Deep sequencing analysis reveals distinctive non-coding RNAs when comparing tumor multidrug-resistant cells and extracellular vesicles with drug-sensitive counterparts

This article is a result of the project NORTE-01-0145-FEDER-000029, supported by the Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was partially financed by the FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). This work is also a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). The authors thank the Portuguese Foundation for Science and Technology (FCT) for the PhD grant of DS (SFRH/BD/98054/2013).Multidrug resistance (MDR) is one of the main limitations of cancer treatment. The overexpression of drug-eux pumps, such as P-glycoprotein (P-gp), is a major cause of MDR. Importantly, di erent studies have shown that extracellular vesicles (EVs) participate in the communication between MDR cells and drug-sensitive counterparts, promoting dissemination of the MDR phenotype. In the present work, we aimed to identify RNA species present in MDR cells and in EVs released by those cells, which may be associated with the MDR phenotype. The RNA content from two pairs (leukemia and lung cancer) of MDR (P-gp overexpressing) cells and their drug-sensitive counterparts, as well as from their EVs, was analyzed by deep sequencing. Our results showed distinctive transcripts for MDR cells and their EVs, when compared with their drug-sensitive counterparts. Remarkably, two pseudogenes (a novel pseudogene and RNA 5.8S ribosomal pseudogene 2) were found to be increased in EVs released by MDR cells in both leukemia and lung cancer models. Moreover, six miRs (miR-204-5p, miR-139-5p, miR-29c-5p, miR-551b-3p, miR-29b-2-5p, and miR-204-3p) exhibited altered levels in lung cancer MDR cells and their EVs. This study provides insights into the contribution of EVs to MDR.publishersversionpublishe

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)

Anti-hepatocellular carcinoma activity using human HepG2 cells and hepatotoxicity of 6-substituted methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate derivatives: in vitro evaluation, cell cycle analysis and QSAR studies

Hepatocellular carcinoma (HCC) is a highly complex cancer, resistant to commonly used treatments and new therapeutic agents are urgently needed. A total of thirty-two thieno[3,2-b]pyridine derivatives of two series: methyl 3-amino-6-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates (1a-1t) and methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates (2a-2n), previously prepared by some of us, were evaluated as new potential anti-HCC agents by studying their in vitro cell growth inhibition on human HepG2 cells and hepatotoxicity using a porcine liver primary cell culture (PLP1). The presence of amino groups linked to a benzene moiety emerges as the key element for the anti-HCC activity. The methyl 3-amino-6-[(3-aminophenyl)ethynyl]thieno[3,2-b]pyridine-2-carboxylate (2f) is the most potent compound presenting GI50 values on HepG2 cells of 1.2 μM compared to 2.9 μM of the positive control ellipticine, with no observed hepatotoxicity (PLP1 GI50>125 μM against 3.3 μM of ellipticine). Moreover this compound changes the cell cycle profile of the HepG2 cells, causing a decrease in the % of cells in the S phase and a cell cycle arrest in the G2/M phase. QSAR studies were also performed and the correlations obtained using molecular and 1D descriptors revealed the importance of the presence of amino groups and hydrogen bond donors for anti-HCC activity, and hydrogen bond acceptors for hepatotoxicity. The best correlations were obtained with 3D descriptors belonging to different subcategories for anti-HCC activity and hepatotoxicity, respectively. These results point to different molecular mechanisms of action of the compounds in anti-HCC activity and hepatotoxicity. This work presents some promising thieno[3,2-b]pyridine derivatives for potential use in the therapy of HCC. These compounds can also be used as scaffolds for further synthesis of more potent analogues.FCT, FEDER/COMPETE/QREN/E

Haemocompatibility of iron oxide nanoparticles synthesized for theranostic applications: a high-sensitivity microfluidic tool

The poor heating efficiency of the most reported magnetic nanoparticles (MNPs), allied to the lack of comprehensive biocompatibility and haemodynamic studies, hampers the spread of multifunctional nanoparticles as the next generation of therapeutic bio-agents in medicine. The present work reports the synthesis and characterization, with special focus on biological/toxicological compatibility, of superparamagnetic nanoparticles with diameter around 18 nm, suitable for theranostic applications (i.e. simultaneous diagnosis and therapy of cancer). Envisioning more insights into the complex nanoparticle-red blood cells (RBCs) membrane interaction, the deformability of the human RBCs in contact with magnetic nanoparticles (MNPs) was assessed for the first time with a microfluidic extensional approach, and used as an indicator of haematological disorders in comparison with a conventional haematological test, i.e. the haemolysis analysis. Microfluidic results highlight the potential of this microfluidic tool over traditional haemolysis analysis, by detecting small increments in the rigidity of the blood cells, when traditional haemotoxicology analysis showed no significant alteration (haemolysis rates lower than 2 %). The detected rigidity has been predicted to be due to the wrapping of small MNPs by the bilayer membrane of the RBCs, which is directly related to MNPs size, shape and composition. The proposed microfluidic tool adds a new dimension into the field of nanomedicine, allowing to be applied as a highsensitivity technique capable of bringing a better understanding of the biological impact of nanoparticles developed for clinical applications.This work was financially supported by: Project POCI-01-0145-FEDER-006984 – Associate Laboratory J Nanopart Res (2016) 18:194 Page 15 of 17 194 123 LSRE-LCM funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalizac¸a˜o (POCI) – and by national funds through FCT - Fundac¸a˜o para a Cieˆncia e a Tecnologia. R.O.R. acknowledges the Ph.D. scholarship SFRH/BD/97658/2013 Granted by FCT. A.M.T.S acknowledges the FCT Investigator 2013 Programme (IF/01501/ 2013), with financing from the European Social Fund and the Human Potential Operational Programme. M.B. would like to thank ERDF (European Regional Development Fund) under grant PO Norte CCDR-N/ON.2 Programme. J.G. also thanks the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 600375.info:eu-repo/semantics/publishedVersio

Polymer microfluidic devices: an overview of fabrication methods

The amount of applications associated with microfluidic devices is increasing since the introduction of Lab-on-a-chip devices in the 1990s, especially regarding biomedical and clinical fields. However, in order for this technology to leave the fundamental research and become a day-life technology (e.g., as point-of-care testing), it needs to be disposable and reasonably less expensive. Polymers, due to their several advantages, such as easier microfabrication and low-cost, fill these needs. Several methods are reported regarding microfabrication and, thus, the main aim of the present work is to provide an overview of the most relevant microfabrication techniques found in literature employing polymers, clarifying also the main advantages and disadvantages of each technique and especially considering their cost and time-consumption. Moreover, a future outlook of low-cost microfabrication techniques and standard methods is provided

A methanolic extract of Ganoderma lucidum fruiting body inhibits the growth of a gastric cancer cell line and affects cellular autophagy and cell cycle

Ganoderma lucidum is one of the most extensively studied mushrooms as a functional food and as a chemopreventive agent due to its recognized medicinal properties. Some G. lucidum extracts have shown promising antitumor potential. In this study, the bioactive properties of various extracts of G. lucidum, from both the fruiting body and the spores, were investigated. The most potent extract identified was the methanolic fruiting body extract, which inhibited the growth of a gastric cancer cell line (AGS) by interfering with cellular autophagy and cell cycle.Fundaçao para a Ciencia e a Tecnologia (FCT, Portugal) and COMPETE/QREN/EU for providing financial support to this work (research project PTDC/AGR-ALI/110062/2009), CIMO (strategic project PEst-OE/AGR/UI0690/2011), FCT for the grant of R.T.L. (SFRH/BPD/68787/2010) and QREN for the grants of F.S.R. and D.S. (NORTE-07-0124-FEDER-000023). The authors also thank Dr Gabriela Almeida for technical assistance in the screening assays and Prof. Anabela Martins from the Polytechnic Institute of Bragança for confrmation of the identification of G. lucidum samples. IPATIMUP, Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, is partially supported by FCT

Study of the cell growth inhibitory effect of aqueous extracts from Tuberaria lignosa in human tumor cell lines

Tuberaria lignosa (Sweet) Samp. (“alcária”), a plant mostly found in Western regions of the Iberian Peninsula, has antioxidant properties due to its composition in ascorbic acid and phenolic compounds [1]. Given these antioxidant properties, together with the traditional use of the plant to treat several diseases, the aims of this work were to: i) investigate if aqueous extracts from this plant, obtained by infusion and decoction, were inhibitors of cell growth in three human tumor cell lines; ii) study the cellular mechanism of action of the most potent extract. A screening of tumor cell growth inhibitory potential was performed with the Sulforhodamine B (SRB) assay using three different human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and HCT-15 (colorectal adenocarcinoma). Results showed that both aqueous extracts of T. lignosa decreased the growth of the cell lines tested and that the T. lignosa infusion extract was the most potent one, particularly in the NCI-H460 and HCT-15 cells. The T. lignosa infusion extract was further tested in the NCI-H460 cells. A determination of its effect on cell cycle profile was carried out, by analyzing cellular DNA content by flow cytometry following incubation with propidium iodide. Determination of cellular apoptosis was also performed, with the Annexin V-FICT and propidium iodide assay, and analyzed by flow cytometry. Preliminary results showed that the selected extract promoted a slight increase in the percentage of cells in the G1 phase of the cell cycle and induced cellular apoptosis. In conclusion, the T. lignosa extract decreased growth of the human tumor cell lines tested and the most potent effect was observed for the T. lignosa infusion extract. Future work will confirm if this effect is due mainly to induction of apoptosis

Insights into the in vitro antitumor mechanism of action of a new pyranoxanthone

Naturally occurring xanthones have been docu mented as having antitumor properties, with some of them presently undergoing clinical trials. In an attempt to improve the biological activities of dihydroxyxanthones, prenylation and other mole cular modifications were performed. All the com pounds reduced viable cell number in a leukemia cell line K-562, with the fused xanthone 3, 4-dihydro-12-hydroxy-2,2-dimethyl-2H,6H-pyrano[3, 2-b]xanthen-6-one (5) being the most potent. The pyranoxanthone 5 was particularly effective in additional leukemia cell lines (HL-60 and BV-173). Furthermore, the pyranoxanthone 5 decreased cel lular proliferation and induced an S-phase cell cycle arrest. In vitro, the pyranoxanthone 5 increased the percentage of apoptotic cells which was confirmed by an appropriate response at the protein level (e.g., PARP cleavage). Using a com puter screening strategy based on the structure of several anti- and pro-apoptotic proteins, it was verified that the pyranoxanthone 5 may block the binding of anti-apoptotic Bcl-xL to pro-apoptotic Bad and Bim. The structure-based screening revealed the pyranoxanthone 5 as a new scaffold that may guide the design of small molecules with better affinity profile for Bcl-xL.info:eu-repo/semantics/publishedVersio

Flower extracts of Filipendula ulmaria (L.) Maxim inhibit cell growth of human tumour cell lines

According to the World Health Organization, cancer is the leading cause of death worldwide and its mortality is expected to rise in the next few years. Despite all efforts, the current therapeutic arsenal is not sufficient to reduce these numbers. Therefore, it is imperative to identify new sources of anticancer drugs. Filipendula ulmaria (L.) Maxim is part of the ethnobothanical patrimony of the Iberian Peninsula. For centuries, it has been used as a medicinal species due to its rich antioxidant content, which includes flavonoids and ascorbic acid [l]. Nonetheless, little is known about its antiproliferative activity in cancer cells. Thus, the aims of this project were to: i) investigate if different flower extracts of F. ulmaria have cell growth inhibitory activity in human tumour cell lines and ii) study the mechanism of action of one of the most potent extracts. Four flower extracts obtained by different extraction methods (decoction, infusion, methanol and methanol:water 80:20, v!v) were screened for tumour cell growth inhibitory activity in three human tumour cell lines: NCI-H460 (non-small cell lung cancer), A375-C5 (melanoma) and MCF-7 (breast adenocarcinoma). One of the most potent extracts (obtained by decoction) was further studied in the NCI-H460 cell line (one of the most sensitive), by investigating its effect on viable cell number, programmed cell death, cellular proliferation and cell cycle profile. Results showed that all extracts have growth inhibitory activity in the studied cell lines, in particular the extract obtained by decoction (GI50 of 70.0 ± 8.6, 96.0 ± 12.4 and 63.3 ± 7.6 flg/mL in the NCI-H460, MCF-7 and A373-C5 cells, respectively). Further studies in the NCI-H460 cell line showed that this extract reduced viable cell number. Moreover, treatment with this extract resulted in a strong reduction of cellular proliferation, with a slight increase in the percentage of cells in the G l phase of the cell cycle. No significant alterations in programmed cell death were observed, although results showed a statistically significant increase in the cellular levels of p53 and p2l. Future work will confirm if this extract is non-toxic to human non-tumour cells