Neutrophils, the most abundant circulating leukocytes, play a well-known role in defense against pathogens through phagocytosis and degranulation. However, a new mechanism involving the release of neutrophil extracellular traps (NETs) composed of DNA, histones, calprotectin, myeloperoxidase, and elastase, among others, has been described. The so-called NETosis process can occur through three different mechanisms: suicidal, vital, and mitochondrial NETosis. Apart from their role in immune defense, neutrophils and NETs have been involved in physiopathological conditions, highlighting immunothrombosis and cancer. Notably, neutrophils can either promote or inhibit tumor growth in the tumor microenvironment depending on cytokine signaling and epigenetic modifications. Several neutrophils’ pro-tumor strategies involving NETs have been documented, including pre-metastatic niche formation, increased survival, inhibition of the immune response, and resistance to oncologic therapies. In this review, we focus on ovarian cancer (OC), which remains the second most incidental but the most lethal gynecologic malignancy, partly due to the presence of metastasis, often omental, at diagnosis and the resistance to treatment. We deepen the state-of-the-art on the participation of NETs in OC metastasis establishment and progression and their involvement in resistance to chemo-, immuno-, and radiotherapies. Finally, we review the current literature on NETs in OC as diagnostic and/or prognostic markers, and their contribution to disease progression at early and advanced stages. The panoramic view provided in this article might pave the way for enhanced diagnostic and therapeutic strategies to improve the prognosis of cancer patients and, specifically, OC patients.

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? 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. 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. 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. 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. 40 µM UA and 20 µM 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 µM, while UB exhibited a mean IC50 of 79.92 µM. Both 40 µM UA and 20 µM 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 µM UB decreased the number of St-T1b adherent cells. TIMP-1 expression was upregulated in response to treating the cells with 40 µM UA (P < 0.05). Regarding the 12Z endometriotic cell line, only 40 µM UA decreased proliferation (P < 0.01); while both 40 µM UA and 20 µM 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 µM, while UB exhibited a mean IC50 of 54.79 µM. UB decreased cell migration (P < 0.05), and decreased the number of adherent cells (P < 0.05). Both 40 µM UA and 20 µM UB significantly decreased the cellular invasion of these cells; and several genes were altered when treating the cells with 40 µM UA and 10 µM 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 µM UA and UB: P < 0.05 both) as well as affecting their area (40 µM UA: P < 0.05, and 80 µM UA: P < 0.01) and integrity (40 µM UA and UB: P < 0.05, 80 µM UA and UB: P < 0.01). On the other hand, UA and UB significantly inhibited organoid development/outgrowth (40 and 80 µM UA: P < 0.0001 both; 40 µM UB: P < ns-0.05-0.001, and 80 µM 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 µM, while UB exhibited a mean IC50 of 52.60 µM). N/A This study was performed on in vitro endometriosis models. 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. This study was funded EU MSCA-RISE-2015 project MOMENDO (691058). The authors have no conflicts of interest to declare.

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway.

In recent years, interest in personalized medicine has considerably increased

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 100 μM) for 24 and 48 h. The results demonstrated that EA arrests an endometrial stromal cell cycle on the G2/M phase, after 48 h. In addition, 100 μM EA treatment significantly decreased ECC-1 cell migration at 20 h and T-HESC cell migration at 10 h and 20 h, while 50 μM EA significantly decreased T-HESC cell migration at 20 h. On the other hand, we proved that the 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 novel and promising candidates for therapeutic treatment of endometriosis.