Antagonizing the spindle assembly checkpoint silencing enhances paclitaxel and Navitoclax-mediated apoptosis with distinct mechanistic

Antimitotic drugs arrest cells in mitosis through chronic activation of the spindle assembly checkpoint (SAC), leading to cell death. However, drug-treated cancer cells can escape death by undergoing mitotic slippage, due to premature mitotic exit. Therefore, overcoming slippage issue is a promising chemotherapeutic strategy to improve the effectiveness of antimitotics. Here, we antagonized SAC silencing by knocking down the MAD2-binding protein p31comet, to delay mitotic slippage, and tracked cancer cells treated with the antimitotic drug paclitaxel, over 3 days live-cell time-lapse analysis. We found that in the absence of p31comet, the duration of mitotic block was increased in cells challenged with nanomolar concentrations of paclitaxel, leading to an additive effects in terms of cell death which was predominantly anticipated during the first mitosis. As accumulation of an apoptotic signal was suggested to prevent mitotic slippage, when we challenged p31comet-depleted mitotic-arrested cells with the apoptosis potentiator Navitoclax (previously called ABT-263), cell fate was shifted to accelerated post-mitotic death. We conclude that inhibition of SAC silencing is critical for enhancing the lethality of antimitotic drugs as well as that of therapeutic apoptosis-inducing small molecules, with distinct mechanisms. The study highlights the potential of p31comet as a target for antimitotic therapies.The authors gratefully acknowledge CESPU—Cooperativa de Ensino Superior Politécnico e Universitário, which financed this work under the projects “ComeTarget_CESPU_2017” and “ComeTax”. Ana C. Henriques acknowledge FCT-Fundação para a Ciência e a Tecnologia for financial support (Grant SFRH/BD/116167/2016)

Generation of Two Paclitaxel-Resistant High-Grade Serous Carcinoma Cell Lines With Increased Expression of P-Glycoprotein

Debulking surgery followed by chemotherapy are the standard of care for high-grade serous carcinoma. After an initial good response to treatment, the majority of patients relapse with a chemoresistant profile, leading to a poor overall survival. Chemotherapy regimens used in high-grade serous carcinomas are based in a combination of classical chemotherapeutic drugs, namely, Carboplatin and Paclitaxel. The mechanisms underlying drug resistance and new drug discovery are crucial to improve patients’ survival. To uncover the molecular mechanisms of chemoresistance and test drugs capable of overcoming this resistant profile, it is fundamental to use good cellular models capable of mimicking the chemoresistant disease. Herein, we established two high-grade serous carcinoma cell lines with intrinsic resistance to Carboplatin and induced Paclitaxel resistance (OVCAR8 PTX R C and OVCAR8 PTX R P) derived from the OVCAR8 cell line. These two chemoresistant cell line variants acquired an enhanced resistance to Paclitaxel-induced cell death by increasing the drug efflux capacity, and this resistance was stable in long-term culture and following freeze/thaw cycles. The mechanism underlying Paclitaxel resistance resides in a significant increase in P-glycoprotein expression and, when this drug efflux pump was blocked with Verapamil, cells re-acquired Paclitaxel sensitivity. We generated two high-grade serous carcinoma cell lines, with a double-chemoresistant (Carboplatin and Paclitaxel) phenotype that mimics the majority of tumor recurrences in ovarian cancer context. This robust tool is suitable for preliminary drug testing towards the development of therapeutic strategies to overcome chemoresistance.This work was developed at i3S/IPATIMUP, an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and partially supported by Funda̧cao para a Cîencia e a Tecnologia (FCT). This research was supported by European Regional Development Funds (ERDF) funds through the COMPETE 2020–Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, Funda̧cao para a Cîencia e a Tecnologia (FCT)/Minist́erio da Cîencia, Tecnologia e Inova̧cao (MCTES), under the project POCI 01-0145-FEDER-029503 (PTDC/MEC-ONC/29503/2017) and CESPU (Cooperativa de Ensino Superior Politécnico e Universitário) under the project ComeTarget_CESPU_2017 (to HB). MN acknowledges FCT/MCTES and UE for financial support through a PhD fellowship (2020.04720.BD) cosponsored by Fundo Social Europeu (FSE) through Programa Operacional Regional Norte (Norte 2020)

Tetracyclic thioxanthene derivatives: Studies on fluorescence and antitumor activity

Thioxanthones are bioisosteres of the naturally occurring xanthones. They have been described for multiple activities, including antitumor. As such, the synthesis of a library of thioxanthones was pursued, but unexpectedly, four tetracyclic thioxanthenes with a quinazoline– chromene scaffold were obtained. These compounds were studied for their human tumor cell growth inhibition activity, in the cell lines A375-C5, MCF-7 and NCI-H460. Photophysical studies were also performed. Two of the compounds displayed GI50 values below 10 µM for the three tested cell lines, and structure–activity relationship studies were established. Three compounds presented similar wavelengths of absorption and emission, characteristic of dyes with a push-pull character. The structures of two compounds were elucidated by X-ray crystallography. Two tetracyclic thioxanthenes emerged as hit compounds. One of the two compounds accumulated intracellularly as a bright fluorescent dye in the green channel, as analyzed by both fluorescence microscopy and flow cytometry, making it a promising theranostic cancer drug candidate.This research was partially supported by the Strategic Funding UIDB/04423/2020, UIDP/04423/2020 (Group of Natural Products and Medicinal Chemistry‐CIIMAR) and under the project PTDC/SAU‐PUB/28736/2017 (reference POCI‐01‐0145‐FEDER‐028736), co‐financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF and by FCT through national funds. Fernando Durães acknowledges his FCT grant (SFRH/BD/144681/2019). Hassan Bousbaa acknowledges funding from CESPU (IMPLDEBRIS‐PL‐3RL‐IINFACTS‐2019). This research was funded by Fundação para a Ciência e a Tecnologia (FCT) through research units UIDB/50006/2020, UID/CTM/50011/2019, UIDB/50011/2020 & UIDP/50011/2020 and by the European Union (FEDER program) through project CENTRO‐01‐0145‐FEDER‐000003

Synthesis of new glycosylated flavonoids with inhibitory activity on cell growth

Natural flavonoids and xanthone glycosides display several biological activities, with the glycoside moiety playing an important role in the mechanism of action of these metabolites. Herein, to give further insights into the inhibitory activity on cell growth of these classes of compounds, the synthesis of four flavonoids (5, 6, 9, and 10) and one xanthone (7) containing one or more acetoglycoside moieties was carried out. Acetyl groups were introduced using acetic anhydride and microwave irradiation. The introduction of one or two acetoglycoside moieties in the framework of 3,7-dihydroxyflavone (4) was performed using two synthetic methods: the Michael reaction and the Koenigs-Knorr reaction. The in vitro cell growth inhibitory activity of compounds 5, 6, 7, 9, and 10 was investigated in six human tumor cell lines: A375-C5 (malignant melanoma IL-1 insensitive), MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and U87MG (glioblastoma astrocytoma). The new flavonoid 3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-β-glucopyranosyl) flavone (10) was the most potent compound in all tumor cell lines tested, with GI50 values < 8 µM and a notable degree of selectivity for cancer cells. © 2018 by the authors.Funding: This work was supported through national funds provided by Foundation for Science and Technology from the Minister of Science, Technology and Higher Education (FCT/MCTES-PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE—Programa Operacional Factores de Competitividade (POFC) (POCI-01-0145-FEDER-016790), PPCDT—Promover a Produção Científica e Desenvolvimento Tecnológico e a Constituição de Redes Temáticas (Project 3599), under the project PTDC/MAR-BIO/4694/2014 in the framework of the programme PT2020 and INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR in the framework of North Portugal Regional Operational Programme (NORTE 2020). Acknowledgments: Marta Correia-da-Silva thanks FCT for the postdoctoral fellowship SFRH/BPD/81878/2011 and Ana R. Neves and Patrícia M.A. Silva for the Ph.D. fellowships SFRH/BD/114856/2016 and SFRH/BD/90744/2012, respectively

Screening a small library of xanthones for antitumor activity and identification of a hit compound which induces apoptosis

Our previous work has described a library of thioxanthones designed to have dual activity as P-glycoprotein modulators and antitumor agents. Some of these compounds had shown a significant cell growth inhibitory activity towards leukemia cell lines, without affecting the growth of non-tumor human fibroblasts. However, their effect in cell lines derived from solid tumors has not been previously studied. The present work aimed at: (i) screening this small series of compounds from an in-house library, for their in vitro cell growth inhibitory activity in human tumor cell lines derived from solid tumors; and (ii) initiate a study of the effect of the most potent compound on apoptosis. The tumor cell growth inhibitory effect of 27 compounds was first analysed in different human tumor cell lines, allowing the identification of a hit compound, TXA1. Its hydrochloride salt TXA1 HCl was then synthesized, to improve solubility and bioavailability. Both TXA1 and TXA1 HCl inhibited the growth of MCF-7, NCI-H460, A375-C5, HeLa, 786-O, Caki-2 and AGS cell lines. The effect of TXA1 HCl in MCF-7 cells was found to be irreversible and was associated, at least in part, with an increase in cellular apoptosis.IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT-Foundation for Science and Technology, under the projects FEDER COMPETE FCOMP-01-0124-FEDER-015752, FCOMP-01-0124-FEDER-011057, 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 Programa Operacional Regional do Norte (ON.2—O Novo Norte), through FEDER funds under the Quadro de Referência Estratégico Nacional (QREN). This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. The authors also thank FCT for the grant of R.T. Lima (SFRH/BPD/68787/2010) and QREN for the grant of D. Sousa (NORTE-07-0124-FEDER-000023)