VALPROIC ACID INDUCES APOPTOSIS AND INCREASES CXCR7 EXPRESSION IN EPITHELIAL OVARIAN CANCER CELL LINE SKOV-3.

Background: The chemokine receptor, CXCR7 is described to play a biologically relevant role in tumor growth and spread. Recently, it was reported that CXCR7 overexpression is associated with an unfavorable prognosis and metastatis of epithelial ovarian cancer (EOC). Aware that, several reports indicated that Histone deacetylases (HDACs) regulate the expression and activity of many proteins involved in both cancer initiation and progression, the aim of this work, was to study the effect of the HDAC inhibitor valproic acid (VPA) on the expression of CXCR7 as well as its impact on survival function in the epithelial ovarian cell line (SKOV-3). Methods: cells were cultured with varying concentrations of VPA (1, 2, 3, 4, 5 and 10 mM) for different durations (0, 12 h, 24 h and 48 h). Cell survival was assessed by Neutral red assay and by colony counting which being stained with crystal violet. CXCR7 expression was determined at mRNA level using quantitative real-time PCR (qRT-PCR) or at the protein level using western blotting. Results: VPA reduces cell survival of SKOV-3 cancer cells. The inhibition effect of VPA was dose and time-dependent. Exposure to VPA at concentrations above 2 mM at 24 h resulted in an increase expression of CXCR7 at both the mRNA and protein levels . Conclusion: These observations provide, for the first time, a better insight into the epigenetic mechanisms involved in regulating CXCR7 expression in EOC and will open new avenues for evaluating drugs that specifically stimulate the apoptosis of EOC with minimal unwanted side effect

VALPROIC ACID INDUCES APOPTOSIS AND INCREASES CXCR7 EXPRESSION IN EPITHELIAL OVARIAN CANCER CELL LINE SKOV-3.

Background: The chemokine receptor, CXCR7 is described to play a biologically relevant role in tumor growth and spread. Recently, it was reported that CXCR7 overexpression is associated with an unfavorable prognosis and metastatis of epithelial ovarian cancer (EOC). Aware that, several reports indicated that Histone deacetylases (HDACs) regulate the expression and activity of many proteins involved in both cancer initiation and progression, the aim of this work, was to study the effect of the HDAC inhibitor valproic acid (VPA) on the expression of CXCR7 as well as its impact on survival function in the epithelial ovarian cell line (SKOV-3). Methods: cells were cultured with varying concentrations of VPA (1, 2, 3, 4, 5 and 10 mM) for different durations (0, 12 h, 24 h and 48 h). Cell survival was assessed by Neutral red assay and by colony counting which being stained with crystal violet. CXCR7 expression was determined at mRNA level using quantitative real-time PCR (qRT-PCR) or at the protein level using western blotting. Results: VPA reduces cell survival of SKOV-3 cancer cells. The inhibition effect of VPA was dose and time-dependent. Exposure to VPA at concentrations above 2 mM at 24 h resulted in an increase expression of CXCR7 at both the mRNA and protein levels . Conclusion: These observations provide, for the first time, a better insight into the epigenetic mechanisms involved in regulating CXCR7 expression in EOC and will open new avenues for evaluating drugs that specifically stimulate the apoptosis of EOC with minimal unwanted side effect

EMT Markers in Locally-Advanced Prostate Cancer: Predicting Recurrence?

Background: Prostate cancer (PCa) is the second most frequent cause of cancer-related death in men worldwide. It is a heterogeneous disease at molecular and clinical levels which makes its prognosis and treatment outcome hard to predict. The epithelial-to-mesenchymal transition (EMT) marks a key step in the invasion and malignant progression of PCa. We sought to assess the co-expression of epithelial cytokeratin 8 (CK8) and mesenchymal vimentin (Vim) in locally-advanced PCa as indicators of EMT and consequently predictors of the progression status of the disease.Methods: Co-expression of CK8 and Vim was evaluated by immunofluorescence (IF) on paraffin-embedded tissue sections of 122 patients with PCa who underwent radical prostatectomies between 1998 and 2016 at the American University of Beirut Medical Center (AUBMC). EMT score was calculated accordingly and then correlated with the patients' clinicopathological parameters and PSA failure.Results: The co-expression of CK8/Vim (EMT score), was associated with increasing Gleason group. A highly significant linear association was detected wherein higher Gleason group was associated with higher mean EMT score. In addition, the median estimated biochemical recurrence-free survival for patients with < 25% EMT score was almost double that of patients with more than 25%. The validity of this score for prediction of prognosis was further demonstrated using cox regression model. Our data also confirmed that the EMT score can predict PSA failure irrespective of Gleason group, pathological stage, or surgical margins.Conclusion: This study suggests that assessment of molecular markers of EMT, particularly CK8 and Vim, in radical prostatectomy specimens, in addition to conventional clinicopathological prognostic parameters, can aid in the development of a novel system for predicting the prognosis of locally-advanced PCa

Rôle du système d'activation du plasminogène dans la différenciation des cellules souches embryonnaires de souris

Le système d’activation du plasminogène (AP) comprenant les protéases uPA et tPA, et leur inhibiteur PAI-1, génère une activité protéolytique dans la matrice extracellulaire et contribue au remodelage tissulaire dans une grande variété de processus physiopathologiques, y compris la myogenèse squelettique, et la différenciation adipocytaire.Nous avons évalué son rôle spécifique dans la différenciation des cellules souches embryonnaires (ES) de souris. On a trouvé que les activités d’uPA et de tPA ainsi que les niveaux protéiques de PAI-1 sont maximaux dans les cellules différenciées, contrairement aux cellules ES indifférenciées où ils sont indétectables et augmentent progressivement dès le jour 3 de la différenciation. La différenciation adipocytaire dans le modèle des cellules ES est inhibée par le traitement par l’amiloride, un inhibiteur spécifique de l’uPA. Egalement, les cellules ES surexprimant une forme active du PAI-1 sous le contrôle d’un système d’expression inductible, montrent des capacités adipogéniques réduites après l’induction du gène. Nos résultats démontrent que le contrôle de l’adipogenèse des cellules ES par le système AP correspond à des étapes successives, différentes, depuis les cellules indifférenciées jusqu’aux cellules bien différenciées. De plus, les capacités de la différenciation adipogénique des cellules pluripotentes induites déficientes en PAI-1 sont augmentées par rapport aux cellules contrôles.Similairement, la myogenèse squelettique est réduite par l’inhibition de l’uPA par l’amiloride ou par la surexpression du PAI-1 durant l’étape terminale de la différenciation du jour 7 au jour 24. Cependant, l’interférence avec l’uPA durant les jours 0 à 3 de la différenciation, stimule la formation des myotubes. Les différenciations cardiomyocyotaire, neuronale, endothéliale et du du muscle lisse ne sont pas affectées par le traitement à l’amiloride ou la surexpression du PAI-1.Nos résultats montrent que le système AP est capable de moduler spécifiquement l’adipogenèse et la myogenèse squelettique des cellules ES par des mécanismes moléculaires successifs différents.Regulation of the extracellular matrix (ECM) plays an important functional biological role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiationin vitro is unknown. We found that uPA and tPA activities and PAI-1 protein are very low in undifferentiated ESCs and increase strongly during the differentiation, reaching a maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system, display reduced adipogenic capacities after induction of the gene. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Furthermore, the adipogenic differentiation capacities of PAI-1-/- induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms

A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells

International audienceEmbryonic stem cells (ESCs) differentiate in vivo and in vitro into all cell lineages, and they have been proposed as cellular therapy for human diseases. However, the molecular mechanisms controlling ESC commitment toward specific lineages need to be specified. We previously found that the p38 mitogen-activated protein kinase (p38MAPK) pathway inhibits neurogenesis and is necessary to mesodermal formation during the critical first 5 days of mouse ESC commitment. This period corresponds to the expression of specific master genes that direct ESC into each of the three embryonic layers. By both chemical and genetic approaches, we found now that, during this phase, the p38MAPK pathway stabilizes the p53 protein level and that interfering directly with p53 mimics the effects of p38MAPK inhibition on ESC differentiation. Anti-p53 siRNA transient transfections stimulate Bcl2 and Pax6 gene expressions, leading to increased ESC neurogenesis compared with control transfections. Conversely, p53 downregulation leads to a strong inhibition of the mesodermal master genes Brachyury and Mesp1 affecting cardiomyogenesis and skeletal myogenesis of ESCs. Similar results were found with p53 À / À ESCs compared with their wild-type counterparts. In addition, knockout p53 ESCs show impaired smooth muscle cell and adipocyte formation. Use of anti-Nanog siRNAs demonstrates that certain of these regulations result partially to p53-dependent repression of Nanog gene expression. In addition to its well-known role in DNA-damage response, apoptosis, cell cycle control and tumor suppression, p53 has also been involved in vivo in embryonic development; our results show now that p53 mediates, at least for a large part, the p38MAPK control of the early commitment of ESCs toward mesodermal and neural lineages

p38 mitogen activated protein kinase controls two successive-steps during the early mesodermal commitment of embryonic stem cells.: P38 CONTROLS THE MESODERMAL COMMITMENT OF ES CELLS

International audienceEmbryonic stem (ES) cells differentiate in vitro into all cell lineages. We previously found that the p38 mitogen activated kinase (p38MAPK) pathway controls the commitment of ES cells toward either cardiomyogenesis (p38 on) or neurogenesis (p38 off ). In this study, we show that p38α knock-out ES cells do not differentiate into cardiac, endothelial, smooth muscle, and skeletal muscle lineages. Reexpression of p38MAPK in these cells partially rescues their mesodermal differentiation defects and corrects the high level of spontaneous neurogenesis of knock-out cells. Wild-type ES cells were treated with a p38MAPK-specific inhibitor during the differentiation process. These experiments allowed us to identify 2 early independent successive p38MAPK functions in the formation of mesodermal lineages. Further, the first one correlates with the regulation of the expression of Brachyury, an essential mesodermal-specific transcription factor, by p38MAPK. In conclusion, by genetic and biochemical approaches, we demonstrate that p38MAPK activity is essential for the commitment of ES cell into cardiac, endothelial, smooth muscle, and skeletal muscle mesodermal lineages

The Use of Stem Cell-Derived Organoids in Disease Modeling: An Update

Organoids represent one of the most important advancements in the field of stem cells during the past decade. They are three-dimensional in vitro culturing models that originate from self-organizing stem cells and can mimic the in vivo structural and functional specificities of body organs. Organoids have been established from multiple adult tissues as well as pluripotent stem cells and have recently become a powerful tool for studying development and diseases in vitro, drug screening, and host–microbe interaction. The use of stem cells—that have self-renewal capacity to proliferate and differentiate into specialized cell types—for organoids culturing represents a major advancement in biomedical research. Indeed, this new technology has a great potential to be used in a multitude of fields, including cancer research, hereditary and infectious diseases. Nevertheless, organoid culturing is still rife with many challenges, not limited to being costly and time consuming, having variable rates of efficiency in generation and maintenance, genetic stability, and clinical applications. In this review, we aim to provide a synopsis of pluripotent stem cell-derived organoids and their use for disease modeling and other clinical applications