Phenobarbital inhibits calpain activity and expression in mouse hepatoma cells.

The antiepileptic drug phenobarbital (PB) exerts hepatic effects related to cell proliferation and tumorigenesis which are closely linked to the Wnt/ß-catenin signaling pathway. This pathway is, amongst others, regulated by calpain proteases. We now identified PB as an inhibitor of Wnt/ß-catenin signaling in mouse hepatoma cells. Further analyses revealed that PB inhibits calpain activity, an effect which is at least in parts mediated by a transcriptional regulation of calpain mRNA levels and which is furthermore independent of the constitutive androstane receptor, the known mediator of most effects of PB in liver cells.

Application of HC-AFW1 Hepatocarcinoma Cells for Mechanistic Studies: Regulation of Cytochrome P450 2B6 Expression by Dimethyl Sulfoxide and Early Growth Response 1.

Various exogenous compounds, for example, the drugs bupropione and propofol, but also various cytostatics, are metabolized in the liver by the enzyme cytochrome P450 (P450) CYP2B6. Transcription from the CYP2B6 gene is regulated mainly via the transcription factors constitutive androstane receptor (CAR) and pregnane-X-receptor (PXR). Most hepatic cell lines express no or only low levels of CYP2B6 because of loss of these two regulators. Dimethyl sulfoxide (DMSO) is frequently used in liver cell cultivation and is thought to affect the expression of various P450 isoforms by inducing or preserving cellular differentiation. We studied the effects of up to 1.5% of DMSO as cell culture medium supplement on P450 expression in hepatocarcinoma cells from line HC-AFW1. DMSO did not induce differentiation of the HC-AFW1 cell line, as demonstrated by unaltered levels of selected mRNA markers important for hepatocyte differentiation, and also by the lack of a DMSO effect on a broader spectrum of P450s. By contrast, CYP2B6 mRNA was strongly induced by DMSO. This process was independent of CAR or PXR activation. Interestingly, elevated transcription of CYP2B6 was accompanied by a simultaneous induction of early growth response 1 (EGR1), a transcription factor known to influence the expression of CYP2B6. Expression of wild-type EGR1 or of a truncated, dominant-negative EGR1 mutant was able to mimic or attenuate the DMSO effect, respectively. These findings demonstrate that EGR1 is involved in the regulation of CYP2B6 by DMSO in HC-AFW1 cells.

HepaChip-MP - a twenty-four chamber microplate for a continuously perfused liver coculture model.

HepaChip microplate (HepaChip-MP) is a microfluidic platform comprised of 24 independent culture chambers with continuous, unidirectional perfusion. In the HepaChip-MP, an automated dielectrophoresis process selectively assembles viable cells into elongated micro tissues. Freshly isolated primary human hepatocytes (PHH) and primary human liver endothelial cells (HuLEC) were successfully assembled as cocultures aiming to mimic the liver sinusoid. Minimal quantities of primary human cells are required to establish micro tissues in the HepaChip-MP. Metabolic function including induction of CYP enzymes in PHH was successfully measured demonstrating a high degree of metabolic activity of cells in HepaChip-MP cultures and sufficient sensitivity of LC-MS analysis even for the relatively small number of cells per chamber. Further, parallelization realized in HepaChip-MP enabled the acquisition of dose-response toxicity data of diclofenac with a single device. Several unique technical features should enable a widespread application of this in vitro model. We have demonstrated fully automated preparation of cell cultures in HepaChip-MP using a pipetting robot. The tubeless unidirectional perfusion system based on gravity-driven flow can be operated within a standard incubator system. Overall, the system readily integrates in workflows common in cell culture labs. Further research will be directed towards optimization of media composition to further extend culture lifetime and study oxygen gradients and their effect on zonation within the sinusoid-like microorgans. In summary, we have established a novel parallelized and scalable microfluidic in vitro liver model showing hepatocyte function and anticipate future in-depth studies of liver biology and applications in pre-clinical drug development.

Inhibition of ß-catenin signaling by phenobarbital in hepatoma cells in vitro.

The antiepileptic drug phenobarbital (PB) exerts hepatic effect based on indirect activation of the constitutive androstane receptor (CAR) via inhibition of the epidermal growth factor receptor (EGFR) and the kinase Src. It has furthermore been observed that in mice PB suppresses the growth of hepatocellular carcinoma with overactive signaling through the oncogenic Wnt/ß-catenin pathway, thus suggesting an interference of PB with ß-catenin signaling. The present work was aimed to characterize effects of PB on ß-catenin signaling at different cellular levels and to elucidate molecular details of the interaction of PB and ß-catenin in an in vitro system of mouse hepatoma cells. PB efficiently inhibited signaling through ß-catenin. This phenomenon was in-depth characterized at the levels of ß-catenin protein accumulation and transcriptional activity. Mechanistic analyses revealed that the effect of PB on ß-catenin signaling was independent of the activation of CAR and also independent of the cytosolic multi-protein complex responsible for physiological post-translation control of the ß-catenin pathway via initiation of ß-catenin degradation. Instead, evidence is provided that PB diminishes ß-catenin protein production by inhibition of protein synthesis via signal transduction through EGFR and Src. The proposed mechanism is well in agreement with previously published activities of PB at the EGFR and Src-mediated regulation of ß-catenin mRNA translation. Inhibition of ß-catenin signaling by PB through the proposed mechanism might explain the inhibitory effect of PB on the growth of specific sub-populations of mouse liver tumors. In conclusion, the present data comprehensively characterize the effect of PB on ß-catenin signaling in mouse hepatoma cells in vitro and provides mechanistic insight into the molecular processes underlying the observed effect.

Coordinate regulation of Cyp2e1 by ß-catenin- and hepatocyte nuclear factor 1α-dependent signaling.

Depending on their position within the liver lobule, hepatocytes fulfill different metabolic functions. Cytochrome P450 (CYP) 2E1 is a drug-metabolizing enzyme which is exclusively expressed in hepatocytes surrounding branches of the hepatic central vein. Previous publications have shown that signaling through the Wnt/ß-catenin pathway, a major determinant of liver zonation, and the hepatocyte-enriched transcription factor HNF (hepatocyte nuclear factor) 1α participate in the regulation of the gene. This study was aimed to decipher the molecular mechanisms by which the two transcription factors, ß-catenin and HNF1α, jointly regulate CYP2E1 at the gene promoter level. Chromatin immunoprecipitation identified a conserved Wnt/ß-catenin-responsive site (WRE) in the murine Cyp2e1 promoter adjacent to a known HNF1α response element (HNF1-RE). In vitro analyses demonstrated that both, activated ß-catenin and HNF1α, are needed for the full response of the promoter. The WRE was dispensable for ß-catenin-mediated effects on the Cyp2e1 promoter, while activity of ß-catenin was integrated into the promoter response via the HNF1-RE. Physical interaction of ß-catenin and HNF1α was demonstrated by co-immunoprecipitation. In conclusion, present data the first time identify and characterize the interplay of HNF1α and ß-catenin and elucidate molecular determinants of CYP2E1 expression in the liver.

A Nexus Consisting of Beta-Catenin and Stat3 Attenuates BRAF Inhibitor Efficacy and Mediates Acquired Resistance to Vemurafenib.

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS. In this study we show that ß-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that ß-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, ß-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of ß-catenin in melanoma progression and therapy resistance. We further demonstrate that ß-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.

Towards multiplexed protein-protein interaction analysis using protein tag-specific nanobodies.

Dynamic protein-protein interactions (PPIs) are an integral part of cellular processes. The discovery of modulators that disrupt or stabilize such interactions is highly important to understand PPIs and address correlating diseases. Bead-based protein assays analyzing PPIs between bait- and prey-proteins exemplify emerging methodologies. To date, most studies employ purified bait-proteins from bacteria. Such proteins are of limited use as they do not undergo eukaryotic folding and lack posttranslational modifications. Here, we present a novel method to generate bead-based protein arrays combining µ-scale purification of bait-proteins combined with site-directed immobilization. First, we express individual bait-proteins as GST- or GFP-fusion constructs in bacterial and mammalian cells. Next, we purify and immobilize these bait-proteins from crude lysates using high affinity tag-specific nanobodies coupled to color-coded beads. Finally, we combined those bait-coupled beads in a protein-array for miniaturized multiplexed GST- and GFP pulldown studies. In a proof-of-principle we study dynamic changes of the endogenous prey-protein ß-catenin following proteasomal inhibition or signaling pathway perturbation. Our strategy enables a fast isolation of highly pure and stable bait-proteins derived from small-scale expression cultures. We propose that this approach enables the generation of bead-based protein arrays comprising hundreds of bait-proteins from different expression systems to study complex PPIs. BIOLOGICAL SIGNIFICANCE: Protein arrays and multiplexed sandwich immunoassays, are widely applied to study protein-protein interaction or to investigate the signaling status of stimulated cells. This study describes for the first time the application of tag-specific nanobodies for site directed immobilization of bait-proteins from different expression systems to generate bead based protein arrays. The analysis of the Wnt-pathway activation by multiplexed µ-scale pulldowns demonstrated the advantages of eukaryotic expression systems regarding the stability and binding properties of individual bait proteins. This article is part of a Special Issue entitled: HUPO 2014.

A bead-based multiplex sandwich immunoassay to assess the abundance and posttranslational modification state of ß-catenin.

A system-wide analysis of cell signaling involves detecting and quantifying a range of proteins and their posttranslational modification states in the same cellular sample. We propose a protocol for a miniaturized, bead-based array and describe its efficiency in characterizing the different forms and functions of ß-catenin. The protocol provides detailed instructions for cell culture and bead array assays that enable insights into complex networks at the systems level.