Identification of aryl hydrocarbon receptor allosteric antagonists from clinically approved drugs.

The human aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, plays a pivotal role in a diverse array of pathways in biological and pathophysiological events. This position AhR as a promising target for both carcinogenesis and antitumor strategies. In this study we utilized computational modeling to screen and identify FDA-approved drugs binding to the allosteric site between α2 of bHLH and PAS-A domains of AhR, with the aim of inhibiting its canonical pathway activity. Our findings indicated that nilotinib effectively fits into the allosteric pocket and forms interactions with crucial residues F82, Y76, and Y137. Binding free energy value of nilotinib is the lowest among top hits and maintains stable within its pocket throughout entire (MD) simulations time. Nilotinib has also substantial interactions with F295 and Q383 when it binds to orthosteric site and activate AhR. Surprisingly, it does not influence AhR nuclear translocation in the presence of AhR agonists; instead, it hinders the formation of the functional AhR-ARNT-DNA heterodimer assembly, preventing the upregulation of regulated enzymes like CYP1A1. Importantly, nilotinib exhibits a dual impact on AhR, modulating AhR activity via the PAS-B domain and working as a noncompetitive allosteric antagonist capable of blocking the canonical AhR signaling pathway in the presence of potent AhR agonists. These findings open a new avenue for the repositioning of nilotinib beyond its current application in diverse diseases mediated via AhR.

Modulation of aryl hydrocarbon receptor activity by tyrosine kinase inhibitors (ponatinib and tofacitinib).

Ponatinib and tofacitinib, established kinase inhibitors and FDA-approved for chronic myeloid leukemia and rheumatoid arthritis, are recently undergoing investigation in diverse clinical trials for potential repurposing. The aryl hydrocarbon receptor (AhR), a transcription factor influencing a spectrum of physiological and pathophysiological activities, stands as a therapeutic target for numerous diseases. This study employs molecular modelling tools and in vitro assays to identify ponatinib and tofacitinib as AhR ligands, elucidating their binding and molecular interactions in the AhR PAS-B domain. Molecular docking analyses revealed that ponatinib and tofacitinib occupy the central pocket within the primary cavity, similar to AhR agonists 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and (benzo[a]pyrene) B[a]P. Our simulations also showed that these compounds exhibit good stability, stabilizing many hot spots within the PAS-B domain, including the Dα-Eα loop, which serves as a regulatory element for the binding pocket. Binding energy calculations highlighted ponatinib's superior predicted affinity, revealing F295 as a crucial residue in maintaining strong interaction with the two compounds. Our in vitro data suggest that ponatinib functions as an AhR antagonist, blocking the downstream signaling of AhR pathway induced by TCDD and B[a]P. Additionally, both tofacitinib and ponatinib cause impairment in AhR-regulated CYP1A1 enzyme activity induced by potent AhR agonists. This study unveils ponatinib and tofacitinib as potential modulators of AhR, providing valuable insights into their therapeutic roles in AhR-associated diseases and enhancing our understanding of the intricate relationship between kinase inhibitors and AhR.

Isolation of a novel quercetin derivative from Terminalia chebula and RT-PCR-assisted probing to investigate its DNA repair in hepatoma cells.

Background and purpose: DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study focused on the purification of a novel quercetin derivative present in Terminalia chebula fruit and studied its protective role in hepatoma cells due to H2O2-DNA damage. Experimental approach: The pure compound obtained from the silica gel column was subjected to structural characterization using spectroscopic techniques. MTT assay was employed to select a non-toxic concentration of the isolated compounds on HepG2 and Chang liver cells. The antigenotoxic property of the compound on HepG2 and Chang liver cells was carried out by alkaline comet assay. Analyses of expression levels of mRNA for two DNA repair enzymes, OGG1 and NEIL1, in HepG2 and Chang liver cells, were carried out using the RT-PCR method. Findings/Results: The pure compound obtained from the fraction-5 of diethyl ether extract was identified as a novel quercetin derivative and named 7-(but-2-en-1-yloxy)-2-(4(but-2-en-1-yloxy)-3-hydroxyphenyl)-3- (hexa-2,4-dien-1-yloxy)-6-hydroxy-4H-chromen-4-one. This compound recorded modest toxicity at the highest concentration tested (percentage cell viability at 100 µg/mL was 64.71 ± 0.38 for HepG2 and 45.32 ± 0.07 for Chang liver cells). The compound has demonstrated noteworthy protection against H2O2-induced DNA damage in both cell lines. Analyses of mRNA expression levels for enzymes OGGI and NEIL1 enzymes in HepG2 and Chang liver cells asserted the protective role of the isolated compound against H2O2-induced DNA damage. Conclusion and implication: The protective effect of a novel quercetin derivative isolated from T. chebula in the hepatoma cells is reported here for the first time.

Exogenous Nucleotides Ameliorate Insulin Resistance Induced by Palmitic Acid in HepG2 Cells through the IRS-1/AKT/FOXO1 Pathways.

Nucleotides (NTs) act as pivotal regulatory factors in numerous biological processes, playing indispensable roles in growth, development, and metabolism across organisms. This study delves into the effects of exogenous NTs on hepatic insulin resistance using palmitic-acid-induced HepG2 cells, administering interventions at three distinct dosage levels of exogenous NTs. The findings underscore that exogenous NT intervention augments glucose consumption in HepG2 cells, modulates the expression of glycogen-synthesis-related enzymes (glycogen synthase kinase 3ß and glycogen synthase), and influences glycogen content. Additionally, it governs the expression levels of hepatic enzymes (hexokinase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase). Moreover, exogenous NT intervention orchestrates insulin signaling pathway (insulin receptor substrate-1, protein kinase B, and forkhead box protein O1) and AMP-activated protein kinase (AMPK) activity in HepG2 cells. Furthermore, exogenous NT intervention fine-tunes the expression levels of oxidative stress-related markers (malondialdehyde, glutathione peroxidase, and NADPH oxidase 4) and the expression of inflammation-related nuclear transcription factor (NF-κB). Lastly, exogenous NT intervention regulates the expression levels of glucose transporter proteins (GLUTs). Consequently, exogenous NTs ameliorate insulin resistance in HepG2 cells by modulating the IRS-1/AKT/FOXO1 pathways and regulate glucose consumption, glycogen content, insulin signaling pathways, AMPK activity, oxidative stress, and inflammatory status.

Influence of alkylthio and arylthio derivatives of tert-butylquinone on the induction of DNA damage in a human hepatocellular carcinoma cell line (HepG2).

The aim of this study was to investigate the effects of tert-butylquinone (TBQ) and its alkylthio and arylthio derivatives on DNA in vitro, using acellular and cellular test systems. Direct interaction with DNA was studied using the plasmid pUC19. Cytotoxic (MTS assay) and genotoxic (comet assay and γH2AX focus assays) effects, and their influence on the cell cycle were studied in the HepG2 cell line. Our results show that TBQ and its derivatives did not directly interact with DNA. The strongest cytotoxic effect on the HepG2 cells was observed for the derivative 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone (IC50 64.68 and 55.64 µM at 24-h and 48-h treatment, respectively). The tested derivatives did not significantly influence the cell cycle distribution in the exposed cellular populations. However, all derivatives showed a genotoxic activity stronger than that of TBQ in the comet assay, with 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone producing the strongest effect. The same derivative also induced DNA double-strand breaks in the γH2AX focus assay.

Effect of iron saturation of bovine lactoferrin on the inhibition of hepatitis B virus in vitro.

Background: Hepatitis B virus (HBV) infection poses a major public health problem worldwide. Bovine lactoferrin (bLf) is a natural product that can inhibit HBV, but the effect of iron saturation on its resistance to HBV is unknown. Aims: The purpose of this study is to investigate the impact of iron saturation of bLf against HBV. Methods: HepG2 cells were cultured in DMEM high glucose containing 10% inactivated fetal calf serum, at 37 °C, in 5% CO2. MTT method was used to detect the cytotoxicity of bLf to HepG2 cells. Apo-bLf and holo-bLf were prepared from bLf. Iron saturation of these proteins was determined by atomic absorption spectrophotometry. Non-cytotoxic concentrations of candidate proteins were used in anti-HBV tests. Fluorescent quantitative polymerase chain reaction was used to detect HBV-DNA. Results: The TC50 and TC0of bLf were 54.570 mg/ml and 1.997 mg/ml, respectively. The iron saturation of bLf, apo-bLf and holo-bLf were 10.29%, 8.42% and 85.32%, respectively. In this study, four non-cytotoxic concentrations of candidate proteins (1.5, 1.0, 0.5, and 0.1 mg/ml, respectively) were used to inhibit HBV in HepG2 cells. The results showed that 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf effectively impaired the HBV-DNA amplification in HBV-infected HepG2 cells (P < 0.05). However, apo-bLf, and Fe3+ did not show the anti-HBV effects. Conclusion: A total of 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf could inhibit HBV-DNA in HepG2 cells. Complete bLf structure, appropriate concentration and iron saturation of bLf are necessary conditions for anti-HBV effects.

Dopamine Inhibits the Expression of Hepatitis B Virus Surface and e Antigens by Activating the JAK/STAT Pathway and Upregulating Interferon-stimulated Gene 15 Expression.

Background and Aims: Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis and liver cancer, and its treatment continues to be difficult. We previously demonstrated that a dopamine analog inhibited the packaging of pregenomic RNA into capsids. The present study aimed to determine the effect of dopamine on the expressions of hepatitis B virus surface and e antigens (HBsAg and HBeAg, respectively) and to elucidate the underlying mechanism. Methods: We used dopamine-treated HBV-infected HepG2.2.15 and NTCP-G2 cells to monitor HBsAg and HBeAg expression levels. We analyzed interferon-stimulated gene 15 (ISG15) expression in dopamine-treated cells. We knocked down ISG15 and then monitored HBsAg and HBeAg expression levels. We analyzed the expression of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway factors in dopamine-treated cells. We used dopamine hydrochloride-treated adeno-associated virus/HBV-infected mouse model to evaluate HBV DNA, HBsAg, and HBeAg expression. HBV virus was collected from HepAD38.7 cell culture medium. Results: Dopamine inhibited HBsAg and HBeAg expression and upregulated ISG15 expression in HepG2.2.15 and HepG2-NTCP cell lines. ISG15 knockdown increased HBsAg and HBeAg expression in HepG2.2.15 cells. Dopamine-treated cells activated the JAK/STAT pathway, which upregulated ISG15 expression. In the adeno-associated virus-HBV murine infection model, dopamine downregulated HBsAg and HBeAg expression and activated the JAK-STAT/ISG15 axis. Conclusions: Dopamine inhibits the expression of HBsAg and HBeAg by activating the JAK/STAT pathway and upregulating ISG15 expression.

Inhibitory nitric oxide production and cytotoxic activities of phenolic compounds from Gnetum montanum Markgr.

One new compound, styrene dimer-type listeanol-4-O-α-ʟ-rhamnopyranosyl-(1→4)-ß-ᴅ-glucopyranoside (1), and four known compounds namely listeanol (2), isorhapotigenin (3), genetifolin E (4), gnetifolin K (5) were isolated from the methanolic extract from the aerial part of the Gnetum montanum Markgr. in Viet Nam. Their chemical structures were determined by modern spectroscopic methods (NMR and HR-ESI-MS) and comparison with those of published data. These compounds were evaluated for their anti-inflammatory and cytotoxic activities. Among them, compound 3 exhibited the NO inhibitory production on the RAW264.7 cell line, and inhibited the HepG2 cell line with respective IC50 values of 79.88 ± 5.51 (µg/mL) (L-NMMA 7.90 ± 0.63 µg/mL), and 63.48 ± 3.63 (µg/mL) (Ellipticine 0.40 ± 0.01 µg/mL).

Cytotoxic potential and metabolomic profiling of alkaloid rich fraction of Tylophora indica leaves.

Tylophora indica (Burm f.) Merrill, belong to family Asclepiadaceae, is considered to be a natural remedy with high medicinal benefits. The objective of this work is to assess the metabolomic profile of T. indica leaves enriched in alkaloids, as well as to evaluate the in vitro cytotoxicity of these leaves using the MTT assay on human breast MCF-7 and liver HepG2 cancer cell lines. Dried leaves of T. indica were extracted by sonication, using methanol containing 2 % (v/v) of acetic acid and obtained fraction was characterized by HPTLC and UPLC-MS. The UPLC-MS study yielded a preliminary identification of 32 metabolites, with tylophorine, tylophorine B, tylophorinine, and tylophorinidine being the predominant metabolites. The cytotoxicity of the extract of T. indica was evaluated on HepG2 and MCF-7 cell lines, yielding inhibitory concentration (IC50) values of 75.71 µg/mL and 69.60 µg/mL, respectively. Data suggested that the phytochemical screening clearly showed presence of numerous secondary metabolites with moderate cytotoxic efficacy. In conclusion, the future prospects of T. indica appear promising for the advancement of phytopharmaceutical-based anticancer medications, as well as for the design of contemporary pharmaceuticals in the field of cancer chemotherapy.

Isobavachalcone induces hepatotoxicity in zebrafish embryos and HepG2 cells via the System Xc--GSH-GPX4 signaling pathway in ferroptosis response.

Isobavachalcone (IBC) is a flavonoid component of the traditional Chinese medicine Psoraleae Fructus, with a range of pharmacological properties. However, IBC causes some hepatotoxicity, and the mechanism of toxicity is unclear. The purpose of this paper was to investigate the possible mechanism of toxicity of IBC on HepG2 cells and zebrafish embryos. The results showed that exposure to IBC increased zebrafish embryo mortality and decreased hatchability. Meanwhile, IBC induced liver injury and increased expression of ALT and AST activity. Further studies showed that IBC caused the increase of ROS and MDA the decrease of CAT, GSH, and GSH-Px; the increase of Fe2+ content; and the changes of ferroptosis related genes (acsl4, gpx4, and xct) and iron storage related genes (tf, fth, and fpn) in zebrafish embryos. Through in vitro verification, it was found that IBC also caused oxidative stress and increased Fe2+ content in HepG2 cells. IBC caused depolarization of mitochondrial membrane potential (MMP) and reduction of mitochondrial ATP, as well as altered expression of ACSl4, SLC7A11, GPX4, and FTH1 proteins. Treatment of HepG2 cells with ferrostatin-1 could reverse the effect of IBC. Targeting the System Xc--GSH-GPX4 pathway of ferroptosis and preventing oxidative stress damage might offer a theoretical foundation for practical therapy and prevention of IBC-induced hepatotoxicity.

Protopanaxadiol Targeting Membrane Induces HepG2 Cell Apoptosis Via Raft-like Formation and Tubulation Disruption.

Protopanaxadiol (PPD), which has a molecular structure similar to cholesterol, is a potent anticancer agent that has been proposed to target the lipid membrane for the pharmacological effects. However, the underlying mechanism by which PPD modulates the cell membrane leading to cancer cell death is not be fully understood. In this work, we used single cell infrared spectroscopy, scanning electron microscopy and confocal microscopy to investigate the effects of PPD on human hepatocellular carcinoma (HepG2) cells, focusing on the change in membrane structure. We found that PPD significantly reduced the number of membrane tubules over the course of treatment. Interestingly, the addition of PPD could promote the formation of lipid raft-like domains (PPD rafts) and even restore the domain disruption caused by methyl-beta-cyclodextrin depletion of membrane cholesterol. In addition, PPD pre-treatment may increase the induction effect of FasL, which impairs cell viability, although it does not appear to be beneficial for Fas clustering in the PPD rafts. Collectively, these results highlight a non-classical mechanism by which PPD induces HepG2 apoptosis by directly affecting the physical properties of the cell membrane, providing a novel insight into understanding membrane-targeted therapy.

Identifying novel aryl hydrocarbon receptor (AhR) modulators from clinically approved drugs: In silico screening and In vitro validation.

The aryl hydrocarbon receptor (AhR) functions as a vital ligand-activated transcription factor, governing both physiological and pathophysiological processes. Notably, it responds to xenobiotics, leading to a diverse array of outcomes. In the context of drug repurposing, we present here a combined approach of utilizing structure-based virtual screening and molecular dynamics simulations. This approach aims to identify potential AhR modulators from Drugbank repository of clinically approved drugs. By focusing on the AhR PAS-B binding pocket, our screening protocol included binding affinities calculations, complex stability, and interactions within the binding site as a filtering method. Comprehensive evaluations of all DrugBank small molecule database revealed ten promising hits. This included flibanserin, butoconazole, luliconazole, naftifine, triclabendazole, rosiglitazone, empagliflozin, benperidol, nebivolol, and zucapsaicin. Each exhibiting diverse binding behaviors and remarkably very low binding free energy. Experimental studies further illuminated their modulation of AhR signaling, and showing that they are consistently reducing AhR activity, except for luliconazole, which intriguingly enhances the AhR activity. This work demonstrates the possibility of using computational modelling as a quick screening tool to predict new AhR modulators from extensive drug libraries. Importantly, these findings hold immense therapeutic potential for addressing AhR-associated disorders. Consequently, it offers compelling prospects for innovative interventions through drug repurposing.

An Optimized P. berghei Liver Stage-HepG2 Infection Model for Simultaneous Quantitative Bioimaging of Host and Parasite Nascent Proteomes.

The Plasmodium parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The parasite liver stage is a key intervention point for antimalarial chemoprophylaxis: successful targeting of liver-stage parasites prevents disease development in individuals and can help to reduce parasite transmission in populations, as the gametocyte forms that transmit infection to mosquitos are exclusively found in the blood stage. Antimalarial drugs that can target multiple parasite stages are thus highly desirable, and one emerging cellular target for such multistage active compounds is the process of protein synthesis or translation. Quantitative study of liver stage translation, and thus mechanistic evaluation of translation inhibitors against liver stage parasites, is not amenable to the methods allowing quantification of asexual blood stage translation, such as radiolabeled amino acid incorporation or lysate-based translation of reporter transcripts. Here, we present a method using o-propargyl puromycin (OPP) labeling of host and parasite nascent proteomes in the P. berghei-HepG2 infection model, followed by automated confocal image acquisition and computational separation of P. berghei vs. H. sapiens nascent proteome signals to allow simultaneous readout of the effects of translation inhibitors on both host and parasite. This protocol details our HepG2 cell culture and infected monolayer handling optimized for microscopy, our OPP labeling workflow, and our approach to automated confocal imaging, image processing, and data analysis. Key features • Uses the o-propargyl puromycin labeling technique developed by Liu et al. to quantitatively analyze protein synthesis in Plasmodium berghei liver-stage parasites in actively translating hepatoma cells. • This quantitative approach should be adaptable for other puromycin-sensitive intracellular pathogens residing in actively translating host cells. • The P. berghei-infected HepG2 recovery and reseeding protocol presented here is of use in applications beyond nascent proteome labeling and quantification.

Comprehensive Transcriptome Profiling of Antioxidant Activities by Glutathione in Human HepG2 Cells.

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 TGFß, 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.

Trimethylamine-N-oxide, a New Risk Factor for Non-alcoholic Fatty Liver Disease Changes the Expression of miRNA-34a, and miRNA-122 in the Fatty Liver Cell Model.

Non-alcoholic fatty liver disease is a multifactorial disorder with complicated pathophysiology ranging from simple steatosis to steatohepatitis and liver fibrosis. Trimethylamine-N-oxide (TMAO) production is believed to be correlated with choline deficiency. This study investigated the expression of miRNA-34a, miRNA-122, and miRNA-192 in the fatty liver cell model treated with different concentrations of TMAO. A fatty liver cell model was developed by exposing HepG2 cells to a mixture of palmitate and oleate in a ratio of 1:2 at a final concentration of 1200 µM for 24 h. The confirmed fatty liver cells were treated with 37.5, 75, 150, and 300 µM of TMAO for 24 h. RT-qPCR was used to quantify the expression of microRNAs in a cellular model. The cellular expression of all microRNAs was significantly higher in treated fatty liver cells compared to normal HepG2 cells (P < 0.05). Only 75 and 150 µM of TMAO significantly increased the expression of miRNA-34a and miRNA-122 compared to both fatty and normal control cells (P < 0.05). Our results provided an experimental documentation for the potential effect of TMAO to change the expression of miR-34a and miR-22 as a mechanism for contributing to the pathogenesis of non-alcoholic fatty liver disease.

The cytotoxic effects of prazosin, chlorpromazine, and haloperidol on hepatocellular carcinoma and immortalized non-tumor liver cells.

Liver cancer annually accounts for over 800,000 cases and 700,000 deaths worldwide. Hepatocellular carcinoma is responsible for over 80% of liver cancer cases. Due to ineffective treatment options and limited surgical interventions, hepatocellular carcinoma is notoriously difficult to treat. Nonetheless, drugs utilized for other medical conditions, such as the antihypertensive medication prazosin, the neuroleptic medication chlorpromazine, and the neuroleptic medication haloperidol, have gained attention for their potential anti-cancer effects. Therefore, this study used these medications for investigating toxicity to hepatocellular carcinoma while testing the adverse effects on a noncancerous liver cell line model THLE-2. After treatment, an XTT cell viability assay, cell apoptosis assay, reactive oxygen species (ROS) assay, apoptotic proteome profile, and western blot were performed. We calculated IC50 values for chlorpromazine and prazosin to have a molar range of 35-65 µM. Our main findings suggest the capability of both of these treatments to reduce cell viability and generate oxidative stress in HepG2 and THLE-2 cells (p value < 0.05). Haloperidol, however, failed to demonstrate any reduction in cell viability revealing no antitumor effect up to 100 µM. Based on our findings, a mechanism of cell death was not able to be established due to lack of cleaved caspase-3 expression. Capable of bypassing many aspects of the lengthy, costly, and difficult cancer drug approval process, chlorpromazine and prazosin deserve further investigation for use in conjunction with traditional chemotherapeutics.

Deciphering the impact of greenhouse pesticides on hepatic metabolism profile: Toxicity experiments on HepG2 cells using chlorpyrifos and emamectin benzoate.

Epidemiological evidence on the health effects of pesticide exposure among greenhouse workers is limited, and the mechanisms are lacking. Building upon our team's previous population study, we selected two pesticides, CPF and EB, with high detection rates, based on the theoretical foundation that the liver serves as a detoxifying organ, we constructed a toxicity model using HepG2 cells to investigate the impact of individual or combined pesticide exposure on the hepatic metabolism profile, attempting to identify targeted biomarkers. Our results showed that CPF and EB could significantly affect the survival rate of HepG2 cells and disrupt their metabolic profile. There were 117 metabolites interfered by CPF exposure, which mainly affected ABC transporter, biosynthesis of amino acids, center carbon metabolism in cancer, fatty acid biosynthesis and other pathways, 95 metabolites interfered by EB exposure, which mainly affected center carbon metabolism in cancer, HIF-1 signaling pathway, valine, leucine and isoleucine biosynthesis, fatty acid biosynthesis and other pathways. The cross analysis and further biological experiments confirmed that CPF and EB pesticide exposure may affect the HIF-1 signaling pathway and valine, leucine and isoleucine biosynthesis in HepG2 cells, providing reliable experimental evidence for the prevention and treatment of liver damage in greenhouse workers.

Preparation and characterization of Sorafenib nano-emulsion: impact on pharmacokinetics and toxicity; an in vitro and in vivo study.

Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer-related deaths worldwide. Current treatment strategies include surgical resection, liver transplantation, liver-directed therapy, and systemic therapy. Sorafenib (Sor) is the first systemic drug authorized by the US Food and Drug Administration (FDA) for HCC treatment. Nevertheless, the conventional oral administration of Sor presents several limitations: poor solubility, low bioavailability, drug resistance development, and off-target tissue accumulation, leading to numerous adverse effects. Nano-emulsion, a nano-delivery system, is a viable carrier for poorly water-soluble drugs. It aims to enhance drug bioavailability, target organ accumulation, and reduce off-target tissue exposure, thus improving therapeutic outcomes while minimizing side effects. This study formulated Sor nano-emulsion (Sor NanoEm) using the homogenization technique. The resultant nano-emulsion was characterized by particle size (121.75 ± 12 nm), polydispersity index (PDI; 0.310), zeta potential (-12.33 ± 1.34 mV), viscosity (34,776 ± 3276 CPs), and pH (4.38 ± 0.3). Transmission Electron Microscopy exhibited spherical nano-droplets with no aggregation signs indicating stability. Furthermore, the encapsulation of Sor within the nano-emulsion sustained its release, potentially reducing the frequency of therapeutic doses. Cytotoxicity assessments on the HepG2 cell line revealed that Sor NanoEm had a significantly (P < 0.05) more potent cytotoxic effect compared to Sor suspension. Subsequent tests highlighted superior pharmacokinetic parameters and reduced dosage requirements of Sor NanoEm in mice. It exhibited an enhanced safety profile, particularly in behavior, brain, and liver, compared to its suspended form. These findings underscore the enhanced pharmacological and toxicological attributes of Sor Nano-emulsion, suggesting its potential utility in HCC treatment.

COSMIC-based mutation database enhances identification efficiency of HLA-I immunopeptidome.

BACKGROUND: Neoantigens have emerged as a promising area of focus in tumor immunotherapy, with several established strategies aiming to enhance their identification. Human leukocyte antigen class I molecules (HLA-I), which present intracellular immunopeptides to T cells, provide an ideal source for identifying neoantigens. However, solely relying on a mutation database generated through commonly used whole exome sequencing (WES) for the identification of HLA-I immunopeptides, may result in potential neoantigens being missed due to limitations in sequencing depth and sample quality. METHOD: In this study, we constructed and evaluated an extended database for neoantigen identification, based on COSMIC mutation database. This study utilized mass spectrometry-based proteogenomic profiling to identify the HLA-I immunopeptidome enriched from HepG2 cell. HepG2 WES-based and the COSMIC-based mutation database were generated and utilized to identify HepG2-specific mutant immunopeptides. RESULT: The results demonstrated that COSMIC-based database identified 5 immunopeptides compared to only 1 mutant peptide identified by HepG2 WES-based database, indicating its effectiveness in identifying mutant immunopeptides. Furthermore, HLA-I affinity of the mutant immunopeptides was evaluated through NetMHCpan and peptide-docking modeling to validate their binding to HLA-I molecules, demonstrating the potential of mutant peptides identified by the COSMIC-based database as neoantigens. CONCLUSION: Utilizing the COSMIC-based mutation database is a more efficient strategy for identifying mutant peptides from HLA-I immunopeptidome without significantly increasing the false positive rate. HepG2 specific WES-based database may exclude certain mutant peptides due to WES sequencing depth or sample heterogeneity. The COSMIC-based database can effectively uncover potential neoantigens within the HLA-I immunopeptidomes.

Purification and Identification of Peptides from Hydrilla verticillata (Linn. f.) Royle with Cytoprotective and Antioxidative Effect against H2O2-Treated HepG2 Cells.

Antioxidant peptides were purified from Hydrilla verticillata (Linn. f.) Royle (HVR) protein hydrolysate by ultrafiltration, gel filtration chromatography, and semipreparative reversed-phase HPLC and identified by UPLC-ESI-MS/MS. Therein, TCLGPK and TCLGER were selected to be synthesized, and they displayed desirable radical-scavenging activity to ABTS (99.20 ± 0.56-99.20 ± 0.43%), DPPH (97.32 ± 0.59-97.56 ± 0.97%), hydroxyl radical (54.32 ± 1.27-70.42 ± 2.01%), and superoxide anion (42.93 ± 1.46-52.62 ± 1.11%) at a concentration of 0.96 µmol/mL. They possessed a cytoprotective effect against H2O2-induced oxidative stress in HepG2 cells in a dose-dependent manner. 1.6 µmol/mL of the two peptides could perfectly protect HepG2 cells from H2O2-induced injury. The TCLGPK exhibited higher antioxidant activity and cytoprotective effect than TCLGER. Western blot and molecular docking results indicated that the two peptides achieved antioxidant ability and cytoprotective effect by combining with Kelch-like ECH-associated protein 1 (Keap1) to activate the Keap1-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements signaling pathway, leading to the activity and expression of the related antioxidases in the pathway significantly up-regulating and the intracellular reactive oxygen species level, lipid peroxidation, and cell apoptosis rate significantly down-regulating.