Effect of urolithins A and B on ectopic endometrial growth in a murine model of endometriosis

Endometriosis is an often painful disease in reproductive-aged women, in which endometrial-like tissue grows outside the uterine cavity. Since the limited current therapeutic alternatives fail in alleviating the symptoms and based on our previous research in in vitro models using the same compounds as the ones used in the present study, we aimed to evaluate the effects of urolithins A (UA) and B (UB) on the growth and survival of endometriotic-like lesions in a murine model of endometriosis. Female BALB/C mice were surgically induced with endometriosis and treated with 2.5 mg kg-1 day-1 intraperitoneal UA or UB. The mice were monitored daily and weighed and the estrous stage was determined. After 28 days of treatment, lesions were counted, measured, excised, and fixed. Both urolithins proved not to affect the estrous cycle or body weight of the mice. UA completely prevented endometriotic-like lesions, while UB diminished the implant volume (p < 0.05). Treatment also reduced epithelial and stromal cell proliferation within the implants (p < 0.001 and p < 0.01, respectively) and apoptosis was enhanced (p < 0.05 and p < 0.01, respectively). These results are promising and reveal that urolithins A and B, separately, have a beneficial effect on the overall endometriotic growth without affecting the body weight or estrous cycle.Fil: Mc Cormack, Bárbara Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Olivares, Carla Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Madanes, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ricci, Analía Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bilotas, Mariela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Barañao, Rosa Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Potential use of ellagic acid for endometriosis treatment: its effect on a human endometrial cell cycle, adhesion and migration

Endometriosis is a common and challenging condition of reproductive-aged women that is defined as the presence of endometrial-like tissue outside the uterine cavity. Despite its prevalence, there is still no effective therapeutics so we aim to evaluate the ellagic acid (EA) effect on the most relevant aspects that are known to be altered in endometriosis. Endometrial primary cultures from women with and without endometriosis and endometrial cell lines were incubated with EA (50 and 100µM) for 24 and 48 h. The results demonstrated that EA arrest endometrial stromal cell cycle on G2 / M phase, after 48 h. In addition, EA 100μM treatment significantly decreased ECC-1 cell migration at 20 h and T-HESC cell migration at 10 h and 20 h; while EA 50μM caused a significant decreased on T-HESC cell migration at 20 h. On the other hand, we proved that treatment with EA for 24 h reduces T-HESC and ECC-1 adhesion to plastic. However, we did not find an effect of EA on cell proliferation. EA has an inhibitory effect on endometrial cell adhesion, migration and cell cycle progression in vitro. These highlight the idea to investigate natural compounds as a novel and promising therapeutic treatment for endometriosis.Fil: Mc Cormack, Bárbara Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bilotas, Mariela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Madanes, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ricci, Analía Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Singla, José Javier. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Barañao, Rosa Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Increased levels of NETosis biomarkers in high-grade serous ovarian cancer patients’ biofluids: Potential role in disease diagnosis and management

Introduction: High-grade serous ovarian cancer (HGSOC) is the second most frequent gynecological malignancy but the most lethal, partially due to the spread of the disease through the peritoneal cavity. Recent evidence has shown that, apart from their role in immune defense through phagocytosis and degranulation, neutrophils are able to participate in cancer progression through the release of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are composed of DNA, histones, calprotectin, myeloperoxidase (MPO) and elastase and the NETosis process has been proposed as a pre-requisite for the establishment of omental metastases in early stages of HGSOC. Nevertheless, its role in advanced stages remains to be elucidated. Therefore, our principal aim is to characterize a NETosis biomarker profile in biofluids from patients with advanced HGSOC and control women. Methods: Specifically, five biomarkers of NETosis (cell-free DNA (cfDNA), nucleosomes, citrullinated histone 3 (citH3), calprotectin and MPO) were quantified in plasma and peritoneal fluid (PF) samples from patients (n=45) and control women (n=40). Results: Our results showed that HGSOC patients presented a higher concentration of cfDNA, citH3 and calprotectin in plasma and of all five NETosis biomarkers in PF than control women. Moreover, these biomarkers showed a strong ability to differentiate the two clinical groups. Interestingly, neoadjuvant treatment (NT) seemed to reduce NETosis biomarkers mainly systemically (plasma) compared to the tumor environment (PF). Discussion: In conclusion, NETosis biomarkers are present in the tumor environment of patients with advanced HGSOC, which might contribute to the progression of the disease. Besides, plasma cfDNA and calprotectin could represent minimally invasive surrogate biomarkers for HGSOC. Finally, NT modifies NETosis biomarkers levels mainly at the systemic level

The hyaluronan synthesis inhibitor 4-methylumbelliferone inhibits cell proliferation and wound healing on in vitro endometriosis models

Objectives: The aim of this study was to evaluate the effect of 4-methylumbelliferone (4MU) on cell proliferation, wound healing process and gelatinase activity on human endometrial stromal (t-HESC) or epithelial (ECC-1) cell lines. Methods: t-HESC and ECC-1 endometrial cell lines were stimulated with different concentrations of 4MU. Cell proliferation was evaluated after 24hs with the cell proliferation reagent WST-1. The area of a scratch closed by the t-HESC was calculated in a wound healing assay, for this purpose photomicrographs were taken at time 0h and time 20hs; and the gelatinase activity in conditioned media of t-HESC was evaluated by zimography. Only p<0.05 was considered as statistically significant. Results: After 24hs of treatment the cell proliferation of ECC-1 was significantly inhibited by 0.5, 1 and 2 mM 4MU (p<0.01, p<0.001 and p<0.01, respectively versus control), whereas cell proliferation of t-HESC was significantly inhibited by 2 and 4 mM 4MU (p<0.01 versus control). Already at 0.5 mM 4MU, the migration of t-HESC cells was significantly inhibited and the scratch was closed in a low percentage (p<0.01 for 0.5 and 1 mM 4MU versus control, p<0.001 for 2 and 4 mM 4MU). Preliminary results of MMP-2 and MMP-9 activities revealed that these gelatinases would not be modulated after 24hs of 4MU treatment. Conclusion: Endometriosis is a benign gynecological disease affecting 10% of women of reproductive age, characterized by the presence of endometriotic foci outside the uterine cavity. Previous studies from our laboratory demonstrated a strong antiangiogenic activity of 4MU. The present results are, in part, in agreement with those reported by other authors. Given these and our background on targeting hyaluronan on endometriosis models we are encouraged to continue investigating on this path to understand the implication of hyaluronan synthesis inhibition on the migratory capacity of the cells and which molecules are involved in this matter.Fil: Olivares, Carla Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Chiappini, Florencia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Mc Cormack, Bárbara Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Madanes, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Randi, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Barañao, Rosa Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Meresman, Gabriela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaInternational Federation of Placenta Associations; VIII Meeting of the Latin American Society of Placenta and Feto-Maternal InteractionCiudad Autónoma de Buenos AiresArgentinaInternational Federation of Placenta AssociationsLatin American Society of Placenta and Feto-Maternal Interactio

Special Issue &ldquo;miRNAs in the Era of Personalized Medicine: From Biomarkers to Therapeutics 2.0&rdquo;

Personalized medicine has become a new paradigm in the management of a variety of diseases [...

Special Issue “miRNAs in the Era of Personalized Medicine: From Biomarkers to Therapeutics 2.0”

Personalized medicine has become a new paradigm in the management of a variety of diseases [...

Identification of novel cyclic nucleotide binding proteins in Trypanosoma cruzi

Cyclic AMP has been implicated as second messenger in a wide range of cellular processes. In the protozoan parasite Trypanosoma cruzi, cAMP is involved in the development of the parasite's life cycle. While cAMP effectors have been widely studied in other eukaryotic cells, little is known about cAMP's mechanism of action in T. cruzi. To date, only a cAMP-dependent protein kinase A (PKA) has been cloned and characterised in this parasite; however experimental evidence indicates the existence of cAMP-dependent, PKA-independent events. In order to identify new cAMP binding proteins as potential cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. Using a combination of search methods 27 proteins with putative cNMP binding domains (CBDs) were identified. Phylogenetic analysis of the CBDs presented a homogeneous distribution, with sequences segregated into two main branches: one containing kinases-like proteins and the other gathering hypothetical proteins with different function or no other known. Comparative modelling of the strongest candidates provides support for the hypothesis that these proteins may give rise to structurally viable cyclic nucleotide binding domains. Pull-down and nucleotide displacement assays strongly suggest that TcCLB.508523.80 could bind cAMP and eventually be a new putative PKA-independent cAMP effector in T. cruzi.Fil: Jager, Adriana Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: de Gaudenzi, Javier Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Mild, Jesica Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Mc Cormack, Bárbara Andrea. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Pantano Gutierrez, Sergio Fabian. Instituto Pasteur de Montevideo; UruguayFil: Altschuler, Daniel Leornardo. University of Pittsburgh; Estados UnidosFil: Edreira, Martin Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. University of Pittsburgh; Estados Unido

The ellagic acid metabolites urolithin A and B differentially affect growth, adhesion, motility, and invasion of endometriotic cells in vitro

STUDY QUESTION: What are the effects of plant-derived antioxidant compounds urolithin A (UA) and B (UB) on the growth and pathogenetic properties of an in vitro endometriosis model? SUMMARY ANSWER: Both urolithins showed inhibitory effects on cell behavior related to the development of endometriosis by differentially affecting growth, adhesion, motility, and invasion of endometriotic cells in vitro. WHAT IS KNOWN ALREADY: Endometriosis is one of the most common benign gynecological diseases in women of reproductive age and is defined by the presence of endometrial tissue outside the uterine cavity. As current pharmacological therapies are associated with side effects interfering with fertility, we aimed at finding alternative therapeutics using natural compounds that can be administered for prolonged periods with a favorable side effects profile. STUDY DESIGN, SIZE, DURATION: In vitro cultures of primary endometriotic stromal cells from 6 patients subjected to laparoscopy for benign pathologies with histologically confirmed endometriosis; and immortalized endometrial stromal (St-T1b) and endometriotic epithelial cells (12Z) were utilized to assess the effects of UA and UB on endometriotic cell properties. Results were validated in three-dimensional (3D) in vitro co-culture spheroids of 12Z and primary endometriotic stroma cells of one patient, and organoids from 3 independent donors with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects on cell growth were measured by non-radioactive colorimetric assay to measure cellular metabolic activity as an indicator of cell viability (MTT assay) and flow cytometric cell cycle assay on primary cultures, St-T1b, and 12Z. Apoptosis analyses, the impact on in vitro adhesion, migration, and invasion were evaluated in the cell lines. Moreover, Real-Time Quantitative Reverse Transcription polymerase chain reaction (RT-qPCR) assays were performed on primary cultures, St- T1b and 12Z to evaluate a plausible mechanistic contribution by factors related to proteolysis (matrix metalloproteinase 2, 3 and 9 -MMP2, MMP3, MMP9-, and tissue inhibitor of metalloproteinases -TIMP-1-), cytoskeletal regulators (Ras-related C3 botulinum toxin substrate 1 -RAC1-, Rho-associated coiled-coil containing protein kinase 2 -ROCK2-), and cell adhesion molecules (Syndecan 1 -SDC1-, Integrin alpha V–ITGAV-). Finally, the urolithins effects were evaluated on spheroids and organoids by formation, viability, and drug screen assays. MAIN RESULTS AND THE ROLE OF CHANCE: 40 mM UA and 20 mM UB produced a significant decrease in cell proliferation in the primary endometriotic cell cultures (P < 0.001 and P < 0.01, respectively) and in the St-T1b cell line (P < 0.001 and P < 0.05, respectively). In St-T1b, UA exhibited a mean half-maximum inhibitory concentration (IC50) of 39.88 mM, while UB exhibited a mean IC50 of 79.92 mM. Both 40 mM UA and 20 mM UB produced an increase in cells in the S phase of the cell cycle (P < 0.01 and P < 0.05, respectively). The same concentration of UA also increased the percentage of apoptotic ST-t1b cells (P < 0.05), while both urolithins decreased cell migration after 24 h (P < 0.001 both). Only the addition of 5 mM UB decreased the number of St-T1b adherent cells. TIMP-1 expression was upregulated in response to treating the cells with 40 mM UA (P < 0.05). Regarding the 12Z endometriotic cell line, only 40 mM UA decreased proliferation (P < 0.01); while both 40 mM UA and 20 mM UB produced an increase in cells in the G2/M phase (P < 0.05 and P < 0.01, respectively). In this cell line, UA exhibited a mean IC50 of 40.46 mM, while UB exhibited a mean IC50 of 54.79 mM. UB decreased cell migration (P < 0.05), and decreased the number of adherent cells (P < 0.05). Both 40 mM UA and 20 mM UB significantly decreased the cellular invasion of these cells; and several genes were altered when treating the cells with 40 mM UA and 10 mM UB. The expression of MMP2 was downregulated by UA (P < 0.001), and expression of MMP3 (UA P < 0.001 and UB P < 0.05) and MMP9 (P < 0.05, both) were downregulated by both urolithins. Moreover, UA significantly downregulated ROCK2 (P < 0.05), whereas UB treatment was associated with RAC1 downregulation (P < 0.05). Finally, the matrix adhesion receptors and signaling (co)receptors SDC1 and ITGAV were downregulated upon treatment with either UA or UB (P < 0.01 and P < 0.05, respectively in both cases). Regarding the effects of urolithins on 3D models, we have seen that they significantly decrease the viability of endometriosis spheroids (80 mM UA and UB: P < 0.05 both) as well as affecting their area (40 mM UA: P < 0.05, and 80 mM UA: P < 0.01) and integrity (40 mM UA and UB: P < 0.05, 80 mM UA and UB: P < 0.01). On the other hand, UA and UB significantly inhibited organoid development/outgrowth (40 and 80 mM UA: P < 0.0001 both; 40 mM UB: P < ns-0.05-0.001, and 80 mM UB: P < 0.01–0.001–0.001), and all organoid lines show urolithins sensitivity resulting in decreasing viability (UA exhibited a mean IC50 of 33.93 mM, while UB exhibited a mean IC50 of 52.60 mM). LARGE-SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: This study was performed on in vitro endometriosis models. WIDER IMPLICATIONS OF THE FINDINGS: These in vitro results provide new insights into the pathogenetic pathways affected by these compounds and mark their use as a potential new therapeutic strategy for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded EU MSCA-RISE-2015 project MOMENDO (691058). The authors have no conflicts of interest to declare.Fil: Mc Cormack, Bárbara Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Maenhoudt, N.. Katholikie Universiteit Leuven; BélgicaFil: Fincke, V.. Munster University Hospital; AlemaniaFil: Stejskalova, A.. Munster University Hospital; AlemaniaFil: Greve, B.. Munster University Hospital; AlemaniaFil: Kiesel, L.. Munster University Hospital; AlemaniaFil: Meresman, Gabriela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vankelecom, H.. Katholikie Universiteit Leuven; BélgicaFil: Götte, M.. Munster University Hospital; AlemaniaFil: Barañao, Rosa Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

New Roles for Old Friends: Involvement of the Innate Immune System in Tumor Progression

The association between the immune system and tumor progression has attracted much interest in the research community in recent years [...