Cytotoxicity of frutalin on distinct cancer cells is independent of its glycosylation

Frutalin is a plant lectin with beneficial immunobiological action, although the access to its active form is still restricted. Moreover, there is a knowledge gap on isoform activity and glycosylation impact on its bioactivity, and recombinant production protocols were seen as ineffective. Here, a simpler and faster production and purification protocol was developed, attaining a yield of purified frutalin 3.3-fold higher than that obtained previously. Hemagglutination assays confirmed that this frutalin isoform could not agglutinate rabbit erythrocytes, while maintaining the native tetrameric structure, as indicated by DLS analysis, and strong interaction with methyl-alpha-galactose, in fluorescence spectroscopy studies. The cytotoxicity of the recombinant frutalin isoform was shown in a broad panel of human cancer cells: colon (HCT116), melanoma (A375), triple-negative breast cancer (MDA-MB-231), and ovarian (IGROV-1). Treatment with 8.511.8 M TrxFTL reduced proliferation of all cancer cells to half in 48 h. This anti-proliferative effect encompasses the p53 pathway since it was significantly reduced in p53-null colon cancer cells (HCT116 p53/; GI50 of 25.0 ± 3.0 M), when compared to the isogenic p53-positive cells (HCT116 p53+/+; GI50 of 8.7 ± 1.8 M; p < 0.002). This recombinantly produced frutalin isoform has relevant cytotoxic effect and its biological activity is not dependent on glycosylation. The developed E. coli production and purification protocol generates high yield of non-glycosylated frutalin isoform with potent cytotoxic activity, enabling the development of novel anticancer p53-targeting therapies.This research was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 and UIDB/50006/2020 (LAQV/REQUIMTE).info:eu-repo/semantics/publishedVersio

Detecção de Yersinia enterocolitica em carne de porco picada - Comparação de três metodologias, com vários tempos de enriquecimento da amostra

Yersinia enterocolitica é um agente patogénico para o Homem, causa importante de zoonose alimentar, responsável por uma grande variedade de sintomatologia intestinal e extra-intestinal. Os suínos são os principais reservatórios da doença, sendo a carne de suíno considerada fonte de infecção. Nas últimas décadas tem-se verificado a emergência de casos de yersiniose, em parte devido à capacidade deste microrganismo para se multiplicar a temperaturas de refrigeração. Com efeito, a prosperidade desta bactéria seguiu o desenvolvimento da globalização da cadeia de frio, assumindo a designação de perigo emergente. Neste trabalho, compararam-se três metodologias: uma que utiliza a técnica BDC (buoyant density centrifugation) como pré-tratamento da amostra, e análise PCR; outra que combina a cultura celular em placa de meio CIN com a lavagem para remoção da biomassa viável, extracção do DNA e análise PCR; e uma metodologia convencional, segundo a ISO 10273:2003. Paralelamente, procurou-se encontrar o ponto ideal no tempo de enriquecimento da amostra, que permitisse obter os melhores resultados na detecção do microrganismo. A sensibilidade dos métodos foi avaliada com amostras de carne de porco picada crua, artificialmente contaminadas com níveis baixos – 3,3 log UFC/g, 2,3 log UFC/g e 1,3 log UFC/g - de uma estirpe de referência de Yersinia enterocolitica. Ametodologia de lavagem da placa CIN foi aquela que permitiu uma melhor detecção de Yersinia enterocolitica quando em baixa concentração, com o menor tempo de enriquecimento da amostra. Os resultados reafirmam a importância das metodologias moleculares ao permitirem uma detecção precoce e para níveis de contaminação bastante baixo

Detection of Yersinia enterocolitica in raw pork by conventional culture methods and PCR based methods

Yersinia enterocolitica is an emerging pathogenic microorganism associated with food. Its ingestion, through contaminated food, may cause different kinds of intestinal disorders. Since there is not much information about the presence of Y. enterocolitica concerning the consumption of food in Portugal and the conventional methodology is not very effective, this study proceeded, by implementing the PCR methodology, in order to detect the pathogenic microorganism in pork meat. One hundred samples of raw minced meat were acquired in supermarkets and butcher’s shops in the Greater Oporto and Braga area, with the purpose of determining the occurrence of Y. enterocolitica. The detection limit of the conventional method (ISO 10273: 2003) was determined to be 105 CFU/g using CIN culture medium and 104 CFU/g using a pre‐treatment step with KOH, which highlights the difficulties in detecting Yersinia using this methodology. A molecular PCR‐based method was implemented, using BDC followed by cellular alkaline lysis to extract the samples’ DNA (BDC‐PCR‐based method). The primers used in this study were the 16S rRNA gene, which allowed the detection of the genera Yersinia, and the yst gene to detect the pathogenic strains of the microorganism. The detection limit was studied in both sets of primers. The values obtained were 102 CFU/g for the 16S rRNA gene and 103 CFU/g for the yst gene for a pre‐enrichment time of 24 h. Nevertheless, we have implemented a combined culture and PCR method for detection of Yersinia that besides ensuring the viability of the cells detected, showed to be more sensitive than the BDC‐PCR‐based method. All the samples were analysed by the BDC‐PCR‐based method and 25 by the three methodologies. The different methodologies will be discussed and the incidence of Y. enterocolitica in raw minced meat pork evaluated. The results of this study show that the molecular methodology and the combined methodology that were here adopted are more sensitive than the common methodology. Therefore, it can become an important tool in food sample control, since it allows quicker results in a smallest time span. It is also more reliable and easier to work with when compared to other conventional methods

Modulation of bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase calpha

Protein kinase Cα (PKCα) is a classical PKC isoform whose involvement in cell death is not completely understood. Bax, a major member of the Bcl-2 family, is required for apoptotic cell death and regulation of Bax translocation and insertion into the outer mitochondrial membrane is crucial for regulation of the apoptotic process. Here we show that PKCα increases the translocation and insertion of Bax c-myc (an active form of Bax) into the outer membrane of yeast mitochondria. This is associated with an increase in cytochrome c (cyt c) release, reactive oxygen species production (ROS), mitochondrial network fragmentation and cell death. This cell death process is regulated, since it correlates with an increase in autophagy but not with plasma membrane permeabilization. The observed increase in Bax c-myc translocation and insertion by PKCα is not due to Bax c-myc phosphorylation, and the higher cell death observed is independent of the PKCα kinase activity. PKCα may therefore have functions other than its kinase activity that aid in Bax c-myc translocation and insertion into mitochondria. Together, these results give a mechanistic insight on apoptosis regulation by PKCα through regulation of Bax insertion into mitochondria.RDS is supported by the FCT grant SFRH/BD/23774/200

The importance of humanized yeast to better understand the role of Bcl-2 family in apoptosis : finding of novel therapeutic opportunities

The Bcl-2 protein family plays a central role in mitochondrial membrane permeabilization. This event and the ensuing release of cytochrome c are decisive in the apoptotic cascade. Therefore, a better knowledge of these processes and their regulation will probably lead to the development of novel therapeutic strategies for treatment of apoptosis-related diseases. However, the mode of action of Bcl-2 protein family and its regulation are not completely understood. Yeast has proved to be a powerful tool to investigate the molecular aspects of several biological processes, including the steps of the apoptotic cascade involving mitochondria. The fact that yeast does not have obvious homologues of the mammalian Bcl-2 family proteins and that these proteins conserve some of their molecular and biochemical functions when expressed in yeast favours the use of this simpler model system to unravel some of the functions of this family. In this review we attempt to encompass the current knowledge regarding Bcl-2 family mode of action and regulation obtained using the yeast model system. Moreover, we discuss how this model system can be used in the future to gain new understanding about the intricate mechanisms of Bcl-2 family protein regulation, and highlight novel therapeutic targets revealed by this system. We believe that the studies here summarized also provide a proof of principle of yeast as an important tool to elucidate some of the complex mechanisms of apoptotic cell death in higher eukaryotes.R. D. Silva (SFRH/BD/23774/2005) have a fellowship from Fundação para a Ciência e Tecnologia, Portugal. This work was supported by a FCT funded project (PTDC/BIA-BCM/ 69448/2006)

The crystal structure of the R280K mutant of human p53 explains the loss of DNA binding

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.We thank Gilberto Fronza (from Mutagenesi e Prevenzione Oncologica, Ospedale Policlinico San Martino, Genova, Italy), for providing us with the pLS76 vector. We acknowledge the European Synchrotron Radiation Facility for the provision of synchrotron radiation facilities and access to beamline ID30B. This work received financial support from the European Union (FEDER, Fundo Europeu de Desenvolvimento Regional, funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade–COMPETE) and National Funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the Partnership Agreement PT2020 UID/MULTI/04378/2013, and projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014–POCI-01-0145-FEDER-016581 and RECI/BBB-BEP/0124/2012. FCT fellowships: PD/BD/114046/2015 (Ana Sara Gomes) and SFRH/BD/96189/2013 (Sara Gomes) (thanks FCT PhD Doctoral Programme BiotechHealth), and SFRH/BPD/110640/2015 (Carla Oliveira).info:eu-repo/semantics/publishedVersio

Design and synthesis of new inhibitors of p53–MDM2 interaction with a chalcone scaffold

The virtual screening of a library of chalcone derivatives led us to the identification of potential new MDM2 ligands. The chalcones with the best docking scores obeying the Lipinski rule of five were subsequently prepared by base-catalyzed aldol reactions. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast-based screening assay. Using this approach two chalcones (3 and 4) were identified as putative small molecule inhibitors of p53–MDM2 interaction. The activity of both chalcones was further investigated in a panel of human tumor cells. Chalcones 3 and 4 revealed a pronounced tumor cell growth inhibitory effect on tumor cell lines. Additionally, chalcone 4 caused alterations in the cell cycle profile, induced apoptosis and increased the levels of p53, p21 and PUMA proteins in NCI-H460 cells. Computational docking studies allowed to predict that, like nutlin-3A (a well-known small-molecule inhibitor of p53–MDM2 interaction), chalcones 3 and 4 bind to the p53-binding site of MDM2. The results here presented will be valuable for the structure-based design of novel and potent p53–MDM2 inhibitors.This research was partially supported by the Strategic Funding UID/Multi/04423/2013 , ERDF , COMPETE , and FCT under the projects PTDC/MAR-BIO/4694/2014, and INNOVMAR – Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035 , Research Line NOVELMAR . This work also received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013 and the FCT project PTDC/DTP-FTO/1981/2014, “PEst-C/SAU/LA0003/2013”, “NORTE-07-0162-FEDER-00018 – Contributos para o reforço da capacidade do IPATIMUP enquanto actor do sistema regional de inovação” and NORTE-07-0162-FEDER-000067 – Reforço e consolidação da capacidade infraestrutural do IPATIMUP para o sistema regional de inovação”, both supported by ON.2 – O Novo Norte, through FEDER funds under the QREN. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. The authors also thank FCT for the grants of R.T. Lima ( SFRH/BPD/68787/2010 ), J. Soares ( SFRH/BD/78971/2011 ), and S. Gomes ( SFRH/BD/96189/2013 ; Doctoral Programme BiotechHealth), L. Raimundo ( PD/BI/113926/2015 , Doctoral Programme BiotechHealth)

Semi-synthesis of small molecules of aminocarbazoles: tumor growth inhibition and potential impact on p53

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1–3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1–3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.The authors thank to national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF) and COMPETE under the Strategic Funding of CIIMAR UIDB/04423/2020 (Natural Products and Medicinal Chemistry) and LAQV/REQUIMTE (UID/QUI/50006/2020), the project PTDC/SAU-PUB/28736/2017 (reference POCI-01–0145-FEDER028736), PTDC/DTP-FTO/1981/2014-POCI-01-0145-FEDER-016581). We also thank FCT for the fellowship SFRH/BD/128673/2017 (J. Loureiro). Ploenthip Puthongking thanks Thailand Research Fund (DBG6080006), Thailand