < 1-128 µg.mL-1) and C. glabrata (MIC 32-64 µg.mL-1). Fluvastatin and atorvastatin were selective for C. albicans in baby hamster kidney (BHK) cells. Moreover, all active statins in the antifungal assay showed high selectivity for fungal cells over bacteria. The combination of atorvastatin, rosuvastatin, and fluvastatin with azoles was associated with a synergistic effect. Active statins do not act on the fungal membrane or wall, but instead stimulate farnesol-dependent pathogenicity factors such as yeast-to-hyphal transition and biofilm generation. Fluvastatin treatment was evaluated in a mouse model of IAC, showing stimulation of the extra-hepatic dissemination of C. albicans but improvements in renal, splenic, and hepatic histological aspects. In conclusion, statins have potent antifungal activity in vitro, but the therapeutic effect in vivo is restricted to their anti-inflammatory activity.

Achyrocline alata potentiates repair of skin full thickness excision in mice.

Plants of the Asteraceae family have been traditionally used as medicinal plants. The species Achyrocline satureioides and Achyrocline alata present anti-inflammatory properties and great chemical similarity. However, no study has been performed to evaluate the influence of these plants on skin wound healing in vivo. Here, we have assessed the effect of these plants extracts on skin wound healing in mice. Mice were randomly arranged into three groups (n = 10), an injury was performed on the dorsal area of the animals, which received the following topical treatment: group 1, control (ointment base); group 2, A. satureioides extract; group 3, A. alata extract. The solution for treatment was prepared as 10% (w/w) concentration. The wound area was measured on days 1, 4, 9, 15 and 17 after treatment and tissues of local lesion were collected on the ninth day for histological analysis. A. alata was more effective since it induced earlier wound closure associated with decreasing initial inflammatory response, faster reepithelialization and collagen remodeling. A. satureioides improved the collagen renovation, but induced slower closure, which may be due to different concentrations of phenolic compounds among the plants here studied. Both plants did not alter the ultrastructural characteristics of cells in the healing process. In conclusion, our findings suggest the potent wound healing capacity of A. alata extracts, as demonstrated by more efficient and faster induction of wound closure. We believe this plant is a potential wound healing treatment for humans and further studies are necessary to assess its clinical practice.

Immunohistochemical Expression of Collagens in the Skin of Horses Treated with Leukocyte-Poor Platelet-Rich Plasma.

This study evaluated the immunohistochemical expression of type I (COL I) and III (COL III) collagens during the healing process of skin treated with leukocyte-poor platelet-rich plasma (LP-PRP). Seven healthy gelding crossbred horses aged 16 to 17 years were used. Two rectangle-shaped wounds were created surgically in the right and left gluteal regions. Twelve hours after wound induction, 0.5 mL of the LP-PRP was administered in each edge of the wounds of one of the gluteal regions. The contralateral region was used as control (CG). Three samples were obtained: after wound induction (T0), 14 days (T1) of healing process, and after complete closure of the skin (T2). The normal skin (T0) showed strong staining for type III and I collagen in papillary and reticular dermis, respectively. In the scar of the treated group, COL III showed important (p < 0.05) increase in immunoreaction in T2 compared with T1. The administration of a single dose of LP-PRP 12 h after induction of wound in horses does not influence formation of collagens I and III. However, the intense labeling for COL III suggests that the tissue was still weak during the macroscopic closure of the wound, demonstrating that healing was not completely finished.

Effects of digoxin and Na, K-ATPase immunoexpression on human oral squamous carcinomas.

AIM: The present study evaluated the expression of α1 and ß1 Na,K-ATPase, as well as the effects of digoxin (DGX) on oral squamous cell carcinomas (OSCCs). PATIENTS AND METHODS: Immunohistochemical expression of α1 and ß1 Na,K-ATPase were evaluated in 60 patients who underwent treatment at the São João de Deus Hospital. SCC-25 viability was assessed by the colorimetric assay. Chi-square or Fisher's exact tests were used to analyze the association of α1 and ß1 Na,K-ATPase expression with the variables. RESULTS: Immunoexpression of α1 and ß1 Na,K-ATPase were observed in 28% and 55% of the tumors, however these proteins were not significant prognostic factors. Tobacco was significantly associated with α1 expression. SCC-25 viability decreased significantly after treatment with 1 µM DGX at 24 h. CONCLUSION: The smoking status of OSCC patients was significantly associated with α1 expression and DGX affected the SCC-25 viability in a dose- and duration-dependent manner.

Evaluation of healing wound and genotoxicity potentials from extracts hydroalcoholic of Plantago major and Siparuna guianensis.

Despite the large use of the Plantago major and Siparuna guianensis in traditional medicine, there are no studies demonstrating the effectiveness from extracts of these plants in the healing process by the present methodology. This study reported the effects and toxicity of the P. major and S. guianensis extracts in the wound healing compared with a commercial product used in Brazil by macroscopic and microscopic analysis. Following injury in cervical dorsal area of the mice, the extract from P. major and S. guianensis and ointment was applied after an injury in cervical dorsal area of the mice. Wound healing rates were calculated at 4, 9, 15 and 21 d after the wounding, and tissues were obtained on the ninth day for histological analysis. Moreover, mutagenic assay of extracts was performed. Mutagenicity studies carried out with plant extracts showed not mutagenic with or without metabolic activations. Reduction of the wound area occurred earlier in mice treated with P. major and control treatment. On the 15th day, the complete wound closure occurred in P. major-treated wounds. Throughout ointment and S. guianensis treatment it was not observed the wound closured. Microscopic analyses of the wound, on the ninth day, showed the more efficient formation of the neoepithelium and skin appendages in animals treated with S. guianensis and P. major, while ointment treatment presented no re-epithelialization and absent skin appendages in wound. Thus, P. major extract showed good effects on wound healing processes rendering it a promising candidate for the treatment of wounds what also justified its traditional usage in wound treatment.

Distribution of laminin ß2, collagen type IV, fibronectin and MMP-9 in ovaries of the teleost fish.

Extracellular matrix in the ovarian follicle has been characterised for several mammalian species but there are no reports that describe the immunolocalisation of the extracellular matrix elements, matrix metalloproteinases, and its relation to plasma 17ß estradiol levels and follicular apoptosis during the teleost's reproductive cycle. The present study used immunohistochemistry to characterise the distribution of laminin ß2, collagen type IV, fibronectin and matrix metalloproteinases-9 (MMP-9). The TUNEL in situ technique was used to quantify apoptosis and indirect immunofluorimetric to determine plasma 17ß estradiol levels. The TUNEL-positive reaction associated with morphological features exhibited follicular apoptosis. During postovulatory follicle involution, the drop in plasma 17ß estradiol levels after spawning contributed to the intense apoptosis observed. By immunohistochemical analysis, laminin ß2 and collagen type IV were identified as the major constituents of the basement membrane. The loss of integrity of the basement membrane occurred due to lyses of the major constituents, and coincides with increased follicular apoptosis. The integrity of the basement membrane is important for the survival of follicular cells. Furthermore, the MMP-9 results suggest that this enzyme is involved in final oocyte maturation and regression of postovulatory follicles. Fibronectin was observed on the surface of follicular cells of the postovulatory follicle in P. argenteus, this being important for maintaining normal cell adhesion to extracellular matrix. In conclusion, our results suggest that the structure and composition of the extracellular matrix, and plasma 17ß estradiol levels related to apoptosis, play an important role during the follicular development and post-spawning involution in teleost fishes.