Mesenchymal stem cell-conditioned medium accelerates skin wound healing:an in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-beta1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

Computer assisted synthesis and in-vitro cytotoxic evaluation of new pyrazole-fused isoquinolinoquinones derivatives as PI3K receptor antagonist with promising antitumoral activity

The importance of pyrazole and isoquinoline-5,8-dione scaffolds in medical chemistry is underlined by the high number of drugs currently on trading that contains these active ingredients. Due to their cytotoxic capability, the interest of medicinal chemists in these heterocyclic rings has grown exponentially especially, for cancer therapy. In this project, the first synthesis of pyrazole-fused isoquinoline-5,8-diones has been developed. 1,3-Dipolar cycloaddition followed by oxidative aromatization, established by our research group, has been employed. Screening of reaction conditions and characterization studies about the regioselectivity have been successfully performed. A remote control of regioselectivity, to achieve the two possible regioisomers has been accomplished. Through Molecular Docking studies, Structure-Activity relationship of differently substituted scaffolds containing our central core proved that a family of PI3K inhibitors have been discovered. Finally, in order to verify the promising antitumor activity, a first test of cell viability in vitro on T98G cell line of a solid brain tumor, the Glioblastoma Multiforme, showed cytotoxic inhibition comparable to currently trade anticancer drugs.

A comparison of immunohistochemical assays and FISH in detecting the ALK translocation in diagnostic histological and cytological lung tumor material.

Introduction: Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH), but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives. 
Methods: Fifteen FISH-positive cases from patients, seven with data on crizotinib therapy and clinical response, were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3, using the proprietary automated system (Ventana), ALK1 (Dako), and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens, 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity, and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification. 
Results: All three IHC assays were 100% specific but sensitivity did vary: D5F3 86%, ALK 79%, 5A4 71%. Intensity was the most discriminating measure overall, with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene.
Conclusions: IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%), specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative, suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein.

Toxicity of hexanic and methanolic fractions from copaifera reticulata ducke (Leguminosae – Caesalpinoidea) against Aedes aegypti Linnaeus (Diptera – Culicidae), in field assays

v. 45, n. 2, abr./jun. 2016.

Anti-MDR and antitumoral action of acetylsalicylic acid on leukaemic cells

ASA (acetylsalicylic acid) is an NSAID (non-steroidal anti-inflammatory drug). ASA has gained attention as a potential chemopreventive and chemotherapeutic agent for several neoplasms. The aim of this study was to analyse the possible antitumoural effects of ASA in two erythroleukaemic cell lines, with or without the MDR (multidrug resistance) phenotype. The mechanism of action of different concentrations of ASA were compared in K562 (non-MDR) and Lucena (MDR) cells by analysing cell viability, apoptosis and necrosis, intracellular ROS (reactive oxygen species) formation and bcl-2, p53 and cox-2 gene expression. ASA inhibited the cellular proliferation or induced toxicity in K562 and Lucena cell lines, irrespective of the MDR phenotype. The ASA treatment provoked death by apoptosis and necrosis in K562 cells and only by necrosis in Lucena cells. ASA also showed antioxidant activity in both cell lines. The bcl-2, p53 and cox-2 genes in both cell lines treated with ASA seem to exhibit different patterns of expression. However, normal lymphocytes treated with the same ASA concentrations were more resistant than tumoral cells. The results of this work show that both cell lines responded to treatment with ASA, demonstrating a possible antitumoral and anti-MDR role for this drug.

Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Síntese de novas 3,4-diidropirimidin-2(1H)-onas graxas e estudo da atividade antitumoral

Neste trabalho foi realizada a síntese de novas 3,4-diidropirimidinonas funcionalizadas com diferentes cadeias graxas. Para a síntese destes compostos os β-cetoésteres graxos foram obtidos a partir da transesterificação do acetoacetato de metila na presença dos álcoois palmítico (2a), esteárico (2b) e oleico (2c), utilizando como catalisador I2 e ácido sulfâmico (NH3SO3). Os compostos foram obtidos com rendimentos de 97%, 90% e 60%, respectivamente. Após, a reação multicomponente de Biginelli foi realizada na presença de InCl3 utilizando os β- cetoésteres graxos 3a-c, os aldeídos aromáticos, benzaldeído, 3-hidroxi benzaldeído e 4-dimetilaminobenzaldeído, na presença de uréia ou tiouréia, levando às 3,4- diidropirimidinonas graxas 5-substituídas 6-11a-c em rendimentos que variaram entre 60–94%. Todos os compostos, ainda inéditos na literatura, foram caracterizados por espectroscopia de infravermelho (IV) e ressonância magnética nuclear de hidrogênio (RMN ¹H) e carbono (RMN ¹³C). A análise do efeito das novas 3,4-diidropirimidin-2(1H)-onas graxas pelo método do MTT na linhagem de glioma C6 rato e UG-138 humana demonstrou uma maior diminuição da viabilidade celular para os compostos graxos contendo as cadeias palmítica e oleica derivados da tiouréia e do 3-hidroxi benzaldeído, respectivamente. Comportamento semelhante foi observado para os compostos contendo as cadeias palmítica e oleica, porém derivados da uréia. Uma diminuição significativa da viabilidade celular, porém em menor grau, foi observada para os compostos graxos contendo as cadeias palmítica e oleica derivados da uréia e do benzaldeído. Os resultados mostraram até o momento que os derivados graxos contendo a cadeia oleica na posição 5 do anel diidropirimídinico derivado do 3- hidroxi benzaldeído e da tiouréia apresentaram maior atividade na diminuição da viabilidade celular de linhagem de glioma.

Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

Background. It has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a traslational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. Methods. A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry have been used in this study. Results. The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a traslational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5' end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. Conclusions. The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we have demonstrated that TSA in fact, differentially regulates both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumoral drugs that are substrates of Pgp. Finally, we have also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.

Crocin and β-Carotene bleaching assays as analytical tools in the optimization of the extraction of hydrophilic and lipophilic antioxidants from tomato

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop worldwide and a rich source of hydrophilic (H) and lipophilic (L) antioxidants. The H fraction is constituted mainly by ascorbic acid and soluble phenolic compounds, while the L fraction contains carotenoids (mostly lycopene), tocopherols, sterols and lipophilic phenolics [1,2]. To obtain these antioxidants it is necessary to follow appropriate extraction methods and processing conditions. In this regard, this study aimed at determining the optimal extraction conditions for H and L antioxidants from a tomato surplus. A 5-level full factorial design with 4 factors (extraction time (I, 0-20 min), temperature (T, 60-180 •c), ethanol percentage (Et, 0-100%) and solid/liquid ratio (S/L, 5-45 g!L)) was implemented and the response surface methodology used for analysis. Extractions were carried out in a Biotage Initiator Microwave apparatus. The concentration-time response methods of crocin and P-carotene bleaching were applied (using 96-well microplates), since they are suitable in vitro assays to evaluate the antioxidant activity of H and L matrices, respectively [3]. Measurements were carried out at intervals of 3, 5 and 10 min (initiation, propagation and asymptotic phases), during a time frame of 200 min. The parameters Pm (maximum protected substrate) and V m (amount of protected substrate per g of extract) and the so called IC50 were used to quantify the response. The optimum extraction conditions were as follows: r~2.25 min, 7'=149.2 •c, Et=99.1 %and SIL=l5.0 giL for H antioxidants; and t=l5.4 min, 7'=60.0 •c, Et=33.0% and S/L~l5.0 g/L for L antioxidants. The proposed model was validated based on the high values of the adjusted coefficient of determination (R2.wi>0.91) and on the non-siguificant differences between predicted and experimental values. It was also found that the antioxidant capacity of the H fraction was much higher than the L one.

Caracterização estrutural e avaliação das atividades farmacológicas da fucana B extraída da alga Dictyota menstrualis

Seaweeds are a major source of biologically active compounds . In the extracellular matrix of these organisms are sulfated polysaccharides that functions as structural components preventing it against dehydration. The fraction 0.9 (FucB) rich in sulfated fucans obtained from brown seaweed Dictyota menstrualis was chemical characterized and evaluated for pharmacological activity by testing anticoagulant activity, stimulatory action on the synthesis of an antithrombotic heparan sulfate, antioxidant activity and its effects in cell proliferation. The main components were FucB carbohydrates (49.80 ± 0.10 %) and sulfate (42.30 ± 0.015 %), with phenolic compounds ( 3.86 ± 0.016 %) and low protein contamination ( 0.58 ± 0.001 % ) . FucB showed polydisperse profile and analysis of signals in the infrared at 1262, 1074 and 930 cm -1 and 840 assigned to S = O bonds sulfate esters , CO bond presence of 3,6- anhydrogalactose , β -D- galactose non- sulfated sulfate and the axial position of fucose C4 , respectively. FucB exhibited moderate anticoagulant activity , the polysaccharides prolonged time (aPTT ) 200 ug ( > 90s ) partial thromboplastin FucB no effect on prothrombin time (PT), which corresponds to the extrinsic pathway of coagulation was observed. This stimulation promoted fraction of about 3.6 times the synthesis of heparan sulfate (HS) by endothelial cells of the rabbit aorta ( RAEC ) in culture compared with cells not treated with FucB . This has also been shown to compete for the binding site with heparin. The rich fraction sulfated fucans exhibited strong antioxidant activity assays on total antioxidant (109.7 and 89.5 % compared with BHT and ascorbic acid standards ) , reducing power ( 71 % compared to ascorbic acid ) and ferric chelation ( 71 , comparing with 5 % ascorbic acid). The fraction of algae showed cytostatic activity on the RAEC cells revealed that the increase of the synthesis of heparan sulfate is not related to proliferation. FucB showed antiproliferative action on cell lines modified as Hela and Hep G2 by MTT assay . These results suggest that FucB Dictyota menstrualis have anticoagulant , antithrombotic , antioxidant potential as well as a possible antitumor action, promoting the stimulation of the synthesis of antithrombotic HS by endothelial cells and is useful in the prevention of thrombosis, also due to its inhibitory action on species reactive oxygen ( ROS ) in some in vitro systems , being involved in promoting a hypercoagulable state

Potencial antitumoral de cogumelos silvestres do Nordeste Transmontano

Os cogumelos são muito apreciados pelo seu valor nutricional [1] assim como pelo seu potencial medicinal [2,3]. Neste trabalho procedeu-se à caracterização química de uma espécie do Nordeste transmontano, avaliando igualmente o seu potencial antitumoral numa linha celular de cancro de mama (MCF-7). Analisar o perfil químico de uma espécie pouco estudada - Leccinum vulpinum Watling, relativamente ao seu conteúdo em compostos bioativos. O seu potencial antitumoral numa linha celular humana de cancro de mama foi também avaliado.