Lipolytic production from solid-state fermentation of the filamentous fungus Penicillium polonicum and its applicability as biocatalyst in the synthesis of ethyl oleate.

Lipases represent versatile biocatalysts extensively employed in transesterification reactions for ester production. Ethyl oleate holds significance in biodiesel production, serving as a sustainable alternative to petroleum-derived diesel. In this study, our goal was to prospect lipase and assess its efficacy as a biocatalyst for ethyl oleate synthesis. For quantitative analysis, a base medium supplemented with Rhodamine B, olive oil, and Tween 80 was used. Solid-state fermentation utilized crambe seeds of varying particle sizes and humidity levels as substrates. In the synthesis of ethyl oleate, molar ratios of 1:3, 1:6, and 1:9, along with a total enzymatic activity of 60 U in n-heptane, were utilized at temperatures of 30 °C, 37 °C, and 44 °C. Reactions were conducted in a shaker at 200 rpm for 60 min. As a result, we first identified Penicillium polonicum and employed the method of solid-state fermentation using crambe seeds as a substrate to produce lipase. Our findings revealed heightened lipolytic activity (22.5 Ug-1) after 96 h of fermentation using crambe cake as the substrate. Optimal results were achieved with crambe seeds at a granulometry of 0.6 mm and a fermentation medium humidity of 60%. Additionally, electron microscopy suggested the immobilization of lipase in the substrate, enabling enzyme reuse for up to 4 cycles with 100% enzymatic activity. Subsequently, we conducted applicability tests of biocatalysts for ethyl oleate synthesis, optimizing parameters such as the acid/alcohol molar ratio, temperature, and reaction time. We attained 100% conversion within 30 min at 37 °C, and our results indicated that the molar ratio proportion did not significantly influence the outcome. These findings provide a methodological alternative for the utilization of biocatalysts in ethyl oleate synthesis.

Biocompatibility of Brazilian native yeast-derived sophorolipids and Trichoderma harzianum as plant-growth promoting bioformulations.

The replacement of agrochemicals by biomolecules is imperative to mitigate soil contamination and inactivation of its core microbiota. Within this context, this study aimed at the interaction between a biological control agent such as Trichoderma harzianum CCT 2160 (BF-Th) and the biosurfactants (BSs) derived from the native Brazilian yeast Starmerella bombicola UFMG-CM-Y6419. Thereafter, their potential in germination of Oryza sativa L. seeds was tested. Both bioproducts were produced on site and characterized according to their chemical composition by HPLC-MS and GC-MS for BSs and SDS-PAGE gel for BF-Th. The BSs were confirmed to be sophorolipids (SLs) which is a well-studied compound with antimicrobial activity. The biocompatibility was examined by cultivating the fungus with SLs supplementation ranging from 0.1 to 2 g/L in solid and submerged fermentation. In solid state fermentation the supplementation of SLs enhanced spore production, conferring the synergy of both bioproducts. For the germination assays, bioformulations composed of SLs, BF-Th and combined (SLT) were applied in the germination of O. sativa L seeds achieving an improvement of up to 30% in morphological aspects such as root and shoot size as well as the presence of lateral roots. It was hypothesized that SLs were able to regulate phytohormones expression such as auxins and gibberellins during early stage of growth, pointing to their novel plant-growth stimulating properties. Thus, this study has pointed to the potential of hybrid bioformulations composed of biosurfactants and active endophytic fungal spores in order to augment the plant fitness and possibly the control of diseases.

Development of a multifunctional and self-preserving cosmetic formulation using sophorolipids and palmarosa essential oil against acne-causing bacteria.

AIMS: The objective of this study was to evaluate the antibacterial effect of sophorolipids in combination with palmarosa essential oil and to develop a cosmetic formulation against acne-causing bacteria. METHODS AND RESULTS: The antibacterial activity of sophorolipids, palmarosa oil and their combined effect was evaluated by broth microdilution and checkerboard methods. Antioxidant activity was determined by the DPPH method. The results showed that the compounds presented antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis. The combination of sophorolipid and palmarosa oil resulted in synergistic and additive interaction reducing the concentration needed for the effectiveness against S. aureus and S. epidermidis, to 98.4% and 50%, respectively. The compounds interaction showed an additive effect for antioxidant activity. The cosmetic formulation without any chemical preservative presents antibacterial activity against S. aureus, S. epidermidis and Cutibacterium acnes. The pH values and organoleptic characteristics of formulations remained stable under all conditions tested. CONCLUSIONS: The association of sophorolipids and palmarosa oil resulted in a self-preserving cosmetic formulation with great stability, and effective antioxidant and antibacterial activities against acne-causing micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed the development of an effective multifunctional cosmetic formulation with natural preservatives to treat acne vulgaris and other skin infections.

The female sex pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster.

Specific mate communication and recognition underlies reproduction and hence speciation. Our study provides new insights in Drosophila melanogaster premating olfactory communication. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues. Female-produced, species-specific volatile pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone (Z)-11-octadecenyl acetate (cVA) interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males. Two isoforms of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. However, only Z4-11Al mediates courtship in experienced males, not (R)-linalool. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds. Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in contrast to Z4-11Al. A blend of the food odour vinegar and cVA attracted females, while a blend of vinegar and female pheromone Z4-11Al attracted males, instead. Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects.

Impact of intravesical instillation of a novel biological response modifier (P-MAPA) on progress of non-muscle invasive bladder cancer treatment in a rat model.

This work describes the effects of immunotherapy with Protein Aggregate Magnesium-Ammonium Phospholinoleate-Palmitoleate Anhydride in the treatment of non-muscle invasive bladder cancer in an animal model. NMIBC was induced by treating female Fischer 344 rats with N-methyl-N-nitrosourea. After treatment with MNU, the rats were distributed into four experimental groups: Control (without MNU) group, MNU (cancer) group, MNU-BCG (Bacillus Calmette-Guerin) group, and MNU-P-MAPA group. P-MAPA intravesical treatment was more effective in histopathological recovery from cancer state in relation to BCG treatment. Western blot assays showed an increase in the protein levels of c-Myc, COUP-TFII, and wild-type p53 in P-MAPA-treated rats in relation to BCG-treated rats. In addition, rats treated with P-MAPA intravesical immunotherapy showed the highest BAX protein levels and the lowest proliferation/apoptotic ratio in relation to BCG-treated rats, pointing out a preponderance of apoptosis. P-MAPA intravesical treatment increased the wild-type p53 levels and enhanced c-Myc/COUP-TFII-induced apoptosis mediated by p53. These alterations were fundamental for histopathological recovery from cancer and for suppress abnormal cell proliferation. This action of P-MAPA on apoptotic pathways may represent a new strategy for treating NMIBC.

Production of a fermented solid containing lipases from Penicillium roqueforti ATCC 10110 and its direct employment in organic medium in ethyl oleate synthesis.

The production and direct employment in organic medium in the ethyl-oleate synthesis of a fermented solid (FS) containing lipases by Penicillium roqueforti ATCC 10110 (PR10110) was investigated. For the production of this FS, the solid-state fermentation of different agroindustrial waste was used, such as: cocoa shell, sugarcane bagasse, sugarcane bagasse with cocoa shell, and cocoa shell with soybean oil and nutrient solution. The response surface methodology was used to study the effect of independent variables of initial moisture content and inductor concentration, as carbon source and inducer on lipase production. The characterization of the fermented solid in organic medium was also carried out. The highest lipase activity (53 ± 5 U g-1 ) was 16% higher than that obtained with the nonoptimized conditions. The characterization studies observed high stability of the FS in organic solvents for 5 h at 30°C, as well as at different temperatures, and the residual activity was measured against triolein. The FS was also able to catalyze ethyl-oleate synthesis maintaining high relative conversion over five reaction cycles of 96 h at 40°C in n-heptane. These results are promising and highlight the use of the FS containing PR10110 lipases for the first time in biocatalytic processes.

12,13-diHOME as a new therapeutic target for metabolic diseases.

Lipokines are bioactive compounds, derived from adipose tissue depots, that control several molecular signaling pathways. Recently, 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), an oxylipin, has gained prominence in the scientific literature. An increase in circulating 12,13-diHOME has been associated with improved metabolic health, and the action of this molecule appears to be mediated by brown adipose tissue (BAT). Scientific evidence indicates that the increase in serum levels of 12,13-diHOME caused by stimuli such as physical exercise and exposure to cold may favor the absorption of fatty acids by brown adipose tissue and stimulate the browning process in white adipose tissue (WAT). Thus, strategies capable of increasing 12,13-diHOME levels may be promising for the prevention and treatment of obesity and metabolic diseases. This review explores the relationship of 12,13-diHOME with brown adipose tissue and its role in the metabolic health context, as well as the signaling pathways involved between 12,13-diHOME and BAT.

Antimicrobial activity of sophorolipids produced by Starmerella bombicola against phytopathogens from cherry tomato.

BACKGROUND: Phytopathogenic microorganisms are the main cause of plant diseases, generating significant economic losses for the agricultural and food supply chain. Cherry tomatoes (Solanum lycopersicum var. cerasiforme) are very perishable plants and highly demanding in the use of pesticides; therefore, alternative solutions such as biosurfactants have aroused as a potent substituent. The main objective of the present study was to investigate the antimicrobial activity of sophorolipids against the phytopathogens Botrytis cinerea, Sclerotium rolfsii, Rhizoctonia solani and Pythium ultimum. RESULTS: The biosurfactant inhibited the mycelial growth in vitro with a minimum concentration of 2 mg mL-1 . The application of sophorolipids at 1, 2 and 4 mg mL-1 in detached leaves of tomato before the inoculation of the fungus B. cinerea was the best treatment, reducing leaf necrosis by up to 76.90%. The use of sophorolipids for washing tomato fruits before the inoculation of B. cinerea was able to inhibit the development of gray mold by up to 96.27%. CONCLUSION: The results for tomato leaves and fruits revealed that the biosurfactant acts more effectively when used preventively. Sophorolipids are stable molecules that show promising action for the potential replacement of pesticides in the field and the post-harvest process against the main tomato phytopathogens. © 2021 Society of Chemical Industry.

In vivo brain levels of acetylcholine and 5-hydroxytryptamine after oleoylethanolamide or palmitoylethanolamide administrations are mediated by PPARα engagement.

The peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that has been linked to the modulation of several physiological functions, including the sleep-wake cycle. The PPARα recognizes as endogenous ligands the lipids oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), which in turn, if systemically injected, they exert wake-promoting effects. Moreover, the activation of PPARα by the administration of OEA or PEA increases the extracellular contents of neurotransmitters linked to the control of wakefulness; however, the role of PPARα activated by OEA or PEA on additional biochemicals related to waking regulation, such as acetylcholine (ACh) and 5-hydroxytryptamine (5-HT), has not been fully studied. Here, we have investigated the effects of treatments of OEA or PEA on the contents of ACh and 5-HT by using in vivo microdialysis techniques coupled to HPLC means. For this purpose, OEA or PEA were systemically injected (5, 10 or 30 mg/kg; i.p.), and the levels of ACh and 5-HT were collected from the basal forebrain, a wake-related brain area. These pharmacological treatments significantly increased the contents of ACh and 5-HT as determined by HPLC procedures. Interestingly, PPARα antagonist MK-886 (30 mg/kg; i.p.) injected before the treatments of OEA or PEA blocked these outcomes. Our data suggest that the activation of PPARα by OEA or PEA produces significant changes on ACh and 5-HT levels measured from the basal forebrain and support the conclusion that PPARα is a suitable molecular element involved in the regulation of wake-related neurotransmitters.

Interaction of an acidic sophorolipid biosurfactant with phosphatidylcholine model membranes.

Sophorolipids (SLs) constitute a group of unique biosurfactants (BS) in the light of their outstanding properties, among which their antimicrobial activities stand out. SLs can exist mainly in an acidic and a lactonic form, both of which display inhibitory activity. Given the amphipathic nature of SLs it is feasible that these antimicrobial actions are the result of the perturbation of the physicochemical properties of targeted membranes. Thus, in this work we have carried out a biophysical study to unveil the molecular details of the interaction of an acidic SL with a model phospholipid membrane made of 1,2-dipalmitoy-sn-glycero-3-phosphocholine (DPPC). Using differential scanning calorimetry it was found that SL altered the phase behaviour of DPPC at low molar fractions, producing fluid phase immiscibility with the result of formation of biosurfactant-enriched domains within the phospholipid bilayer. Fourier-transform infrared spectroscopy showed that SL interacted with DPPC increasing ordering of the phospholipid acyl chain palisade and hydration of the lipid/water interface. Small angle X-ray scattering showed that SL did not modify bilayer thickness in the biologically relevant Lα fluid phase. SL was found to induce contents leakage in 1-palmitoy-2-oleoy-sn-glycero-3-phosphocholine (POPC) unilamellar liposomes, at sublytic concentrations below the cmc. This SL-induced membrane permeabilization at concentrations below the onset for membrane solubilization can be the result of the formation of laterally segregated domains, which might contribute to provide a molecular basis for the reported antimicrobial actions of SLs.

Antimicrobial effects of sophorolipid in combination with lactic acid against poultry-relevant isolates.

The objective of this study was to evaluate the antibacterial effect of sophorolipid in combination with lactic acid against relevant bacteria isolated from the poultry industry. Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, and Escherichia coli were isolated from chicken meat and antibacterial tests with sophorolipid and lactic acid were performed. Checkerboard, time-kill, and scanning electron microscopy analyses were used to confirm the antibacterial action and the combined effects. Although no inhibitory effects were observed for E. coli and Salmonella, these compounds presented antibacterial activity against L. monocytogenes and S. aureus. Additionally, sophorolipid and lactic acid were not cytotoxic at the concentrations used in the tests. The combination of sophorolipid and lactic acid resulted in an additive interaction, reducing the concentration of the active compounds needed for effectiveness against S. aureus and L. monocytogenes, to 50% and 75%, respectively. These findings lead to the possibility of developing a new, sustainable, and natural antimicrobial solution that is considered noncytotoxic and has wide applicability in the poultry industry to reduce substantial losses in this sector.

Modulation of SCD1 activity in hepatocyte cell lines: evaluation of genomic stability and proliferation.

Stearoyl-CoA desaturase (SCD) is a central lipogenic enzyme for the synthesis of monounsaturated fatty acids (MUFA). SCD1 overexpression is associated with a genetic predisposition to hepatocarcinogenesis in mice and rats. This work hypothesized possible roles of SCD1 to genomic stability, lipogenesis, cell proliferation, and survival that contribute to the malignant transformation of non-tumorigenic liver cells. Therefore, HepG2 tumor cells were treated with the SCD1 inhibitor (CAY10566) to ensure a decrease in proliferation/survival, as confirmed by a lipidomic analysis that detected an efficient decrease in the concentration of MUFA. According to that, we switched to a model of normal hepatocytes, the HepaRG cell line, where we: (i) overexpressed SCD1 (HepaRG-SCD1 clones), (ii) inhibited the endogenous SCD1 activity with CAY10566, or (iii) treated with two monounsaturated (oleic OA and/or palmitoleic PA) fatty acids. SCD1 overexpression or MUFA stimulation increased cell proliferation, survival, and the levels of AKT, phospho-AKT(Ser473), and proliferating cell nuclear antigen (PCNA) proteins. By contrast, opposite molecular and cellular responses were observed in HepaRG cells treated with CAY10566. To assess genomic stability, HepaRG-SCD1 clones were treated with ionizing radiation (IR) and presented reduced levels of DNA damage and higher survival at doses of 5 Gy and 10 Gy compared to parental cells. In sum, this work suggests that modulation of SCD1 activity not only plays a role in cell proliferation and survival, but also in maintaining genomic stability, and therefore, contributes to a better understanding of this enzyme in molecular mechanisms of hepatocarcinogenesis projecting SCD1 as a potential translational target.

Macrocystis pyrifera Extract Residual as Nutrient Source for the Production of Sophorolipids Compounds by Marine Yeast Rhodotorula rubra.

Seaweed processing generates liquid fraction residual that could be used as a low-cost nutrient source for microbial production of metabolites. The Rhodotorula strain is able to produce antimicrobial compounds known as sophorolipids. Our aim was to evaluate sophorolipid production, with antibacterial activity, by marine Rhodotorula rubra using liquid fraction residual (LFR) from the brown seaweed Macrocystis pyrifera as the nutrient source. LFR having a composition of 32% w/w carbohydrate, 1% w/w lipids, 15% w/w protein and 52% w/w ash. The best culture condition for sophorolipid production was LFR 40% v/v, without yeast extract, artificial seawater 80% v/v at 15 °C by 3 growth days, with the antibacterial activity of 24.4 ± 3.1 % on Escherichia coli and 21.1 ± 3.8 % on Staphylococcus aureus. It was possible to identify mono-acetylated acidic and methyl ester acidic sophorolipid. These compounds possess potential as pathogen controllers for application in the food industry.

In vitro and in vivo effects of P-MAPA immunomodulator on schistosomiasis.

Schistosomiasis is an infectious disease caused by helminth parasites of the genus Schistosoma; it is transmitted in over 78 countries. The main strategy for schistosomiasis control is treatment of infected people with praziquantel (PZQ). As PZQ-resistant strains have emerged, new anti-schistosomal agents have become necessary. We evaluated the in vitro and in vivo effect of P-MAPA, an aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride with immunomodulatory properties; it is produced by Aspergillus oryzae fermentation. In vitro, P-MAPA (5, 50, and 100 µg/mL) damaged the Schistosoma mansoni tegument, causing thorn losses and tuber destruction in male worms and peeling and erosion in females after 24-h incubation. In vivo, P-MAPA (5 and 100 mg/kg, alone and combined with PZQ - 50 mg/kg) reduced the number of eggs by up to 69.20% in the liver and 88.08% in the intestine. Furthermore, granulomas were reduced up to 83.13%, and there was an increase in the number of dead eggs and a reduction of serum aspartate aminotransferase levels. These data suggest that P-MAPA activity can help improve schistosomiasis treatment and patients' quality of life.

Evaluation of sclerotherapy of benign oral vascular lesions with monoethanolamine oleate.

OBJECTIVE: To investigate the efficacy of sclerotherapy with monoethanolamine oleate (MEO) in a series of cases of benign oral vascular lesions (BOVL). MATERIAL AND METHODS: Clinical records and images were retrieved (2015-2019), and data regarding age, gender, location, size, symptomatology, treatment and outcomes of patients were collected. All patients were diagnosed according to the classification of International Society for the Study of Vascular Anomalies and received the same treatment protocol (MEO 0.05 g/mL). The collected data were submitted to descriptive analysis and Pearson's chi-square test (p ≤ 0.05). RESULTS: Thirty-seven patients were treated. Most were female (70.3%) aged 9 to 88 years (median, 57.5 ± 17.4 years). Lower lip (54.1%) was the most affected site followed by buccal mucosa (16.2%). Thirty-two lesions were asymptomatic and 35.1% showed ≤ 0.5 cm in size. In 48.6% of the patients, only one application of MEO was performed. Complete regression occurred in 62.2% of cases, whereas 27% showed partial regression. One patient showed hypersensitivity during treatment. There was no significant difference between clinical outcome and age, anatomic site, size, and number of applications of MEO. CONCLUSIONS: Sclerotherapy with MEO is an acceptable and affordable treatment and can provide satisfactory results in BOVL, especially where other treatment options could compromise the esthetic aspects. CLINICAL RELEVANCE: As it is a non-invasive therapy leading, in most cases, to adequate clinical results, safety, and tolerability, sclerotherapy with MEO can be considered an effective treatment for BOVL.

The P-MAPA Immunomodulator Partially Prevents Apoptosis Induced by Zika Virus Infection in THP-1 Cells.

BACKGROUND: Zika virus is an emerging arbovirus of global importance. ZIKV infection is associated with a range of neurological complications such as the Congenital Zika Syndrome and Guillain Barré Syndrome. Despite the magnitude of recent outbreaks, there is no specific therapy to prevent or to alleviate disease pathology. OBJECTIVE: To investigate the role of P-MAPA immunomodulator in Zika-infected THP-1 cells. METHODS: THP-1 cells were subjected to Zika virus infection (Multiplicity of Infection = 0.5) followed by treatment with P-MAPA for until 96 hours post-infection. After that, the cell death was analyzed by annexin+/ PI+ and caspase 3/ 7+ staining by flow cytometry. In addition, virus replication and cell proliferation were accessed by RT-qPCR and Ki67 staining, respectively. RESULTS: We demonstrate that P-MAPA in vitro treatment significantly reduces Zika virus-induced cell death and caspase-3/7 activation on THP-1 infected cells, albeit it has no role in virus replication and cell proliferation. CONCLUSION: Our study reveals that P-MAPA seems to be a satisfactory alternative to inhibit the effects of Zika virus infection in mammalian cells.

P-MAPA, a Fungi-Derived Immunomodulatory Compound, Induces a Proinflammatory Response in a Human Whole Blood Model.

P-MAPA is a complex compound, derived from Aspergillus oryzae cultures, that has shown immunomodulatory properties in infection and cancer animal models. Despite promising results in these models, the mechanisms of cellular activation by P-MAPA, suggested to be Toll-like receptor- (TLR-) dependent, and its effect on human immune cells, remain unclear. Using an ex vivo model of human whole blood, the effects of P-MAPA on complement system activation, production of cytokines, and the expression of complement receptors (CD11b, C5aR, and C3aR), TLR2, TLR4, and the coreceptor CD14 were analyzed in neutrophils and monocytes. P-MAPA induced complement activation in human blood, detected by increased levels of C3a, C5a, and SC5b-9 in plasma. As a consequence, CD11b expression increased and C5aR decreased upon activation, while C3aR expression remained unchanged in leukocytes. TLR2 and TLR4 expressions were not modulated by P-MAPA treatment on neutrophils, but TLR4 expression was reduced in monocytes, while CD14 expression increased in both cell types. P-MAPA also induced the production of TNF-α, IL-8, and IL-12 and oxidative burst, measured by peroxynitrite levels, in human leukocytes. Complement inhibition with compstatin showed that P-MAPA-induced complement activation drives modulation of C5aR, but not of CD11b, suggesting that P-MAPA acts through both complement-dependent and complement-independent mechanisms. Compstatin also significantly reduced the peroxynitrite generation. Altogether, our results show that P-MAPA induced proinflammatory response in human leukocytes, which is partially mediated by complement activation. Our data contribute to elucidate the complement-dependent and complement-independent mechanisms of P-MAPA, which ultimately result in immune cell activation and in its immunomodulatory properties in infection and cancer animal models.

Oleamide Induces Cell Death in Glioblastoma RG2 Cells by a Cannabinoid Receptor-Independent Mechanism.

The endocannabinoid system has been associated with antiproliferative effects in several types of tumors through cannabinoid receptor-mediated cell death mechanisms. Oleamide (ODA) is a CB1/CB2 agonist associated with cell growth and migration by adhesion and/or ionic signals associated with Gap junctions. Antiproliferative mechanisms related to ODA remain unknown. In this work, we evaluated the effects of ODA on cell viability and morphological changes in a rat RG2 glioblastoma cell line and compared these effects with primary astrocyte cultures from 8-day postnatal rats. RG2 and primary astrocyte cultures were treated with ODA at increasing concentrations (25, 50, 100, and 200 µM) for different periods of time (12, 24, and 48 h). Changes in RG2 cell viability and morphology induced by ODA were assessed by viability/mitochondrial activity test and phase contrast microscopy, respectively. The ratios of necrotic and apoptotic cell death, and cell cycle alterations, were evaluated by flow cytometry. The roles of CB1 and CB2 receptors on ODA-induced changes were explored with specific receptor antagonists. ODA (100 µM) induced somatic damage, detachment of somatic bodies, cytoplasmic polarization, and somatic shrinkage in RG2 cells at 24 and 48 h. In contrast, primary astrocytes treated at the same ODA concentrations exhibited cell aggregation but not cell damage. ODA (100 µM) increased apoptotic cell death and cell arrest in the G1 phase at 24 h in the RG2 line. The effects induced by ODA on cell viability of RG2 cells were independent of CB1 and CB2 receptors or changes in intracellular calcium transient. Results of this novel study suggest that ODA exerts specific antiproliferative effects on RG2 glioblastoma cells through unconventional apoptotic mechanisms not involving canonical signals.

P-MAPA activates TLR2 and TLR4 signaling while its combination with IL-12 stimulates CD4+ and CD8+ effector T cells in ovarian cancer.

AIMS: Ovarian cancer (OC) is the most lethal gynecological malignancies and many women develop chemoresistance associated with the inflammatory process. We investigated the effects of P-MAPA and IL-12 on the inflammatory and immune responses in a chemically-induced OC model. MAIN METHODS: OCs were induced with 7,12-dimethylbenz(a)anthracene into the ovarian bursa, and the animals were given P-MAPA (5 mg/kg bw., i.p., twice a week), or IL-12 (300 ng/kg bw., i.p., one a week) for 60 days, or both P-MAPA and IL-12. Immunohistochemistry, western blot, flow cytometry, and multiplex assay were used to examine the effectiveness of immunotherapies in OC. KEY FINDINGS: The combinatory therapy improved the general OC features, reducing inflammatory cells and adipocyte accumulation, in addition to revealing a soft and mobile tissue with no adherences and peritoneal implants. P-MAPA treatment increased the levels of TLR2, TLR4 and TRIF in OCs while decreasing the number of regulatory T (Treg) cells. Additionally, the association of P-MAPA with IL-12 significantly increased the number of CD4+ and CD8+ T effector cells in draining lymph nodes. Regarding the inflammatory mediators, P-MAPA enhanced the levels of the pro-inflammatory cytokine IL-17 while P-MAPA+IL-12 increased the levels of IL-1ß. Treatment with IL-12 enhanced the cytokine levels of IL-17, TNF-α, IL-1ß, and IL-2 in addition to the chemokine MIP-1α. SIGNIFICANCE: We conclude that P-MAPA upregulated TLR2 and TLR4 signaling, possibly activating the non-canonical pathway, while attenuating the tumor immunosuppression. Also, the combination of P-MAPA with IL-12 improves the antitumor immunoresponse, opening a new therapeutic approach for fighting OC.