Human Hepatoma Huh7 Cells Research Articles

4-Hydroxyestrogen Metabolites Strongly Prevent Chemically-Induced Ferroptotic Hepatocyte Injury In Vitro and In Vivo.

Ferroptosis is a regulated cell death characterized by excessive accumulation of toxic lipid reactive oxygen species (ROS). Ferroptosis is an underlying cause in some human diseases, including the drug-induced liver injury. The present study aims to determine whether 4-hydroxyestrone (4-OH-E1) and 4-hydroxyestradiol (4-OH-E2), two endogenous catechol estrogens, can prevent chemically-induced ferroptotic hepatocyte injury in vitro and in vivo. The induction of ferroptotic cell death by erastin and RSL3 in rat H-4-II-E and human HuH-7 hepatoma cells is used as in vitro models. 4-OH-E1 and 4-OH-E2 each exhibit a strong protection against erastin/RSL3-induced ferroptosis in H-4-II-E hepatoma cells, and they also strongly abrogate erastin/RSL3-induced accumulation of cellular NO, ROS and lipid-ROS. A similar protective effect is observed with 4-OH-E1 and 4-OH-E2 in RSL3-induced ferroptosis in HuH-7 cells. Mechanistically, these two catechol estrogens protect hepatoma cells against chemically-induced ferroptosis mainly through binding to cellular PDI protein with a high affinity, which leads to inhibition of PDI-catalyzed NOS dimerization (activation), thereby preventing the accumulation of cellular NO, ROS and lipid-ROS. In addition, the direct antioxidant activity of these two estrogens may also partially contribute to their cytoprotective effect. In vivo animal studies show that 4-OH-E1 and 4-OH-E2 also have a strong protective effect against acetaminophen-induced liver injury in a mouse model. Together, the results of this study demonstrate that 4-OH-E1 and 4-OH-E2 are endogenous factors with a strong protective activity against chemically-induced hepatocyte injury both in vitro and in vivo.

Casz1 and Znf101/Zfp961 differentially regulate apolipoproteins A1 and B, alter plasma lipoproteins, and reduce atherosclerosis.

High apolipoprotein B-containing (apoB-containing) low-density lipoproteins (LDLs) and low apoA1-containing high-density lipoproteins (HDLs) are associated with atherosclerotic cardiovascular diseases. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both significantly decreases apoB and increases apoA1 expression by inducing mRNA degradation of 2 different transcription factors, Znf101 and Casz1. We found that Znf101 enhances apoB expression, while Casz1 represses apoA1 expression. The hepatic knockdown of Casz1 in mice increased plasma apoA1, HDL, and cholesterol efflux capacity. The hepatic knockdown of Zfp961, an ortholog of Znf101, reduced lipogenesis and production of triglyceride-rich lipoproteins and atherosclerosis, without causing hepatic lipid accumulation. This study identifies hepatic Znf101/Zfp961 and Casz1 as potential therapeutic targets to alter plasma lipoproteins and reduce atherosclerosis without causing liver steatosis.

Unveiling the Cytotoxic and Apoptosis-Inducing Abilities of An Edible Bird’s Nest Extract in Human Hepatocellular Carcinoma HUH-7 Cells

Unveiling the Cytotoxic and Apoptosis-Inducing Abilities of An Edible Bird’s Nest Extract in Human Hepatocellular Carcinoma HUH-7 Cells

Gallein, G protein βγ subunits inhibitor, suppresses the TGF-α-induced migration of hepatocellular carcinoma cells via inhibition of the c-Jun N-terminal kinase.

G protein-coupled receptor (GPCR) signaling regulates a wide range of pathophysiological cell functions via G protein α and βγ subunits. Small molecules targeting the subunits of Gα and Gβγ have been developed as cancer therapeutics. We have previously reported that transforming growth factor-α (TGF-α) induces the migration of human hepatocellular carcinoma (HCC) HuH7 cells through the activation of AKT, p38 mitogen-activated protein kinase (MAPK), Rho-kinase and c-Jun N-terminal kinase (JNK). This study aims to determine whether Gβγ subunits regulate the TGF-α-induced migration of HCC HuH7 cells using gallein, a Gβγ subunits inhibitor. The Janus family of tyrosine kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway was also involved in the regulation of the migration. Gallein significantly reduced the TGF-α-induced cell migration. In contrast, fluorescein, a gallein-related compound that has no effect on Gβγ subunits, failed to affect the cell migration. Gallein suppressed the TGF-α-stimulated phosphorylation of JNK without affecting the phosphorylation of epidermal growth factor receptor, AKT, p38 MAPK, target protein of Rho-kinase and STAT3. Conversely, fluorescein did not attenuate the phosphorylation of JNK. These results strongly suggest that Gβγ subunits act as positive regulators in TGF-α-induced migration of HCC cells via the JNK signalling pathway.

6824 Bile Acid Elevation in Nonalcoholic Steatohepatitis Correlates With Fibrogenesis and Mitochondrial Dysfunction In Vitro

Abstract Disclosure: N. Patel: None. Y. Li: None. M. Raul: None. C. Sottas: None. V. Papadopoulos: None. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease. It is estimated that over 115 million adults are affected by NASH worldwide. The causes of NASH are unclear, and no treatment is yet available. Bile acids (BAs) are steroids synthesized in the liver, promoting lipid absorption. The levels of BAs are tightly regulated in the body. Patients diagnosed with NASH frequently exhibit increased levels of BAs in both liver tissue and plasma, indicating a potential association between elevated BA concentrations and the pathogenesis of NASH. In an in vivo study using a high-fat diet rat model of NASH, we measured plasma BA composition and levels. The results obtained indicated substantial increases in the concentrations of glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), and taurodeoxycholic acid (TDCA) in NASH compared to controls. These findings suggested their potential implication in the development of NASH. To elucidate the mechanistic link between changes in BA synthesis and fibrogenesis, in vitro experiments were conducted using the LX-2 humane hepatic stellate and Huh7 human hepatocellular carcinoma cells. First, the concentrations of the selected bile acids (GCA, GCDCA, GDCA, and TDCA) used in our studies were found to be non-toxic to the cells using the MTT assay. Subsequent qPCR analyses demonstrated the upregulation of key fibrotic genes (TGFβ, COL1A1, ACTA2) in LX-2 cells following exposure to individual bile acids GCA, GCDCA, GDCA, and TDCA. Immunocytochemistry (ICC) studies further demonstrated fibrogenesis by revealing increased expression of COL1A1 following treatment with each of the four bile acids. These data suggest that changes in BA formation are linked to fibrogenesis. We subsequently explored the impact of BAs on mitochondrial function using the Seahorse XF Cell Mito Stress Test in Huh7 cells. The results obtained revealed a reduction in the oxygen consumption rate and ATP production in response to BA treatment. This observation suggests a potential role for modified BAs in disrupting mitochondrial homeostasis, a recognized contributor to the progression of NASH. In summary, the data presented provide valuable insights into the complex effects of modified BAs on NASH pathogenesis, establishing connections between changes in BA composition and fibrogenesis, mitochondrial dysfunction. Presentation: 6/2/2024

MicroRNA-615-3p decreases apo B expression in human liver cells

Plasma lipids are mainly carried in apolipoprotein B (apoB) containing lipoproteins. High levels of these lipoproteins are associated with several metabolic diseases and lowering their plasma levels is associated with reduced incidence of atherosclerotic cardiovascular disease. MicroRNAs (miRs) are small non-coding RNAs that reduce the protein expression of their target mRNAs and are potential therapeutic agents. Here, we identified a novel miR-615-3p that interacts with human 3′-UTR of apoB mRNA, induces post-transcriptional mRNA degradation, and reduces cellular and secreted apoB100 in human hepatoma Huh-7 cells. Reducing cellular miR-615-3p levels by CRISPR-sgRNA increased cellular and secreted apoB100 indicating endogenous miR regulates apoB expression. Overexpression of miR-615-3p along with or without palmitic acid treatment decreased cellular and media apoB and increased cellular triglyceride levels without inducing endoplasmic reticulum stress. These studies have identified miR-615-3p as a negative regulator of apoB expression in human liver-derived cells. It is likely that there are more miRs that regulate apoB-containing lipoprotein assembly and secretion. Discovery of additional miRs may uncover novel mechanisms that control lipoprotein assembly and secretion.

SIRT1 regulates hepatic vldlr levels

BackgroundEndoplasmic reticulum (ER) stress-mediated increases in the hepatic levels of the very low-density lipoprotein (VLDL) receptor (VLDLR) promote hepatic steatosis by increasing the delivery of triglyceride-rich lipoproteins to the liver. Here, we examined whether the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) regulates hepatic lipid accumulation by modulating VLDLR levels and the subsequent uptake of triglyceride-rich lipoproteins.MethodsRats fed with fructose in drinking water, Sirt1−/− mice, mice treated with the ER stressor tunicamycin with or without a SIRT1 activator, and human Huh-7 hepatoma cells transfected with siRNA or exposed to tunicamycin or different inhibitors were used.ResultsHepatic SIRT1 protein levels were reduced, while those of VLDLR were upregulated in the rat model of metabolic dysfunction-associated steatotic liver disease (MASLD) induced by fructose-drinking water. Moreover, Sirt1−/− mice displayed increased hepatic VLDLR levels that were not associated with ER stress, but were accompanied by an increased expression of hypoxia-inducible factor 1α (HIF-1α)-target genes. The pharmacological inhibition or gene knockdown of SIRT1 upregulated VLDLR protein levels in the human Huh-7 hepatoma cell line, with this increase abolished by the pharmacological inhibition of HIF-1α. Finally, SIRT1 activation prevented the increase in hepatic VLDLR protein levels in mice treated with the ER stressor tunicamycin.ConclusionsOverall, these findings suggest that SIRT1 attenuates fatty liver development by modulating hepatic VLDLR levels.

N-acetylcysteine alleviates arsenic trioxide-induced reductions in hepatic catalase gene expression both in vitro and in vivo

Arsenic trioxide (ATO), one of the oldest and most frequently used poisons, is well-known in forensic science for inducing hepatotoxicity. The regulation of peroxisomal antioxidative enzyme catalase (CAT) involves intricate mechanisms at both transcriptional and post-transcriptional levels. However, the molecular mechanisms underlying the regulation of CAT gene expression in hepatic cells remain elusive. Furthermore, the regulation of CAT gene expression evident in animals administered with ATO in vivo is not well-explored, although several studies have revealed ATO-induced reductions in CAT enzymatic activity in rat livers. In this study, we revealed ATO-dependent reductions in CAT gene expression in both rat liver and Huh-7 human hepatoma cells. Our results indicate that the decline in CAT enzymatic activity can be attributed, at least in part, to the downregulation of its gene expression. The ATO-induced reduction in CAT expression was concurrent with the reduction in peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator (PGC)-1α and inactivation of PPARγ, both considered as positive regulators of CAT gene expression. Moreover, antioxidant N-acetylcysteine (NAC) demonstrated the capability to alleviate the downregulation of CAT gene expression both in vivo and in vitro. Additionally, NAC played a role in alleviating ATO-induced hepatotoxicity, potentially by mitigating the transcriptional downregulation of the CAT gene. Altogether, these results indicate that ATO exerts toxicity by inhibiting the antioxidant defense mechanism, which may be useful for forensic diagnosis of arsenic poisoning and clinical treatment of mitigating ATO-induced hepatotoxicity.

Abstract 1022: Microrna-615-3p Decreases Apolipoprotein B Expression In Human Liver Cells And Its Plasma Concentrations Are Inversely Correlated With Plasma Lipid Levels

Plasma lipids are mainly carried in apolipoprotein B (apoB) containing lipoproteins. High levels of these lipoproteins are associated with several metabolic diseases and lowering their plasma levels is associated with reduced incidence of atherosclerotic cardiovascular disease. MicroRNAs (miRs) are small non-coding RNAs that reduce protein expression of their target genes and are potential therapeutic agents. Here, we identified a novel miR-615-3p that interacts with human 3′-UTR of apoB mRNA, induces post-transcriptional mRNA degradation, and reduces cellular and secreted apoB100 in human hepatoma Huh-7 cells. Reducing cellular miR-615-3p levels by CRISPR-sgRNA increased cellular and secreted apoB100 indicating endogenous miR regulates apoB expression. Furthermore, we found that obese subjects have lower levels of miR-615-3p than healthy individuals. Moreover, there was a significant negative correlation between miR-615-3p and plasma lipids and apoB levels. These studies indicate that miR-615-3p negatively regulates apoB and its plasma levels could serve as a biomarker for obesity and possibly other metabolic diseases.

Abstract 3041: APOB Gene Deletion Increases The Expression Of Vitamin D Binding Protein In Human Hepatoma Cells

High plasma apolipoprotein B (apoB)-containing lipoproteins are both a biomarker and a causal mediator of many metabolic diseases. Inhibition of APOB mRNA decreases plasma lipid levels but at a risk of lipid accumulation in tissues. To understand the global consequences of reduced apoB synthesis, we generated a human hepatoma (Huh-7) cell culture model system deficient in apoB. ApoB deficient Huh-7 cells (Ako) were generated using the CRISPR/Cas9 system. Total RNA for transcriptomics and overnight conditioned media for proteomics and lipidomics was obtained from the Ako and Huh-7 cells (n=3). Statistical analysis was done using Student’s t-test while comparing two groups, p < 0.05 was considered significant. APOB gene deletion significantly reduced APOB mRNA and protein levels in the Ako cells compared to control cells. Ako cells supported apoB48 secretion when transfected with apoB48 expression plasmids. These cells did not accumulate significant amounts of triglyceride. KEGG pathway analysis of RNA-Seq data showed a significant increase in DNA replication/repair pathway and complement/coagulation cascade in the Ako cells. The most downregulated pathways included the PI3k-Akt signaling and MAPK signaling pathways. Differential gene expression analysis identified vitamin D binding protein (VDB, gene GC ) mRNA as the most highly upregulated transcript in Ako cells. The Ako media secretome contained significantly higher amounts of VDB compared to control Huh-7. Furthermore, targeted lipidomics of the secretome showed significant increases in media ergocalciferol. Our qPCR and western blot analysis confirmed increased expression of VDB mRNA and protein secretion in the Ako cells as compared to the Huh-7 cells. We established a cell culture model deficient in secreting apoB-containing lipoproteins. These cells, however, support the assembly and secretion of apoB48 when transfected with apoB48 expression plasmid. A major consequence of apoB deficiency was upregulation of the VDB expression. We interpret these data to suggest that human hepatoma cells have two differentially regulated pathways for the transport of vitamin D. In the absence of apoB-lipoprotein assembly, these cells upregulate VDB to augment secretion of vitamin D.

Effects of plumbagin on proliferation and apoptosis of human hepatocellular carcinoma cells based on CKB/p53 signaling pathway

To investigate the effects of plumbagin on the proliferation and apoptosis of human hepatoma Huh-7 cells and its mechanism based on the creatine kinase B(CKB)/p53 signaling pathway. Huh-7 cells were treated with plumbagin from 1 to 12 μmol·L~(-1) for cell counting kit-8(CCK-8) assay, and 1, 3, and 6 μmol·L~(-1) were determined as low, medium, and high concentrations of plumbagin for subsequent experiments. CKB gene was knocked out in Huh-7 cells by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated proteins(Cas)-9 gene editing technology. CKB overexpression lentivirus was transfected into Huh-7 cells to up-regulate the expression of CKB. Cell proliferation and apoptosis were detected by plate cloning assay and flow cytometry. The mRNA expression of CKB was detected by quantitative real-time PCR(qRT-PCR). CKB, p53, mouse double minute 2 homolog(MDM2), B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), and caspase-3 protein were detected by Western blot(WB). The results showed that plumbagin significantly inhibited the proliferation of Huh-7 cells and induced cell apoptosis. Compared with the control group, the apoptosis level was significantly increased in the plumbagin group, while the apoptosis level was significantly decreased in the plumbagin combined with the apoptosis inhibitor group. Plumbagin significantly down-regulated the protein expression levels of CKB, Bcl-2, and MDM2 and up-regulated the protein expression levels of p53, Bax, and caspase-3. Knockdown of the CKB gene decreased the proliferative ability of Huh-7 cells, increased the apoptotic rate, decreased the expression levels of Bcl-2 and MDM2 proteins, and increased the expression levels of p53, Bax, and caspase-3 proteins. After up-regulation of CKB expression, the proliferation ability of Huh-7 cells was enhanced, and the protein expression levels of Bcl-2 and MDM2 were elevated. The protein expression levels of p53, Bax, and caspase-3 were decreased. In addition, plumbagin reversed the effect of overexpression of CKB on the proliferation and apoptosis of Huh-7 cells. In conclusion, plumbagin significantly inhibited the proliferative ability of Huh-7 cells, and the mechanism may be related to the inhibition of CKB expression, activation of the p53 signaling pathway, and regulation of the expression of mitochondrial-associated apoptotic proteins, ultimately inducing cell apoptosis.

Platelets Affect the Activity of Amino Acid Transporter SNAT4 in HuH-7 Human Hepatoma Cells

Platelets have been reported to exert diverse actions besides hemostasis and thrombus formation in the body. However, whether platelets affect transporter activity remains to be determined. In this study, we examined the effects of platelets on the activity of amino acid transporter system A, which is known to be changed by various factors, and we clarified the mechanism by which platelets affect system A activity. Among system A subtypes, we found that sodium-coupled neutral amino acid transporter (SNAT) 4 played a central role in the transport activity of system A in HuH-7 human hepatoma cells. Interestingly, platelets showed a biphasic effect on system A activity: activated platelet supernatants (APS) including the granule contents released from platelets downregulated system A activity at lower concentrations and the downregulation was suppressed at higher concentrations. The downregulation was due to a decrease in the affinity of SNAT4 for its substrate and not a decrease in the SNAT4 abundance on the plasma membrane. In addition, APS did not decrease the expression level of SNAT4 mRNA. On the other hand, platelets did not affect system A activity when the platelet suspension was added to HuH-7 cells. These results indicate that platelets indirectly affect the transport activity of system A by releasing bioactive substances but do not directly affect it by binding to HuH-7 cells.

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl β-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Network pharmacology, molecular docking and experimental verification reveal the mechanism of Yiguanjian decoction in treating acute liver failure

Introduction: The potential targets and action mechanism of Yiguanjian (YGJ) decoction in treating acute liver failure (ALF) were determined using network pharmacology, molecular docking and cell experiments.Methods: The Pharmacology of Traditional Chinese Medicine Systems (TCMSP) and BATMAN-TCM databases were used to find YGJ decoction targets. The Genecard database was employed to predict targets associated with ALF. Cytascape software was utilised to visualise and select common targets along with establishing a protein–protein interaction (PPI) network. Core targets were screened and putative function was assessed via gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyzes. Molecular docking was performed using AutodockTools, PyMoL and Discovery Studio software to verify the correlation between the YGJ decoction and selected targets. MTT assay was conducted to determine the effect of the YGJ decoction on the viability of Huh-7 human hepatoma cells. The effects of the YGJ decoction on the expression of putative targets in Huh-7 cells were determined via western blot and quantitative real-time polymerase chain reaction analyzes.Results: Overall, 9153 YGJ decoction and 576 ALF-related targets were obtained, and 469 YGJ decoction and ALF cross targets were obtained. Of these, 20 key targets, including AKT1, estrogen receptor 1 (ESR1), catalase (CAT), interleukin-1β (IL-1β) and discs large homolog 4 (DLG4), were selected from the PPI network. GO function enrichment analysis revealed that these targets were primarily associated with regulating system processes, cell body, oxidoreductase activity and other processes. An enrichment analysis using the KEGG pathway database revealed that the treatment of ALF using the YGJ decoction was primarily associated with the AGE-RAGE, cGMP-PKG and HIF-1 signalling pathways. Molecular docking indicated that quercetin, stigmasterol and β- sitosterol as well as AKT1, ESR1, CAT, IL-1β and DLG4 exhibited good binding affinity. In vitro experiments revealed that the YGJ decoction significantly reversed lipopolysaccharide (LPS)-induced apoptosis (P < 0.01), inhibited LPS-induced increase in IL-1β and ESR1 levels and upregulated LPS-induced decrease in AKT1, CAT and DLG4 levels (P < 0.05).Conclusions: The YGJ decoction alleviates ALF by regulating multiple targets and its action mechanism may be associated with ameliorating oxidative stress and inflammation.

Behavior of intracellular lipid droplets during cell division in HuH7 hepatoma cells

Intracellular lipid droplets (LDs) are ubiquitous organelles found in many cell types. During mitosis, membranous organelles, including mitochondria, are divided into small pieces and transferred to daughter cells; however, the process of LD transfer to daughter cells is not fully elucidated. Herein, we investigated the behavior of LDs during mitosis in HuH7 human hepatoma cells. While fragments of the Golgi apparatus were scattered in the cytosol during mitosis, intracellular LDs retained their size and spherical morphology as they translocated to the two daughter cells. LDs were initially distributed throughout the cell during prophase but positioned outside the spindle in metaphase, aligning at the far sides of the centrioles. A similar distribution of LDs during mitosis was observed in another hepatocarcinoma HepG2 cells. When the spindle was disrupted by nocodazole treatment or never in mitosis gene A-related kinase 2A knockdown, LDs were localized in the area outside the chromosomes, suggesting that spindle formation is not necessary for LD localization at metaphase. The amount of major LD protein perilipin 2 reduced while LDs were enriched in perilipin 3 during mitosis, indicating the potential alteration of LD protein composition. Conclusively, the behavior of LDs during mitosis is distinct from that of other organelles in hepatocytes.

Inactivation of human coronaviruses using an automated room disinfection device

The emergence of more virulent and epidemic strains of viruses, especially in the context of COVID-19, makes it more important than ever to improve methods of decontamination. The objective of this study was to evaluate the potential of on-demand production of chlorine species to inactivate human coronaviruses. The commercial prototype disinfection unit was provided by Unipolar Water Technologies. The Unipolar device generates active chlorine species using an electrochemical reaction and dispenses the disinfectant vapour onto surfaces with an aspirator. The minimum effective concentration and exposure time of disinfectant were evaluated on human hepatoma (Huh7) cells using 50% tissue culture infectious dose (TCID50) assay and human coronavirus 229E (HCoV-229E), a surrogate for pathogenic human coronaviruses. We showed that chlorine species generated in the Unipolar device inactivate HCoV-229E on glass surfaces at ≥ 400 parts per million active chlorine concentration with a 5 min exposure time. Here, inactivation refers to the inability of the virus to infect the Huh7 cells. Importantly, no toxic effect was observed on Huh7 cells for any of the active chlorine concentrations and contact times tested.

Hepatoma Derived Growth Factor (HDGF) Targeting as a Therapeutic Approach for Radio-Naive and Radioresistant Hepatocellular Carcinoma

The role of radiotherapy in treating hepatocellular carcinoma (HCC) has gained importance. Hepatoma-derived growth factor (HDGF) is a nuclear growth factor involved in the development of HCC and its overexpression has been linked to tumor progression and radiation treatment failure. The aim of this study is to investigate the potential therapeutic benefits of HDGF-targeting treatment in combination with or without radiation for both radio-naive and radioresistant HCC cells. A series of radiation doses (2Gy, 4Gy, 8Gy, 12Gy, and 20Gy in one fraction) were administered to Huh7 human HCC cells. The gene expression profiles of radiation-treated Huh7 cells were analyzed. The radioresistant Huh7 cell line (Huh7-IR) was established by repeatedly irradiating with 2-week intervals with 8 Gy of X-ray to a total dose of 80 Gy. After acquiring radioresistance, the expression levels of HDGF, cancer stem cell (CSC) markers, and epithelial-mesenchymal transition (EMT) factors were validated. The pShuttle-shHDGF plasmid with adenovirus vector was generated for HDGF targeting treatment (HDGF knockdown). The effect of the HDGF targeting treatment on tumorigenic processes and cell cycle distribution in the radio-naive and radioresistant cells with or without irradiation were explored. After radiation treatment, qPCR analysis showed that the mRNA expression of HDGF, CSC markers (CD133, CD44, and ABCG2), and EMT markers (TWIST and SNAIL) increased over time, with the most significant upregulation observed between 48-72 hours. In the radioresistant Huh7-IR cells, HDGF, CSC markers (CD133 and CD44), and the EMT transcriptional factor (TWIST) were significantly higher than in the parental Huh7 cells. The Huh7-IR cells also showed a significant arrest in the G0/G1 phase. X-ray radiation alone significantly reduced cell proliferation and invasion in the Huh7 cells compared to the control group. The HDGF targeting treatment significantly enhanced the X-ray-mediated inhibition of colony formation and cell invasion in both the Huh7 and Huh7-IR cells. Targeting HDGF leads to cell cycle arrest (G2/M phase) in Huh7 and Huh7-IR cells. The HDGF targeting treatment in combination with X-ray irradiation results in extensive G2/M phase arrest. These results indicate the crucial role of HDGF in radiation therapy for radio-naive and radioresistant HCC cells. Targeting HDGF effectively suppressed colony formation and invasion, and enhanced cell cycle arrest in the G2/M phase in combination with radiation. HDGF may serve as a promising therapeutic target for improving HCC management strategies. Further investigation is required to fully comprehend the role of HDGF in improving radiation therapy efficacy for HCC.

AN1284 attenuates steatosis, lipogenesis, and fibrosis in mice with pre-existing non-alcoholic steatohepatitis and directly affects aryl hydrocarbon receptor in a hepatic cell line.

Non-alcoholic steatohepatitis (NASH) is an aggressive form of fatty liver disease with hepatic inflammation and fibrosis for which there is currently no drug treatment. This study determined whether an indoline derivative, AN1284, which significantly reduced damage in a model of acute liver disease, can reverse steatosis and fibrosis in mice with pre-existing NASH and explore its mechanism of action. The mouse model of dietary-induced NASH reproduces most of the liver pathology seen in human subjects. This was confirmed by RNA-sequencing analysis. The Western diet, given for 4 months, caused steatosis, inflammation, and liver fibrosis. AN1284 (1 mg or 5 mg/kg/day) was administered for the last 2 months of the diet by micro-osmotic-pumps (mps). Both doses significantly decreased hepatic damage, liver weight, hepatic fat content, triglyceride, serum alanine transaminase, and fibrosis. AN1284 (1 mg/kg/day) given by mps or in the drinking fluid significantly reduced fibrosis produced by carbon tetrachloride injections. In human HUH7 hepatoma cells incubated with palmitic acid, AN1284 (2.1 and 6.3 ng/ml), concentrations compatible with those in the liver of mice treated with AN1284, decreased lipid formation by causing nuclear translocation of the aryl hydrocarbon receptor (AhR). AN1284 downregulated fatty acid synthase (FASN) and sterol regulatory element-binding protein 1c (SREBP-1c) and upregulated Acyl-CoA Oxidase 1 and Cytochrome P450-a1, genes involved in lipid metabolism. In conclusion, chronic treatment with AN1284 (1mg/kg/day) reduced pre-existing steatosis and fibrosis through AhR, which affects several contributors to the development of fatty liver disease. Additional pathways are also influenced by AN1284 treatment.

Iron Chelation Property, Antioxidant Activity, and Hepatoprotective Effect of 6-Gingerol-Rich Ginger (Zingiber officinale) Extract in Iron-Loaded Huh7 Cells.

Iron is essential for numerous biological processes; however, an iron imbalance can contribute to a number of diseases. An excess of iron can accumulate in the body and subsequently induce the production of reactive oxygen species (ROS), leading to oxidative tissue damage and organ dysfunction. The liver, a major iron storage site, is vulnerable to this iron-induced oxidative damage; however, this issue can be overcome by the chelation of excess iron. This study aimed to investigate the effect of 6-gingerol-rich ginger (Zingiber officinale) extract on iron chelation, antioxidation, and hepatoprotective function in protecting against iron-induced oxidative liver cell injury. In experiments, 6-gingerol was confirmed to be a main bioactive component of the ginger extract and possessed free radical scavenging activity, decreasing ABTS•+ and DPPH• radical levels, and inhibiting AAPH-induced red blood cell hemolysis. Interestingly, the extract significantly reduced the levels of labile cellular iron (LCI), intracellular ROS, and lipid peroxidation products (TBARS) in iron-loaded human hepatoma (Huh7) cells. In conclusion, this work highlights the iron chelation property of 6-gingerol-rich ginger extract and its antioxidant activity, which could potentially protect the liver from iron-induced oxidative tissue damage.

Excessive N-acetylcysteine exaggerates glutathione redox homeostasis and apoptosis during acetaminophen exposure in Huh-7 human hepatoma cells

Acetaminophen (APAP) hepatotoxicity is one of the biggest drawbacks of this relatively safe and widely used drug. In addition to its hepatotoxicity, APAP also cause comparable levels of toxicity on human hepatoma cells. Here we show activation of the intrinsic caspase-9/3 pathway of apoptosis followed by gasdermin E (GSDME) cleavage and subsequent ballooning in APAP (10 mM, 72 h)-treated Huh-7 human hepatocarcinoma cells. N-acetylcysteine (NAC), an antioxidant currently used as an antidote for APAP overdose, does not alleviate APAP toxicity in Huh-7 cells; NAC overdose (10 mM) rather aggravates APAP toxicity. NAC overdose not only aggravates cell death, but also decreases the cellular GSH/GSSG ratio, an indicator of redox homeostasis of glutathione. These results show for the first time that APAP-induced apoptosis in hepatoma cells is followed by secondary necrosis via the caspase-3/GSDME pathway. NAC overdose (10 mM) not only worsens the glutathione redox status, but also accelerates this pathway.