Bovine milk lactoferrin selectively kills highly metastatic prostate cancer PC-3 and osteosarcoma MG-63 cells in vitro

Prostate cancer and osteosarcoma are the second most common type of cancer affecting men and the fifth most common malignancy among adolescents, respectively. The use of non-toxic natural or natural-derived products has been one of the current strategies for cancer therapy, owing to the reduced risks of induced-chemoresistance development and absence of secondary effects. In this perspective, lactoferrin (Lf), a natural protein derived from milk, emerges as a promising anticancer agent due to its well-recognized cytotoxicity and anti-metastatic activity. Here, we aimed to ascertain the potential activity of bovine Lf (bLf) against highly metastatic cancer cells. The bLf effect on prostate PC-3 and osteosarcoma MG-63 cell lines, both displaying plasmalemmal V-ATPase, was studied and compared with the breast cancer MDA-MB-231 and the non-tumorigenic BJ-5ta cell lines. Cell proliferation, cell death, intracellular pH, lysosomal acidification and extracellular acidification rate were evaluated. Results show that bLf inhibits proliferation, induces apoptosis, intracellular acidification and perturbs lysosomal acidification only in highly metastatic cancer cell lines. In contrast, BJ-5ta cells are insensitive to bLf. Overall, our results establish a common mechanism of action of bLf against highly metastatic cancer cells exhibiting plasmalemmal V-ATPase. This study opens promising perspectives for further research on the anticancer role of Lf, which ultimately will contribute to its safer and more rational application in the human therapy of these life-threatening cancers.This study was supported by national funds through Fundação para a Ciência e Tecnologia (FCT) under the scope of the projects: UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), UID/ BIO/04469/2013 (POCI-01-0145-FEDER-006684), FCT-ANR/ BEX-BCM/0175/2012, PEstOE/BIA/UI4050/2014, RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and PTDC/ SAU-BMA/121028/2010.info:eu-repo/semantics/publishedVersio

Deciphering the interaction of lactoferrin with V-ATPase towardsa deeper understanding of its mechanisms of action

Lactoferrin (Lf), a bioactive milk protein, exhibits strong anticancer and antifungal activities. The search for Lf targets and mechanisms of action is of utmost importance to enhance its effective applications. A common feature among Lf-treated cancer and fungal cells is the inhibition of a proton pump called V-ATPase. Lf-driven V-ATPase inhibition leads to cytosolic acidification, ultimately causing cell death of cancer and fungal cells. Given that a detailed elucidation of how Lf and V-ATPase interact is still missing, in this work we aimed to fill this gap by employing a multilevel computational approach. Molecular dynamics (MD) simulations of both proteins were performed to obtain a robust sampling of their conformational landscape, followed by clustering and protein-protein docking. Subsequently, MD simulations of the docked complexes and free binding energy calculations were carried out to evaluate the dynamic binding process and build the final ranking. This computational pipeline allowed the unraveling of the putative mechanism by which Lf inhibits V-ATPase and the identification of key binding residues that will certainly aid in the rational design of follow-up experimental studies, bridging in this way computational and experimental biochemistry.info:eu-repo/semantics/publishedVersio

Selection of a new peptide homing SK-BR-3 breast cancer cells

Breast cancer diagnosis remains a challenge, mostly due to its heterogeneity. This reality translates in delayed treatments, increasing treatment aggressiveness and lower chances of overall survival. The conventional detection techniques, although becoming increasingly sophisticated each year, still lack the ability to provide reliable conclusions without being time consuming, expensive and uncomfortable for the patients. The identification of novel biomarkers for breast cancer research is therefore of utmost relevance for an early diagnosis. Moreover, breast cancer specific peptide moieties can be used to develop novel targeted drug delivery systems. In this work we used phage display to identify a novel peptide with specificity to the SK-BR-3 breast cancer cell line. Cytometry assays confirmed its specificity, while bioinformatics and docking studies predicted the potential biomarkers at the SK-BR-3 cells surface. These findings can be potentially useful in the clinical context, contributing to more specific and targeted therapeutic solutions against HER2-positive breast cancer subtypes.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Débora Ferreira and Ana Cláudia Pereira are recipient of fellowships supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral programme in Applied and Environmental Microbiology (DP_AEM). The authors also acknowledge César Pimenta from NOVA Institute of Chemical and Biological Technology António Xavier (NOVA ITQB) for the docking insights.info:eu-repo/semantics/publishedVersio

Synthesis and cytotoxicity assessment of citrate-coated calcium and manganese ferrite nanoparticles for magnetic hyperthermia

Calcium-doped manganese ferrite nanoparticles (NPs) are gaining special interest in the biomedical field due to their lower cytotoxicity compared with other ferrites, and the fact that they have improved magnetic properties. Magnetic hyperthermia (MH) is an alternative cancer treatment, in which magnetic nanoparticles promote local heating that can lead to the apoptosis of cancer cells. In this work, manganese/calcium ferrite NPs coated with citrate (CaxMn1−xFe2O4 (x=0, 0.2, 1), were synthesized by the sol-gel method, followed by calcination, and then characterized regarding their crystalline structure (by X-ray diffraction, XRD), size and shape (by Transmission Electron Microscopy, TEM), hydrodynamic size and zeta potential (by Dynamic Light Scattering, DLS), and heating efficiency (measuring the Specific Absorption Rate, SAR, and Intrinsic Loss Power, ILP) under an alternating magnetic field. The obtained NPs showed a particle size within the range of 10 nm to 20 nm (by TEM) with a spherical or cubic shape. Ca0.2Mn0.8Fe2O4 NPs exhibited the highest SAR value of 36.3 W/g at the lowest field frequency tested, and achieved a temperature variation of ~7 °C in 120 s, meaning that these NPs are suitable magnetic hyperthermia agents. In vitro cellular internalization and cytotoxicity experiments, performed using the human cell line HEK 293T, confirmed cytocompatibility over 0–250 µg/mL range and successful internalization after 24 h. Based on these studies, our data suggest that these manganese-calcium ferrite NPs have potential for MH application and further use in in vivo systems.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of CEB (UIDB/04469/2020), CBMA (UIDB/04050/2020) and CF-UM-UP (UIDB/04650/2020) units. R.G.D. Andrade and S.R.S. Veloso acknowledge FCT for the PhD grants 2020.05781.BD and SFRH/BD/144017/2019, respectively.info:eu-repo/semantics/publishedVersio

How can a milk protein selectively kill cancer cells? Mechanisms underlying lactoferrin-induced apoptosis

Lactoferrin (Lf) is an iron-binding protein abundant in milk that has been shown to exhibit anticancer activity. Since Lf is non-toxic to cancer cells (1) and is well tolerated in humans (2), this protein has a huge potential to be used in cancer therapy. However, the targets and mechanisms underlying its selective anticancer activity are poorly elucidated, which limits its clinical exploitation. The recruitment of the proton pump V-ATPase to the plasma membrane, where it mediates the acidification of the tumor microenvironment, is a recognized feature involved in the acquisition of a metastatic phenotype in different cancers, including breast cancer. Therefore, inhibitors of this pump have emerged as promising anticancer drugs. Here we show that bovine lactoferrin (bLf) preferentially inhibits cell proliferation and induces apoptosis in two highly metastatic breast cancer cell lines, which display a prominent localization of V-ATPase at the plasma membrane, but not in a lowly metastatic or a non-tumorigenic cell lines (3). We then characterized the mechanism underlying bLf-induced apoptosis and demonstrated that bLf selective cytotoxicity is caused by the inhibition of extracellular acidification rate and the ensuing intracellular acidification in the highly metastatic breast cancer cells. Accordingly, bLf, like the well-known proton pump inhibitors concanamycin A and bafilomycin A1, inhibits V-ATPase proton pumping and hydrolytic activities in sub-cellular fractions enriched in this proton pump. We recently also demonstrated that bLf preferentially induces apoptosis in other types of highly metastatic cancer cells other than breast (4). Altogether, our data demonstrated for the first time that bLf acts as a V-ATPase inhibitor and established a common mechanism of action of bLf against highly metastatic cancer cell exhibiting this proton pump at the plasma membrane. This study opens promising perspectives for the safer and more rational application of bLf in the therapy of these life-threatening cancers. 1. Gibbons JA et al. (2015) BMC Cancer doi: 10.1186/s12885-015-1441-4. 2. Hayes TG et al. (2010) Invest New Drugs doi: 10.1007/s10637-009-9233-9. 3. Pereira CS et al. (2016) Oncotarget doi: 10.18632/oncotarget.11394. 4. Guedes JP et al. (2018) Frontiers in Oncology doi.org/10.3389/fonc.2018.00200info:eu-repo/semantics/publishedVersio

Insights on the molecular mechanisms underlying the anticancer activity of lactoferrin in metastatic cancer cell lines

Lactoferrin (Lf) is an iron-binding protein derived from milk that is present in many tissues and biological fluids. It has been shown that this natural compound exihibits anticancer and anti-metastatic activities as well as cytotoxicity against several cancer cell lines. We have recently found that bovine lactoferrin (bLf) selectively triggers cell death in highly metastatic breast cancer cells through inhibition of the plasmalemmal proton pump V-H+-ATPase.1 In the present study we aim to determine whether this mechanism of action underlies the anticancer/anti-metastatic activity of bLf in metastatic cancer cells other than breast cancer cells. To this end, we assessed bLf-induced inhibition of cell proliferation and intracellular acidification of a prostate and an osteosarcoma metastatic cell line and compared it with the effects on the previously used metastatic breast cancer cell line. The possibility of a common molecular target/mechanism of action of bLf underpinning its anticancer/anti-metastatic activity will be discussed

Modulation of butyrate-degrading methanogenic communities by conductive materials

Butyrate is a volatile fatty acid commonly present in anaerobic bioreactors. Previous research showed that methane production (MP) rates from butyrate, by lake sediment microbiomes, doubled by addition of carbon nanotubes, which was accompanied by changes in the microbial community composition, with enrichment of typical fatty-acid degrading bacteria (Syntrophomonas spp.), well known to exchange electrons with methanogens via hydrogen or formate formation1. But the authors suggested that electrons exchange via conductive materials (CM) may take place instead. In our study, anaerobic butyrate-degrading enrichment cultures were developed with other CM, namely activated carbon (AC) and magnetite (Mag) at 0.5 g/L. MP started earlier in AC enrichment and complete degradation was achieved faster in Mag enrichment. Syntrophomonas spp. were enriched in all cultures (representing 60 to 80 % of the total bacterial community), but hydrogenotrophic methanogens were highly stimulated by AC (78 % of Methanomicrobiales), while the methanogenic community of Mag culture was more diverse in acetoclastic methanogens (43% of Methanosarcina and Methanosaeta). It is still unclear if the improvement on butyrate degradation is associated to the role of CM in interspecies electron transfer, but it is undoubtful that they differentially modulate the methanogenic communities towards faster MP.info:eu-repo/semantics/publishedVersio

Efetividade do serviço móvel de urgência (Samu): uso de séries temporais interrompidas

OBJETIVO: Avaliar o desempenho do serviço de atendimento móvel de urgência (Samu) na região do Grande ABC, utilizando como condição traçadora o infarto agudo do miocárdio. MÉTODOS: A análise de séries temporais interrompidas foi a abordagem de escolha para testar efeitos imediatos e graduais da intervenção na população de estudo. A pesquisa compreendeu séries temporais mensais ajustadas da taxa de mortalidade hospitalar por infarto agudo do miocárdio no período entre 2000 e 2011. Os dados foram extraídos do Sistema de Informações sobre Mortalidade, usando a análise de regressão segmentada para avaliar o nível e tendência da intervenção antes e após sua implementação. Para fortalecer a validade interna do estudo, foi incluída uma região controle. RESULTADOS: A análise de séries temporais interrompidas mostrou redução de 0,04 mortes por 100.000 habitantes na taxa de mortalidade em relação à tendência subjacente desde a implantação do serviço de atendimento médico de urgência (p = 0,0040; IC95% -0,0816 – -0,0162) e uma redução no nível de 2,89 mortes por 100.000 habitantes (p = 0,0001; IC95% -4,3293 – -1,4623), ambos com significância estatística. Em relação à região controle, a Baixada Santista, a diferença da tendência do resultado entre desfecho de intervenção e controle pós-intervenção de -0,0639 mortes por 100.000 habitantes mostrou-se estatisticamente significativa (p = 0,0031; IC95% -0,1060 – -0,0219). Não podemos excluir confundimentos, mas limitamos sua presença no estudo incluindo séries de região controle. CONCLUSÕES: Embora a análise de séries temporais interrompidas tenha limitações, essa modelagem pode ser útil para a análise de desempenho de políticas e programas. Apesar de a intervenção estudada não ser uma condição que por si só implica na efetividade, a efetividade não estaria presente sem essa intervenção, que, integrada a outras condições, gera um resultado positivo. O Samu é uma estratégia cuja expansão precisa ser levada em consideração ao formular e consolidar políticas com foco nas urgências e emergências.OBJECTIVE: To evaluate the performance of the Mobile Emergency Medical Services (SAMU) in the ABC Region, using myocardial infarction as tracer condition. METHODS: The analysis of interrupted time series was the approach chosen to test immediate and gradual effects of the intervention on the study population. The research comprised adjusted monthly time series of the hospital mortality rate by myocardial infarction in the period between 2000 and 2011. Data were extracted from the Mortality Information System (SIM), using segmented regression analysis to evaluate the level and trend of the intervention before and after its implementation. To strengthen the internal validity of the study, a control region was included. RESULTS: The analysis of interrupted time series showed a reduction of 0.04 deaths per 100,000 inhabitants in the mortality rate compared to the underlying trend since the implementation of the Emergency Medical Services (p = 0.0040; 95%CI: −0.0816 – −0.0162) and a reduction in the level of 2.89 deaths per 100,000 inhabitants (p = 0.0001; 95%CI: −4.3293 – −1.4623), both with statistical significance. Regarding the control region, Baixada Santista, the difference in the result trend between intervention outcome and post-intervention control of −0.0639 deaths per 100,000 inhabitants was statistically significant (p = 0.0031; 95%CI: −0.1060 – −0.0219). We cannot exclude confounders, but we limited their presence in the study by including control region series. CONCLUSIONS: Although the analysis of interrupted time series has limitations, this modeling can be useful for analyzing the performance of policies and programs. Even though the intervention studied is not a condition that in itself implies effectiveness, the latter would not be present without the former, which, integrated with other conditions, generates a positive result. SAMU is a strategy that must be expanded when formulating and consolidating policies focusing on emergency care

Meglumine antimoniate and miltefosine combined with allopurinol sustain pro-inflammatory immune environments during canine leishmaniosis treatment

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic disease of global concern. Antileishmanial drug therapies commonly used to treat sick dogs improve their clinical condition, although when discontinued relapses can occur. Thus, the current study aims to evaluate the effect of CanL treatments in peripheral blood, lymph node, and bone marrow cytokine profile associated with clinical recovery. Two groups of six dogs diagnosed with CanL were treated with miltefosine combined with allopurinol and meglumine antimoniate combined with allopurinol (MT+A and MG+A), respectively. At diagnosis and after treatment, during a 3-month follow-up, clinical signs, hematological and biochemical parameters, urinalysis results and antileishmanial antibody titers were registered. Furthermore, peripheral blood, popliteal lymph node, and bone marrow samples were collected to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β, and IFN-γ by qPCR. In parallel, were also evaluated samples obtained from five healthy dogs. Both treatment protocols promoted the remission of clinical signs as well as normalization of hematological and biochemical parameters and urinalysis values. Antileishmanial antibodies returned to non-significant titers in all dogs. Sick dogs showed a generalized upregulation of IFN-γ and downregulation of IL-2, IL-4, and TGF-β, while gene expression of IL-12, TNF-α, IL-5, and IL-10 varied between groups and according to evaluated tissue. A trend to the normalization of cytokine gene expression was induced by both miltefosine and meglumine antimoniate combined therapies. However, IFN-γ gene expression was still up-regulated in the three evaluated tissues. Furthermore, the effect of treatment in the gene expression of cytokines that were not significantly changed by infection, indicates that miltefosine and meglumine antimoniate combined therapy directly affects cytokine generation. Both combined therapies are effective in CanL treatment, leading to sustained pro-inflammatory immune environments that can compromise parasite survival and favor dogs' clinical cure. In the current study, anti-inflammatory and regulatory cytokines do not seem to play a prominent role in CanL or during clinical recovery.publishersversionpublishe

Image processing tool for the detection and quantification of xylanase activity in a metagenomic study

The vast diversity of unexplored microbial communities inhabiting the planet drives the continuous screening for promising biocatalysts. Until recently, the strategies to find new microorganisms and their enzymes were mainly focused on laboratory studies of pure microbial cultures. However, a great amount of environmental microorganisms cannot be cultivated under laboratorial conditions [1]. Metagenomics has emerged as an innovative approach to explore these uncultivable microorganisms through the analysis of DNA extracted from environmental samples [2]. It is considered a powerful tool for the discovery of novel biocatalysts and two different approaches have been proposed. Sequence-based studies recognize candidate genes but do not provide direct conclusions about the functionality of the encoded enzymes. On the other hand, the function-based approach allows the identification of new enzymes and also leads to preliminary information about their activities and physicochemical parameters. Indeed, function-based screenings have been successfully used in different environments to find genes encoding lignocellulose-degrading enzymes, such as xylanases [2]. These enzymes are considered important catalysts in the biological decomposition of lignocellulosic residues. In this study, a fosmid library previously prepared in Escherichia coli with genomic DNA extracted from a compost sample collected in a national composting unit (Lipor) [3] was evaluated through a functional screening. To assess the xylanase activity of all the clones, a fast and simple chromogenic screening test using AZCL-xylan was performed in 96-well microplates at room temperature. Afterwards, the positive clones were selected and incubated at different temperatures (25, 37, 45 and 60 ºC) with the same substrate in Petri plates, for three days, to identify the most fast and promising clones. The presence of blue color was assumed as positive responses correlated with areas size. Area boundaries were extracted automatically by analyzing color images of the samples using MATLABs in-house functions. At 60 ºC, no positive clones were detected. Two positive clones simultaneously exhibited enzymatic activity under 25, 37 and 45 ºC. In general, 37 ºC proved to be the most suitable temperature for the detection of xylanase activity. The method herein reported can be further optimized for the automatic detection of different enzymatic activities in high throughput screenings.The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project LIGNOZYMES-Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01-0145-FEDER-029773). J.S. and A.M.A.C. acknowledge their research grants UMINHO/BIM/2020/28 and UMINHO/BPD/37/2018, respectively, under the scope of the project LIGNOZYMES. The authors also acknowledge the Portuguese composting unit Lipor for kindly supplying the compost sample.info:eu-repo/semantics/publishedVersio