scholarly journals Positive allosteric modulation of P2X7 promotes apoptotic cell death over lytic cell death responses in macrophages

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Stefan Bidula ◽  
Kshitija Dhuna ◽  
Ray Helliwell ◽  
Leanne Stokes
Keyword(s):  
Cell Death ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Allosteric Modulation ◽  
Cell Swelling ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
Membrane Rupture ◽  
Cell Death Pathways ◽  
Intrinsic Apoptosis Pathway

AbstractP2X7 is an ATP-gated ion channel that is highly expressed by leukocytes, such as macrophages. Here, P2X7 has been demonstrated to be involved in the regulation of various cell death pathways; including apoptosis, pyroptosis, necrosis, and autophagy. However, cell death induction via P2X7 is complex and is reliant upon the nature of the stimulus, the duration of the stimulus, and the cell type investigated. Previous reports state that high extracellular ATP concentrations promote osmotic lysis, but whether positive allosteric modulation of P2X7 in the presence of lower concentrations of ATP condemns cells to the same fate is unknown. In this study, we compared cell death induced by high ATP concentrations, to cell death induced by compound K, a recently identified and potent positive allosteric modulator of P2X7. Based on our observations, we propose that high ATP concentrations induce early cell swelling, loss of mitochondrial membrane potential, plasma membrane rupture, and LDH release. Conversely, positive allosteric modulation of P2X7 primarily promotes an intrinsic apoptosis pathway. This was characterised by an increase in mitochondrial Ca2+, accelerated production of mitochondrial ROS, loss of mitochondrial membrane permeability in a Bax-dependent manner, the potential involvement of caspase-1, and caspase-3, and significantly accelerated kinetics of caspase-3 activation. This study highlights the ability of positive allosteric modulators to calibrate P2X7-dependent cell death pathways and may have important implications in modulating the antimicrobial immune response and in the resolution of inflammation.

scholarly journals Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and the Bcl-2 Protein Family

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Christopher Platen ◽  
Stephan Dreschers ◽  
Jessica Wappler ◽  
Andreas Ludwig ◽  
Stefan Düsterhöft ◽  
...  
Keyword(s):  
Cell Death ◽  
Bacterial Infections ◽  
Caspase 3 ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Dependent Manner ◽  
Intrinsic Apoptosis ◽  
Caspase 9 ◽  
Apoptosis Pathway ◽  
Intrinsic Apoptosis Pathway

Neonates are extremely susceptible to bacterial infections, and evidences suggest that phagocytosis-induced cell death (PICD) is less frequently triggered in neonatal monocytes than in monocytes from adult donors. An insufficient termination of the inflammatory response, leading to a prolonged survival of neonatal monocytes with ongoing proinflammatory cytokine release, could be associated with the progression of various inflammatory diseases in neonates. Our previous data indicate that amphiregulin (AREG) is increasingly expressed on the cell surface of neonatal monocytes, resulting in remarkably higher soluble AREG levels after proteolytic shedding. In this study, we found that E. coli-infected neonatal monocytes show an increased phosphorylation of ERK, increased expression of Bcl-2 and Bcl-XL, and reduced levels of cleaved caspase-3 and caspase-9 compared to adult monocytes. In both cell types, additional stimulation with soluble AREG further increased ERK activation and expression of Bcl-2 and Bcl-XL and reduced levels of cleaved caspase-3 and caspase-9 in an EGFR-dependent manner. These data suggest that reduced PICD of neonatal monocytes could be due to reduced intrinsic apoptosis and that AREG can promote protection against PICD. This reduction of the intrinsic apoptosis pathway in neonatal monocytes could be relevant for severely prolonged inflammatory responses of neonates.

scholarly journals The Multifaceted Roles of Pyroptotic Cell Death Pathways in Cancer

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1313
Author(s):  
Man Wang ◽  
Shuai Jiang ◽  
Yinfeng Zhang ◽  
Peifeng Li ◽  
Kun Wang
Keyword(s):  
Cell Death ◽  
Biological Significance ◽  
Chemotherapeutic Agents ◽  
The Body ◽  
Cell Swelling ◽  
Pathogen Invasion ◽  
Membrane Rupture ◽  
Cancer Management ◽  
Cell Death Pathways ◽  
Cancer Pathogenesis

Cancer is a category of diseases involving abnormal cell growth with the potential to invade other parts of the body. Chemotherapy is the most widely used first-line treatment for multiple forms of cancer. Chemotherapeutic agents act via targeting the cellular apoptotic pathway. However, cancer cells usually acquire chemoresistance, leading to poor outcomes in cancer patients. For that reason, it is imperative to discover other cell death pathways for improved cancer intervention. Pyroptosis is a new form of programmed cell death that commonly occurs upon pathogen invasion. Pyroptosis is marked by cell swelling and plasma membrane rupture, which results in the release of cytosolic contents into the extracellular space. Currently, pyroptosis is proposed to be an alternative mode of cell death in cancer treatment. Accumulating evidence shows that the key components of pyroptotic cell death pathways, including inflammasomes, gasdermins and pro-inflammatory cytokines, are involved in the initiation and progression of cancer. Interfering with pyroptotic cell death pathways may represent a promising therapeutic option for cancer management. In this review, we describe the current knowledge regarding the biological significance of pyroptotic cell death pathways in cancer pathogenesis and also discuss their potential therapeutic utility.

scholarly journals Endothelial cell death subroutines at physiological and supraphysiological concentrations of calcium phosphate bions

2020 ◽  
pp. 19-30
Author(s):  
В.Е. Маркова ◽  
Д.К. Шишкова ◽  
А.Г. Кутихин
Keyword(s):  
Cell Death ◽  
Calcium Phosphate ◽  
Optical Density ◽  
Caspase 3 ◽  
Considerable Proportion ◽  
Colorimetric Determination ◽  
Apoptosis Pathway ◽  
Caspase Inhibitors ◽  
Regulated Cell Death ◽  
Intrinsic Apoptosis Pathway

Актуальность. Формирующиеся при перенасыщении крови ионами кальция и фосфора и циркулирующие в кровотоке кальций-фосфатные бионы (КФБ) вызывают дисфункцию эндотелия вследствие гибели части артериальных эндотелиальных клеток (ЭК). Цель исследования. Оценить типы гибели первичных артериальных ЭК человека под воздействием физиологических и супрафизиологических концентраций сферических КФБ (СКФБ) и игольчатых КФБ (ИКФБ). Материалы и методы. К конфлюэнтным культурам первичных ЭК коронарной и внутренней грудной артерии человека в 96-луночных планшетах были добавлены равные объемы (10 мкл на лунку) суспензий СКФБ и ИКФБ с оптической плотностью 0,08-0,10 (физиологическая концентрация) или 0,42-0,45 (супрафизиологическая концентрация) на длине волны 650 нм. Во всех экспериментальных группах также производилось селективное ингибирование каспазы-3 (Z-D(OMe)E(OMe)VD(OMe)-FMK, 100 мкмоль/л) или ингибирование всех каспаз (Z-VAD(OMe)-FMK, 100 мкмоль/л) для оценки типа клеточной гибели (регулируемая или моментальная). Жизнеспособность клеток определялась посредством последовательного колориметрического определения их метаболической активности через 4, 24 и 48 часов после добавления КФБ. Результаты. При добавлении супрафизиологических концентраций КФБ уже на первой временной точке большинство (60-85%) эндотелиальных клеток погибало вне зависимости от типа добавленных КФБ и воздействия ингибиторов каспаз, при этом через 24 и 48 часов экспозиции ингибиторы каспаз оказывали некоторое цитопротективное действие на незначительное количество выживших клеток. При добавлении физиологических концентраций КФБ ингибиторы каспаз оказывали выраженное цитопротективное действие через 24 и 48 часов экспозиции, при этом ИКФБ демонстрировали существенно более высокую токсичность для ЭК в сравнении с СКФБ. Независимо от временной точки пан-каспазный ингибитор оказывал значительно более выраженное цитопротективное действие по сравнению с селективным ингибитором каспазы-3, что свидетельствует о кумулятивном эффекте ингибирования каспаз, возникающем, вероятно, вследствие запуска внутреннего пути апоптоза. Заключение. В супрафизиологических концентрациях КФБ вызывают моментальную гибель абсолютного большинства ЭК, однако в физиологических концентрациях ингибиторы каспаз существенно повышают выживаемость ЭК, что свидетельствует о регулируемом направлении их клеточной гибели. Дальнейшие исследования в этом направлении должны расшифровать молекулярные пути регулируемой клеточной гибели ЭК под воздействием физиологических концентраций КФБ. Background. Calcium phosphate bions (CPB) formed and circulating in the blood at its supersaturation with calcium and phosphate provoke endothelial dysfunction by causing the demise of arterial endothelial cells (ECs). Aim. To examine cell death subroutines of human primary arterial ECs exposed to physiological and supraphysiological concentrations of spherical CPB (CPB-S) and needle-shaped CPB (CPB-N). Materials and methods. Equal volumes (10 μL) of CPB-S and CPB-N at physiological concentration (optical density at 650 nm wavelength = 0.08-0.10) or supraphysiological amounts (optical density at 650 nm wavelength = 0.42-0.45) were added to the confluent primary human coronary artery and internal thoracic artery ECs cultured in 96-well plates. In all experimental groups, we selectively inhibited caspase-3 by adding Z-D(OMe)E(OMe)VD(OMe)-FMK (100 μmol/L) or all caspases (Z-VAD(OMe)-FMK, 100 μmol/L) to assess whether the CPB-induced cell death is regulated or accidental. Cell viability was evaluated by sequential colorimetric determination of metabolic activity at 4, 24, and 48 hours of incubation with CPB. Results. At supraphysiological CPB concentrations, the majority (60-85%) of ECs died regardless of CPB type and caspase inhibitors, albeit at 24- and 48-hour time points the latter had minor cytoprotective action. However at physiological CPB levels, caspase inhibitors rescued a considerable proportion of ECs after 24 or 48 hours of exposure, and CPB-N had significantly higher toxicity than CPB-S. Regardless of the time point, the cytoprotective effect of the pan-caspase inhibitor was significantly higher than that of the selective caspase-3 inhibitor indicating a cumulative caspase inhibition and suggesting that cell death was precipitated by an intrinsic apoptosis pathway. Conclusion. At supraphysiological concentrations, CPB cause instant cell death; yet at physiological amounts, caspase inhibitors rescue the majority of ECs testifying to the regulated cell death. Further studies in this field should decipher the molecular pathways of CPB-induced regulated cell death of ECs.

scholarly journals Isoharringtonine Induces Apoptosis of Non-Small Cell Lung Cancer Cells in Tumorspheroids via the Intrinsic Pathway

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1521
Author(s):  
Ji Hae Lee ◽  
So-Young Park ◽  
Wonbin Hwang ◽  
Jee Young Sung ◽  
Myoung-Lae Cho ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Cancer Cells ◽  
Natural Product ◽  
Nuclear Receptor ◽  
Small Cell ◽  
Dependent Manner ◽  
Small Cell Lung ◽  
Apoptosis Pathway ◽  
Intrinsic Apoptosis Pathway

Lung cancer is the major cause of cancer-associated death worldwide, and development of new therapeutic drugs is needed to improve treatment outcomes. Three-dimensional (3D) tumorspheroids offer many advantages over conventional two-dimensional cell cultures due to the similarities to in vivo tumors. We found that isoharringtonine, a natural product purified from Cephalotaxus koreana Nakai, significantly inhibited the growth of tumorspheroids with NCI-H460 cells in a dose-dependent manner and induced apoptotic cell death in our 3D cell culture system. On the other hand, A549 tumorspheroids displayed low sensitivity to isoharringtonine-induced apoptosis. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor known to regulate proliferation and apoptosis of cancer cells. We observed that knockdown of NR4A1 dramatically increased isoharringtonine-induced cancer cell death in A549 tumorspheroids by activating the intrinsic apoptosis pathway. Furthermore, treatment with combined isoharringtonine and iNR4A1 significantly inhibited multivulva formation in a Caenorhabditis elegans model and tumor development in a xenograft mouse model. Taken together, our data suggest that isoharringtonine is a potential natural product for treatment of non-small cell lung cancers, and inhibition of NR4A1 sensitizes cancer cells to anti-cancer treatment.

scholarly journals The caspase-3/GSDME signal pathway as a switch between apoptosis and pyroptosis in cancer

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Mingxia Jiang ◽  
Ling Qi ◽  
Lisha Li ◽  
Yanjing Li
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Caspase 3 ◽  
Suppressor Gene ◽  
Signal Pathway ◽  
Cell Swelling ◽  
Tumor Cell Death ◽  
Cell Death Pathways ◽  
Apoptotic Pathways ◽  
New Strategies

Abstract Apoptosis has long been recognized as a mechanism that kills the cancer cells by cytotoxic drugs. In recent years, studies have proved that pyroptosis can also shrink tumors and inhibit cells proliferation. Both apoptosis and pyroptosis are caspase-dependent programmed cell death pathways. Cysteinyl aspartate specific proteinase-3 (Caspase-3) is a common key protein in the apoptosis and pyroptosis pathways, and when activated, the expression level of tumor suppressor gene Gasdermin E (GSDME) determines the mechanism of tumor cell death. When GSDME is highly expressed, the active caspase-3 cuts it and releases the N-terminal domain to punch holes in the cell membrane, resulting in cell swelling, rupture, and death. When the expression of GSDME is low, it will lead to the classical mechanism of tumor cell death, which is apoptosis. More interestingly, researchers have found that GSDME can also be located upstream of caspase-3, connecting extrinsic, and intrinsic apoptotic pathways. Then, promoting caspase-3 activation, and forming a self-amplifying feed-forward loop. GSDME-mediated pyroptosis is correlated with the side effects of chemotherapy and anti-tumor immunity. This article mainly reviews the caspase-3/GSDME signal pathway as a switch between apoptosis and pyroptosis in cancer, to provide new strategies and targets for cancer treatment.

scholarly journals Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells

2019 ◽  
Vol 20 (17) ◽  
pp. 4254 ◽  
Author(s):  
Lien Van Hoecke ◽  
Laurens Raes ◽  
Stephan Stremersch ◽  
Toon Brans ◽  
Juan C. Fraire ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cells ◽  
Protein Delivery ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Kinase Domain ◽  
Cell Swelling ◽  
Caspase 8 ◽  
Mixed Lineage Kinase ◽  
Membrane Rupture

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

scholarly journals Recovering Drug-Induced Apoptosis Subnetwork from Connectivity Map Data

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Jiyang Yu ◽  
Preeti Putcha ◽  
Jose M. Silva
Keyword(s):  
Cell Death ◽  
Cancer Cell ◽  
Specific Cell ◽  
Connectivity Map ◽  
Drug Induced ◽  
Significant Differential Expression ◽  
Apoptosis Pathway ◽  
Cell Death Pathways ◽  
Intrinsic Apoptosis Pathway ◽  
Extrinsic Apoptosis

The Connectivity Map (CMAP) project profiled human cancer cell lines exposed to a library of anticancer compounds with the goal of connecting cancer with underlying genes and potential treatments. Since the therapeutic goal of most anticancer drugs is to induce tumor-selective apoptosis, it is critical to understand the specific cell death pathways triggered by drugs. This can help to better understand the mechanism of how cancer cells respond to chemical stimulations and improve the treatment of human tumors. In this study, using CMAP microarray data from breast cancer cell line MCF7, we applied a Gaussian Bayesian network modeling approach and identified apoptosis as a major drug-induced cellular-pathway. We then focused on 13 apoptotic genes that showed significant differential expression across all drug-perturbed samples to reconstruct the apoptosis network. In our predicted subnetwork, 9 out of 15 high-confidence interactions were validated in the literature, and our inferred network captured two major cell death pathways by identifying BCL2L11 and PMAIP1 as key interacting players for the intrinsic apoptosis pathway and TAXBP1 and TNFAIP3 for the extrinsic apoptosis pathway. Our inferred apoptosis network also suggested the role of BCL2L11 and TNFAIP3 as “gateway” genes in the drug-induced intrinsic and extrinsic apoptosis pathways.

scholarly journals Analysis of BNIP3 and BNIP3L/Nix expression in cybrid cell lines harboring two LHON-associated mutations.

2019 ◽  
Author(s):  
Agata Kodroń ◽  
Parvana Hajieva ◽  
Agata Kulicka ◽  
Bohdan Paterczyk ◽  
Elona Jankauskaite ◽  
...  
Keyword(s):  
Cell Death ◽  
Optic Nerve ◽  
Cellular Response ◽  
Nerve Degeneration ◽  
Apoptosis Pathway ◽  
Mtdna Mutations ◽  
Cell Death Pathways ◽  
Intrinsic Apoptosis Pathway ◽  
Response To Stress ◽  
In Cells

Mitochondria are key players in cell death through the activation of the intrinsic apoptosis pathway. BNIP3 and BNIP3L/Nix are outer mitochondrial membrane bifunctional proteins which because of containing both BH3 and LIR domains play a role in cellular response to stress by regulation of apoptosis and selective autophagy. Leber’s Hereditary Optic Neuropathy (LHON) is the most common mitochondrial disease in adults, characterized by painless loss of vision caused by atrophy of the optic nerve. The disease in over 90% of cases is caused by one of three mutations in the mitochondrial genome: 11778G>A, 3460G>A or 14484T>C. The pathogenic processes leading to optic nerve degeneration are largely unknown, however, the most common explanation is that mtDNA mutations increase the apoptosis level in this tissue. Here we present the results of analysis of BNIP3 and BNIP3L/Nix proteins in cells harboring a combination of the 11778G>A and the 3460G>A LHON mutations. Experiments performed on cybrids revealed that BNIP3 protein level is decreased in LHON cells compared to controls. CCCP treatment resulted in apoptosis induction only in control cells. Moreover, we also noticed reduced level of autophagy in LHON cybrids. The presented results suggest that in cells carrying LHON mutations expression of proteins involved in regulation of apoptosis and autophagy is decreased what in turn may disturb cell death pathways in those cells and affect cellular response to stress.  

scholarly journals The Novel ALG-2 Target Protein CDIP1 Promotes Cell Death by Interacting with ESCRT-I and VAPA/B

2021 ◽  
Vol 22 (3) ◽  
pp. 1175
Author(s):  
Ryuta Inukai ◽  
Kanako Mori ◽  
Keiko Kuwata ◽  
Chihiro Suzuki ◽  
Masatoshi Maki ◽  
...  
Keyword(s):  
Cell Death ◽  
Essential Gene ◽  
Susceptibility Gene ◽  
Target Protein ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Endosomal Sorting ◽  
Cell Death Pathways ◽  
Apoptotic Protein

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

scholarly journals Anticancer effects of mifepristone on human uveal melanoma cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Prisca Bustamante Alvarez ◽  
Alexander Laskaris ◽  
Alicia A. Goyeneche ◽  
Yunxi Chen ◽  
Carlos M. Telleria ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Progesterone Receptor ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Caspase 3 ◽  
Annexin V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free Dna

Abstract Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

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