Expression In HepG2 Research Articles

Xanthone-loaded niosomes as innovative potential delivery platform: formulation, characterization, and cytotoxic effect on human cancer cell lines

Cancer poses a perpetual threat to public health, presenting challenges in therapy due to the unregulated proliferation and intricate nature of tumors. Xanthone (Xan) exhibits anti-cancer activity, yet its therapeutic potential is hindered by its limited aqueous solubility and intestinal permeability. To address this issue, we attempted to formulate Xan into niosomes using a (21.41) full factorial design, investigating lipid amount and surfactant/stabilizer ratio impact on vesicle size (VS), entrapment efficiency percentage (EE%) and release efficacy percentage (RE%). The optimal formula was characterized for its ex-vivo permeation, morphology, and in-vitro cytotoxicity using a panel of eight human cancer cell lines. Cell cycle analysis was employed to determine the optimal formula's potential for inducing cell cycle arrest. Annexin V-FITC binding, Bax (BCL2-Associated X Protein), and Bcl-xl (B-cell lymphoma-extra-large) protein expression levels were assessed to determine the degree of apoptosis. Cell migration was assessed by the wound healing assay. The optimal formula (F1), with a desirability value of 0.924, demonstrated minimized VS (83.21±4.37 nm), maximized EE% (66.50±4.43%), and maximized RE% (77.05±1.19%). F1 exhibited enhanced ex-vivo permeation (by 4.96-fold compared to Xan suspension), and morphological integrity. F1 displayed notable antiproliferative effects, particularly in HepG2 and MCF-7 cells with IC50 values of 5.4±0.11 μM and 15.6±0.22 μM, respectively. The anticancer effect was further evidenced by its ability to inhibit cell migration, G1 phase cell cycle arrest, apoptosis induction, and increased Bax/Bcl-xl expression in HepG2 and MCF-7 cells. Accordingly, Xan-loaded niosomes exhibit encouraging potential as a potent anticancer therapy, warranting further exploration.

Nucleos(t)ide analogues potentially activate T lymphocytes through inducing interferon expression in hepatic cells and patients with chronic hepatitis B

Chronic hepatitis B (CHB) leads to liver inflammation and dysfunction, resulting in liver fibrosis and cancer. Nucleos(t)ide analogues (NAs), inhibitors of hepatitis B virus (HBV), specifically suppress HBV replication. We proposed that immune modulation benefits seroconversion by HBsAg loss. However, activation of T lymphocytes also deteriorates hepatic inflammation. Therefore, we intended to investigate the T cell status and its relationship with hepatic functions in CHB patients treated with NAs. Serum markers, including liver function markers AST, ALT, and HBV-infected markers HBV DNA, HBsAg, HBeAg, and HBsAb were measured in the clinical routine. The T cell levels and markers, including CD69, CD107a, CXCR3, and PD-1 were investigated using flow cytometry. Meanwhile, IFNγ, IL-2, and CXCL10 as immune activation markers in the PBMCs were investigated using qPCR. To validate the effects of NAs on T cell status, qPCR and flow cytometry were used to investigate the gene expression in the HepG2 and PLC5 cells treated with NAs, and in the healthy PBMCs treated with the cell-cultured supernatant. We found that NAs significantly suppressed HBV DNA and reduced AST and ALT levels in the CHB patients. Meanwhile, AST and ALT were both positively correlated with activation marker CD107a in CD8+ T cells. In addition, we found that the CHB patients with seroconversion exhibited a higher CD4/CD8 ratio (p < 0.05) compared to non-seroconversion. We demonstrated that NAs potentially induced IFNs and PD-L1 expression in HepG2 and PLC5 cells. Moreover, the collected supernatant from NAs-treated HepG2 significantly activated PBMCs. This study revealed that the reduction of HBV by NAs may be the reason leading to less AST and ALT levels. We further demonstrated that NAs induced IFN expression in hepatic cells to potentially activate T lymphocytes, which was positively associated with AST and ALT levels in the CHB patients. The results may explain the phenomena in clinical that when the virus is reactivated by aborted use of NAs, it causes consequent T cells-mediated severe acute-on-chronic liver injury.

Methyltransferase Like-3-Mediated N6-Methyladenosine Modification of Long Noncoding RNA Hepatocyte Nuclear Factor 1a Antisense RNA 1/Hepatocyte Nuclear Factor 4a Antisense RNA 1 Regulates Cytochrome P450 Enzyme Expression.

Interindividual variations in the expression and activity of cytochrome P450 enzymes (CYPs) led to lower therapeutic efficacy or adverse drug events. We previously demonstrated that CYPs are regulated by the long noncoding RNAs (lncRNAs) hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and HNF4A-AS1 via transcription factors (TFs) including hepatocyte nuclear factor 1a (HNF1A), hepatocyte nuclear factor 4a (HNF4A), and pregnane X receptor (PXR). However, the upstream mechanisms regulating HNF1A-AS1 and HNF4A-AS1 are poorly understood. N6-methyladenosine (m6A) is a prevalent epitranscriptomic modification in mammalian RNA. Therefore, the aim of this study was to investigate whether m6A modification regulates the expression of HNF1A-AS1 and HNF4A-AS1 and affects CYP expression in HepG2 and Huh7 cells. The methyltransferase-like 3 (METTL3) inhibitor, STM2457, significantly suppressed the expression of HNF1A-AS1 and induced HNF4A-AS1 expression. Consistent with this, a loss-of-function assay of METTL3 in the cell lines resulted in the downregulation of HNF1A-AS1 and its downstream HNF1A, PXR, and CYPs at the RNA level, as well as the downregulation of some CYPs proteins, and upregulation of HNF4A-AS1. The results of gain-of-function experiments showed the opposite trend. Mechanistically, subsequent RNA stability experiments confirmed that METTL3 affected the stability of both lncRNAs, but in opposite ways; that is, METTL3 reduced HNF1A-AS1 stability and increased HNF4A-AS1 stability. Rescue experiments confirmed that the regulation of METTL3 on TFs and CYPs may require the involvement of these two lncRNAs. Altogether, our study demonstrates that METTL3 is involved in TFs-mediated CYP expression by affecting HNF1A-AS1/HNF4A-AS1 stability. SIGNIFICANCE STATEMENT: Although the impact of long noncoding RNAs (lncRNAs) including hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and hepatocyte nuclear factor 4a antisense RNA 1 (HNF4A-AS1) on the downstream transcription factor (TF) and cytochrome P450 enzyme (CYP) expression is well studied, the upstream regulation of these two lncRNAs by methyltransferase-like 3 (METTL3) remains unexplored. This study reveals that METTL3 is involved in the regulation of lncRNA-TF-CYP expression by affecting the stability of HNF1A-AS1 and HNF4A-AS1 in HepG2 and Huh7 cells.

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.

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. 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. 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. Dopamine inhibits the expression of HBsAg and HBeAg by activating the JAK/STAT pathway and upregulating ISG15 expression.

Xanthohumol, a prenylated chalcone, regulates lipid metabolism by modulating the LXRα/RXR-ANGPTL3-LPL axis in hepatic cell lines and high-fat diet-fed zebrafish models

Angiopoietin-like 3 (ANGPTL3) acts as an inhibitor of lipoprotein lipase (LPL), impeding the breakdown of triglyceride-rich lipoproteins (TGRLs) in circulation. Targeting ANGPTL3 is considered a novel strategy for improving dyslipidemia and atherosclerotic cardiovascular diseases (ASCVD). Hops (Humulus lupulus L.) contain several bioactive prenylflavonoids, including xanthohumol (Xan), isoxanthohumol (Isoxan), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN), with the potential to manage lipid metabolism. The aim of this study was to investigate the lipid-lowering effects of Xan, the effective prenylated chalcone in attenuating ANGPTL3 transcriptional activity, both in vitro using hepatic cells and in vivo using zebrafish models, along with exploring the underlying mechanisms. Xan (10 and 20 μM) significantly reduced ANGPTL3 mRNA and protein expression in HepG2 and Huh7 cells, leading to a marked decrease in secreted ANGPTL3 proteins via hepatic cells. In animal studies, orally administered Xan significantly alleviated plasma triglyceride (TG) and cholesterol levels in zebrafish fed a high-fat diet. Furthermore, it reduced hepatic ANGPTL3 protein levels and increased LPL activity in zebrafish models, indicating its potential to modulate lipid profiles in circulation. Furthermore, molecular docking results predicted that Xan exhibits a higher binding affinity to interact with liver X receptor α (LXRα) and retinoic acid X receptor (RXR) than their respective agonists, T0901317 and 9-Cis-retinoic acid (9-Cis-RA). We observed that Xan suppressed hepatic ANGPTL3 expression by antagonizing the LXRα/RXR-mediated transcription. These findings suggest that Xan ameliorates dyslipidemia by modulating the LXRα/RXR-ANGPTL3-LPL axis. Xan represents a novel potential inhibitor of ANGPTL3 for the prevention or treatment of ASCVD.

Dimethyloxalylglycine Suppresses SREBP1c and Lipogenic Gene Expressions in Hepatocytes Independently of HIF1A.

Dimethyloxalylglycine (DMOG) is a representative inhibitor of the prolyl hydroxylase domain (PHD), which mediates the degradation of hypoxia-inducible factor-1-alpha (HIF1A). DMOG exerts its pharmacological effects via the canonical pathway that involves PHD inhibition; however, it remains unclear whether DMOG affects lipogenic gene expression in hepatocytes. We aimed to elucidate the effects of DMOG on sterol regulatory element-binding protein-1c (SREBP1c), a master regulator of fatty acid synthesis in hepatocytes. DMOG treatment inhibited SREBP1c mRNA and protein expression in HepG2 and AML12 hepatocytes and reduced the transcript levels of SREBP1c-regulated lipogenic genes. A luciferase reporter assay revealed that DMOG inhibited the transcriptional activity of SREBP1c. Moreover, DMOG suppressed SREBP1c expression in mice liver. Mechanistically, treatment with DMOG enhanced the expression of HIF1A and insulin-induced gene 2 (INSIG2), which inhibits the activation of SREBP1c. However, HIF1A or INSIG2 knockdown failed to reverse the inhibitory effect of DMOG on SREBP1c expression, suggesting a redundant role of HIF1A and INSIG2 in terms of repressing SREBP1c. DMOG did not function through the canonical pathway involving inhibition of SREBP1c by PHD, highlighting the presence of non-canonical pathways that mediate its anti-lipogenic effect.

Hepatoprotective effects of aspirin on diethylnitrosamine-induced hepatocellular carcinoma in rats by reducing inflammation levels and PD-L1 expression

Aspirin, as a widely used anti-inflammatory drug, has been shown to exert anti-cancer effects in a variety of cancers. PD-L1 is widely expressed in tumor cells and inhibits anti-tumor immunity. This study aims to clarify whether aspirin exerts its anti-hepatocellular carcinoma (HCC) effect by inhibiting PD-L1 expression. The rat model of HCC was established by drinking 0.01% diethylnitrosamine (DEN), and aspirin was given by gavage. The gross and blood biochemical indexes of rats were analyzed. CD4 and CD8 expression in liver tissues were investigated by immunohistochemistry. CCK8 assay was used to detect the inhibitory effect of aspirin on the proliferation of HCC cells. The regulatory effect of aspirin on PD-L1 expression was analyzed by western blot. As a result, the tumor number and liver weight ratio in the DEN + ASA group were lower than those in the DEN group (P = 0.006, P = 0.046). Compared with the DEN group, the expression of CD4 in the DEN + ASA group was significantly increased, while CD8 was decreased (all P < 0.01). Biochemical indexes showed that there were differences in all indexes between the DEN and control group (P < 0.05). The levels of DBIL, ALP, and TT in the DEN + ASA group were lower than those in the DEN group (P = 0.038, P = 0.042, P = 0.031). In the DEN group, there was an obvious fibrous capsule around the tumor, and the portal vein was dilated. The pathological changes were mild in the DEN + ASA group. Compared with the DEN group, the expression of PD-L1 in liver tissue of the DEN + ASA group was decreased (P = 0.0495). Cytological experiments further showed that aspirin could inhibit the proliferation and PD-L1 expression in Hep G2 and Hep 3B cells. In conclusion, aspirin can inhibit the proliferation of HCC cells and reduce tumor burden by reducing inflammation and targeting PD-L1.

Targeting oncogenic TERT promoter variants by allele-specific epigenome editing

BackgroundActivation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers.ResultsSequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2.ConclusionsWe applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.

Tazemetostat decreases β-catenin and CD13 protein expression in HEPG-2 and Hepatitis B virus-transfected HEPG-2 with decreased cell viability

Hepatocellular carcinoma (HCC) is one of the global health concerns. Hepatitis B virus (HBV) is one of the major causes of HCC. Poor clinical outcome of HCC patients is attributed to a small population of cancer cells known as cancer stem cells (CSCs). In this work, we studied the effect of inhibiting the enhancer of zeste homologue 2 (EZH2), a histone methyltransferase known to be overexpressed in CSCs, using tazemetostat (Taz). The effect of Taz was assessed in the HCC cell line (HEPG2) and Hepatitis B virus-transfected HEPG2 (HBV/HEPG2) cells. MTT assay showed a significant decrease in HEPG2 cells viability after 48 h treatment with either 0.5, 1, 4 or 6 μM Taz. HEPG2 and HBV/HEPG2 cells were incubated with either 0.5 or 1 μM Taz for 48 h, and then, the cells and supernatants were collected for protein expression analysis of EZH2, CD13, epithelial cell adhesion molecule (EpCAM) and β-catenin using enzyme-linked immunosorbent assay (ELISA). Taz showed a significant dose-dependent inhibition of EZH2, CD13 and β-catenin in HEPG2 and HBV/HEPG2 cells. Also, EpCAM protein levels were significantly decreased in HBV/HEPG2 but not in HEPG2 cell line alone. Our results indicate that Taz inhibition of EZH2 leads to downregulation of β-catenin signaling and eventually decreased expression of CD13 and EpCAM, which are characteristic for CSCs. The present study suggests that Taz could be a promising treatment for HCC including HBV-induced HCC that might be used in combination with radio/chemotherapy to target CSCs and prevent tumor relapse.

An integrative analysis reveals the prognostic value and potential functions of MTMR2 in hepatocellular carcinoma

Abnormal expression of myotubularin-related protein 2 (MTMR2) has been identified in certain types of cancer, leading to varying effects on tumor genesis and progression. However, the various biological significances of MTMR2 in hepatocellular carcinoma (HCC) have not been systematically and comprehensively studied. The aim of this study was to explore the role of MTMR2 in HCC. We obtained the raw data from Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Afterward, we analyzed the data using R and cBioPortal. We investigated the connection between MTMR2 and its expression, prognosis, clinical significance, methylation, genetic alterations, tumor microenvironment (TME), tumor mutation burden (TMB), and drug reactivity in HCC patients. MTMR2 expression levels in HCC cells were validated through western blotting and RT-qPCR. MTMR2 exhibits high levels of expression across a wide range of cancer types, including HCC. MTMR2 is diagnostically valuable in detecting HCC, with its up-regulated expression often being indicative of poor prognosis among HCC patients. The in vitro experiments confirmed elevated MTMR2 expression in HepG2, HUH-7, and MHCC-97H cells. Univariate and multivariate Cox analysis demonstrated that MTMR2 was an independent prognostic factor in HCC patients. The cg20195272 site has the highest degree of methylation in MTMR2, and it is positively correlated with MTMR2 expression. Patients with high levels of methylation at the cg20195272 site show poor prognosis. Analysis of the TME indicates that high expression of MTMR2 is associated with elevated ESTIMATE score and that MTMR2 expression correlates positively with infiltration by resting memory CD4 T cells, activated dendritic cells, as well as several immune checkpoints. There is a negative correlation between MTMR2 expression and TMB, and drug sensitivity analyses have shown that higher MTMR2 expression is associated with lower IC50 values. This study indicates that increased expression of MTMR2 may play a crucial role in the occurrence, progression, diagnosis, prognostic prediction and drug therapy of HCC.

Saikosaponin-b2 Inhibits Primary Liver Cancer by Regulating the STK4/IRAK1/NF-κB Pathway.

The development of primary liver cancer (PLC) is associated with chronic liver inflammation and the loss of associated tumor suppressor genes, which characterizes inflammation-related tumors. In this study, we aimed to explore the effect of saikosaponin-b2 (SS-b2) on the development of PLC and its effect of the STK4 expression and IRAK1/NF-κB signaling axis. In vitro and in vivo experiments showed that SS-b2 exerted potent anti-inflammatory and antitumor effects. A PLC model was induced in vivo by treating male BALB/c mice with diethylnitrosamine, while an inflammatory model was induced in vitro by exposing RAW 264.7 macrophages to lipopolysaccharides (LPS). After treating cancer mice with SS-b2, the serum levels of alpha-fetoprotein, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly reduced. Ki67 expression also decreased. The carcinomatous lesions of the liver were attenuated. Similar results were observed in liver tissue and RAW 264.7 macrophages, where SS-b2 significantly elevated serine/threonine protein kinase 4 (STK4) expression and decreased the expression of interleukin-1 receptor-associated kinase 1 (IRAK1), nuclear factor-kappaB (NF-κB), and downstream inflammatory cytokines, thus exerting anti-cancer and anti-inflammatory effects. Moreover, we employed siRNA to silence the STK4 expression in HepG2 to investigate the anti-tumor effect of SS-b2 in vitro. The STK4 knockdown would upregulate IRAK1 and thus the activation of NF-κB activity revealed by the increase in the levels of proinflammatory cytokines, consequently impairing SS-b2-induced inhibition of liver cancer development. Consequently, SS-b2 effectively inhibited PLC by upregulating STK4 to suppress the IRAK1/NF-κB signaling axis and is a promising agent for treating this disease.

Phellinus linteus mycelia extract in COVID-19 prevention and identification of its key metabolic compounds profiling using UPLC-QTOF-MS/MS spectrometry

For centuries, food, herbal medicines, and natural products have been valuable resources for discovering novel antiviral drugs, uncovering new structure-activity relationships, and developing effective strategies to prevent/treat viral infections. One such resource is Phellinus linteus, a mushroom used in folk medicine in Taiwan, Japan, Korea, and China. In this rich historical context, the key metabolites of Phellinus linteus mycelia ethanolic extract (GKPL) impacting the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at multiple stages have yet to be explored. Thus, this study systematically identifies and assesses the inhibitory effect of GKPL on the SARS-CoV-2 virus. Initially, the concentrations and contact times of GKPL against SARS-CoV-2 pseudovirus were assessed in HepG2 cells. Subsequently, utilizing the Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry method, potential biomarkers in the fungal extract were discerned. Metabolomic analysis identified 18 compounds in GKPL, with hispidin and hypholomine B present in the highest amounts. These compounds were isolated using chromatographic techniques and further identified through 1D NMR spectroscopic and mass spectrometry analysis. Hispidin and hypholomine B were found to inhibit the infection of SARS-CoV-2 pseudovirus by reducing angiotensin-converting enzyme 2 gene expression in HepG2, thereby decreasing viral entry. Moreover, hispidin and hypholomine B effectively block the spike receptor-binding domain, while hypholomine B, for the first time, showed significant inhibition of 3CL protease. This suggests that GKPL, enriched with hispidin and hypholomine B, has the potential to be used as an active ingredient against SARS-CoV-2.

Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis

BackgroundHepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells.MethodsIC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated.ResultsN-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 μg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 μg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h.ConclusionN-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.

Design, Synthesis, and Antiproliferative Activities of 1,3‐Disubstituted 2‐Thioxoimidazolidin‐4‐one Derivatives against HepG2 and MCF‐7 Cells

AbstractA series of 1,3‐disubstituted‐2‐thioxoimidazolidin‐4‐one and diazine derivatives were synthesized and confirmed through IR, NMR, and mass spectrometry. The in vitro cytotoxic activity of synthesized compounds against HePG2 and MCF‐7 cells were assessed by MTT assay. Furthermore, Aromatase, VEGFR‐2, B‐Raf, cell cycle, Annexin V, caspase‐3 and sulphatase expression by PCR, and β‐catenin expression by western blot were estimated. The results revealed that the synthesized compounds possess cytotoxic activity against HePG2 and MCF‐7 cell lines. Compound 11 (2,2′‐(1E,1′E)‐1,1′‐(hydrazine‐1,2‐diylidene) bis (ethan‐1‐yl‐1‐ylidene) bis (4‐(phenyldiazenyl) phenol) had greater cytotoxic activity than staurosporine (6.24 μg/mL), with IC50 of 2.55 μg/mL against HepG2 cells and (4.21 μg/mL) against MCF‐7 cells. Moreover, the inhibitory activity against aromatase was (0.156 μg/mL), (0.23 μg/mL) against VEGFR2, and (0.25 μg/mL) against B‐Raf. Also, this compound decreased DNA content of HepG2 cells in the S phase and the G2/M phase, and increased the apoptosis in Annexin measurement. By real‐time PCR, This compound up‐regulated the expression of caspase‐3 in HepG2 cells, reduced the expression of sulphatase gene in HepG2 cells, and reduced β‐catenin expression in HepG2 by western blot analysis. Consequently, the synthesized derivatives could be a promising scaffold for the development of potent anti‐cancer agents due to their apoptotic and anti‐inflammatory activities.

Protective Effect of Fucoxanthin on Zearalenone-Induced Hepatic Damage through Nrf2 Mediated by PI3K/AKT Signaling.

Hepatotoxic contaminants such as zearalenone (ZEA) are widely present in foods. Marine algae have a wide range of potential applications in pharmaceuticals, cosmetics, and food products. Research is ongoing to develop treatments and products based on the compounds found in algae. Fucoxanthin (FXN) is a brown-algae-derived dietary compound that is reported to prevent hepatotoxicity caused by ZEA. This compound has multiple biological functions, including anti-diabetic, anti-obesity, anti-microbial, and anti-cancer properties. Furthermore, FXN is a powerful antioxidant. In this study, we examined the effects of FXN on ZEA-induced stress and inflammation in HepG2 cells. MTT assays, ROS generation assays, Western blots, and apoptosis analysis were used to evaluate the effects of FXN on ZEA-induced HepG2 cell inflammation. Pre-incubation with FXN reduced the cytotoxicity of ZEA toward HepG2 cells. FXN inhibited the ZEA-induced production of pro-inflammatory cytokines, including IL-1 β, IL-6, and TNF-α. Moreover, FXN increased HO-1 expression in HepG2 by activating the PI3K/AKT/NRF2 signaling pathway. In conclusion, FXN inhibits ZEA-induced inflammation and oxidative stress in hepatocytes by targeting Nrf2 via activating PI3K/AKT signaling.

A systematic study on the treatment of hepatitis B-related hepatocellular carcinoma with drugs based on bioinformatics and key target reverse network pharmacology and experimental verification

BackgroundChronic hepatitis B virus (HBV) infection is the major etiology of hepatocellular carcinoma (HCC). However, the mechanism of hepatitis B-related hepatocellular carcinoma (HBV-related HCC) is still unclear. Therefore, understanding the pathogenesis and searching for drugs to treat HBV-related HCC was an effective strategy to treat this disease.PurposeBioinformatics was used to predict the potential targets of HBV-related HCC. The reverse network pharmacology of key targets was used to analyze the clinical drugs, traditional Chinese medicine (TCM) and small molecules of TCM in the treatment of HBV-related HCC.MethodsIn this study, three microarray datasets totally containing 330 tumoral samples and 297 normal samples were selected from the GEO database. These microarray datasets were used to screen DEGs. And the expression profile and survival of 6 key genes were analyzed. In addition, Comparative Toxicogenomics Database and Coremine Medical database were used to enrich clinical drugs and TCM of HBV-related HCC by the 6 key targets. Then the obtained TCM were classified based on the Chinese Pharmacopoeia. Among these top 6 key genes, CDK1 and CCNB1 had the most connection nodes and the highest degree and were the most significantly expressed. In general, CDK1 and CCNB1 tend to form a complex, which is conducive to cell mitosis. Hence, this study mainly studied CDK1 and CCNB1. HERB database was used to predict small molecules TCM. The inhibition effect of quercetin, celastrol and cantharidin on HepG2.2.15 cells and Hep3B cells was verified by CCK8 experiment. The effects of quercetin, celastrol and cantharidin on CDK1 and CCNB1 of HepG2.2.15 cells and Hep3B cells were determined by Western Blot.ResultsIn short, 272 DEGs (53 upregulated and 219 downregulated) were identified. Among these DEGs, 6 key genes with high degree were identified, which were AURKA, BIRC5, CCNB1, CDK1, CDKN3 and TYMS. Kaplan–Meier plotter analysis showed that higher expression levels of AURKA, BIRC5, CCNB1, CDK1, CDKN3 and TYMS were associated with poor OS. According to the first 6 key targets, a variety of drugs and TCM were identified. These results showed that clinical drugs included targeted drugs, such as sorafenib, palbociclib and Dasatinib. and chemotherapy drugs, such as cisplatin and doxorubicin. TCM, such as the TCM flavor was mainly warm and bitter, and the main meridians were liver and lung. Small molecules of TCM included flavonoids, terpenoids, alkaloids and glycosides, such as quercetin, celastrol, cantharidin, hesperidin, silymarin, casticin, berberine and ursolic acid, which have great potential in anti-HBV-related HCC. For molecular docking of chemical components, the molecules with higher scores were flavonoids, alkaloids, etc. Three representative types of TCM small molecules were verified respectively, and it was found that quercetin, celastrol and cantharidin inhibited the proliferation of HepG2.2.15 cells and Hep3B cells along concentration gradient. Quercetin, celastrol and cantharidin decreased CDK1 expression in HepG2.2.15 and Hep3B cells, but for CCNB1, only cantharidin decreased CCNB1 expression in the two strains of cells.ConclusionIn conclusion, AURKA, BIRC5, CCNB1, CDK1, CDKN3 and TYMS could be potential targets for the diagnosis and prognosis of HBV-related HCC. Clinical drugs include chemotherapeutic and targeted drug, traditional Chinese medicine is mainly bitter and warm TCM. Small molecular of TCM including flavonoids, terpenoids and glycosides and alkaloids, which have great potential in anti-HBV-related HCC. This study provides potential therapeutic targets and novel strategies for the treatment of HBV-related HCC.

MESP1-knockdown inhibits the proliferation and epithelial mesenchymal transition of hepatocellular carcinoma and enhances the tumor-suppressive effect of 5-fluorouracil

Primary liver hepatocellular carcinoma (HCC) is the third most deadly malignancy worldwide,in part, because it is often diagnosed at an advanced stage. Thus, molecular markers are needed to aid in the early diagnosis and treatment of HCC. Expression of abnormal mesoderm posterior-1 (MESP1) promotes tumorigenesis; however,its role in the regulation of HCC proliferation, apoptosis,and invasion is unknown. Here,we analyzed data in The Cancer Genome Atlas (TCGA)and Genotype Tissue Expression (GTEx) databases on the pan-cancer expression of MESP1 and its relationship with clinical characteristics and prognosis of patients with HCC. The expression of MESP1 was measured in 48 HCC tissues using immunohistochemical staining,and the results were correlated with clinical stage, tumor differentiation, tumor size,and metastasis. MESP1 expression was downregulated using small interfering RNA (siRNA) in the HCC cell lines HepG2 and Hep3B,and cell viability, proliferation,cell cycle, apoptosis,and invasion were analyzed. Finally,we also evaluated the tumor suppression effect of MESP1 downregulation combined with 5-fluorouracil (5-FU) treatment. Our results showed that MESP1 is a pan-oncogene associated with poor prognosis in patients with HCC. siRNA-induced downregulation of MESP1 expression in HepG2 and Hep3B cells exhibited downregulation of β-catenin and GSK3β expression 48h after transfection, along with an increase in apoptosis rate, arrest in the G1-S phase,and a decrease in mitochondrial membrane potential. Moreover,the expression levels of c-Myc, PARP1, bcl2, Snail1, MMP9, and immune checkpoint genes (TIGIT, CTLA4,LAG3,CD274,and PDCD1) were downregulated, while those of caspase3 and E-cadherin were upregulated. Tumor cells also showed decreased migration ability. Furthermore, siRNA interference of MESP1 expression combined with 5-FU-treatment of HCC cells significantly enhanced the G1-S phase block and apoptosis. MESP1 showed an aberrant high expression in HCC and was associated with poor clinical outcomes; therefore, MESP1 may be a potential target for the diagnosis and treatment of HCC.

Autologous blood transfusion promotes autophagy and inhibits hepatocellular carcinoma progression through HIF-1α signalling pathway.

To explore the molecular mechanism of autologous blood transfusion promoting autophagy of hepatocellular carcinoma (HCC) cells and inhibiting the HCC progression through HIF-1α signalling pathway. This is a research paper. Rat hepatocellular carcinoma model and HepG2 cell model were built. The rats with HCC were conducted a surgery, and their blood was collected for detection to detect the recurrence and metastasis of the rats. Western blot was used to analysed the expression of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein. The apoptosis rate of HepG2 cells was detected by flow cytometry, and autophagosomes were observed by transmission electron microscopy. HIF-1α expression was measured by immunofluorescence assay. The expressions of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein were highest in model + autoblood group compared with the model group. HIF-1α content of model group was higher, but content of TP53, MDM2, ATG5 and ATG14 in the model group is the second. The highest apoptosis rate was found in HepG2 + autoblood group. The number of autophagosomes in HepG2 + autoblood was obviously larger than that of HepG2 + autoblood + inhibitor. HIF-1α expression of immunofluorescence assay showed that high expression of HIF-1α was clearly observed in HepG2 and HepG2 + autoblood group from confocal observation. However, there was no HIF-1α protein expression in HepG2 + autoblood + inhibitor group. The migration rate in HepG2 group, HepG2 + autoblood group and HepG2 + autoblood + inhibitor group was 85.71 ± 7.38%, 14.36 ± 6.54% and 61.25 ± 5.39%, respectively. Autologous blood transfusion promotes autophagy of HCC cells through HIF-1α signalling pathway, which further inhibits HCC migration and erosion.

The Potential Inhibitory Role of Acetyl-L-Carnitine on Proliferation, Migration, and Gene Expression in HepG2 and HT29 Human Adenocarcinoma Cell Lines.

Malignancies of the liver and colon are the most prevalent forms of digestive system cancer globally. Chemotherapy, one of the most significant treatments, has severe side effects. Chemoprevention using natural or synthetic medications can potentially reduce cancer severity. Acetyl-L-carnitine (ALC) is an acetylated derivative of carnitine essential for intermediate metabolism in most tissues. This study aimed to investigate the effects of ALC on the proliferation, migration, and gene expression of human liver (HepG2) and colorectal (HT29) adenocarcinoma cell lines. The cell viability and half maximal inhibitory concentration of both cancer cell lines were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing after treatment was assessed using a migration assay. Morphological changes were imaged using brightfield and fluorescence microscopy. Post treatment, apoptotic DNA was detected using a DNA fragmentation assay. The relative mRNA expressions of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor (VEGF) were evaluated using RT-PCR. The results showed that ALC treatment affects the wound-healing ability of HepG2 and HT29 cell lines. Changes in nuclear morphology were detected under fluorescent microscopy. ALC also downregulates the expression levels of MMP9 and VEGF in HepG2 and HT29 cell lines. Our results indicate that the anticancer action of ALC is likely mediated by a decrease in adhesion, migration, and invasion.

Farnesoid X Receptor Activation Decreases Toll-like Receptor 2 Expression by Upregulating HBeAg Production

Background: Previous investigations have demonstrated that hepatitis B virus (HBV) infection leads to elevated serum bile acid levels, which is considered to cause liver damage. Thus, we suppose that bile acids may be of considerable significance in inducing immune tolerance. Methods: In this investigation, we explored the functions of the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids, in modulating hepatitis B e antigen (HBeAg) production and toll-like receptor (TLR) expression in vitro and in vivo. Results: The results showed that FXR activation promoted secreted and intracellular HBeAg expression in HepG2 and HEK293T cells. However, FXR antagonist Z-guggulsterone (Z-g) decreased the bile acid-mediated HBeAg production. Meanwhile, TLR2 expression significantly reduced in HepG2 cells transfected with pAAV/HBV1.2 plasmid comprising whole HBV genome and treated with bile acids, but not with mutant pAAV/HBV1.2 plasmid with defected HBeAg product. In the hydrodynamic injection HBV mouse model, the level of serum HBeAg was decreased, but intrahepatic TLR2 expression was elevated in FXR-/- mice. Conclusions: In conclusion, FXR activation inhibits TLR2-mediated innate immunity by upregulating HBeAg production. Our data indicate that a mild elevation of serum bile acids may cause immune tolerance and lead to virus persistence in HBV-infected patients.