In vitro cytotoxicity of Selol-loaded magnetic nanocapsules against neoplastic cell lines under AC magnetic field activation

The goals of this study are to evaluate in vitro compatibility of magnetic nanomaterials and their therapeutic potential against cancer cells. Highly stable ionic magnetic fluid sample (maghemite, gamma-Fe2O3) and Selol were incorporated into polymeric nanocapsules by nanoprecipitation method. The cytotoxic effect of Selol-loaded magnetic nanocapsules was assessed on murine melanoma (B16-F10) and oral squamous cell carcinoma (OSCC) cell lines following AC magnetic field application. The influence of different nanocapsules on cell viability was investigated by colorimetric MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. In the absence of AC magnetic field Selol-loaded magnetic nanocapsules, containing 100 mu g/mL Selol plus 5 x 10(12) particle/mL, showed antitumoral activity of about 50% on B16-F10 melanoma cells while OSCC carcinoma cells demonstrated drug resistance at all concentrations of Selol and magnetic fluid (range of 100-500 mu g/mL Selol and 5 x 10(12) -2.5 x 10(13) particle/mL). On the other hand, under AC applied fields (1 MHz and 40 Oe amplitude) B16-F10 cell viability was reduced down to 40.5% (+/- 3.33) at the highest concentration of nanoencapsulated Selol. The major effect, however, was observed on OSCC cells since the cell viability drops down to about 33.3% (+/- 0.38) under application of AC magnetic field. These findings clearly indicate that the Selol-loaded magnetic nanocapsules present different toxic effects on neoplastic cell lines. Further, the cytotoxic effect was maximized under AC magnetic field application on OSCC, which emphasizes the effectiveness of the magnetohyperthermia approach. (C) 2012 American Institute of Physics. [doi: 10.1063/1.3680541]

The In Vitro and In Vivo Antitumour Activities of Nitrosyl Ruthenium Amine Complexes

Ruthenium compounds of the type trans-[Ru(NO)(NH3)(4)(L)] X-3, L = N-heterocyclic ligands, P(OEt)(3), SO32-, X BF4- or PF6-, or [Ru(NO)Hedta], were tested for antitumour activity in vitro against murine melanoma and human tumour cells. The ruthenium complexes induced DNA fragmentation and morphological alterations suggestive of necrotic tumour cell death. The calculated IC50 values were lower than 100 mu M. Complexes for which L = isn or imN were partially effective in vivo in a syngeneic model of murine melanoma B16F10, increasing animal survival. In addition, the same ruthenium complexes effectively inhibited angiogenesis of HUVEC cells in vitro. The results suggest that these nitrosyl complexes are a promising platform to be explored for the development of novel antitumour agents.

Influence of caffeine on the survival of leaf-cutting ants Atta sexdens rubropilosa and in vitro growth of their mutualistic fungus

BACKGROUND: Leaf-cutting ants collect plant fresh material for the cultivation of their mutualistic fungus. Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) cause great economic losses through their foraging activity, mainly in agriculture. The main control method is the application of granulated toxic baits incorporated with an active ingredient (AI). The present goal is to evaluate the effect of caffeine on in vitro growth of the mutualistic fungus and on the survival of the leaf-cutting ants, aiming to verify the potential toxicity of this secondary metabolite over these organisms. RESULTS: Three distinct patterns of fungal growth correlated with caffeine concentration were observed: (1) no effect (0.01% caffeine); (2) intermediate growth reduction (0.05% caffeine); (3) drastic growth reduction (0.10 and 0.50% caffeine). The highest caffeine concentration causes fungus death in the first week. As for insect survival, caffeine does not seem to exert any effect. The treatments with diet containing caffeine showed similar values of M50, irrespective of caffeine concentration. CONCLUSION: As caffeine was shown to reduce growth of the mutualistic fungus of Atta sexdens rubropilosa, but with no conclusive effect on insect survival, a hypothetical explanation for the selection of different Coffea species by this leaf-cutting ant species might be associated with caffeine toxicity to the fungus. Copyright (C) 2011 Society of Chemical Industry

Synthesis and characterization of a diruthenium(II,III)-ketoprofen compound and study of the in vitro effects on CRC cells in comparison to the naproxen and ibuprofen derivatives

A new diruthenium(II,III) complex, of formula [Ru2Cl(ket)(4)], Ruket, containing the non-steroidal anti-inflammatory drug ketoprofen was synthesized and mainly characterized by electrospray ionization mass spectrometry (ESI-MS), UV-Vis-IR electronic spectroscopy and FTIR and Raman vibrational spectroscopies. The four drug-carboxylato bridging ligands stabilize a Ru-2(II,III) mixed valent core in a paddlewheel type structure as confirmed by ESI mass spectra, electronic and vibrational spectroscopies and magnetic measurements. Ruket and the analogous compounds containing ibuprofen, Ruibp, and naproxen, Runpx, were tested for the biological effects in the human colon carcinoma cells HT-29 and Caco-2 expressing high and low levels of COX-2 respectively. All compounds only weakly affected the proliferation of the colorectal cancer cells HT-29 and Caco-2, and similarly only partially inhibited the production/activity of MMP-2 and MMP-9 by HT-29 cells, suggesting that COX-2 inhibition by these drugs can only partially be involved in the pharmacological effects of these derivatives. (c) 2012 Elsevier Ltd. All rights reserved.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 degrees C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K-m = 37.53 mM S-1), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K-i = 0.196 nM), indicating that this protease is a potential target for pest control. (C) 2011 Elsevier Ltd. All rights reserved.

Identification of archaeal proteins that affect the exosome function in vitro

Abstract Background The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Abstract Background Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods Eosinophils were purified using a percoll gradient followed by immunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion.

Two series of new semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and mechanism of action

Background The discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation. Results Among the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC6(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of β-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series. Conclusions Three new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.

Formation and stability of oil-in-water nanoemulsions containing rice bran oil: in vitro and in vivo assessments

Abstract Background Nanoemulsions have practical application in a multitude of commercial areas, such as the chemical, pharmaceutical and cosmetic industries. Cosmetic industries use rice bran oil in sunscreen formulations, anti ageing products and in treatments for skin diseases. The aim of this study was to create rice bran oil nanoemulsions using low energy emulsification methods and to evaluate their physical stability, irritation potential and moisturising activity on volunteers with normal and diseased skin types. Results The nanoemulsion developed by this phase diagram method was composed of 10% rice bran oil, 10% surfactants sorbitan oleate/PEG-30 castor oil, 0.05% antioxidant and 0.50% preservatives formulated in distilled water. The nanoemulsion was stable over the time course of this study. In vitro assays showed that this formulation has a low irritation potential, and when applied to human skin during in vivo studies, the nanoemulsion improved the skin's moisture and maintained normal skin pH values. Conclusion The results of irritation potential studies and in vivo assessments indicate that this nanoemulsion has potential to be a useful tool to treat skin diseases, such as atopic dermatitis and psoriasis.

In vitro trypanocidal evaluation of pinane derivatives from essential oils of ripe fruits from Schinus terebinthifolius Raddi (Anacardiaceae)

Essential oils of ripe fruits from Schinus terebinthifolius (Anacardiaceae), obtained using a pilot extractor and a Clevenger apparatus were chemically characterized. Due the high amount of (-)- α-pinene in both oils, this monoterpene was tested against the protozoan parasite Trypanosoma cruzi, showing a moderate potential (IC50 63.56 µg/mL) when compared to benznidazole (IC50 43.14 µg/mL). Otherwise, (-)- α-pinene oxide did not showed anti-trypanosomal activity (IC50 > 400 µg/mL) while (-)-pinane showed an IC50 of 56.50 µg/mL. The obtained results indicated that the epoxydation of α-pinene results to the loss of the anti-parasitic activity while its hydrogenation product, contributed slightly to the increased activity.

Effects of in vitro selenium supplementation on blood and milk neutrophils from dairy cows

The study was designed to assess the effects of in vitro selenium addition on intracellular hydrogen peroxide production by neutrophils from the milk and blood of dairy cows. Blood from 10 dairy cows and 20 milk samples from five dairy cows were incubated with 0 mg (control) or 10μM of sodium selenite. Then, milk and blood neutrophils were submitted for evaluation of intracellular hydrogen peroxide production by flow cytometry using 2',7'-dichlorofluorescein diacetate as a probe. The selenium status of the animals was evaluated by determination of the blood glutathione peroxidase activity. The results of the present work showed that in vitro selenium supplementation leads to an enhancement in intracellular hydrogen peroxide production, which indicates an improvement in the bactericidal effects of blood and milk neutrophils even in cows with a selenium-adequate status. Thus, the present study showed that in vitro Se supplementation leads to an enhancement in intracellular hydrogen peroxide production, indicating an improvement in the bactericidal effects of blood and milk neutrophils in cows with Se-adequate status.

In vitro schistosomicidal effects of the essential oil of Tagetes erecta

The in vitro schistosomicidal effects of the essential oil obtained from Tagetes erecta L. Asteraceae, leaves (TE-EO) collected in Brazil against Schistosoma mansoni worms are reported in this paper. The oil caused a significant decrease in the motor activity at 50 µg/mL as minimal concentration after 24 h. This oil also caused death of all the parasites and the separation of coupled pairs into individual male and female at 100 µg/mL after 24 h. The viability of adult worm groups treated with the TE-EO at 100 µg/mL was similar to that of groups treated with praziquantel (positive control). In addition, the oil promoted the inhibition of eggs development at all the tested concentrations. These data indicate that the TE-EO could be considered as a promising source for the development of new schistosomicidal agents.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

The role of interleuchin 6 (IL-6) in the promotion of an aggressive and stem cell-like phenotype in human breast cancer cells and in stem/progenitor cells expanded in vitro as mammospheres

High serum levels of Interleukin-6 (IL-6) correlate with poor outcome in breast cancer patients. However no data are available on the relationship between IL-6 and stem/progenitor cells which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in mammospheres (MS), multi-cellular structures enriched in stem/progenitor cells of the mammary gland, and also in MCF-7 breast cancer cells. We show that MS from node invasive breast carcinoma tissues express IL-6 mRNA at higher levels than MS from matched non-neoplastic mammary glands. We find that IL-6 mRNA is detectable only in basal-like breast carcinoma tissues, an aggressive variant showing stem cell features. Our results reveal that IL-6 triggers a Notch-3-dependent up-regulation of the Notch ligand Jagged-1, whose interaction with Notch-3 promotes the growth of MS and MCF-7 derived spheroids. Moreover, IL-6 induces a Notch-3-dependent up-regulation of the carbonic anhydrase IX gene, which promotes a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, an autocrine IL-6 loop relies upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, our data support the hypothesis that IL-6 induces malignant features in Notch-3 expressing, stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

In vivo- und In vitro-DNA-Verpackung in virusähnliche Partikel des Humanen Papillomvirus Typ 33

Die Rolle der DNA-Bindungsdomäne der Kapsidproteine L1 und L2 humaner Papillomviren (HPV) wird bezüglich der in vitro DNA-Verpackung kontrovers diskutiert und ist für die in vivo DNA-Verpackung noch ungeklärt. Ich konnte zeigen, dass die L1 Proteine der HPV Typen 16, 18 und 33 DNA binden, nicht aber das HPV33 L2 Protein. Die DNA-Bindungsdomäne habe ich auf die letzten sieben Aminosäuren des Carboxyterminus eingegrenzt. In Funktionsanalysen zeigte ich, dass die DNA-Bindungsdomäne des L1 Proteins für den Einschluss von Markerplasmid DNA in Kapside in einem in vivo Ansatz essentiell ist, nicht aber für eine in vitro DNA-Verpackung. Das L2 Protein, das in Kapside eingebaut wurde, denen die L1 DNA-Bindungsdomäne fehlte, konnte die DNA-Verpackung nicht aufrechterhalten.Zusätzlich habe ich die Infektiösität in vitro und in vivo hergestellter DNA-haltiger Kapside (Pseudovirionen) verglichen. Dabei konnte ich zeigen, dass in vivo gewonnene Pseudovirionen, die DNA in Form von Chromatin enthalten, bis zu fünffach infektiöser sind als Pseudovirionen, die in vitro hergestellt wurden und histonfreie DNA enthalten. Biochemische und strukturelle Unterschiede konnten zwischen den zwei Arten von Pseudovirionen nicht festgestellt werden. Chromatin scheint demzufolge die Infektiösität der Pseudovirionen zu verstärken.