Effects of clopidogrel bisulfate on B16-F10 cells and tumor development in a murine model of melanoma.

Metastatic melanoma is a very aggressive skin cancer. Platelets are constituents of the tumor microenvironment and, when activated, contribute to cancer progression, especially metastasis and inflammation. P2Y12 is an adenosine diphosphate receptor that triggers platelet activation. Inhibition of P2Y12 by clopidogrel bisulfate (CB) decreases platelet activation, which is also controlled by the extracellular concentration and the metabolism of purines by purinergic enzymes. We evaluated the effects of CB on the viability and proliferation of cultured B16-F10 cells. We also used a metastatic melanoma model with C57BL-6 mice to evaluate cancer development and purine metabolism modulation in platelets. B16-F10 cells were administered intraperitoneally to the mice. Two days later, the animals underwent a 12-day treatment with CB (30 mg/kg by gavage). We have found that CB reduced cell viability and proliferation in B16-F10 culture in 72 h at concentrations above 30 µm. In vivo, CB decreased tumor nodule counts and lactate dehydrogenase levels and increased platelet purine metabolism. Our results showed that CB has significant effects on melanoma progression.

Kinetic properties of E-NTPDase activity in lymphocytes isolated from bone marrow, thymus and mesenteric lymph nodes of Wistar rats.

Plasma membrane anchored nucleotidases (E-ATPDases), as the E-NTPDase family, could hydrolyze and regulate the pericellular levels of nucleotides in lymphocytes. Each immune organ has a different microenvironment and display lymphocytes with different functions and phenotypes. Considering the different functions of each resident subpopulations of lymphocytes, the E-ATPDases activities in bone marrow (BML), thymus (TL) and mesenteric lymph node (MLL) lymphocytes of Wistar rats were characterized. The hydrolysis of extracellular nucleotides (eATP and eADP) showed linearity in time of reaction between 0 and 120 min, and concentration of lymphocytes expressed in proteins between 1 and 6 µg protein in the reaction medium. The optimal activity was attained at 37 °C in a pH value of 8.0. The necessity of the cofactors Ca2+ and Mg2+ for the enzymatic activity was confirmed through a curve of concentration of 0-1000 µM in the reaction medium, with both cofactors showing similar effects in the enzymatic activity. The Chevillard plot revealed that the hydrolysis of eATP and eADP occurred at the same active site of the enzyme. The analyses of E-ATPDases inhibitor and enzyme specificity showed possible involvement of E-NTPDase isoforms - 1 and - 2 in the isolated cells. Furthermore, different kinetic behavior of the nucleotide hydrolysis in each resident subpopulation lymphocyte was observed in this study, as MLL showed the higher Vmax with the lowest km values, while TL had the lowest Vmax and high km values. The kinetic values for E-NTPDase activity of each immune tissue lymphocytes can be an important therapeutic target for numeral immune-related diseases.

Istradefylline modulates purinergic enzymes and reduces malignancy-associated factors in B16F10 melanoma cells.

ATP and adenosine exert pivotal roles in the development, maintenance, and metastatic spreading of melanoma. The action of such key melanoma tumor microenvironment (TME) constituents might be complementary or opposed, and their effects are not exclusive to immune cells but also to other host cells and tumor cells. The effects of ATP are controlled by the axis CD39/73, resulting in adenosine, the main actor in the TME, and A2A is the crucial mediator of its effects. We evaluated ATP and adenosine signaling through A2A on B16F10 melanoma cells using istradefylline (IST) (antiparkinsonian A2A antagonist) and caffeine (CAF) treatments after exposure to ATP and adenosine. Adenosine increased melanoma cell viability and proliferation in a concentration-dependent manner. ATP increases viability only as a substrate by CD39 to produce adenosine. Both IST and CAF are toxic to B16F10 cells, but only IST potentialized paclitaxel-induced cytotoxic effects, even decreasing its IC50 value. IST positively modulated CD39 and CD73 expression. CD39 activity was increased, and E-ADA was reduced, indicating that the melanoma cells promoted compensatory feedback in the production and maintenance of adenosine levels. A2A antagonism by IST reduced the factors associated with malignancy, like migration, adhesion, colony formation, and the capacity to produce melanin. Moreover, IST significantly increases nitric oxide (NO) production, which correlates to a decline in melanoma cell viability by apoptotic events. Altogether, our results suggest that adenosine signaling through A2A is essential for B16F10 cells, and its inhibition by IST causes compensatory purinergic enzymatic modulations. Furthermore, IST is a promising therapy that provides new ways to improve current melanoma treatments.

Antioxidant, hepatoprotective, genoprotective, and cytoprotective effects of quercetin in a murine model of arthritis.

Rheumatoid arthritis is a highly debilitating inflammatory autoimmune disease which is characterized by joint destruction. The present study sought to investigate the effect of quercetin in rats with complete Freund's adjuvant-induced arthritis. Animals were divided into control/saline, control/quercetin (5 mg/kg, 25 mg/kg, and 50 mg/kg) arthritis/saline, and arthritis/quercetin (5 mg/kg, 25 mg/kg, and 50 mg/kg); the treatments were administered for 45 days. Biochemical, oxidative stress, genotoxicity, and cytotoxicity parameters were evaluated. All doses of quercetin reduced the levels of aspartate aminotransferase, thiobarbituric acid-reactive substances, and reactive oxygen species; however, only treatment with 25 or 50 mg/kg increased catalase activity. Total thiol and reduced glutathione levels were not significantly affected by the induction nor by the treatments. Genotoxicity assessed by DNA damage, and cytotoxicity through picogreen assay, decreased after treatments with quercetin. Our results present evidence of the antioxidant, cytoprotective, genoprotective and hepatoprotective, and effects of quercetin, demonstrating its potential as a candidate for coadjuvant therapy.

Hydroalcoholic extract of leaf of Arachis hypogaea L. (Fabaceae) did not induce toxic effects in the repeated-dose toxicity study in rats.

Arachis hypogaea L. (peanut) leaf is traditionally used for the treatment of insomnia in Asia. However, studies describing the safety and toxicity profile for this plant preparation are limited. Thus, the goal of this study was to investigate the toxicity of peanut leaf hydroalcoholic extract (PLHE) repeated treatment. The extract was administered orally (100, 300 or 1000 mg/kg) in male and female Wistar rats for 28 days (OECD guideline 407). PLHE treatment did not cause mortality or weight variation in the animals. Also, there was no alteration on locomotor activity (open field test), motor coordination (rotarod test), or anxiety behaviour (elevated plus-maze test). Male rats had a reduction in relative liver weight (100 mg/kg) and an increase in total kidney weight (1000 mg/kg), but there was no change in biochemical and haematological parameters after PLHE treatment. Free extracellular double-stranded DNA (dsDNA) levels was also evaluated, but PLHE treatment did not increase this parameter in rat organs. Also, the dose of 1000 mg/kg of PLHE significantly increased the total thiols in the liver of females compared with the control animals. Thus, PLHE did not induce toxicity after repeated exposure for 28 days in rats.

Effect of quercetin on E-NTPDase/E-ADA activities and cytokine secretion of complete Freund adjuvant-induced arthritic rats.

The effect of quercetin was assessed in rats induced with complete Freund adjuvant (CFA). Arthritis scores, paw oedema, latency, activities of myeloperoxidase (MPO), ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), and ectoadenosine deaminase (E-ADA) in lymphocytes were determined. Furthermore, nucleotide and nucleoside levels as well as the secretion of pro- and anti-inflammatory cytokines were evaluated. Animals were treated with saline and quercetin in doses of 5, 25, and 50 mg/kg for 45 days. The result revealed that quercetin (50 mg/kg) reduced arthritis score and paw oedema, and increased the latency in the thermal hyperalgesia test. Histopathological analysis showed that all the doses of quercetin reduced infiltration of inflammatory cells. MPO activity was increased in the arthritis group; however, quercetin reduced this activity. E-NTPDase activity was increased in lymphocytes of arthritis rats, and treatment with quercetin reversed this increase. However, E-ADA activity was reduced in the arthritis group, and treatment with quercetin modulated the activity of this enzyme in arthritis rat groups. Serum adenosine levels were increased in arthritis, and the levels were lowered with quercetin treatment. Quercetin treatment in arthritis groups decreased the elevated levels of cytokines in the arthritis control group. Thus, quercetin demonstrated an anti-inflammatory effect, and this flavonoid may be a promising natural compound for the treatment of arthritis. SIGNIFICANCE OF THE STUDY: Quercetin may represent a potential therapeutic compound in the treatment of rheumatoid arthritis. Findings from this study indicate that quercetin suppresses swelling and attenuates the underlying inflammatory responses. This is the first report where quercetin was shown to modulate the immune response to arthritis via attenuation of the purinergic system (E-NTPDase and E-ADA activities) and the levels of IFN-gamma and IL-4. Thus, this work is relevant to basic research and may be translated into clinical practice.

Adenosine signaling and adenosine deaminase regulation of immune responses: impact on the immunopathogenesis of HIV infection.

Infection by human immunodeficiency virus (HIV) causes the acquired immune deficiency syndrome (AIDS), which has devastating effects on the host immune system. HIV entry into host cells and subsequent viral replication induce a proinflammatory response, hyperactivating immune cells and leading them to death, disfunction, and exhaustion. Adenosine is an immunomodulatory molecule that suppresses immune cell function to protect tissue integrity. The anti-inflammatory properties of adenosine modulate the chronic inflammation and immune activation caused by HIV. Lack of adenosine contributes to pathogenic events in HIV infection. However, immunosuppression by adenosine has its shortcomings, such as impairing the immune response, hindering the elimination of the virus and control of viral replication. By attempting to control inflammation, adenosine feeds a pathogenic cycle affecting immune cells. Deamination of adenosine by ADA (adenosine deaminase) counteracts the negative effects of adenosine in immune cells, boosting the immune response. This review comprises the connection between adenosinergic system and HIV immunopathogenesis, exploring defects in immune cell function and the role of ADA in protecting these cells against damage.

Characterization of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) activity in mouse peritoneal cavity cells.

This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E-NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca2+ . A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E-NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.

Free and nanoencapsulated vitamin D3 : effects on E-NTPDase and E-ADA activities in an animal model with induced arthritis.

UNLABELLED: The effect of vitamin D3 in oral solution (VD3 ) and vitamin D3 -loaded nanocapsules (NC-VD3 ) was analysed in animals with complete Freund's adjuvant (CFA) induced arthritis (AR). For this purpose, we evaluated scores for arthritis, thermal hyperalgesia and paw oedema, as well as histological analyses and measurements of the activity of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) enzymes in rat lymphocytes. Haematological and biochemical parameters were also determined. The doses administered were 120 UI/day of VD3 and 15.84 UI/day of NC-VD3 . Fifteen days after the induction of AR, the groups were treated for 15 days with vitamin D3 . The results demonstrated that VD3 was able to reduce arthritis scores, thermal hyperalgesia and paw oedema in rats with CFA-induced arthritis. However, treatment with NC-VD3 did not reduce arthritis scores. The histological analyses showed that both formulations were able to reduce the inflammatory changes induced by CFA. The activity of E-NTPDase in rat lymphocytes was higher in the AR compared with the control group, while the activity of E-ADA was lower. This effect was reversed after the 15-day treatment. Data from this study indicates that both forms of vitamin D3 seem to contribute to decreasing the inflammatory process induced by CFA, possibly altering the activities of ectoenzymes. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: The effects promoted by both formulations of vitamin D3 , either in oral solution or nanoencapsulated form, strongly suggests the softening of the inflammatory process induced by complete Freund's adjuvant (CFA), possibly altering the E-NTPDase and E-ADA activities. However, it is known that vitamin D has a beneficial effect on the modulation of the immune system components responsible for the inflammatory process. Moreover, the establishment of responses to treatment with vitamin D3 may provide an alternative for inhibiting the proinflammatory response, assisting in our understanding of the immunopathology of this disease and possibly improving the signs and symptoms that hinder the quality of life of patients with rheumatoid arthritis. HIGHLIGHTS: Evaluation of the effects on the E-NTPDase and E-ADA activities in an animal model of induced arthritis. Two formulations of vitamin D3 were used: form oral solution and nanoencapsulated. Vitamin D3 seems to contribute to the inflammatory process induced by CFA. Vitamin D3 possibly alters the E-NTPDase and E-ADA activities. Vitamin D3 may be an alternative supplementary treatment for chronic arthritis.

Vanadium(V) complexes derived from triphenylphosphonium and hydrazides: cytotoxicity evaluation and interaction with biomolecules.

The development of coordination compounds with antineoplastic therapeutic properties is currently focused on non-covalent interactions with deoxyribonucleic acid (DNA). Additionally, the interaction profiles of these compounds with globular plasma proteins, particularly serum albumin, warrant thorough evaluation. In this study, we report on the interactions between biomolecules and complexes featuring hydrazone-type imine ligands coordinated with vanadium. The potential to enhance the therapeutic efficiency of these compounds through mitochondrial targeting is explored. This targeting is facilitated by the derivatization of ligands with triphenylphosphonium groups. Thus, this work presents the synthesis, characterization, interactions, and cytotoxicity of dioxidovanadium(V) complexes (C1-C5) with a triphenylphosphonium moiety. These VV-species are coordinated to hydrazone-type iminic ligands derived from (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride ([AH]Cl) and aromatic hydrazides ([H2L1]Cl-[H2L5]Cl). The structures of the five complexes were elucidated through single-crystal X-ray diffraction and vibrational spectroscopies, confirming the presence of dioxidovanadium(V) species in various geometries with degrees of distortion (τ = 0.03-0.50) and highlighting their zwitterionic characteristics. The molecular structural stability of C1-C5 in solution was ascertained using 1H, 19F, 31P, and 51V-nuclear magnetic resonance. Moreover, their interactions with biomolecules were evaluated using diverse spectroscopic methodologies and molecular docking, indicating moderate interactions (Kb ≈ 104 M-1) with calf thymus DNA in the minor groove and with human serum albumin, predominantly in the superficial IB subdomain. Lastly, the cytotoxic potentials of these complexes were assessed in keratinocytes of the HaCaT lineage, revealing that C1-C5 induce a reduction in metabolic activity and cell viability through apoptotic pathways.

Free and nanoencapsulated curcumin prevents cigarette smoke-induced cognitive impairment and redox imbalance.

Cigarette smoke-exposure promotes neurobiological changes associated with neurocognitive abnormalities. Curcumin, a natural polyphenol, have shown to be able to prevent cigarette smoke-induced cognitive impairment. Here, we investigated possible mechanisms involved in curcumin protection against cigarette smoke-induced cognitive impairment and, due to its poor bioavailability, we investigated the potential of using curcumin-loaded lipid-core nanocapsules (C-LNC) suspension. Rats were treated with curcumin and cigarette smoke, once a day, 5 days each week, for 30 days. Animals were divided into ten groups: I, control (vehicle/corn oil); II, curcumin 12.5mg/kg; III, curcumin 25mg/kg; IV, curcumin 50mg/kg; V, C-LNC 4 mg/kg; VI, tobacco exposed; VII, curcumin 12.5mg/kg along with tobacco exposure; VIII, curcumin 25mg/kg along with tobacco exposure; IX, curcumin 50mg/kg along with tobacco exposure; X, C-LNC 4 mg/kg along with tobacco exposure. Cigarette smoke-exposure impaired object recognition memory (P<0.001), indicated by the low recognition index, increased biomarkers of oxidative/nitrosative stress such as TBARS (P<0.05) and NOx (P<0.01), decreased antioxidant defenses such as NPSH content (P<0.01) and SOD activity (P<0.01) and inhibited the activities of enzymes involved in ion homeostasis such as Na(+),K(+)-ATPase and Ca(2+)-ATPase. Both curcumin formulations (free and nanoencapsulated) prevented the memory impairment, the redox imbalance and the alterations observed in the ATPases activities. Maintenance of ion homeostasis and redox balance is involved in the protective mechanism of curcumin against tobacco-induced cognitive impairment. Our results suggest that curcumin is a potential therapeutic agent for neurocognition and that C-LNC may be an alternative to its poor bioavailability.

Ectoenzymes and cholinesterase activity and biomarkers of oxidative stress in patients with lung cancer.

We aimed to examine the nucleoside triphosphate diphosphohydrolases (NTPDase) in lymphocytes; adenosine deaminase (ADA) and butyrylcholinesterase (BChE) in serum; and acetylcholinesterase (AChE), superoxide dismutase (SOD), and catalase (CAT) activity in whole blood; since these enzymes are involved in inflammation responses as well as in oxidative stress conditions. We also checked the levels of total thiols (T-SH), non-protein thiols (NPSH), and thiobarbituric acid reactive substances (TBARS) in serum of patients with lung cancer. We collected blood samples from patients (n = 31) previously treated for lung cancer with chemotherapy. Patients were classified as stage IIIb and IV according to the Union for International Cancer Control (UICC). The results showed a significant increase in the hydrolysis of ATP, ADP, and adenosine in patients when compared with the control group. The activity of AChE, SOD, and CAT as well as the T-SH and NPSH levels were higher in patients group and TBARS levels were lower in patients compared with the control group. These findings demonstrated that the enzymes activity involved in the control of inflammatory and immune processes as well as the oxidative stress parameters are altered in patients with lung cancer.

Activities of enzymes that hydrolyze adenine nucleotides in lymphocytes from patients with rheumatoid arthritis.

The purpose of this study was to investigate the activities of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and adenosine deaminase (E-ADA; EC 3.5.4.4) in lymphocytes from patients with rheumatoid arthritis (RA). Thirty patients diagnosed with RA through American College of Rheumatology criteria as well as 30 healthy patients were selected. Peripheral blood lymphocytes were isolated, and E-NTPDase and E-ADA activities were assayed. The results demonstrated an increased E-NTPDase activity (both ATP and ADP as substrates) and a decreased E-ADA activity in RA patients. These data suggest an organic effort to preserve the adenosine level, which is known to have anti-inflammatory and analgesic properties, working as a potent suppressor of immune response.

E-ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy.

Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2-like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto-adenosine deaminase (E-ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E-ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E-ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E-ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy.

Influence of Toxoplasma gondii acute infection on cholinesterase activities of Wistar rats.

Several studies have shown the mechanisms and importance of immune responses against Toxoplasma gondii infection and the notable role of cholinesterases in inflammatory reactions. However, the association between those factors has not yet been investigated. Therefore, the aim of this study was to evaluate the acetylcholinesterase (AChE) activity in blood and lymphocytes and the activity of butyrylcholinesterase (BChE) in serum of rats experimentally infected with T. gondii during the acute phase of infection. For that, an in vivo study was performed with evaluations of AChE and BChE activities on days 5 and 10 post-infection (PI). The activity of AChE in blood was increased on day 5 PI, while in lymphocytes its activity was enhanced on days 5 and 10 PI (P<0.05). No significant difference was observed between groups regarding to the activity of BChE in serum. A positive (P<0.01) correlation was observed between AChE activity and number of lymphocytes. The role of AChE as an inflammatory marker is well known in different pathologies; thus, our results lead to the hypothesis that AChE has an important role in modulation of early immune responses against T. gondii infection.

Antimicrobial activity of water-soluble tetra-cationic porphyrins on Pseudomonas aeruginosa.

This manuscript presents the cytotoxicity, antimicrobial activity, antibiofilm preliminary properties, and associated therapy with commercial drugs using water-soluble tetra-cationic porphyrins against Pseudomonas aeruginosa. Two commercial tetra-cationic porphyrins were tested against a standard strain of P. aeruginosa 01 (PA01) in antibacterial activity assays under dark conditions and irradiated with white light for 120 min. Porphyrin 4-H2TMePor showed better antimicrobial activity and was chosen for further tests. Increased minimum inhibitory concentration was observed in the presence of reactive oxygen species, suggesting that photooxidation was mediated by the singlet oxygen production. In the time-kill curve assay, 4-H2TMePor inhibited bacterial growth in 90 min of irradiation. The checkerboard assay revealed synergistic interactions. Biofilms of the standard PA01 strain and three clinical isolates were formed. The biofilm destruction assay was more efficient for PA01, significantly reducing the biofilm biomass formed compared to the positive control. The associated treatment to destroy the biofilm potentiated a significant decrease in the biofilm biomass compared to the positive control. The photosensitizer did not damage human keratinocytes or mouse fibroblasts in the cytotoxicity assays, demonstrating the safety of using 4-H2TMePor. Atomic force microscopy indicated lower adhesion force, higher cell wall deformation, and higher dissipation energy in the treated control compared to untreated PA01. Given our findings, it is evident that water-soluble tetra-cationic porphyrins have excellent antimicrobial and a preliminary antibiofilm activity against Gram-negative bacteria, proving to be a potential photosensitizer for clinical use.

The First Report of In Vitro Antifungal and Antibiofilm Photodynamic Activity of Tetra-Cationic Porphyrins Containing Pt(II) Complexes against Candida albicans for Onychomycosis Treatment.

Onychomycosis is a prevalent nail fungal infection, and Candida albicans is one of the most common microorganisms associated with it. One alternative therapy to the conventional treatment of onychomycosis is antimicrobial photoinactivation. This study aimed to evaluate for the first time the in vitro activity of cationic porphyrins with platinum(II) complexes 4PtTPyP and 3PtTPyP against C. albicans. The minimum inhibitory concentration of porphyrins and reactive oxygen species was evaluated by broth microdilution. The yeast eradication time was evaluated using a time-kill assay, and a checkerboard assay assessed the synergism in combination with commercial treatments. In vitro biofilm formation and destruction were observed using the crystal violet technique. The morphology of the samples was evaluated by atomic force microscopy, and the MTT technique was used to evaluate the cytotoxicity of the studied porphyrins in keratinocyte and fibroblast cell lines. The porphyrin 3PtTPyP showed excellent in vitro antifungal activity against the tested C. albicans strains. After white-light irradiation, 3PtTPyP eradicated fungal growth in 30 and 60 min. The possible mechanism of action was mixed by ROS generation, and the combined treatment with commercial drugs was indifferent. The 3PtTPyP significantly reduced the preformed biofilm in vitro. Lastly, the atomic force microscopy showed cellular damage in the tested samples, and 3PtTPyP did not show cytotoxicity against the tested cell lines. We conclude that 3PtTPyP is an excellent photosensitizer with promising in vitro results against C. albicans strains.

Karaya/Gellan-Gum-Based Bilayer Films Containing 3,3'-Diindolylmethane-Loaded Nanocapsules: A Promising Alternative to Melanoma Topical Treatment.

This study aimed to incorporate nanocapsules containing 3,3'-diindolylmethane (DIM) with antitumor activity into a bilayer film of karaya and gellan gums for use in topical melanoma therapy. Nanocarriers and films were prepared by interfacial deposition of the preformed polymer and solvent casting methods, respectively. Incorporating DIM into nanocapsules increased its antitumor potential against human melanoma cells (A-375) (IC50 > 24.00 µg/mL free DIM × 2.89 µg/mL nanocapsules). The films were transparent, hydrophilic (θ < 90°), had homogeneous thickness and weight, and had a DIM content of 106 µg/cm2. Radical ABTS+ scavenger assay showed that the DIM films presented promising antioxidant action. Remarkably, the films showed selective bioadhesive potential on the karaya gum side. Considering the mechanical analyses, the nanotechnology-based films presented appropriate behavior for cutaneous application and controlled DIM release profile, which could increase the residence time on the application site. Furthermore, the nanofilms were found to increase the permeation of DIM into the epidermis, where melanoma develops. Lastly, the films were non-hemolytic (hemolysis test) and non-irritant (HET-CAM assay). In summary, the combination of karaya and gellan gum in bilayer films that contain nanoencapsulated DIM has demonstrated potential in the topical treatment of melanoma and could serve as a viable option for administering DIM for cutaneous melanoma therapy.

A more accurate profile of Achyrocline satureioides hypocholesterolemic activity.

The aim of this study was to investigate the effect of the aqueous extract (AE) of Achyrocline satureioides on serum lipid profile, liver oxidative profile and Na(+),K(+)-ATPase activity of rats submitted to a hyperlipidic diet. The animals were divided into four groups: control (C), AE 10% (A(10)), hyperlipidic (H) and hyperlipidic/AE 10% (HA(10)). In serum, we measured the levels of total cholesterol (TC), high-density lipoprotein, very-low-density lipoprotein, low-density lipoprotein (LDL) and triglyceride (TG). In liver homogenates, we measured the thiobarbituric acid reactive substances, the carbonyl proteins, the non-protein thiols (NPSHs) and the activity of superoxide dismutase, catalase (CAT) and Na(+),K(+)-ATPase. We observed a significant increase in the TC and LDL levels in the H group. A. satureioides prevented these effects, decreased the TG levels in the HA(10) group and increased the NPSH levels in the A(10) and HA(10) groups. The H group showed an increase in the carbonyl protein level and a decrease in CAT and Na(+),K(+)-ATPase activities. With the use of this model, results show that increased levels of lipids are related to a redox imbalance in the liver, which is also related to the inhibition of Na(+),K(+)-ATPase activity, and that chronic administration of the AE of A. satureioides is capable of changing this profile.

Increased NTPDase activity in lymphocytes during experimental sepsis.

We investigated in rats induced to sepsis the activity of ectonucleoside triphosphate diphosphohydrolase (NTPDase; CD39; E.C. 3.6.1.5), an enzyme involved in the modulation of immune responses. After 12 hours of surgery, lymphocytes were isolated from blood and NTPDase activity was determined. It was also performed the histology of kidney, liver, and lung. The results demonstrated an increase in the hydrolysis of adenosine-5'-triphosphate (ATP) (P < 0.01), but no changes regarding adenosine-5'-monophosphate (ADP) hydrolysis (P > 0.05). Histological analysis showed several morphological changes in the septic group, such as vascular congestion, necrosis, and infiltration of mononuclear cells. It is known that the intracellular milieu contains much more ATP nucleotides than the extracellular. In this context, the increased ATPasic activity was probably induced as a dynamic response to clean up the elevated ATP levels resulting from cellular death.