In Vitro Study of Cytotoxic Mechanisms of Alkylphospholipids and Alkyltriazoles in Acute Lymphoblastic Leukemia Models.

This study investigates the efficacy of miltefosine, alkylphospholipid, and alkyltriazolederivative compounds against leukemia lineages. The cytotoxic effects and cellular and molecular mechanisms of the compounds were investigated. The inhibitory potential and mechanism of inhibition of cathepsins B and L, molecular docking simulation, molecular dynamics and binding free energy evaluation were performed to determine the interaction of cathepsins and compounds. Among the 21 compounds tested, C9 and C21 mainly showed cytotoxic effects in Jurkat and CCRF-CEM cells, two human acute lymphoblastic leukemia (ALL) lineages. Activation of induced cell death by C9 and C21 with apoptotic and necrosis-like characteristics was observed, including an increase in annexin-V+propidium iodide-, annexin-V+propidium iodide+, cleaved caspase 3 and PARP, cytochrome c release, and nuclear alterations. Bax inhibitor, Z-VAD-FMK, pepstatin, and necrostatin partially reduced cell death, suggesting that involvement of the caspase-dependent and -independent mechanisms is related to cell type. Compounds C9 and C21 inhibited cathepsin L by a noncompetitive mechanism, and cathepsin B by a competitive and noncompetitive mechanism, respectively. Complexes cathepsin-C9 and cathepsin-C21 exhibited significant hydrophobic interactions, water bridges, and hydrogen bonds. In conclusion, alkyltriazoles present cytotoxic activity against acute lymphoblastic lineages and represent a promising scaffold for the development of molecules for this application.

First generation of multifunctional peptides derived from latarcin-3a from Lachesana tarabaevi spider toxin.

The need for discovering new compounds that can act selectively on pathogens is becoming increasingly evident, given the number of deaths worldwide due to bacterial infections or tumor cells. New multifunctional biotechnological tools are being sought, including compounds present in spider venoms, which have high biotechnological potential. The present work aims to perform the rational design and functional evaluation of synthetic peptides derived from Lachesana tarabaevi spider toxin, known as latarcin-3a. The antimicrobial activity was tested against Gram-positive and -negative bacteria, with minimum inhibitory concentrations (MIC) between 4 and 128 µg.ml-1. Anti-biofilm tests were then performed to obtain MICs, where the peptides demonstrated activity from 4 to 128 µg.ml-1. In vitro cell cytotoxicity assays were carried out from tumor cell lines, lineages C1498, Kasumi-1, K-562, Jurkat, MOLT4, and Raji. Erythrocyte integrity was evaluated in the presence of synthetic peptides analog, which did not promote hemolysis at 128 µg.ml-1. The peptide that showed the best antibacterial activity was Lt-MAP3 and the best antitumor was Lt-MAP2. In conclusion, rational design of multifunctional antimicrobial peptides may be promising alternative tools in the treatment of emerging diseases such as bacterial infections and tumor cells.

Chemodiversity and Anti-Leukemia Effect of Metabolites from Penicillium setosum CMLD 18.

Penicillium setosum represents a Penicillium species recently described, with little up-to-date information about its metabolic and biological potential. Due to this scenario, we performed chemical and biological studies of P. setosum CMLD18, a strain isolated from Swinglea glutinosa (Rutaceae). HRMS-MS guided dereplication strategies and anti-leukemia assays conducted the isolation and characterization of six compounds after several chromatographic procedures: 2-chloroemodic acid (2), 2-chloro-1,3,8-trihydroxy-6- (hydroxymethyl)-anthraquinone (7), 7-chloroemodin (8), bisdethiobis(methylthio)acetylaranotine (9), fellutanine C (10), and 4-methyl-5,6-diihydro-2H-pyran-2-one (15). From the assayed metabolites, (10) induced cellular death against Kasumi-1, a human leukemia cell line, as well as good selectivity for it, displaying promising cytotoxic activity. Here, the correct NMR signal assignments for (9) are also described. Therefore, this work highlights more detailed knowledge about the P. setosum chemical profile as well as its biological potential, offering prospects for obtaining natural products with anti-leukemia capabilities.

Distribution of monocytes subpopulations in the peripheral blood from patients with Behçet's disease - Impact of disease status and colchicine use.

The innate immune response has a predominant role in Behçet's disease (BD) pathogenesis, but few studies have assessed monocytes in BD. This study aims to evaluate the profile of monocytes subsets in the peripheral blood of BD patients and healthy controls (HC). Monocytes subsets were identified as classical (CD14+CD16-), intermediate (CD14+CD16dim), and non-classical (CD14dimCD16high) subsets. Patients with BD presented a lower number of total monocytes (p = 0.020) and a lower number (p < 0.0001) of circulating classical monocytes than HC. In contrast, the number of intermediate monocytes was higher in BD patients than HC (p < 0.0001). In BD patients, no associations were observed with the severity of clinical manifestations or therapy. Colchicine was associated with a higher number of non-classical monocytes (p = 0.035). In conclusion, BD patients present an altered distribution of monocytes subsets with a reduction of classical and an increase of intermediate subsets.

α-amyrin-loaded nanocapsules produce selective cytotoxic activity in leukemic cells.

INTRODUCTION: Amyrins are triterpenes that have attractive pharmacological potential; however, their low water solubility and erratic stomach absorption hinders their use as a drug. The aim of this paper was to develop a novel α-amyrin-loaded nanocapsule for intestinal delivery and evaluate, preliminarily, its cytotoxic ability against leukemic cells. MATERIAL AND METHODS: Five nanocapsule formulations were designed by the solvent displacement-evaporation method. Poly-ε-caprolactone, Eudragit® E100, and Kollicoat® Mae 100 P were used as film-former materials. Particle size, polydispersity index (PdI), zeta potential, and the pH of all formulations were measured. The cytotoxic potential of the nanocapsules was evaluated in vitro using different leukemic lineages RESULTS: Nanocapsules coated with Kollicoat® Mae 100 P presented the smallest particle size (130 nm), the lowest zeta-potential (-38 mV), and the narrowest size distribution (PdI = 0.100). The entrapment efficiency was 65.47%, while the loading capacity was 2.40%. Nanocapsules release 100% of α-amyrin in 40 min (pH 7.4), by using a possible mechanism of swelling-diffusion. The formulation showed excellent on-shelf physicochemical stability during one year. Additionally, nanocapsules produced a selective cytotoxic effect on a human leukemia lineage Kasumi-1, an acute myeloid leukemia cell line, and produced cell death by apoptosis CONCLUSION: α-amyrin-loaded nanocapsules appear to be a promising nanoformulation that could be used against leukemia.

Methanolic extract of Cariniana rubra Gardner ex Miers stem bark negatively regulate the leukocyte migration and TNF-α and up-regulate the annexin-A1 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Cariniana rubra Gardner ex Miers (Lecythidaceae), is a native and endemic tree in Brazil, whose inner stem bark decoction preparation is used in folk medicine to treat various inflammatory disorders. Previous scientific reports confirmed its popular use as an anti-inflammatory, without, however, evaluating its action mechanisms. AIM: The objective of this study was to determine the cytotoxicity and anti-inflammatory mechanism of action of the methanolic extract of Cariniana rubra (MECr), using experimental models in vivo and in vitro, as well as to identify secondary metabolites present in the extract. MATERIAL AND METHODS: The MECr was prepared by maceration of inner stem bark powder in methanol (1:10 w/v). The in vitro cytotoxicity effect was evaluated in CHO-k1 cells. The Hippocratic screening test was conducted to evaluate the acute toxicity of MECr in mice. The actions of MECr on leukocyte migration, cytokine levels (IL-1ß and TNF-α) and annexin-A1 (AnxA1) expression, were carried out on lambda-type carrageenan air pouch inflammation model in Swiss mice. Additionally, the phytochemical analysis of MECr was carried out by thin-layer chromatography (TLC) and spectrometric mass analysis with electrospray ionization ESI(-)/MS and gas chromatography-mass spectrometry (GC-MS). RESULTS: Treatment of CHO-k1 cells for 24 h with MECr did not cause cytotoxicity (IC50 > 200 µg/mL), however, the MECr was shown to be cytotoxic after 72 h of cell exposure (IC50 = 19.90 ± 3.51 µg/mL). In the Hippocratic test, oral treatment of mice with 750, 1500, or 3000 mg/kg of MECr did not show any histopathological changes and mortality during the 14 days of observation. In the carrageenan air pouch inflammation model, MECr reduced (p < 0.001) polymorphonuclear migration (57.7% and 57.8%), leukocyte monocyte migration (74.5% and 61.8%) in the air pouch cavity and in the skin tissue, respectively. MECr also inhibited TNF-α concentration in the air cavity wash (3.2%, p < 0.01) and increased expression of the AnxA1 protein (26.9%, p < 0.01) in the skin tissue, particularly in neutrophils. ß-sitosterol (1.95%), gallic acid (1.24%), ß-amyrin (0.87%) and stigmasterol (0.66%) were identified as the major constituents in methanolic extract. CONCLUSION: MECr exhibits significant anti-inflammatory action at least by increasing AnxA1 expression and by inhibiting the release of TNF-α pro-inflammatory cytokine and leukocyte migration, which is probably linked to the presence of identified biologically active compounds, especially gallic acid and terpenes. We believe that the results of this study provide a pharmacological basis for the MECr to be considered as a potential therapeutic agent for the treatment of inflammatory diseases.

Gemfibrozil Induces Anemia, Leukopenia and Reduces Hematopoietic Stem Cells via PPAR-α in Mice.

Hypercholesterolemia, also called high cholesterol, is a form of hyperlipidemia, which may be a consequence of diet, obesity or diabetes. In addition, increased levels of low-density lipoprotein (LDL) and reduced levels of high-density lipoprotein (HDL) cholesterol are associated with a higher risk of atherosclerosis and coronary heart disease. Thus, controlling cholesterol levels is commonly necessary, and fibrates have been used as lipid-lowering drugs. Gemfibrozil is a fibrate that acts via peroxisome proliferator-activated receptor alpha to promote changes in lipid metabolism and decrease serum triglyceride levels. However, anemia and leukopenia are known side effects of gemfibrozil. Considering that gemfibrozil may lead to anemia and that gemfibrozil acts via peroxisome proliferator-activated receptor alpha, we treated wild-type and peroxisome proliferator-activated receptor alpha-knockout mice with gemfibrozil for four consecutive days. Gemfibrozil treatment led to anemia seven days after the first administration of the drug; we found reduced levels of hemoglobin, as well as red blood cells, white blood cells and a reduced percentage of hematocrits. PPAR-alpha-knockout mice were capable of reversing all of those reduced parameters induced by gemfibrozil treatment. Erythropoietin levels were increased in the serum of gemfibrozil-treated animals, and we also observed an increased expression of hypoxia-inducible factor-2 alpha (HIF-2α) and erythropoietin in renal tissue, while PPAR-alpha knockout mice treated with gemfibrozil did not present increased levels of serum erythropoietin or tissue HIF-2α and erythropoietin mRNA levels in the kidneys. We analyzed bone marrow and found that gemfibrozil reduced erythrocytes and hematopoietic stem cells in wild-type mice but not in PPAR-alpha-knockout mice, while increased colony-forming units were observed only in wild-type mice treated with gemfibrozil. Here, we show for the first time that gemfibrozil treatment leads to anemia and leukopenia via peroxisome proliferator-activated receptor alpha in mice.

Antimicrobial and antileukemic effects: in vitro activity of Calyptranthes grandifolia aqueous leaf extract.

Natural products are still a promising source of bioactive molecules. Food and Drug Administration data showed that approximately 49% of the approved molecules originate naturally or chemically-resemble these substances, of which more than 70% are being used in anticancer therapy. It is noteworthy that at present there are no scientific studies to prove the effectiveness and safety of a number of plants used in folk medicine such as in the case of Calyptranthes grandifolia O. Berg (Myrtaceae) originally from South America. The aim of the present study was to determine the biological potential and toxicological effects of the aqueous leaf extract of C. grandifolia. The main detected phytoconstituents were condensed tannins and flavonoids and a high quantity of polyphenols. Regarding the antimicrobial potential, the extract exerted inhibitory activity against Pseudomonas aeruginosa. The results also revealed the extract induced DNA damage in a concentration-dependent manner in RAW 264.7 cells. In addition, C. grandifolia produced cytotoxicity in leukemia cell lines (HL60 and Kasumi-1) without affecting isolated human lymphocytes but significantly inhibited JAK3 and p38α enzyme activity. Taken together, these findings add important information on the biological and toxicological effects of C. grandifolia, indicating that aqueous extract may be a source of natural antimicrobial and antileukemic constituents.

Involvement of P2 receptors in hematopoiesis and hematopoietic disorders, and as pharmacological targets.

Several reports have shown the presence of P2 receptors in hematopoietic stem cells (HSCs). These receptors are activated by extracellular nucleotides released from different sources. In the hematopoietic niche, the release of purines and pyrimidines in the milieu by lytic and nonlytic mechanisms has been described. The expression of P2 receptors from HSCs until maturity is still intriguing scientists. Several reports have shown the participation of P2 receptors in events associated with modulation of the immune system, but their participation in other physiological processes is under investigation. The presence of P2 receptors in HSCs and their ability to modulate this population have awakened interest in exploring the involvement of P2 receptors in hematopoiesis and their participation in hematopoietic disorders. Among the P2 receptors, the receptor P2X7 is of particular interest, because of its different roles in hematopoietic cells (e.g., infection, inflammation, cell death and survival, leukemias and lymphomas), making the P2X7 receptor a promising pharmacological target. Additionally, the role of P2Y12 receptor in platelet activation has been well-documented and is the main example of the importance of the pharmacological modulation of P2 receptor activity. In this review, we focus on the role of P2 receptors in the hematopoietic system, addressing these receptors as potential pharmacological targets.

Ethanolic Extract of Senna velutina Roots: Chemical Composition, In Vitro and In Vivo Antitumor Effects, and B16F10-Nex2 Melanoma Cell Death Mechanisms.

Cutaneous melanoma is among the most aggressive types of cancer, and its rate of occurrence increases every year. Current pharmacological treatments for melanoma are not completely effective, requiring the identification of new drugs. As an alternative, plant-derived natural compounds are described as promising sources of new anticancer drugs. In this context, the objectives of this study were to identify the chemical composition of the ethanolic extract of Senna velutina roots (ESVR), to assess its in vitro and in vivo antitumor effects on melanoma cells, and to characterize its mechanisms of action. For these purposes, the chemical constituents were identified by liquid chromatography coupled to high-resolution mass spectrometry. The in vitro activity of the extract was assessed in the B16F10-Nex2 melanoma cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and based on the apoptotic cell count; DNA fragmentation; necrostatin-1 inhibition; intracellular calcium, pan-caspase, and caspase-3 activation; reactive oxygen species (ROS) levels; and cell cycle arrest. The in vivo activity of the extract was assessed in models of tumor volume progression and pulmonary nodule formation in C57Bl/6 mice. The chemical composition results showed that ESVR contains flavonoid derivatives of the catechin, anthraquinone, and piceatannol groups. The extract reduced B16F10-Nex2 cell viability and promoted apoptotic cell death as well as caspase-3 activation, with increased intracellular calcium and ROS levels as well as cell cycle arrest at the sub-G0/G1 phase. In vivo, the tumor volume progression and pulmonary metastasis of ESVR-treated mice decreased over 50%. Combined, these results show that ESVR had in vitro and in vivo antitumor effects, predominantly by apoptosis, thus demonstrating its potential as a therapeutic agent in the treatment of melanoma and other types of cancer.

Could a plant derived protein potentiate the anticancer effects of a stem cell in brain cancer?

Glioblastoma is the most aggressive brain tumor with poor overall survival bellow 2 years. The natural compounds with anti-cancer properties, are thus gaining attention for possible adjuvant GBM treatment. In various cancer models Enterolobium contortisiliquum Trypsin Inhibitor (EcTI) proved to have anti-cancer effects. Here, we investigated the EcTI effects on GBM U87 cells and on mesenchymal stem cells (MSC) compared to their direct coculture (MSC/U87). MSC are present in tumor stroma, modulating GBM cells phenotype, and also represent potential drug delivery vehicle due to their tumor tropism. We showed that in p53-wild type U87 cells, metabolic activity was less affected by EcTI as in MSC monocuture, but the metabolic rate of mixed coculture was significantly reduced at lower EcTI concentration. Under coculture condition, EcTI potentiated MSC induced cell cycle arrest, possible due to highly increased p53, p21 and lower D1 expression, but there was no effect on apoptosis. Accordingly, in the coculture EcTI also enhanced Ca2+ signalling mediated via bradykinin receptor 2, being associated with nitric oxide release that highly impaired proliferation and invasion. The mechanism did not seem to involve changes in cell adhesion but rather it down-regulated the ß1 integrin signaling with associated p-FAK in U87 cells, both supporting inhibition of invasion. Finally, some cytokines were down-regulated, indicating that EcTI inhibition of signalling might be mediated by cytokines. In conclusion, these results indicate that in cocultured MSC/U87 cells EcTI impairs the metabolic activity, proliferation, and reduced invasion, possibly associated with observed cytokines secretion. In this context, we confirmed that the plant derived protein potentiated the anticancer effects, induced by MSC, as represented by GBM U87 cell line.

Antioxidant and cytotoxic activity of propolis of Plebeia droryana and Apis mellifera (Hymenoptera, Apidae) from the Brazilian Cerrado biome.

Propolis is a complex bioactive mixture produced by bees, known to have different biological activities, especially in countries where there is a rich biodiversity of plant species. The objective of this study was to determine the chemical composition and evaluate the antioxidant and cytotoxic properties of Brazilian propolis from the species Plebeia droryana and Apis mellifera found in Mato Grosso do Sul, Brazil. In the ethanolic extracts of P. droryana propolis (ExEP-P) and A. mellifera (ExEP-A) acids, phenolic compounds, terpenes and tocopherol were identified as major compounds. Both extracts presented antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, the maximum activities being 500 µg/mL (ExEP-P) and 300 µg/mL (ExEP-A). However, only ExEP-A was able to inhibit lipid peroxidation induced by the oxidizing agent 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which inhibited oxidative hemolysis and reduced the levels of malondialdehyde (MDA) in human erythrocytes for 4 h of incubation. The extracts also reduced the cell viability of the K562 erythroleukemia tumour line, with a predominance of necrotic death. Thus, it is concluded that the propolis produced by P. droryana and A. mellifera contain important compounds capable of minimizing the action of oxidizing substances in the organism and reducing the viability of erythroleukemia cells.