Margarita Hadzopoulou-Cladaras

An HNF4α-miRNA Inflammatory Feedback Circuit Regulates Hepatocellular Oncogenesis

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.

The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression.

ABSTRACT The orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function.

The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B‐100 (apoB‐100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5′ and 3′ untranslated regions which are 128 and 301 nucleotides respectively. The DNA‐derived protein sequence shows that apoB‐100 is 513,000 daltons and contains 4560 amino acids including a 24‐amino‐acid‐long signal peptide. The mol. wt of apoB‐100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non‐amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid‐binding domains of apoB‐100. The protein contains 25 cysteines and 20 potential N‐glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor‐binding domain of apoB‐100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15‐kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB‐100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.

CREB-binding Protein Is a Transcriptional Coactivator for Hepatocyte Nuclear Factor-4 and Enhances Apolipoprotein Gene Expression

Hepatocyte nuclear factor-4 (HNF-4) is a liver-enriched transcription factor that is crucial in the regulation of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism and in determining the hepatic phenotype. We have previously shown that HNF-4 contains transcription activation functions at the N terminus (AF-1) and the C terminus (AF-2) which work synergistically to confer full HNF-4 activity. Here, we show that HNF-4 recruits the CREB-binding protein (CBP) coactivator on promoters of genes that contain functional HNF-4 sites. HNF-4 interacts with the N-terminal region of CBP (amino acids 1–771) and the C-terminal region of CBP (amino acids 1812–2441). The two activating functions of HNF-4, AF-1 and AF-2, interact with the N terminus and the N and C terminus of CBP, respectively. In addition, we show that in contrast to the other nuclear hormone receptors the interaction between HNF-4 and CBP is ligand-independent. Recruitment of CBP by HNF-4 results in an enhancement of the transcriptional activity of the latter. CBP does not activate gene expression in the absence of HNF-4, and dominant negative forms of HNF-4 prevent transcriptional activation by CBP, suggesting that the mere recruitment of CBP by HNF-4 is not sufficient for enhancement of gene expression. These findings demonstrate that CBP acts as a transcriptional coactivator for HNF-4 and provide new insights into the regulatory function of HNF-4.

Camphene, a Plant-Derived Monoterpene, Reduces Plasma Cholesterol and Triglycerides in Hyperlipidemic Rats Independently of HMG-CoA Reductase Activity

Background Central to the pathology of coronary heart disease is the accumulation of lipids, cholesterol and triglycerides, within the intima of arterial blood vessels. The search for drugs to treat dislipidemia, remains a major pharmaceutical focus. In this study, we evaluated the hypolipidemic properties of the essential oil from Chios mastic gum (MGO). Methodology/Principal Findings The hypolipidemic effect of MGO was investigated in naïve as well as in rats susceptible to detergent-induced hyperlipidemia. Serum cholesterol and triglycerides were determined using commercial kits. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity was measured in HepG2 cell extracts using a radioactive assay; cellular cholesterol and cholesterol esters were assessed using gas chromatography. MGO administration into naïve rats resulted in a dose-dependent reduction in the constitutive synthesis of serum cholesterol and triglycerides. In hyperlipidemic rats, MGO treatment had also a strong hypolipidemic effect. By testing various components of MGO, we show for the first time that the hypolipidemic action is associated with camphene. Administration of camphene at a dose of 30 µg/gr of body weight in hyperlipidemic rats resulted in a 54.5% reduction of total cholesterol (p<0.001), 54% of Low Density Lipoprotein (LDL)-cholesterol (p<0.001) and 34.5% of triglycerides (p<0.001). Treatment of HepG2 cells with camphene led to a decrease in cellular cholesterol content to the same extend as mevinolin, a known HMG-CoA reductase inhibitor. The hypolipidemic action of camphene is independent of HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. Conclusions Given the critical role that the control of hyperlipidemia plays in cardiovascular disease, the results of our study provide insights into the use of camphene as an alternative lipid lowering agent and merits further evaluation.

Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

Differential expression of IL-17, 22 and 23 in the progression of colorectal cancer in patients with K-ras mutation: Ras signal inhibition and crosstalk with GM-CSF and IFN-γ.

Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future.

Non-genomic effects of thyroid hormone in adult cardiac myocytes: relevance to gene expression and cell growth

Besides the well-characterized genomic action of thyroid hormone (TH), mediated by thyroid hormone receptors (TRs), accumulating data support the so-called non-genomic action of TH, which is often related to activation of signalling pathways. In this study, we sought to determine whether TH activates intracellular signalling pathways in the adult cardiac myocytes and whether such activation modulates cell growth and the expression of target proteins important in cardiac function. We demonstrate that TH promotes a rapid increase in the phosphorylation of several kinases, ERK1/2, PKCδ, p38-MAPK and Akt. This activation is inhibited by triiodothyroacetic acid (triac), which is a TH analogue known to displace the hormone from membrane bound receptors, indicating that this TH effect is mediated through a cell membrane-initiated mechanism. Furthermore, using specific inhibitors of the TH-activated kinases, we show that the long-term effects of TH on the expression of sarcoplasmic reticulum Ca2+-ATPase (SERCA), α- and β-myosin heavy chain (MHC) and cell growth are reverted, implying that what is initiated as a non-genomic action of the hormone interfaces with genomic effects. These data provide further insights into the underlying mechanisms of TH action in the heart with potentially important implications in the management of cardiac pathology.

In vitro activity of dietary flavonol congeners against human cancer cell lines

BackgroundFlavonoids have physiological activity and a variety of pharmacological properties, including anticancer activity in vitro, but structure–anticancer activity relationships are unclear.AimThe objectives of this work were to investigate the activity of dietary flavonol congeners against cell lines derived from human solid tumours and to examine whether the in vitro activity was associated with specific structural feature(s) of the molecules.MethodsAntiproliferative activity of the flavonol congeners was investigated against eight different human cancer cell lines representing different types of human solid tumour, using the sulforhodamine B (SRB) assay in accordance with the instructions published by the NCI. Cell cycle perturbations caused by the congeners were monitored by flow-cytometric analysis of DNA stained with propidium iodide.ResultsMost of the flavonols examined had weak antiproliferative and cytotoxic activity. Of all the flavonol congeners tested peracetylated tiliroside found to be the most powerful, with significant antiproliferative and cytotoxic activity. Most flavonols induced similar cell cycle perturbations, whereas induction of apoptosis was significant only for cells treated with peracetylated tiliroside.ConclusionsThese findings indicated that the –OH groups of aromatic ring B were not linked to the cytotoxic and antiproliferative activity of the tested flavonols whereas peracetylation of the glycosides resulted in moderate improvement. In contrast, acetylation of tiliroside esterified with coumaric acid at position 5 of the sugar moiety greatly improved the activity of this congener. Overall, the results of this study suggest a critical role of sugar moiety substituents in the anticancer activity of the flavonols.

Cadmium modulates H-ras expression and caspase-3 apoptotic cell death in breast cancer epithelial MCF-7 cells

Cadmium (Cd) is a well-known metal carcinogen associated with tumor formation and carcinogenesis. It has been shown to induce cancer through various cellular mechanisms involving inhibition of DNA repair, abnormal gene expression, induction of oxidative stress, and triggering apoptosis. It is well-established that the H-ras oncogene is involved in the process of carcinogenesis with direct effects on cellular proliferation and tumorigenesis. Given the biotoxicity of cadmium and its association with carcinogenesis, the effect of that metal ion (Cd(II)) was investigated, in a concentration-dependent fashion, on cell viability, cell proliferation, caspase-3 mediated apoptosis and H-ras gene expression in human breast cancer epithelial MCF-7 cells transfected with the H-ras oncogene (wild type and G12V mutation). The findings show a significant modulation effect of cadmium on H-ras gene expression accompanied by up-regulation of caspase-3-related apoptosis in the concentration range of 100-1000 nΜ cadmium. Concurrently, there is a decrease in MCF-7 proliferation. Collectively, the results a) indicate an interplay of cadmium with H-ras(wt and G12V), with cadmium exhibiting a significant concentration-dependent effect on the modulation of H-ras expression, cell viability and proliferation, and b) project distinctly interwoven roles for both cadmium and H-ras in aberrant physiologies in cancer cells.