Co-Activation of TGFβ and Wnt Signalling Pathways Abrogates EMT in Ovarian Cancer Cells

The aggressive property of ovarian cancer (OC) in terms of epithelialmesenchymal transition (EMT), proliferation and metastasis are of major concern. Different growth factors including TGFβ are associated with regulating these molecular events but the underlying mechanisms remain unclear. The aim of this report is to decipher the regulation of EMT by co-activation of TGFβ and Wnt signalling cascades in gaining malignancy. Methods: The expression of the different components of signalling events were analyzed by QPCR, Western blot, Immunofluorescence microscopy and flow cytometry. β-catenin promoter activity was checked by luciferase assay. Results: We observed reduced EMT in ovarian cancer cells upon co-activation with TGFβ1 and LiCl as shown by the expressions of epithelial/ mesenchymal markers and the EMT promoting factor, Snail1, accompanied by decrease in the invasion and migration of the cells compared to individual pathway activation. A detailed study of the mechanism suggested reduction in the β-catenin and p-GSK3b (Ser 9) levels to be the driving cause of this phenomenon, which was reversed upon co-activation with higher concentrations of LiCl. Conclusions: Therefore, tumourigenesis might be affected by the concentration of ligand/ growth factors for the respective signalling pathways activated in the tumour microenvironment and interaction between them might alter tumourigenesis

Lysophosphatidic Acid Promotes Epithelial to Mesenchymal Transition in Ovarian Cancer Cells by Repressing SIRT1

Epithelial-to-mesenchymal transition (EMT) plays an essential role in the transition from early to invasive phenotype, however the underlying mechanisms still remain elusive. Herein, we propose a mechanism through which the class-III deacetylase SIRT1 regulates EMT in ovarian cancer (OC) cells. Methods: Expression analysis was performed using Q-PCR, western blot, immunofluorescence and fluorescence-IHC study. Matrigel invasion assay was used for the invasion study. Morphological alterations were observed by phalloidin-staining. Co-immunoprecipitation study was performed to analyze protein-protein interaction. Results: Overexpression of SIRT1-WT as well as Resveratrol-mediated SIRT1 activation antagonized the invasion of OC cells by suppressing EMT. SIRT1 deacetylates HIF1α, to inactivate its transcriptional activity. To further validate HIF1α inactivation, its target gene, i.e. ZEB1, an EMTinducing factor was found to attenuate upon SIRT1 activation. To uncover the regulatory factor governing SIRT1 expression, lysophosphatidic acid (LPA), a highly enriched oncolipid in ascites/ serum of OC patients, was found to down-regulate SIRT1 expression. Importantly, LPA was found to induce the mesenchymal switch in OC cells through suppression of SIRT1. Decreased level of SIRT1 was further validated in ovarian tissue samples of OC patients. Conclusion: We have identified a mechanism that relates SIRT1 down-regulation to LPA-induced EMT in OC cells and may open new arenas on developing novel anti-cancer therapeutics

Чинники ефективності антикризового управління суб'єктами господарювання в економіці України

У статті розглядаються проблеми формування підходів організації антикризового управління суб'єктами господарювання в економіці України. На ґрунті вітчизняного та зарубіжного досвіду й результатів власних досліджень автора запропоновано психологічний тип антикризового менеджера. (The article is devoted to the problems of forming of approaches of organization of anticrisis management by the subjects of menage in the economy of Ukraine. On the base of domestic and foreign experience and results of own researches of author the psychological type of anticrisis manager is offered.

The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis

Background/Aims: The conformation, folding and lipid binding properties of the intestinal fatty acid binding proteins (IFABP) have been extensively investigated. In contrast, the functional aspects of these proteins are not understood and matter of debates. In this study, we aim to address the deleterious effects of FA overload on cellular components, particularly mitochondria; and how IFABP helps in combating this stress by restoring the mitochondrial dynamics. Methods: In the present study the functional aspect of IFABP under conditions of lipid stress was studied by a string of extensive in-cell studies; flow cytometry by fluorescence-activated cell sorting (FACS), confocal imaging, western blotting and quantitative real time PCR. We deployed ectopic expression of IFABP in rescuing cells under the condition of lipid stress. Again in order to unveil the mechanistic insights of functional traits, we arrayed extensive computational approaches by means of studying centrality calculations along with protein-protein association and ligand induced cluster dissociation. While addressing its functional importance, we used FCS and in-silico computational analyses, to show the structural distribution and the underlying mechanism of IFABP’s action. Results: Ectopic expression of IFABP in HeLa cells has been found to rescue mitochondrial morphological dynamics and restore membrane potential, partially preventing apoptotic damage induced by the increased FAs. These findings have been further validated in the functionally relevant intestinal Caco-2 cells, where the native expression of IFABP protects mitochondrial morphology from abrogation induced by FA overload. However, this native level expression is insufficient to protect against apoptotic cell death, which is rescued, at least partially in cells overexpressing IFABP. In addition, shRNA mediated IFABP knockdown in Caco-2 cells compromises mitochondrial dynamics and switches on intrinsic apoptotic pathways under FA-induced metabolic stress. Conclusion: To summarize, the present study implicates functional significance of IFABP in controlling ligand-induced damage in mitochondrial dynamics and apoptosis

Invasion of ovarian cancer cells is induced by PITX2-mediated activation of TGF-β and Activin-A

Background:Most ovarian cancers are highly invasive in nature and the high burden of metastatic disease make them a leading cause of mortality among all gynaecological malignancies. The homeodomain transcription factor, PITX2 is associated with cancer in different tissues. Our previous studies demonstrated increased PITX2 expression in human ovarian tumours. Growing evidence linking activation of TGF-β pathway by homeodomain proteins prompted us to look for the possible involvement of this signalling pathway in PITX2-mediated progression of ovarian cancer. Methods: The status of TGF-β signalling in human ovarian tissues was assessed by immunohistochemistry. The expression level of TGFB/INHBA and other invasion-associated genes was measured by quantitative-PCR (Q-PCR) and Western Blot after transfection/treatments with clones/reagents in normal/cancer cells. The physiological effect of PITX2 on invasion/motility was checked by matrigel invasion and wound healing assay. The PITX2- and activin-induced epithelial-mesenchymal transition (EMT) was evaluated by Q-PCR of respective markers and confocal/phase-contrast imaging of cells. Results: Human ovarian tumours showed enhanced TGF-β signalling. Our study uncovers the PITX2-induced expression of TGFB1/2/3 as well as INHBA genes (p < 0.01) followed by SMAD2/3-dependent TGF-β signalling pathway. PITX2-induced TGF-β pathway regulated the expression of invasion-associated genes, SNAI1, CDH1 and MMP9 (p < 0.01) that accounted for enhanced motility/invasion of ovarian cancers. Snail and MMP9 acted as important mediators of PITX2-induced invasiveness of ovarian cancer cells. PITX2 over-expression resulted in loss of epithelial markers (p < 0.01) and gain of mesenchymal markers (p < 0.01) that contributed significantly to ovarian oncogenesis. PITX2-induced INHBA expression (p < 0.01) contributed to EMT in both normal and ovarian cancer cells. Conclusions: Overall, our findings suggest a significant contributory role of PITX2 in promoting invasive behaviour of ovarian cancer cells through up-regulation of TGFB/INHBA. We have also identified the previously unknown involvement of activin-A in promoting EMT. Our work provides novel mechanistic insights into the invasive behavior of ovarian cancer cells. The extension of this study have the potential for therapeutic applications in future

ETS-1 Protein Regulates Vascular Endothelial Growth Factor-induced Matrix Metalloproteinase-9 and Matrix Metalloproteinase-13 Expression in Human Ovarian Carcinoma Cell Line SKOV-3

The mechanism of vascular endothelial growth factor (VEGF)-regulated expression of MMPs followed by cancer cell scattering/invasion is poorly understood. VEGF induces MMP-9, MMP-13, and ETS-1 through PI3K/AKT and p38 MAPK pathways in SKOV-3 cells. VEGF induces ETS-1, which activates specific MMPS, leading to the invasion/scattering in SKOV-3 cells. This study provides useful information that reveals the molecular mechanism of ovarian cancer metastasis

Fatty acid Represses Insulin Receptor Gene Expression by Impairing HMGA1 through Protein Kinase Ce

It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p < 0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKCe was also inhibited. Surprisingly, phosphorylation of cytosolic PKCe was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKCe translocation inhibitor peptide, eV1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKCe migration to implement palmitate effect. Experimental evidences indicate that phospho-PKCe adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity

Procollagen Synthesis is Increased in Hypothyroid Rat Ovary by a Parallel and Compensatory Pathway

Collagen biosynthesis is a multistep process that starts with the transcription and translation of the individual collagen gene. It is characterized by the presence of a large number of co- and posttranslational modifications. Hydroxylysine is found only in animal proteins and mostly in collagens. Procollagen lysyl hydroxylation is the first step in collagen biosynthetic pathway and lysyl hydroxylases (Plod isoforms) are responsible for this enzymatic process. Previously we showed the down regulation of Plod isoforms in hypothyroid ovary. As hypothyroidism is a stress for normal animals, we wanted to explore whether any compensatory pathway exists to balance the reduced lysyl hydroxylation of collagen in hypothyroid rat ovary. In this report we have shown that procollagen I and III are increased in hypothyroid condition and subsequently decreased upon T3 addback. Heat Shock Protein-47 is a collagen-specific molecular chaperone and its existence in ovary has been documented. The genes encoding HSP-47,prolyl-4-hydroxylase-α and -β (P4H- α and - β) are increased in hypothyroid condition. Down regulation of lysyl hydroxylase in hypothyroid condition results less collagen formation. At the same time over production of procollagens, HSP-47 and P4H is very significant as they may compensate the damage whatsoever caused due to hypothyroidism in ovarian tissue

Fenofibrate Reverses Palmitate Induced Impairment in Glucose Uptake in Skeletal Muscle Cells by Preventing Cytosolic Ceramide Accumulation

Backgrounds/Aims: The lipid induced insulin resistance is a major pathophysiologic mechanism underlying glucose intolerance of varying severity. PPARα-agonists are proven as effective hypolipidemic agents. The aim of this study was to see if impaired glucose uptake in palmitate treated myotubes is reversed by fenofibrate. Methods: Palmitate-treated myotubes were used as a model for insulin resistance, impaired glucose uptake, fatty acid oxidation and ceramide synthesis. mRNA levels of CPT1 and CPT2 were determined by PCR array and Q-PCR. Results: The incubation of myotubes with 750 uM palmitate not only reduced glucose uptake but also impaired fatty acid oxidation and cytosolic ceramide accumulation. Palmitate upregulated CPT1b expression in L6 myotubes, while CPT2 expression level remained unchanged. The altered stoichiometric ratio between the two CPT isoforms led to reduced fatty acid oxidation (FAO), ceramide accumulation and impaired glucose uptake, whereas administration of 200 µM fenofibrate signifcantly reversed the above abnormalities by increasing CPT2 mRNA levels and restoring CPT1b to CPT2 ratio. Conclusion: Palmitate-induced alteration in the stoichiometric ratio of mitochondrial CPT isoforms leads to incomplete FAO and enhanced cytosolic ceramide accumulation that lead to insulin resistance. Fenofibrate ameliorated insulin resistance by restoring the altered stoichiometry by upregulating CPT2 and preventing, cytoplasmic ceramide accumulation