The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system.

Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 µm (P < 0.05), while not affecting cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 µm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 µm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.

Computational model for fetal skeletal defects potentially linked to disruption of retinoic acid signaling.

All-trans retinoic acid (ATRA) gradients determine skeletal patterning morphogenesis and can be disrupted by diverse genetic or environmental factors during pregnancy, leading to fetal skeleton defects. Adverse Outcome Pathway (AOP) frameworks for ATRA metabolism, signaling, and homeostasis allow for the development of new approach methods (NAMs) for predictive toxicology with less reliance on animal testing. Here, a data-driven model was constructed to identify chemicals associated with both ATRA pathway bioactivity and prenatal skeletal defects. The phenotype data was culled from ToxRefDB prenatal developmental toxicity studies and produced a list of 363 ToxRefDB chemicals with altered skeletal observations. Defects were classified regionally as cranial, post-cranial axial, appendicular, and other (unspecified) features based on ToxRefDB descriptors. To build a multivariate statistical model, high-throughput screening bioactivity data from >8,070 chemicals in ToxCast/Tox21 across 10 in vitro assays relevant to the retinoid signaling system were evaluated and compared to literature-based candidate reference chemicals in the dataset. There were 48 chemicals identified for effects on both in vivo skeletal defects and in vitro ATRA pathway targets for computational modeling. The list included 28 chemicals with prior evidence of skeletal defects linked to retinoid toxicity and 20 chemicals without prior evidence. The combination of thoracic cage defects and DR5 (direct repeats of 5 nucleotides for RAR/RXR transactivation) disruption was the most frequently occurring phenotypic and target disturbance, respectively. This data model provides valuable AOP elucidation and validates current mechanistic understanding. These findings also shed light on potential avenues for new mechanistic discoveries related to ATRA pathway disruption and associated skeletal dysmorphogenesis due to environmental exposures.

Advances in the Detection of Pancreatic Cancer Through Liquid Biopsy.

Pancreatic cancer refers to the development of malignant tumors in the pancreas: it is associated with high mortality rates and mostly goes undetected in its early stages for lack of symptoms. Currently, surgical treatment is the only effective way to improve the survival of pancreatic cancer patients. Therefore, it is crucial to diagnose the disease as early as possible in order to improve the survival rate of patients with pancreatic cancer. Liquid biopsy is a unique in vitro diagnostic technique offering the advantage of earlier detection of tumors. Although liquid biopsies have shown promise for screening for certain cancers, whether they are effective for early diagnosis of pancreatic cancer is unclear. Therefore, we reviewed relevant literature indexed in PubMed and collated updates and information on advances in the field of liquid biopsy with respect to the early diagnosis of pancreatic cancer.

Tumor Development and Angiogenesis in Adult Brain Tumor: Glioblastoma.

Angiogenesis is the growth of new capillaries from the preexisting blood vessels. Glioblastoma (GBM) tumors are highly vascularized tumors, and glioma growth depends on the formation of new blood vessels. Angiogenesis is a complex process involving proliferation, migration, and differentiation of vascular endothelial cells (ECs) under the stimulation of specific signals. It is controlled by the balance between its promoting and inhibiting factors. Various angiogenic factors and genes have been identified that stimulate glioma angiogenesis. Therefore, attention has been directed to anti-angiogenesis therapy in which glioma proliferation is inhibited by inhibiting the formation of new tumor vessels using angiogenesis inhibitory factors and drugs. Here, in this review, we highlight and summarize the various molecular mediators that regulate GBM angiogenesis with focus on recent clinical research on the potential of exploiting angiogenic pathways as a strategy in the treatment of GBM patients.

Elucidating the microRNA-203 specific biological processes in glioblastoma cells from comprehensive RNA-sequencing transcriptome profiling.

Glioblastoma (GBM) is the most common primary malignant intracranial adult brain tumor. Allelic deletion on chromosome 14q plays an essential role in GBM pathogenesis, and this chromosome 14q site was thought to harbor multiple tumor suppressor genes associated with GBM, a region that also encodes microRNA-203 (miR-203). This study was conducted to identify whole transcriptome profile changes associated with miR-203 expression by high-throughput RNA sequencing. Enrichment analyses for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that miR-203 expression had a strong, negative effect on a number of fundamental and interconnected biological processes involved in cell growth and proliferation. The biological processes mostly influenced were p53 signaling pathway, FoxO signaling pathway, DNA replication, cell cycle, MAPK signaling pathway, and apoptosis. In total, 847 upregulated and 345 downregulated differentially expressed genes were identified in control versus miR-203 expressing glioma cells. After GO enrichment, the downregulated differentially expressed genes such as BCL2, SPARC were found to be mainly enriched in cell cycle regulation and apoptosis processes, whereas the upregulated differentially expressed genes such as CCND1, E2F1 were involved in the DNA replication and cell cycle regulation. We also performed miR-203 target analysis and found BCL2, AKT, SPARC, ROBO1, c-JUN, PDGFA, and CREB were predicted target of miR-203 and miR-203 expression suppressed the protein and mRNA levels of these target genes by western blotting and qRT-PCR analysis. Moreover, co-transfection experiments using a luciferase-based reporter assay demonstrated that miR-203 directly regulated BCL-2 expression and BCL-2 overexpression suppressed miR-203 mediated glioma cell apoptosis. These results indicate that overexpression of miR-203 coordinately regulates several oncogenic pathways in GBM.

Single nucleotide polymorphism of rs2596542 and the risk of hepatocellular carcinoma development: A meta-analysis.

BACKGROUND: Major histocompatibility complex class I-related chain A (MICA) is considered as a tumor antigen, and its expression is affected by its genetic polymorphisms. However, the relationship between rs2596542 polymorphisms in MICA promoter region and hepatocellular carcinoma (HCC) is not fully elucidated so far. This study aims to explore the relationship between single nucleotide polymorphism of rs2596542 and the risk of HCC development through meta-analysis. METHODS: MEDLINE, Web of Science, and EMBASE databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between MICA rs2596542 polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: Fourteen case-control studies involving 4,900 HCC cases and 19,519 controls were included. The MICA rs2596542C allele was significantly associated with decreased risk of HCC based on allelic contrast (OR = 0.76, 95% CI = 0.69-0.83, P < .001), homozygote comparison (OR = 0.57, 95% CI = 0.48-0.69, P < .001), and a recessive genetic model (OR = 0.77, 95% CI = 0.65-0.91, P < .001), whereas patients carrying the MICA rs2596542TT genotype had significantly higher risk of HCC than those with the CT or CC genotype (TT vs CT + CC, OR = 1.57, 95% CI = 1.36-1.81, P < .001). Subgroups analyses based on the ethnic or the source of control groups found very similar findings. CONCLUSION: The C allele in MICA rs2596542 is a protective factor for hepatocarcinogenesis, whereas the T allele is a risk factor. Further large and well-designed studies are needed to confirm this conclusion.

Updates and advancements in the management of hepatocellular carcinoma patients after hepatectomy.

Introduction: The 5-year recurrence rate of hepatocellular carcinoma (HCC) after hepatic resection or local ablation is up to 70%. Adjuvant therapies to prevent HCC recurrence have been reported but are not currently recommended by EASL or AASLD guidelines. This review examined evidence from randomized controlled trials, meta-analyses and systematic reviews on the safety and efficacy of adjuvant therapies and chemotherapies in HCC patients after resection or local ablation.Areas covered: PubMed was searched through 15 June 2019. Available evidence was assessed based on the GRADE system.Expert commentary: Transarterial chemoembolization is the best adjuvant therapy for HCC patients at high risk of recurrence, antiviral therapy with nucleoside analogs is effective for preventing recurrence of HBV-related HCC, and interferon-α is effective for preventing recurrence of HCV-related HCC. Further studies are needed to clarify the efficacy of adjuvant immune checkpoint inhibitors. Adjuvant sorafenib appears to offer negligible clinical benefit and high risk of adverse effects.

SPARC overexpression alters microRNA expression profiles involved in tumor progression.

Medulloblastoma is the most common malignant brain tumor in children. SPARC (secreted protein acidic and rich in cysteine), a multicellular non-structural glycoprotein is known to be involved in multiple processes in various cancers. Previously, we reported that SPARC expression significantly impairs medulloblastoma tumor growth in vitro and in vivo and also alters chemo sensitivity. MicroRNAs are a class of post-transcriptional gene regulators with critical functions in tumor progression. In addition, microRNA (miRNA) expression changes are also involved in chemo-resistance. Herein, we assessed microRNA (miRNA) profiling to identify the functional network and biological pathways altered in SPARC-overexpressed medulloblastoma cells. A total of 27 differentially expressed miRNAs were identified between the control and SPARC-overexpressed samples. Potential messenger RNA (mRNA) targets of the differentially expressed miRNA were identified using Ingenuity Pathway Analysis (IPA). Network-based functional analyses were performed on the available human protein interaction and miRNA-gene association data to highlight versatile miRNAs among the significantly deregulated miRNAs using the IPA, and the biological pathway analysis using the PANTHER web-based tool. We have identified six miRNAs (miR-125b1*, miR-146a-5p, miR-181a-5p, miR-204-5p, miR-219-5p and miR-509-3p) that are associated with SPARC sensitivity by comparison of miRNA expression patterns from the SPARC treated cells with the control cells. Furthermore, pathway enrichment analysis outline that these six microRNAs mainly belong to biological processes related to cancer related signaling pathways. Collectively, these studies have the potential to indicate novel biomarkers for treatment response and can also be applied to develop novel therapeutic treatment for medulloblastoma.

MicroRNAs in glioblastoma pathogenesis and therapy: A comprehensive review.

Glioblastoma (GBM), also known as grade IV astrocytoma, is the most aggressive primary intracranial tumor of the adult brain. MicroRNAs (miRNAs), a class of small non-coding RNA species, have critical functions across various biological processes. A great deal of progress has been made recently in dissecting miRNA pathways associated with the pathogenesis of GBM. miRNA expression signatures called gene signatures also characterize and contribute to the phenotypic diversity of GBM subclasses through their ability to regulate developmental growth and differentiation. miRNA molecules have been identified as diagnostic and prognostic biomarkers for patient stratification and may also serve as therapeutic targets and agents. This review summarizes: (i) the current understanding of the roles of miRNAs in the pathogenesis of GBM, (ii) the potential use of miRNAs in GBM diagnosis and glioma grading, (iii) further prospects of developing miRNAs as novel biomarkers and therapeutic targets for GBM, and (iv) important practical considerations when considering miRNA therapy for GBM patients.

Developmental cardiotoxicity effects of four commonly used antiepileptic drugs in embryonic chick heart micromass culture and embryonic stem cell culture systems.

Antiepileptic drugs (AEDs) are commonly used drugs in pregnant women with epilepsy. Prenatal exposure to AEDs increases the risk of major or minor congenital malformation during embryonic development. The precise mode of action and intracellular mechanisms of these AEDs during embryonic development remains unclear. To determine relative teratogenic risk of AEDs, four AED drugs including valproic acid (VPA), phenytoin (PHT), phenobarbital (PB), and trimethadione (TMD) were tested using two in vitro systems (the embryonic chick heart micromass (MM) culture and the in vitro differentiating mouse embryonic stem cells into cardiomyocytes culture systems). Cardiomyocyte cultures (the heart MM and ES cell-derived cardiomyocytes) were treated with or without different concentrations of PHT, PB, TMD (10-100 µM), and VPA (100-2000 µM). 5-Fluorouracil (5-FU) (1-10 µM) and l-ascorbic acid (10-1000 µM) were used as positive and negative controls. It was found that these four commonly used AEDs and 5-FU tested have the potential to inhibit embryonic heart cell differentiation (p<0.05) without inducing any cytotoxicity. VPA at higher concentrations (≥800 µM), and 5-FU at all doses proved to be cytotoxic in the differentiating ES cell culture system. The results demonstrated in this study suggest that the use of these four commonly prescribed AEDs during pregnancy may have an effect on embryonic heart development, and may be associated with congenital cardiovascular defects.