Function of caspase-14 in trophoblast differentiation

<p>Abstract</p> <p>Background</p> <p>Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored.</p> <p>Methods</p> <p>Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways.</p> <p>Results</p> <p>Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted.</p> <p>Conclusion</p> <p>Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.</p

Mechanisms of cisplatin-induced cell death in malignant mesothelioma cells: Role of inhibitor of apoptosis proteins (IAPs) and caspases

Malignant mesothelioma (MM) is an aggressive and highly chemoresistant tumour. Although cisplatin is used in frontline therapy of this disease treatment remains palliative at best. The biochemical pathways activated by cisplatin and the mechanisms of resistance in mesothelioma cells are poorly understood. Overexpression of inhibitor of apoptosis proteins (IAPs) has been described in clinical mesothelioma tumours and proposed as therapeutic targets. In this study, we examined cisplatin-induced cell death pathways and IAPs in three mesothelioma-derived cell lines. Cisplatin induced cell death in mesothelioma cell lines was characterised by biochemical mechanisms classically associated with apoptosis including: mitochondrial depolarisation, phosphatidylserine translocation and caspase activation. Surprisingly mRNA expression of IAPs in mesothelioma was not upregulated relative to primary mesothelial cells except for survivin which was higher in the most resistant cell line. In contrast, protein expression of both XIAP and survivin was upregulated in all mesothelioma cells, consistent with post-translational regulation. Knockdown of either XIAP or survivin by RNAi did not affect the sensitivity to cisplatin in any of the cell lines. Survivin RNAi did, however, inhibit proliferation in the highest expressing cell line, ONE58. The pan-caspase inhibitor z-VAD and the more selective caspase 3/7 inhibitor z-DEVD had no effect upon the sensitivity of any of the cell lines to cisplatin indicating that caspase-independent pathways predominate. The findings of the present study provide insights into cisplatin-induced mechanisms in mesothelioma cells and show that alternative pathways are operating which may provide new options for targeting this extremely resistant tumour

Wnt antagonist secreted frizzled-related protein 4 upregulates adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells

With more than 1.4 billion overweight or obese adults worldwide, obesity and progression of the metabolic syndrome are major health and economic challenges. To address mechanisms of obesity, adipose tissue-derived mesenchymal stem cells (ADSCs) are being studied to detail the molecular mechanisms involved in adipogenic differentiation. Activation of the Wnt signalling pathway has inhibited adipogenesis from precursor cells. In our study, we examined this anti-adipogenic effect in further detail stimulating Wnt with lithium chloride (LiCl) and 6-bromo indirubin 3'oxime (BIO). We also examined the effect of Wnt inhibition using secreted frizzled-related protein 4 (sFRP4), which we have previously shown to be pro-apoptotic, anti-angiogenic, and anti-tumorigenic. Wnt stimulation in LiCl and BIOtreated ADSCs resulted in a significant reduction (2.7-fold and 12-fold respectively) in lipid accumulation as measured by Oil red O staining while Wnt inhibition with sFRP4 induced a 1.5-fold increase in lipid accumulation. Furthermore, there was significant 1.2-fold increase in peroxisome proliferator-activated receptor gamma (PPAR ?) and CCAAT/enhancer binding protein alpha (C/EBPa), and 1.3-fold increase in acetyl CoA carboxylase protein levels. In contrast, the expression of adipogenic proteins (PPAR?, C/EBPa, and acetyl CoA carboxylase) were decreased significantly with LiCl (by 1.6, 2.6, and 1.9-fold respectively) and BIO (by 7, 17, and 5.6-fold respectively) treatments. These investigations demonstrate interplay between Wnt antagonism and Wnt activation during adipogenesis and indicate pathways for therapeutic intervention to control this process

Multifunctional Properties of Chicken Embryonic Prenatal Mesenchymal Stem Cells- Pluripotency, Plasticity, and Tumor Suppression

The chick embryo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis, and loss/gain of function experiments. In the present study, we assessed and characterized bone marrow derived mesenchymal stem cells from prenatal day 13 chicken embryos (chBMMSCs) and determined some novel properties. After assessing the mesenchymal stem cell (MSC) properties of these cells by the presence of their signature markers (CD 44, CD 73, CD 90, CD 105, and vimentin), we ascertained a very broad spectrum of multipotentiality as these MSCs not only differentiated into the classic tri-lineages of MSCs but also into ectodermal, endodermal, and mesodermal lineages such as neuron, hepatocyte, islet cell, and cardiac. In addition to wide plasticity, we detected the presence of several pluripotent markers such as Oct4, Sox2, and Nanog. This is the first study characterizing prenatal chBMMSCs and their ability to not only differentiate into mesenchymal lineages but also into all the germ cell layer lineages. Furthermore, our studies indicate that prenatal chBMMSCs derived from the chick provide an excellent model for multi-lineage development studies because of their broad plasticity and faithful reproduction of MSC traits as seen in the human. Here, we also present evidence for the first time that media derived from prenatal chBMMSC cultures have an anti-tumorigenic, anti-migratory, and pro-apoptotic effect on human tumors cells acting through the Wnt-ß-catenin pathway. These data confirm that chBMMSCs are enriched with factors in their secretome that are able to destroy tumor cells. This suggests a commonality of properties of MSCs across species between human and chicken

The effects of phenoxodiol on the cell cycle of prostate cancer cell lines

Background: Prostate cancer is associated with a poor survival rate. The ability of cancer cells to evade apoptosis and exhibit limitless replication potential allows for progression of cancer from a benign to a metastatic phenotype. The aim of this study was to investigate in vitro the effect of the isoflavone phenoxodiol on the expression of cell cycle genes. Methods: Three prostate cancer cell lines-LNCaP, DU145, and PC3 were cultured in vitro, and then treated with phenoxodiol (10 μM and 30 μM) for 24 and 48 h. The expression of cell cycle genes p21WAF1, c-Myc, Cyclin-D1, and Ki-67 was investigated by Real Time PCR. Results: Here we report that phenoxodiol induces cell cycle arrest in the G1/S phase of the cell cycle, with the resultant arrest due to the upregulation of p21WAF1 in all the cell lines in response to treatment, indicating that activation of p21WAF1 and subsequent cell arrest was occurring via a p53 independent manner, with induction of cytotoxicity independent of caspase activation. We found that c-Myc and Cyclin-D1 expression was not consistently altered across all cell lines but Ki-67 signalling expression was decreased in line with the cell cycle arrest. Conclusions: Phenoxodiol demonstrates an ability in prostate cancer cells to induce significant cytotoxicity in cells by interacting with p21WAF1 and inducing cell cycle arrest irrespective of p53 status or caspase pathway interactions. These data indicate that phenoxodiol would be effective as a potential future treatment modality for both hormone sensitive and hormone refractory prostate cancer

Cancer stem cell metabolism: A potential target for cancer therapy

© 2016 The Author(s). Cancer Stem cells (CSCs) are a unipotent cell population present within the tumour cell mass. CSCs are known to be highly chemo-resistant, and in recent years, they have gained intense interest as key tumour initiating cells that may also play an integral role in tumour recurrence following chemotherapy. Cancer cells have the ability to alter their metabolism in order to fulfil bio-energetic and biosynthetic requirements. They are largely dependent on aerobic glycolysis for their energy production and also are associated with increased fatty acid synthesis and increased rates of glutamine utilisation. Emerging evidence has shown that therapeutic resistance to cancer treatment may arise due to dysregulation in glucose metabolism, fatty acid synthesis, and glutaminolysis. To propagate their lethal effects and maintain survival, tumour cells alter their metabolic requirements to ensure optimal nutrient use for their survival, evasion from host immune attack, and proliferation. It is now evident that cancer cells metabolise glutamine to grow rapidly because it provides the metabolic stimulus for required energy and precursors for synthesis of proteins, lipids, and nucleic acids. It can also regulate the activities of some of the signalling pathways that control the proliferation of cancer cells. This review describes the key metabolic pathways required by CSCs to maintain a survival advantage and highlights how a combined approach of targeting cellular metabolism in conjunction with the use of chemotherapeutic drugs may provide a promising strategy to overcome therapeutic resistance and therefore aid in cancer therapy

Cloning and characterization of an apoptosis-associated gene in the human placenta

Placenta is a transient feto-maternal association that develops during mammalian pregnancies. Human placental tissue during the first trimester of pregnancy is an actively dividing and differentiating tissue, while near term, it represents a fully differentiated unit performing many life-sustaining functions for the fetus. Previous studies have demonstrated that the percentage of placental cells that undergo apoptosis is greater at full term as compared to the first trimester of pregnancy. In this study, we undertook a study aimed at gaining an insight into the kind of genes expressed in the two developmentally distinct stages of gestation ie, the first trimester and term using Differential Display RT-PCR. Cloning and sequencing of one of the differentially expressed cDNAs from term placental tissue revealed that it is a novel gene, referred to as T-18 in the text. In this study, we also examined the regulation of this gene during apoptosis in the human placenta. A model for analysis of placental apoptosis was established by incubating placental villi in serum-free culture medium. It was observed that apoptosis occurred rapidly following incubation of placental villi without tropic support, and the proposed free-radical scavenger, superoxide dismutase (SOD) suppressed apoptosis in the placenta. Interestingly, the levels of T-18 mRNA increased significantly during spontaneous induction of apoptosis and decreased when apoptosis was blocked by SOD. These data clearly suggest that there is a strong correlation between the expression of T-18 and placental apoptosis and that T-18, may play a significant role in this process. Furthermore, the establishment of a defined in vitro explant culture model should facilitate elucidation of factors, which regulate apoptosis in human placenta

Cloning and characterization of an apoptosis-associated gene in the human placenta

Placenta is a transient feto-maternal association that develops during mammalian pregnancies. Human placental tissue during the first trimester of pregnancy is an actively dividing and differentiating tissue, while near term, it represents a fully differentiated unit performing many life-sustaining functions for the fetus. Previous studies have demonstrated that the percentage of placental cells that undergo apoptosis is greater at full term as compared to the first trimester of pregnancy. In this study, we undertook a study aimed at gaining an insight into the kind of genes expressed in the two developmentally distinct stages of gestation ie, the first trimester and term using Differential Display RT-PCR. Cloning and sequencing of one of the differentially expressed cDNAs from term placental tissue revealed that it is a novel gene, referred to as T-18 in the text. In this study, we also examined the regulation of this gene during apoptosis in the human placenta. A model for analysis of placental apoptosis was established by incubating placental villi in serum-free culture medium. It was observed that apoptosis occurred rapidly following incubation of placental villi without tropic support, and the proposed free-radical scavenger, superoxide dismutase (SOD) suppressed apoptosis in the placenta. Interestingly, the levels of T-18 mRNA increased significantly during spontaneous induction of apoptosis and decreased when apoptosis was blocked by SOD. These data clearly suggest that there is a strong correlation between the expression of T-18 and placental apoptosis and that T-18, may play a significant role in this process. Furthermore, the establishment of a defined in vitro explant culture model should facilitate elucidation of factors, which regulate apoptosis in human placenta

Number and brightness analysis of sFRP4 domains in live cells demonstrates vesicle association signal of the NLD domain and dynamic intracellular responses to Wnt3a.

The Wnts are secreted, lipidated glycoproteins that play a role in cellular processes of differentiation, proliferation, migration, survival, polarity and stem cell self-renewal. The majority of Wnts biological effects are through binding to specific frizzled (Fzd) receptor complexes leading to activation of downstream pathways. Secreted frizzled-related proteins (sFRPs) were first identified as antagonists of Wnt signalling by binding directly to Wnts. They comprise two domains, a Fzd-like cysteine rich domain (CRD) and a netrin-like domain (NLD). Subsequently sFRPs have been shown to also interact with Fzd receptors and more diverse functions have been identified, including potentiation of Wnt signalling. Many aspects of the biology of this family remain to be elucidated. We used the number and brightness (N&amp;B) method, a technique based on fluorescence fluctuation analysis, to characterise the intracellular aggregation and trafficking of sFRP4 domains. We expressed sFRP4 and its' domains as EGFP fusions and then characterised the effect of endogenous Wnt3a by fluorescence confocal imaging. We observed vesicular trafficking of sFRP4 and that the NLD domain has a vesicular association signal. We found that sFRP4 and the CRD formed oligomeric aggregates in the perinuclear region while the NLD was distributed evenly throughout the cell with a larger proportion of aggregates. Most significantly we observed intracellular redistribution of sFRP4 in response to Wnt3a suggesting that Wnt3a can modulate intracellular localisation and secretion of sFRP4. Our results reveal a number of novel findings regarding sFRP4 which are likely to have relevance to this wider family