Protection of catalpol against triptolide-induced hepatotoxicity by inhibiting excessive autophagy via the PERK-ATF4-CHOP pathway

Catalpol significantly reduces triptolide-induced hepatotoxicity, which is closely related to autophagy. The aim of this study was to explore the unclear protective mechanism of catalpol against triptolide. The detoxification effect of catalpol on triptolide was investigated in HepaRG cell line. The detoxification effects were assessed by measuring cell viability, autophagy, and apoptosis, as well as the endoplasmic reticulum stress protein and mRNA expression levels. We found that 5–20 µg/L triptolide treatments increased the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), as well as the expression of autophagy proteins including LC3 and Beclin1. The expression of P62 was downregulated and the production of autophagosomes was increased, as determined by transmission electron microscope and monodansylcadaverine staining. In contrast, 40 µg/L catalpol reversed these triptolide-induced changes in the liver function index, autophagy level, and apoptotic protein expression, including Cleaved-caspase3 and Cleaved-caspase9 by inhibiting excessive autophagy. Simultaneously, catalpol reversed endoplasmic reticulum stress, including the expression of PERK, which regulates autophagy. Moreover, we used the PERK inhibitor GSK2656157 to prove that the PERK-ATF4-CHOP pathway of the unfolded protein response is an important pathway that could induce autophagy. Catalpol inhibited excessive autophagy by suppressing the PERK pathway. Altogether, catalpol protects against triptolide-induced hepatotoxicity by inhibiting excessive autophagy via the PERK-ATF4-CHOP pathway. The results of this study are beneficial to clarify the detoxification mechanism of catalpol against triptolide-induced hepatotoxicity and to promote the application of triptolide.

A Minimal Subset of Seven Genes Associated with Tumor Hepatocyte Differentiation Predicts a Poor Prognosis in Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a deadly cancer worldwide as a result of a frequent late diagnosis which limits the therapeutic options. Tumor progression in HCC is closely correlated with the dedifferentiation of hepatocytes, the main parenchymal cells in the liver. Here, we hypothesized that the expression level of genes reflecting the differentiation status of tumor hepatocytes could be clinically relevant in defining subsets of patients with different clinical outcomes. To test this hypothesis, an integrative transcriptomics approach was used to stratify a cohort of 139 HCC patients based on a gene expression signature established in vitro in the HepaRG cell line using well-controlled culture conditions recapitulating tumor hepatocyte differentiation. The HepaRG model was first validated by identifying a robust gene expression signature associated with hepatocyte differentiation and liver metabolism. In addition, the signature was able to distinguish specific developmental stages in mice. More importantly, the signature identified a subset of human HCC associated with a poor prognosis and cancer stem cell features. By using an independent HCC dataset (TCGA consortium), a minimal subset of seven differentiation-related genes was shown to predict a reduced overall survival, not only in patients with HCC but also in other types of cancers (e.g., kidney, pancreas, skin). In conclusion, the study identified a minimal subset of seven genes reflecting the differentiation status of tumor hepatocytes and clinically relevant for predicting the prognosis of HCC patients.

Assessing hepatoprotective effects of antioxidants on amiodarone-induced cytotoxicity in human hepatoma HepaRG cell line

Effective therapy of amiodarone-induced hepatotoxicity requires studying the mechanisms of the toxic effects of amiodarone on hepatocytes and assessing the potential impact of hepotoprotective agents. The study was aimed to assess hepatoprotective effects of antioxidants on the amiodarone-induced hepatotoxicity with the use of immortalized human hepatoma cells of the HepaRG cell line. Cell viability was evaluated upon exposure to amiodarone and in the mixture with vitamin Е, N-acetylcysteine and S-adenosylmethionine by impedance measurement; the levels of some hepatotoxicity biomarkers were defined using the Luminex xMAP technology. As a result of the research, the dose-dependent toxic effects of amiodarone were established. The IC50 value of amiodarone in the HepaRG cell line was 3.5 μМ. It is shown that cytotoxic effects decrease and the IC50 value increases in the presence of vitamin Е, N-acetylcysteine and S-adenosylmethionine. Amiodarone reduces the activity of cell cycle regulators: AKT, JNK kinases, and p53 protein. Exposure to amiodarone results in reduced intracellular ATP levels and the release of intracellular enzymes (malate dehydrogenase 1, glutathione S-transferase, sorbitol dehydrogenase, 5'-nucleotidase) into conditioned medium, indicating the necrotic cell death. Thus, vitamin Е, S-adenosylmethionine and N-acetylcysteine reduce amiodarone cytotoxicity in the model of amiodarone-induced damage to hepatocytes and can be considered as hepatoprotective agents in case of the need to protect liver against the hepatotoxic effects of amiodarone.

A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

Inhibition of nuclear factor (erythroid-derived 2)-like 2 promotes hepatic progenitor cell activation and differentiation

The stem cell ability to self-renew and lead regeneration relies on the balance of complex signals in their microenvironment. The identification of modulators of hepatic progenitor cell (HPC) activation is determinant for liver regeneration and may improve cell transplantation for end-stage liver disease. This investigation used different models to point out the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) as a key regulator of the HPC fate. We initially proved that in vivo models of biliary epithelial cells (BECs)/HPC activation show hepatic oxidative stress, which activates primary BECs/HPCs in vitro. NRF2 downregulation and silencing were associated with morphological, phenotypic, and functional modifications distinctive of differentiated cells. Furthermore, NRF2 activation in the biliary tract repressed the ductular reaction in injured liver. To definitely assess the importance of NRF2 in HPC biology, we applied a xenograft model by inhibiting NRF2 in the human derived HepaRG cell line and transplanting into SCID/beige mice administered with anti-Fas antibody to induce hepatocellular apoptosis; this resulted in effective human hepatocyte repopulation with reduced liver injury. To conclude, NRF2 inhibition leads to the activation and differentiation of liver progenitors. This redox-dependent transcription factor represents a potential target to regulate the commitment of undifferentiated hepatic progenitors into specific lineages.

The antiretroviral 2',3'-dideoxycytidine causes mitochondrial dysfunction in proliferating and differentiated HepaRG human cell cultures

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus infection, and their use can cause mitochondrial toxicity, including mitochondrial DNA (mtDNA) depletion in several cases. The first generation NRTIs, including 2',3'-dideoxycytidine (ddC), were originally and are still pursued as anticancer agents. NRTI-sensitive DNA polymerases localizing to mitochondria allow for the opportunity to poison proliferating cancer cell mtDNA replication as certain cancers rely heavily on mitochondrial functions. However, mtDNA replication is independent of the cell cycle creating a significant concern that toxicants such as ddC impair mtDNA maintenance in both proliferating and non-proliferating cells. To examine this possibility, we tested the utility of the HepaRG cell line to study ddC-induced toxicity in isogenic proliferating (undifferentiated) and non-proliferating (differentiated) cells. Following ddC exposures, we measured cell viability, mtDNA copy number, and mitochondrial bioenergetics utilizing trypan blue, Southern blotting, and extracellular flux analysis, respectively. After 13 days of 1 μM ddC exposure, proliferating and differentiated HepaRG harbored mtDNA levels of 0.9% and 17.9% compared to control cells, respectively. Cells exposed to 12 μM ddC contained even less mtDNA. By day 13, differentiated cell viability was maintained but declined for proliferating cells. Proliferating HepaRG bioenergetic parameters were severely impaired by day 8, with 1 and 12 μM ddC, while differentiated cells displayed defects of spare and maximal respiratory capacities (day 8) and proton-leak linked respiration (day 14) with 12 μM ddC. These results indicate HepaRG is a useful model to study proliferating and differentiated cell mitochondrial toxicant exposures.

Synthesis of Poly(Dimethylmalic Acid) Homo- and Copolymers to Produce Biodegradable Nanoparticles for Drug Delivery: Cell Uptake and Biocompatibility Evaluation in Human Heparg Hepatoma Cells

Hydrophobic and amphiphilic derivatives of the biocompatible and biodegradable poly(dimethylmalic acid) (PdiMeMLA), varying by the nature of the lateral chains and the length of each block, respectively, have been synthesized by anionic ring-opening polymerization (aROP) of the corresponding monomers using an initiator/base system, which allowed for very good control over the (co)polymers’ characteristics (molar masses, dispersity, nature of end-chains). Hydrophobic and core-shell nanoparticles (NPs) were then prepared by nanoprecipitation of hydrophobic homopolymers and amphiphilic block copolymers, respectively. Negatively charged NPs, showing hydrodynamic diameters (Dh) between 50 and 130 nm and narrow size distributions (0.08 < PDI < 0.22) depending on the (co)polymers nature, were obtained and characterized by dynamic light scattering (DLS), zetametry, and transmission electron microscopy (TEM). Finally, the cytotoxicity and cellular uptake of the obtained NPs were evaluated in vitro using the hepatoma HepaRG cell line. Our results showed that both cytotoxicity and cellular uptake were influenced by the nature of the (co)polymer constituting the NPs.

mRNA Expression and Activity of Nucleoside Transporters in Human Hepatoma HepaRG Cells

The HepaRG cell line is a highly differentiated human hepatoma cell line, displaying the expression of various drug transporters. However, functional expression of nucleoside transporters remains poorly characterized in HepaRG cells, although these transporters play a key role in hepatic uptake of antiviral and anticancer drugs. The present study was, therefore, designed to characterize the expression, activity and regulation of equilibrative (ENT) and concentrative (CNT) nucleoside transporter isoforms in differentiated HepaRG cells. These cells were found to exhibit a profile of nucleoside transporter mRNAs similar to that found in human hepatocytes, i.e., notable expression of ENT1, ENT2 and CNT1, with very low or no expression of CNT2 and CNT3. ENT1 activity was, next, demonstrated to be the main uridine transport activity present in HepaRG cells, like in cultured human hepatocytes. Various physiological factors, such as protein kinase C (PKC) activation or treatment by inflammatory cytokines or hepatocyte growth factor (HGF), were additionally found to regulate expression of ENT1, ENT2 and CNT1; PKC activation and HGF notably concomitantly induced mRNA expression and activity of ENT1 in HepaRG cells. Overall, these data suggest that HepaRG cells may be useful for analyzing cellular pharmacokinetics of nucleoside-like drugs in human hepatic cells, especially of those handled by ENT1.