Zonation related function and ubiquitination regulation in human hepatocellular carcinoma cells in dynamic vs. static culture conditions

<p>Abstract</p> <p>Background</p> <p>Understanding hepatic zonation is important both for liver physiology and pathology. There is currently no effective systemic chemotherapy for human hepatocellular carcinoma (HCC) and its pathogenesis is of special interest. Genomic and proteomic data of HCC cells in different culture models, coupled to pathway-based analysis, can help identify HCC-related gene and pathway dysfunctions.</p> <p>Results</p> <p>We identified zonation-related expression profiles contributing to selective phenotypes of HCC, by integrating relevant experimental observations through gene set enrichment analysis (GSEA). Analysis was based on gene and protein expression data measured on a human HCC cell line (HepG2/C3A) in two culture conditions: dynamic microfluidic biochips and static Petri dishes. Metabolic activity (HCC-related cytochromes P450) and genetic information processing were dominant in the dynamic cultures, in contrast to kinase signaling and cancer-specific profiles in static cultures. That, together with analysis of the published literature, leads us to propose that biochips culture conditions induce a periportal-like hepatocyte phenotype while standard plates cultures are more representative of a perivenous-like phenotype. Both proteomic data and GSEA results further reveal distinct ubiquitin-mediated protein regulation in the two culture conditions.</p> <p>Conclusions</p> <p>Pathways analysis, using gene and protein expression data from two cell culture models, confirmed specific human HCC phenotypes with regard to CYPs and kinases, and revealed a zonation-related pattern of expression. Ubiquitin-mediated regulation mechanism gives plausible explanations of our findings. Altogether, our results suggest that strategies aimed at inhibiting activated kinases and signaling pathways may lead to enhanced metabolism-mediated drug resistance of treated tumors. If that were the case, mitigating inhibition or targeting inactive forms of kinases would be an alternative.</p

Predictive toxicology using systemic biology and liver microfluidic "on chip" approaches: Application to acetaminophen injury

International audienceWe have analyzed transcriptomic, proteomic and metabolomic profiles of hepatoma cells cultivated inside a microfluidic biochip with or without acetaminophen (APAP). Without APAP, the results show an adaptive cellular response to the microfluidic environment, leading to the induction of anti-oxidative stress and cytoprotective pathways. In presence of APAP, calcium homeostasis perturbation, lipid peroxidation and cell death are observed. These effects can be attributed to APAP metabolism into its highly reactive metabolite. N-acetyl-p-benzoquinone imine (NAPQI). That toxicity pathway was confirmed by the detection of GSH-APAP, the large production of 2-hydroxybutyrate and 3-hydroxybutyrate, and methionine, cystine, and histidine consumption in the treated biochips. Those metabolites have been reported as specific biomarkers of hepatotoxicity and glutathione depletion in the literature. In addition, the integration of the metabolomic, transcriptomic and proteomic collected profiles allowed a more complete reconstruction of the APAP injury pathways. To our knowledge, this work is the first example of a global integration of microfluidic biochip data in toxicity assessment. Our results demonstrate the potential of that new approach to predictive toxicology

Integrated Proteomic and Transcriptomic Investigation of the Acetaminophen Toxicity in Liver Microfluidic Biochip

Microfluidic bioartificial organs allow the reproduction of in vivo-like properties such as cell culture in a 3D dynamical micro environment. In this work, we established a method and a protocol for performing a toxicogenomic analysis of HepG2/C3A cultivated in a microfluidic biochip. Transcriptomic and proteomic analyses have shown the induction of the NRF2 pathway and the related drug metabolism pathways when the HepG2/C3A cells were cultivated in the biochip. The induction of those pathways in the biochip enhanced the metabolism of the N-acetyl-p-aminophenol drug (acetaminophen-APAP) when compared to Petri cultures. Thus, we observed 50% growth inhibition of cell proliferation at 1 mM in the biochip, which appeared similar to human plasmatic toxic concentrations reported at 2 mM. The metabolic signature of APAP toxicity in the biochip showed similar biomarkers as those reported in vivo, such as the calcium homeostasis, lipid metabolism and reorganization of the cytoskeleton, at the transcriptome and proteome levels (which was not the case in Petri dishes). These results demonstrate a specific molecular signature for acetaminophen at transcriptomic and proteomic levels closed to situations found in vivo. Interestingly, a common component of the signature of the APAP molecule was identified in Petri and biochip cultures via the perturbations of the DNA replication and cell cycle. These findings provide an important insight into the use of microfluidic biochips as new tools in biomarker research in pharmaceutical drug studies and predictive toxicity investigations

Validation d'un microsystème hépatique dédié aux études pharmaco-toxicologiques

COMPIEGNE-BU (601592101) / SudocSudocFranceF

Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor

International audienceAn important issue in toxicity studies is the development of pertinent new in vitro tests that will be able to provide an alternative to in vivo testing methods. Current developments in the fields of tissue engineering and microtechnology make it possible to propose the use of microfluidic bioreactors as a tool for enhanced in vitro investigations. However, both the cells' behavior in complex environments and their response to chemicals need to be better understood, especially for future validation of any new assay. To characterize the sensitivity of this approach, we investigated the behavior of a liver cell model with respect to variations of two cell culture parameters in a microfluidic bioreactor: inoculated cell density (0.35x106, 0.45x106 and 0.65x106 cells/bioreactor) and microfluidic flow rates (0, 10 and 25µL/min). We also investigated an environmental pollutant modeled with three ammonia concentrations (0, 5 and 10mM). Proliferation in the bioreactor was found to be flow rate and inoculated cell density dependent. This led to a mean value of 1.2±0.2x106 cells in the 3D microenvironment of the bioreactor without ammonia loadings after 96h of cultures. Cell metabolism rates, such as glucose and glutamine consumption or CYP1A detoxification, were found to be higher in dynamic conditions than in static conditions. Furthermore, increased ammonium chloride concentration in turn increased glucose and glutamine consumptions and CYP1A activity. Inhibition of 50% of cell proliferation (IC50) during the ammonium chloride analysis was found at 5mM when cell concentrations of 0.35x106 cells/bioreactor were inoculated. In contrast, no effect could be detected at 5mM for larger cell densities of 0.65x106 cells/bioreactor, demonstrating concentration and cell density dependence in the bioreactors. This study highlighted the sensitivity of the HepG2/C3A cells to microfluidic culture conditions and illustrated the potential for larger in vitro toxicity studies using microfluidic bioreactors

A cocktail of metabolic probes demonstrates the relevance of primary human hepatocyte cultures in a microfluidic biochip for pharmaceutical drug screening

International audienceIn this paper, we compare the biotransformation capacities of cryopreserved primary human hepatocytes cultivated in a liver microfluidic biochip and in plates. The hepatocytes were exposed to the CIME cocktail (Carte d'Identité MEtabolique), a mixture of seven probes (acetaminophen, amodiaquine, caffeine, dextromethorphan, midazolam, omeprazole and tolbutamide) for key enzymes involved in the xenobiotic metabolism and pharmacokinetics. The purpose of the cocktail was to give an overview of the metabolic profile of the hepatocytes due to concomitant exposure and a simultaneous mass spectrometric detection method of the metabolites. The results showed a greater activity for CYP1A2, CYP2C9, CYP2C19 CYP2D6, CYP3A and UGT1A1 after 4 h of incubation in the microfluidic biochip when compared to the plate cultures. Furthermore, the metabolic ratio time-course measured at 1 h, 3 h and 4 h indicated that the enzymatic activity increased when the hepatocytes were cultivated in the microfluidic biochip, in contrast with their response in the plate cultures. These results illustrated the functional relevance of liver culture in the PDMS microfluidic biochip. The original method based on a microfluidic culture coupled with CIME cocktail analysis allowed the maintenance and the evaluation of the metabolic performances of the primary human hepatocytes through a new rapid assay. This metabolic analysis can thus become the reference situation when parallel studies of drug metabolism and toxicities are planned with functional hepatocytes in biochips

Microfabricated Mammalian Organ Systems And Their Integration Into Models Of Whole Animals And Humans

While in vitro cell based systems have been an invaluable tool in biology, they often suffer from a lack of physiological relevance. The discrepancy between the in vitro and in vivo systems has been a bottleneck in drug development process and biological sciences. The recent progress in microtechnology has enabled manipulation of cellular environment at a physiologically relevant length scale, which has led to the development of novel in vitro organ systems, often termed \u27organ-on-a-chip\u27 systems. By mimicking the cellular environment of in vivo tissues, various organ-on-a-chip systems have been reported to reproduce target organ functions better than conventional in vitro model systems. Ultimately, these organ-on-a-chip systems will converge into multi-organ \u27body-on-a-chip\u27 systems composed of functional tissues that reproduce the dynamics of the whole-body response. Such microscale in vitro systems will open up new possibilities in medical science and in the pharmaceutical industry. © 2013 The Royal Society of Chemistry