scholarly journals Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

2020 ◽  
Vol 22 (1) ◽  
pp. 202
Author(s):  
Josephin Glück ◽  
Julia Waizenegger ◽  
Albert Braeuning ◽  
Stefanie Hessel-Pras
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Pyrrolizidine Alkaloids ◽  
Defense Mechanism ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

scholarly journals In Vitro Cytotoxicity of Oleanolic/Ursolic Acids-Loaded in PLGA Nanoparticles in Different Cell Lines

Pharmaceutics ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 362 ◽  
Author(s):  
Amélia M. Silva ◽  
Helen L. Alvarado ◽  
Guadalupe Abrego ◽  
Carlos Martins-Gomes ◽  
Maria L. Garduño-Ramirez ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Specific Activity ◽  
Polymeric Nanoparticles ◽  
Hepatoma Cell ◽  
Plga Nanoparticles ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell

Oleanolic (OA) and ursolic (UA) acids are recognized triterpenoids with anti-cancer properties, showing cell-specific activity that can be enhanced when loaded into polymeric nanoparticles. The cytotoxic activity of OA and UA was assessed by Alamar Blue assay in three different cell lines, i.e., HepG2 (Human hepatoma cell line), Caco-2 (Human epithelial colorectal adenocarcinoma cell line) and Y-79 (Human retinoblastoma cell line). The natural and synthetic mixtures of these compounds were tested as free and loaded in polymeric nanoparticles in a concentration range from 2 to 32 µmol/L. The highest tested concentrations of the free triterpene mixtures produced statistically significant cell viability reduction in HepG2 and Caco-2 cells, compared to the control (untreated cells). When loaded in the developed PLGA nanoparticles, no differences were recorded for the tested concentrations in the same cell lines. However, in the Y-79 cell line, a decrease on cell viability was observed when testing the lowest concentration of both free triterpene mixtures, and after their loading into PLGA nanoparticles.

scholarly journals In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 617-623 ◽  
Author(s):  
J Fandrey ◽  
HF Bunn
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Human Hepatoma Cell ◽  
Competitive Polymerase Chain Reaction ◽  
Polymerase Chain

Abstract The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis

1993 ◽  
Vol 104 (2) ◽  
pp. 307-315 ◽  
Author(s):  
A.C. Bayly ◽  
N.J. French ◽  
C. Dive ◽  
R.A. Roberts
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferators ◽  
Hepatoma Cell Lines

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens. In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death. Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocytochemistry. Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium. The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

PEGylation potentiates hepatoma cell targeted liposome-mediated in vitro gene delivery via the asialoglycoprotein receptor

2017 ◽  
Vol 72 (7-8) ◽  
pp. 293-301 ◽  
Author(s):  
Nkosiyethu K. Mkhwanazi ◽  
Charles B. de Koning ◽  
Willem A.L. van Otterlo ◽  
Mario Ariatti ◽  
Moganavelli Singh
Keyword(s):  
Gene Delivery ◽  
Hepatoma Cell ◽  
Asialoglycoprotein Receptor ◽  
Sub Saharan Africa ◽  
Treatment Modalities ◽  
Condensed State ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell

AbstractHepatocellular carcinoma is a burgeoning health issue in sub-Saharan Africa and East Asia where it is most prevalent. The search for gene medicine treatment modalities for this condition represents a novel departure from current treatment options and is gaining momentum. Here we report on nonPEGylated and on sterically stabilized PEGylated cationic liposomes decorated with D-galacto moieties linked to 24.1 Å spacers for asialoglycoprotein receptor (ASGP-R)-targeted vehiculation of pCMV-luc plasmid DNA. Cargo DNA is fully liposome associated at N/P ratio=3:1 and is partially protected from the effects of serum nucleases. Moreover, at this ratio, lipoplex dimensions (89–97 nm) are compatible with the requirements for extravasation in vivo. Ethidium displacement assays show that the reporter DNA is in a less condensed state when bound to PEGylated liposomes than with nonPEGylated liposomes. PEGylated lipoplexes were well tolerated by both HEK293 (ASGP-R-negative) and HepG2 (ASGP-R-positive) cell lines and delivered DNA to the human hepatoma cell line HepG2 by ASGP-R mediation at levels three-fold greater than nonPEGylated lipoplexes. PEGylated ASGP-R-targeted liposomes reported in this study possess the required characteristics for hepatotropic gene delivery and may be considered for further application in vivo.

scholarly journals In Vivo and In Vitro Effect of a Nutrient Mixture on Human Hepatoma Cell Line SK‐Hep‐1

2007 ◽  
Vol 21 (6) ◽  
Author(s):  
M. Waheed Roomi ◽  
Vadim Ivanov ◽  
Aleksandra Niedzwiecki ◽  
Matthias Rath
Keyword(s):  
Cell Line ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Human Hepatoma Cell Line ◽  
Vitro Effect

scholarly journals Active Transport of Hepatotoxic Pyrrolizidine Alkaloids in HepaRG Cells

2021 ◽  
Vol 22 (8) ◽  
pp. 3821
Author(s):  
Anne-Margarethe Enge ◽  
Florian Kaltner ◽  
Christoph Gottschalk ◽  
Albert Braeuning ◽  
Stefanie Hessel-Pras
Keyword(s):  
Pyrrolizidine Alkaloids ◽  
Hepatoma Cell ◽  
Metabolic Activation ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Food Contaminants ◽  
Plant Metabolites ◽  
Human Hepatoma Cell ◽  
Secondary Plant Metabolites ◽  
Heparg Cells

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites occurring as food contaminants that can cause severe liver damage upon metabolic activation in hepatocytes. However, it is yet unknown how these contaminants enter the cells. The role of hepatic transporters is only at the beginning of being recognized as a key determinant of PA toxicity. Therefore, this study concentrated on assessing the general mode of action of PA transport in the human hepatoma cell line HepaRG using seven structurally different PAs. Furthermore, several hepatic uptake and efflux transporters were targeted with pharmacological inhibitors to identify their role in the uptake of the PAs retrorsine and senecionine and in the disposition of their N-oxides (PANO). For this purpose, PA and PANO content was measured in the supernatant using LC-MS/MS. Also, PA-mediated cytotoxicity was analyzed after transport inhibition. It was found that PAs are taken up into HepaRG cells in a predominantly active and structure-dependent manner. This pattern correlates with other experimental endpoints such as cytotoxicity. Pharmacological inhibition of the influx transporters Na+/taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter 1 (SLC22A1) led to a reduced uptake of retrorsine and senecionine into HepaRG cells, emphasizing the relevance of these transporters for PA toxicokinetics.

scholarly journals In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 617-623 ◽  
Author(s):  
J Fandrey ◽  
HF Bunn
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Human Hepatoma Cell ◽  
Competitive Polymerase Chain Reaction ◽  
Polymerase Chain

The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo

2006 ◽  
Vol 291 (4) ◽  
pp. E737-E744 ◽  
Author(s):  
Heidi A. Parkes ◽  
Elaine Preston ◽  
Donna Wilks ◽  
Mercedes Ballesteros ◽  
Lee Carpenter ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Hepg2 Cells ◽  
Hepatoma Cell ◽  
Acid Oxidation ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Chain Acyl

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 ± 3 vs. 67 ± 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 ± 6 vs. 100 ± 6 nmol/g protein in controls, P < 0.05) and increased [14C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 ± 4 vs. 20 ± 2 μmol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-14C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.

scholarly journals The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide, an In Vitro Study

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3886
Author(s):  
Stefania Sut ◽  
Irene Ferrarese ◽  
Maria Giovanna Lupo ◽  
Nicola De Zordi ◽  
Elisa Tripicchio ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Density Lipoprotein ◽  
In Vitro Study ◽  
Volatile Constituent ◽  
Dependent Manner ◽  
Human Hepatoma Cell ◽  
Concentration Dependent Manner

In the present study the ability of supercritical carbon dioxide (SCO2) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO2 extracts, one oil (ML-SCO2) and a semisolid (MW-SCO2), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO2 was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO2 reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO2 (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO2 extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.

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