Development and cytotoxicity evaluation of multiple nanoemulsions for oral co-delivery of 5-fluorouracil and short chain triglycerides for colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer in the world, but current chemotherapy options are limited due to adverse effects and low oral bioavailability of drugs. In this study, we investigated the obtainment parameters and composition of new multiple nanoemulsions (MN) based on microemulsions for oral co-delivery of 5-fluorouracil (5FU) and short-chain triglycerides (SCT, either tributyrin or tripropionin). The area of microemulsion formation was increased from 14% to 38% when monocaprylin was mixed with tricaprylin as oil phase. Addition of SCT reduced this value to 24-26%. Using sodium alginate aqueous dispersion as internal aqueous phase (to avoid phase inversion) did not further affected the area but increased microemulsion viscosity by 1.5-fold. To obtain the MN, selected microemulsions were diluted in an external aqueous phase; droplet size was 500 nm and stability improved using polyoxyethylene oleyl ether at 1-2.5% as surfactant in the external phase and a dilution ratio of 1:1 (v/v). 5FU in vitro release could be better described by the Korsmeyer-Peppas model. No pronounced changes in droplet size were observed when selected MNs were incubated in buffers mimicking gastrointestinal fluids. The 5FU cytotoxicity in monolayer cell lines presenting various mutations was influenced by its incorporation in the nanocarrier, presence of SCT and cell mutation status. The MNs selected reduced the viability of tumor spheroids (employed as 3D tumor models) by 2.2-fold compared to 5FU solution and did not affect the survival of the G. mellonella, suggesting effectiveness and safety.

Hyaluronic acid nanoemulsions improve piplartine cytotoxicity in 2D and 3D breast cancer models and reduce tumor development after intraductal administration.

Nanoemulsions modified with chitosan (NE-Q) or hyaluronic acid (NE-HA), developed for intraductal administration of piplartine (piperlongumine) and local breast cancer treatment, were evaluated for cytotoxic effects in vitro in 2D and 3D breast cancer models and in vivo in a chemically induced carcinogenesis model. Droplet size was lower than 100 nm, and zeta potential varied from +17.9 to -25.5 mV for NE-Q and NE-HA, respectively. Piplartine nanoencapsulation reduced its IC50 up to 3.6-fold in T-47D and MCF-7 monolayers without differences between NE-Q and NE-HA, and up to 6.6-fold in cancer spheroids. Cytotoxicity improvement may result from a more efficient NE-mediated delivery, as suggested by stronger fluorescent staining of cells and spheroids. In 1-methyl-1-nitrosourea -induced breast cancer models, intraductal administration of piplartine-loaded NE-HA inhibited breast tumor development and histological alterations. These results support the potential applicability of piplartine-loaded NE-HA for intraductal treatment of breast cancer.

Embelin potentiates venetoclax-induced apoptosis in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) belongs to a group of hematological cancer whose relapse cases are often associated with chemoresistance that impairs treatment success and contributes to a poor outcome. For this reason, there is an urgent need for the development of new therapeutic strategies. Herein, we explore the combination of venetoclax, a BCL2 inhibitor, and embelin, an XIAP inhibitor, in the AML cell lines. Combinatory treatment of venetoclax and embelin potentiated cytotoxic effects of these drugs, demonstrating that both in combination present lower IC50 values than single treatment of either venetoclax or embelin alone in both cell lines analyzed. The combinatory treatment further increased the apoptosis-inducing properties of both compounds. Computer simulations suggest that embelin binds to both BIR2 and BIR3 domains of XIAP, reinforcing this inhibitory apoptosis protein as an embelin target. Although all AML cell lines presented similar basal levels of XIAP, the combinatory treatment effectively inhibited XIAP expression in OCI-AML3 cells. In conclusion, the inhibition of both apoptosis inhibitory players, BCL2 and XIAP, by venetoclax and embelin, respectively, potentiated their cytotoxic effects in AML cell lines.

Does the heterotrophic system influence the cellular immune response of Litopenaeus vannamei shrimp? In vitro phagocytosis indices and superoxide anion production comparisons.

Aquaculture production has increased in the last decades, with crustacean production contributing with 9.8% of the total production. However, fisheries and aquaculture sectors present several challenges, such as fish stocks fished beyond biological sustainability, animal diseases, biosecurity, and environmental impact. It is important to improve shrimp production with healthy animals, avoiding environmental impacts, e.g. with the use of heterotrophic rearing system. It is known that the heterotrophic system can stimulate the activation of immune genes, but how it affects the shrimp immune system is unknown. To assess if a heterotrophic system influences the cellular immune response in shrimp, Litopenaeus vannamei shrimp were reared in heterotrophic and clear water systems. Cellular immune response parameters such as total and differential hemocyte counts, phagocytosis indices and the production of the superoxide anion were evaluated after 60, 120 and 180 days. After 60 days, total haemocyte counts were higher in shrimps reared in the clear water system, while after 120 days it was higher in shrimps reared in the heterotrophic system. No significant difference was observed after 180 days. Hyaline, granular and semi-granular cells showed similar behavior, peaking after 120 days in the heterotrophic system. By the 60th day, phagocytic capacity was higher in the heterotrophic system, while no differences were found for the 120th and 180th day. No differences were detected concerning the phagocytic index or superoxide anion production. The heterotrophic system can affect total and differential shrimp haemocyte counts and phagocytic capacity, depending on the period of time they were maintained in this system. However, the phagocytic index and superoxide anion production are not affected by the heterotrophic system at the time points evaluated herein.

Anticancer Potential of Compounds from the Brazilian Blue Amazon.

"Blue Amazon" is used to designate the Brazilian Economic Exclusive Zone, which covers an area comparable in size to that of its green counterpart. Indeed, Brazil flaunts a coastline spanning 8000 km through tropical and temperate regions and hosting part of the organisms accredited for the country's megadiversity status. Still, biodiversity may be expressed at different scales of organization; besides species inventory, genetic characteristics of living beings and metabolic expression of their genes meet some of these other layers. These metabolites produced by terrestrial creatures traditionally and lately added to by those from marine organisms are recognized for their pharmaceutical value, since over 50% of small molecule-based medicines are related to natural products. Nonetheless, Brazil gives a modest contribution to the field of pharmacology and even less when considering marine pharmacology, which still lacks comprehensive in-depth assessments toward the bioactivity of marine compounds so far. Therefore, this review examined the last 40 years of Brazilian natural products research, focusing on molecules that evidenced anticancer potential-which represents ~ 15% of marine natural products isolated from Brazilian species. This review discusses the most promising compounds isolated from sponges, cnidarians, ascidians, and microbes in terms of their molecular targets and mechanisms of action. Wrapping up, the review delivers an outlook on the challenges that stand against developing groundbreaking natural products research in Brazil and on a means of surpassing these matters.

Survivin modulation in the antimelanoma activity of prodiginines.

Melanoma is a type of skin cancer with an elevated incidence of metastasis and chemoresistance. Such features hamper treatment success of these neoplasms, demanding the search for new therapeutic options. Using a two-step resin-based approach, we recently demonstrated that cytotoxic prodiginines bind to the inhibitor of apoptosis protein, survivin. Herein, we explore the role of survivin in melanoma and whether its modulation is related to the antimelanoma properties of three cytotoxic prodiginines (prodigiosin, cyclononylprodigiosin, and nonylprodigiosin) isolated from marine bacteria. In melanoma patients and cell lines, survivin is overexpressed, and higher levels negatively impact survival. All three prodiginines caused a decrease in cell growth with reduced cytotoxicity after 24 h compared to 72 h treatment, suggesting that low concentrations promote cytostatic effects in SK-Mel-19 (BRAF mutant) and SK-Mel-28 (BRAF mutant), but not in SK-Mel-147 (NRAS mutant). An increase in G1 population was observed after 24 h treatment with prodigiosin and cyclononylprodigiosin in SK-Mel-19. Further studies indicate that prodigiosin induced apoptosis and DNA damage, as detected by increased caspase-3 cleavage and histone H2AX phosphorylation, further arguing for the downregulation of survivin. Computer simulations suggest that prodigiosin and cyclononylprodigiosin bind to the BIR domain of survivin. Moreover, knockdown of survivin increased long-term toxicity of prodigiosin, as observed by reduced clonogenic capacity, but did not alter short-term cytotoxicity. In summary, prodiginine treatment provoked cytostatic rather than cytotoxic effects, cell cycle arrest at G0/G1 phase, induction of apoptosis and DNA damage, downregulation of survivin, and decreased clonogenic capacity in survivin knockdown cells.

Targeting the Oncogenic TBX2 Transcription Factor With Chromomycins.

The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic processes including the promotion of proliferation and bypass of senescence. Importantly, based on compelling biological evidences, TBX2 has been considered as a potential target for new anticancer therapies. There has therefore been a substantial interest to identify molecules with TBX2-modulatory activity, but no such substance has been found to date. Here, we adopt a targeted approach based on a reverse-affinity procedure to identify the ability of chromomycins A5 (CA5) and A6 (CA6) to interact with TBX2. Briefly, a TBX2-DNA-binding domain recombinant protein was N-terminally linked to a resin, which in turn, was incubated with either CA5 or CA6. After elution, bound material was analyzed by UPLC-MS and CA5 was recovered from TBX2-loaded resins. To confirm and quantify the affinity (KD) between the compounds and TBX2, microscale thermophoresis analysis was performed. CA5 and CA6 modified the thermophoretic behavior of TBX2, with a KD in micromolar range. To begin to understand whether these compounds exerted their anti-cancer activity through binding TBX2, we next analyzed their cytotoxicity in TBX2 expressing breast carcinoma, melanoma and rhabdomyosarcoma cells. The results show that CA5 was consistently more potent than CA6 in all tested cell lines with IC50 values in the nM range. Of the cancer cell types tested, the melanoma cells were most sensitive. The knockdown of TBX2 in 501mel melanoma cells increased their sensitivity to CA5 by up to 5 times. Furthermore, inducible expression of TBX2 in 501mel cells genetically engineered to express TBX2 in the presence of doxycycline, were less sensitive to CA5 than the control cells. Together, the data presented in this study suggest that, in addition to its already recognized DNA-binding properties, CA5 may be binding the transcription factor TBX2, and it can contribute to its cytotoxic activity.

Enriching cancer pharmacology with drugs of marine origin.

Marine natural products have proven, over the last half-century, to be effective biological modulators. These molecules have revealed new targets for cancer therapy as well as dissimilar modes of action within typical classes of drugs. In this scenario, innovation from marine-based pharmaceuticals has helped advance cancer chemotherapy in many aspects, as most of these are designated as first-in-class drugs. Here, by examining the path from discovery to development of clinically approved drugs of marine origin for cancer treatment-cytarabine (Cytosar-U®), trabectedin (Yondelis®), eribulin (Halaven®), brentuximab vedotin (Adcetris®), and plitidepsin (Aplidin®)- together with those in late clinical trial phases-lurbinectedin, plinabulin, marizomib, and plocabulin-the present review offers a critical analysis of the contributions given by these new compounds to cancer pharmacotherapy.

Determinants of elephant foraging behaviour in a coupled human-natural system: Is brown the new green?

Crop raiding by wildlife poses major threats to both wildlife conservation and human well-being in agroecosystems worldwide. These threats are particularly acute in many parts of Africa, where crop raiders include globally threatened megafauna such as elephants, and where smallholder agriculture is a primary source of human livelihood. One framework for understanding herbivore feeding behaviour, the forage-maturation hypothesis, predicts that herbivores should align their movements with intermediate forage biomass (i.e., peak green-up); this phenomenon is known as "surfing the green wave." Crop-raiding elephants, however, often consume not just foliage, but also fruits and tubers (e.g., maize and potatoes), which generally mature after seasonal peaks in photosynthetic activity. Thus, although elephants have been reported to surf the green wave in natural habitats, they may utilize a different strategy in cultivated landscapes by selecting crops that are "browning down." We sought to understand the factors that underpin movement of elephants into agricultural landscapes. In Mozambique's Gorongosa National Park, we used movement data from GPS-collared elephants, together with precipitation records, remotely sensed estimates of landscape greenness (NDVI), DNA-based diet analysis, measurements of plant nutritional quality and survey-based metrics of crop availability to understand spatiotemporal variation in elephant crop-raiding behaviour. Elephants tracked peak NDVI while foraging inside the Park. During the dry season, however, when NDVI within the Park declined and availability of mature crops was high, crop raiding increased dramatically, and elephants consistently selected crop plants that were browning down while foraging in cultivated landscapes. Crops contained significantly higher digestible energy than wild food plants, but comparable (and sometimes lower) levels of digestible protein, suggesting that this foraging strategy maximized energy rather than protein intake. Our study is the first to combine GPS tracking data with high-resolution diet analysis and community-based reporting of crop availability to reveal fine-scale plasticity in foraging behaviour of elephants at the human-wildlife interface. Our results extend the forage-maturation hypothesis by showing that elephants surf waves of plant brown-down in cultivated landscapes. These findings can aid efforts to reduce human-elephant conflict by enabling wildlife managers to prioritize mitigation actions in time and space with limited resources.

Synthesis and Cytotoxic Evaluation of 1H-1,2,3-Triazol-1-ylmethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-diones.

The 1,2-naphthoquinone compound was previously considered active against solid tumors. Moreover, glycosidase inhibitors such as 1,2,3-1H triazoles has been pointed out as efficient compounds in anticancer activity studies. Thus, a series of eleven 1,2-naphthoquinones tethered in C2 to 1,2,3-1H-triazoles 9a-k were designed, synthesized and their cytotoxic activity evaluated using HCT-116 (colon adenocarcinoma), MCF-7 (breast adenocarcinoma) and RPE (human nontumor cell line from retinal epithelium). The chemical synthesis was performed from C-3 allylation of lawsone followed by iodocyclization with subsequent nucleophilic displacement with sodium azide and, finally, the 1,3-dipolar cycloaddition catalyzed by Cu(I) with terminal alkynes led to the formation of 1H-1,2,3-Triazol-1-ylmethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-diones in good yields. Compounds containing aromatic group linked to 1,2,3-triazole ring (9c, 9d, 9e, 9i) presented superior cytotoxic activity against cancer cell lines with IC50 in the range of 0.74 to 4.4 µM indicating that the presence of aromatic rings substituents in the 1,2,3-1H-triazole moiety is probably responsible for the improved cytotoxic activity.

Soluble Uric Acid Activates the NLRP3 Inflammasome.

Uric acid is a damage-associated molecular pattern (DAMP), released from ischemic tissues and dying cells which, when crystalized, is able to activate the NLRP3 inflammasome. Soluble uric acid (sUA) is found in high concentrations in the serum of great apes, and even higher in some diseases, before the appearance of crystals. In the present study, we sought to investigate whether uric acid, in the soluble form, could also activate the NLRP3 inflammasome and induce the production of IL-1ß. We monitored ROS, mitochondrial area and respiratory parameters from macrophages following sUA stimulus. We observed that sUA is released in a hypoxic environment and is able to induce IL-1ß release. This process is followed by production of mitochondrial ROS, ASC speck formation and caspase-1 activation. Nlrp3-/- macrophages presented a protected redox state, increased maximum and reserve oxygen consumption ratio (OCR) and higher VDAC protein levels when compared to WT and Myd88-/- cells. Using a disease model characterized by increased sUA levels, we observed a correlation between sUA, inflammasome activation and fibrosis. These findings suggest sUA activates the NLRP3 inflammasome. We propose that future therapeutic strategies for renal fibrosis should include strategies that block sUA or inhibit its recognition by phagocytes.

Ocean acidification affects parameters of immune response and extracellular pH in tropical sea urchins Lytechinus variegatus and Echinometra luccunter.

The rising concentration of atmospheric CO2 by anthropogenic activities is changing the chemistry of the oceans, resulting in a decreased pH. Several studies have shown that the decrease in pH can affect calcification rates and reproduction of marine invertebrates, but little attention has been drawn to their immune response. Thus this study evaluated in two adult tropical sea urchin species, Lytechinus variegatus and Echinometra lucunter, the effects of ocean acidification over a period of 24h and 5days, on parameters of the immune response, the extracellular acid base balance, and the ability to recover these parameters. For this reason, the phagocytic capacity (PC), the phagocytic index (PI), the capacity of cell adhesion, cell spreading, cell spreading area of phagocytic amebocytes in vitro, and the coelomic fluid pH were analyzed in animals exposed to a pH of 8.0 (control group), 7.6 and 7.3. Experimental pH's were predicted by IPCC for the future of the two species. Furthermore, a recovery test was conducted to verify whether animals have the ability to restore these physiological parameters after being re-exposed to control conditions. Both species presented a significant decrease in PC, in the pH of coelomic fluid and in the cell spreading area. Besides that, Echinometra lucunter showed a significant decrease in cell spreading and significant differences in coelomocyte proportions. The recovery test showed that the PC of both species increased, also being below the control values. Even so, they were still significantly higher than those exposed to acidified seawater, indicating that with the re-establishment of the pH value the phagocytic capacity of cells tends to restore control conditions. These results demonstrate that the immune system and the coelomic fluid pH of these animals can be affected by ocean acidification. However, the effects of a short-term exposure can be reversible if the natural values ​​are re-established. Thus, the effects of ocean acidification could lead to consequences for pathogen resistance and survival of these sea urchin species.

The impact of rising sea temperature on innate immune parameters in the tropical subtidal sea urchin Lytechinus variegatus and the intertidal sea urchin Echinometra lucunter.

Ocean temperatures are rising throughout the world, making it necessary to evaluate the impact of these temperature changes on sea urchins, which are well-known bioindicators. This study evaluated the effect of an increase in temperature on the immune response of the subtidal Lytechinus variegatus and the intertidal Echinometra lucunter sea urchins. Both species were exposed to 20 (control), 25 and 30 °C temperatures for 24 h, 2, 7 and 14 days. Counting of coelomocytes and assays on the phagocytic response, adhesion and spreading of coelomocytes were performed. Red and colorless sphere cells were considered biomarkers for heat stress. Moreover, a significant decrease in the phagocytic indices and a decrease in both cell adhesion and cell spreading were observed at 25 and 30 °C for L. variegatus. For E. lucunter, the only alteration observed was for the cell proportions. This report shows how different species of sea urchins respond immunologically to rising temperatures.