Project description:Cocaine toxicity has been a subject of study because cocaine is one of the most common and potent drugs of abuse. In the current study the effect of cocaine on human liver cancer cell line (HepG2) was assessed. Cocaine toxicity (IC50) on HepG2 cells was experimentally calculated using an XTT assay at 2.428 mM. The metabolic profile of HepG2 cells was further evaluated to investigate the cytotoxic activity of cocaine at 2 mM at three different time points. Cell medium and intracellular material samples were analyzed with a validated HILIC-MS/MS method for targeted metabolomics on an ACQUITY Amide column in gradient mode with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring. About 106 hydrophilic metabolites from different metabolic pathways were monitored. Multivariate analysis clearly separated the studied groups (cocaine-treated and control samples) and revealed potential biomarkers in the extracellular and intracellular samples. A predominant effect of cocaine administration on alanine, aspartate, and glutamate metabolic pathway was observed. Moreover, taurine and hypotaurine metabolism were found to be affected in cocaine-treated cells. Targeted metabolomics managed to reveal metabolic changes upon cocaine administration, however deciphering the exact cocaine cytotoxic mechanism is still challenging.
Project description:Barley bran has potential bioactivities due to its high content of polyphenols and dietary fiber, etc. Fermentation has been considered as an effective way to promote the functional activity of food raw materials. In this study, polysaccharides from barley bran extract fermented by Lactiplantibacillus plantarum dy-1 (FBBE-PS) were analyzed, and its effects on lipid accumulation and oxidative stress in high-fat HepG2 cells induced by sodium oleate were evaluated. The results showed that the molecular weight decreased and monosaccharide composition of polysaccharides changed significantly after fermentation. In addition, 50 μg/mL FBBE-PS could reduce the triglyceride (TG) content and reaction oxygen species (ROS) level in high-fat HepG2 cells by 21.62% and 30.01%, respectively, while increasing the activities of superoxide dismutase (SOD) and catalase (CAT) represented by 64.87% and 22.93%, respectively. RT-qPCR analysis revealed that FBBE-PS could up-regulate the lipid metabolism-related genes such as ppar-α, acox-1 and cpt-1α, and oxidation-related genes such as nrf2, ho-1, nqo-1, sod1, cat, etc. The metabolomics analysis indicated that FBBE-PS could alleviate lipid deposition by inhibiting the biosynthesis of unsaturated fatty acids, which is consistent with the downregulation of scd-1 expression. It is demonstrated that fermentation can alter the properties and physiological activities of polysaccharides in barley bran, and FBBE-PS exhibited an alleviating effect on lipid deposition and oxidative stress in high-fat cells.
Project description:Hepatocellular carcinoma (HCC) is the most common primary liver cancer and poor prognosis. Emerging evidence suggests that epigenetic alterations play a crucial role in HCC, suggesting epigenetic inhibition as a promising therapeutic approach. Indeed, the bromodomain and extra-terminal (BET) inhibitors inhibit the proliferation and invasion of various cancers but still lack a strong mechanistic rationale. Here, we identified the differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) in human HCC cell line HepG2 treated with the BET inhibitors, JQ1, OTX015, or ABBV-075. We analyzed the correlation between DEmRNAs and DElncRNAs in common for the three inhibitors based on their expression profiles and performed functional annotation pathway enrichment analysis. Most of these shared DEmRNAs and DElncRNAs, including some novel transcripts, were downregulated, indicating decreased proliferation/adhesion and increased apoptosis/inflammation. Our study suggests that BET proteins play a crucial role in regulating cancer progression-related genes and provide a valuable resource for novel putative biomarkers and therapeutic targets in HCC.
Project description:Nonalcoholic fatty liver disease (NAFLD) is characterized by an increase in hepatic lipid accumulation due to impaired lipid metabolism. Although a correlation was found between NAFLD and sphingosine-1-phosphate (S1P), the role of the sphingolipid remains controversial. The aim of this study was to investigate any involvement of S1P in steatosis using its analog FTY720P and HepG2 cells. Lipid accumulation was induced by incubating the cells in a mixture of oleic and palmitic acid, and was quantified using Oil Red O. The involvement of signaling mediators was studied using pharmacological inhibitors and western blot analysis. FTY720P increased lipid accumulation, but this increase wasn't maintained in the presence of inhibitors of S1PR3, Gq, SREBP, mTOR, PI3K, and PPARγ indicating their involvement in the process. The results revealed that FTY720P binds to S1PR3 which activates sequentially Gq, PI3K, and mTOR leading to an increase in SREBP expression and PPARγ activation. It was concluded that in presence of a high level of fatty acids, lipid accumulation is increased in hepatocytes by the exogenously added FTY720P.
Project description:Crocetin is a carotenoid extracted from Gardenia jasminoides, one of the most popular traditional Chinese medicines, which has been used in the prevention and treatment of various diseases. The present study is aimed at clarifying the effect of crocetin on gene expression profiling of HepG2 cells by RNA-sequence assay and further investigating the molecular mechanism underlying the multiple biofunctions of crocetin based on bioinformatics analysis and molecular evidence. Among a total 23K differential genes identified, crocetin treatment upregulated the signals of 491 genes (2.14% of total gene probes) and downregulated the signals of 283 genes (1.24% of total gene probes) by ≥2-fold. The Gene Ontology analysis enriched these genes mainly on cell proliferation and apoptosis (BRD4 and DAXX); lipid formation (EHMT2); cell response to growth factor stimulation (CYP24A1 and GCNT2); and growth factor binding (ABCB1 and ABCG1), metabolism, and signal transduction processes. The KEGG pathway analysis revealed that crocetin has the potential to regulate transcriptional misregulation, ABC transporters, bile secretion, alcoholism, systemic lupus erythematosus (SLE), and other pathways, of which SLE was the most significantly disturbed pathway. The PPI network was constructed by using the STRING online protein interaction database and Cytoscape software, and 21 core proteins were obtained. RT-qPCR datasets serve as the solid evidence that verified the accuracy of transcriptome sequencing results with the same change trend. This study provides first-hand data for comprehensively understanding crocetin targeting on hepatic metabolism and its multiple biofunctions.
Project description:Glutathione (GSH) has long been recognised for its antioxidant and detoxifying effects on the liver. The hepatoprotective effect of GSH involves the activation of antioxidative systems such as NRF2; however, details of the mechanisms remain limited. A comparative analysis of the biological events regulated by GSH under physiological and oxidative stress conditions has also not been reported. In this study, DNA microarray analysis was performed with four experiment arms including Control, GSH, hydrogen peroxide (HP), and GSH + HP treatment groups. The GSH-treated group exhibited a significant upregulation of genes clustered in cell proliferation, growth, and differentiation, particularly those related to MAPK, when compared with the Control group. Additionally, liver functions such as alcohol and cholesterol metabolic processes were significantly upregulated. On the other hand, in the HP-induced oxidative stress condition, GSH (GSH + HP group) demonstrated a significant activation of cell proliferation, cell cycle, and various signalling pathways (including TGFβ, MAPK, PI3K/AKT, and HIF-1) in comparison to the HP group. Furthermore, several disease-related pathways, such as chemical carcinogenesis-reactive oxygen species and fibrosis, were significantly downregulated in the GSH + HP group compared to the HP group. Collectively, our study provides a comprehensive analysis of the effects of GSH under both physiological and oxidative stress conditions. Our study provides essential insights to direct the utilisation of GSH as a supplement in the management of conditions associated with oxidative stress.
Project description:Protein array technology not only identifies a large number of proteins but also determines their expression levels. In the present study, antibody array analysis is used to decipher the proteins involved in hesperidin-induced cell death in HepG2 cells. Altered proteins in hesperidin treated cells were compared with that of untreated control cells by using a RayBio® Label‑based (L series) human antibody array kit. The identified proteins were further confirmed using western blot analysis. STRING software based analysis was used to determine the protein‑protein interactions. Many proteins related to signal transduction, cellular mechanisms, cell growth and proliferation regulatory proteins were identified. Among the proteins identified Hsp90, Smac/DIABLO, Prdx6 and FRK were significantly reduced in hesperidin treated cells. To the best of the authors' knowledge, the present study is the first to use antibody array for identifying proteins marker in hesperidin‑induced cell death in HepG2 cells. The present study provides a novel insight into the anticancer mechanism of hesperidin.
Project description:Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.
Project description:Impairment of hepatic fatty acid metabolism can lead to liver steatosis and injury. Testing drugs for interference with hepatic fatty acid metabolism is therefore important. To find out whether HepG2 cells are suitable for this purpose, we investigated the effect of three established fatty acid metabolism inhibitors and of three test compounds on triglyceride accumulation, palmitate metabolism, the acylcarnitine pool and dicarboxylic acid accumulation in the cell supernatant and on ApoB-100 excretion in HepG2 cells. The three established inhibitors [etomoxir, methylenecyclopropylacetic acid (MCPA), and 4-bromocrotonic acid (4-BCA)] depleted mitochondrial ATP at lower concentrations than cytotoxicity occurred, suggesting mitochondrial toxicity. They inhibited palmitate metabolism at similar or lower concentrations than ATP depletion, and 4-BCA was associated with cellular fat accumulation. They caused specific changes in the acylcarnitine pattern and etomoxir an increase of thapsic (C18 dicarboxylic) acid in the cell supernatant, and did not interfere with ApoB-100 excretion (marker of VLDL export). The three test compounds (amiodarone, tamoxifen, and the cannabinoid WIN 55,212-2) depleted the cellular ATP content at lower concentrations than cytotoxicity occurred. They all caused cellular fat accumulation and inhibited palmitate metabolism at similar or higher concentrations than ATP depletion. They suppressed medium-chain acylcarnitines in the cell supernatant and amiodarone and tamoxifen impaired thapsic acid production. Tamoxifen and WIN 55,212-2 decreased cellular ApoB-100 excretion. In conclusion, the established inhibitors of fatty acid metabolism caused the expected effects in HepG2 cells. HepG cells proved to be useful for the detection of drug-associated toxicities on hepatocellular fatty acid metabolism.