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Clinical and Translational Medicine Dec 2023Sex disparities constitute a significant issue in hepatocellular carcinoma (HCC). However, the mechanism of gender dimorphism in HCC is still not completely understood.
BACKGROUND
Sex disparities constitute a significant issue in hepatocellular carcinoma (HCC). However, the mechanism of gender dimorphism in HCC is still not completely understood.
METHODS
5-Hydroxymethylcytosine (5hmC)-Seal technology was utilised to detect the global 5hmC levels from four female and four male HCC samples. Methylation of XIST was detected by Sequenom MassARRAY methylation profiling between HCC tissues (T) and adjacent normal liver tissues (L). The role of Tet methylcytosine dioxygenase 2 (TET2) was investigated using diethylnitrosamine (DEN)-administered Tet2 female mice, which regulated XIST in hepatocarcinogenesis. All statistical analyses were carried out by GraphPad Prism 9.0 and SPSS version 19.0 software.
RESULTS
The results demonstrated that the numbers of 5hmC reads in the first exon of XIST from female HCC tissues (T) were remarkably lower than that in female adjacent normal liver tissues (L). Correspondingly, DNA methylation level of XIST first exon region was significantly increased in female T than in L. By contrast, no significant change was observed in male HCC patients. Compared to L, the expression of XIST in T was also significantly downregulated. Female patients with higher XIST in HCC had a higher overall survival (OS) and more extended recurrence-free survival (RFS). Moreover, TET2 can interact with YY1 binding to the promoter region of XIST and maintain the hypomethylation state of XIST. In addition, DEN-administered Tet2 mice developed more tumours than controls in female mice.
CONCLUSIONS
Our study provided that YY1 and TET2 could interact to form protein complexes binding to the promoter region of XIST, regulating the methylation level of XIST and then affecting the expression of XIST. This research will provide a new clue for studying sex disparities in hepatocarcinogenesis.
HIGHLIGHTS
XIST was significantly downregulated in HCC tissues and had gender disparity. Methylation levels in the XIST first exon were higher in female HCC tissues, but no significant change in male HCC patients. The TET2-YY1 complex regulate XIST expression in female hepatocytes. Other ways regulate XIST expression in male hepatocytes.
Topics: Animals; Female; Humans; Male; Mice; Carcinoma, Hepatocellular; Chromosomes; DNA Methylation; Liver Neoplasms; Sex Characteristics
PubMed: 38148658
DOI: 10.1002/ctm2.1518 -
Ecotoxicology and Environmental Safety Jan 2024N-nitrosodiethylamine (ND) is an extremely toxic unavoidable environmental contaminant. Copper-albumin (CuAB) complex, a newly developed Cu complex, showed antioxidant...
Modulatory mechanisms of copper-albumin complex toward N-nitrosodiethylamine-induced neurotoxicity in mice via regulating oxidative damage, inflammatory, and apoptotic signaling pathways.
N-nitrosodiethylamine (ND) is an extremely toxic unavoidable environmental contaminant. Copper-albumin (CuAB) complex, a newly developed Cu complex, showed antioxidant and anti-inflammatory potential. Hereby, we explored the plausible neuroprotective role of CuAB complex toward ND-evoked neurotoxicity in mice. Twenty-four male mice were sorted into 4 groups (6 mice each). Control group, mice were administered oral distilled water; and CuAB group, mice received CuAB complex at a dose of 817 µg/kg orally, three times weekly. In ND group, ND was given intraperitoneally (50 mg/kg body weight, once weekly for 6 w). CuAB+ND group, mice were administered a combination of CuAB and ND. The brain was quickly extracted upon completion of the experimental protocol for the evaluation of the oxidative/antioxidative markers, inflammatory cytokines, and histopathological examination. Oxidative stress was induced after ND exposure indicated by a reduction in GSH and SOD1 level, with increased MDA level. In addition, decreased expression of SOD1 proteins, Nrf2, and 5-HT mRNA expression levels were noticed. An apoptotic cascade has also been elicited, evidenced by overexpression of Cyt c, Cl. Casp 3. In addition, increased regulation of proinflammatory genes (TNF-α, IL-6, iNOS, Casp1, and NF-κB (p65/p50); besides, increment of protein expression of P-IKBα and reduced expression of IKBα. Pretreatment with CuAB complex significantly ameliorated ND neuronal damage. Our results recommend CuAB complex supplementation because it exerts neuroprotective effects against ND-induced toxicity.
Topics: Mice; Male; Animals; Copper; Diethylnitrosamine; Superoxide Dismutase-1; NF-kappa B; Oxidative Stress; Signal Transduction; Antioxidants; Neurotoxicity Syndromes; NF-E2-Related Factor 2
PubMed: 38113799
DOI: 10.1016/j.ecoenv.2023.115841 -
Mutagenesis Mar 2024The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug...
The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.
Topics: Humans; Animals; Cricetinae; Nitrosamines; Mutagens; Diethylnitrosamine; Mutagenesis; Mutagenicity Tests; Carcinogens
PubMed: 38112628
DOI: 10.1093/mutage/gead033 -
Journal of Ethnopharmacology Mar 2024Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its...
ETHNOPHARMACOLOGICAL RELEVANCE
Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its underlying functional mechanism still remains unknown.
AIM OF THE STUDY
The study aimed to explore the anti-hepatocarcinogenic effects of M-BJJW, specifically its influence on PD-L1-mediated immune evasion in hypoxic conditions, and elucidate the related molecular mechanisms in HCC.
MATERIALS AND METHODS
To investigate the therapeutic efficacy and mechanisms underlying M-BJJW's effects on HCC, we employed a diethylnitrosamine (DEN)-induced rat model maintained for 120 days. Following model establishment, flow cytometry was utilized to assess the distribution of immune cell populations in peripheral blood, spleens, and tumor tissues after M-BJJW administration. Simultaneously, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze cytokine profiles in serum samples. Immunohistochemistry was employed to determine the expression levels of crucial proteins within tumor tissues. Furthermore, HCC cells exposed to CoCl underwent Western blot analysis to validate the expression levels of HIF-1α, PD-L1, STAT3, and nuclear factor kappa B (NF-κB) p65. The modulatory effects of STAT3 and NF-κB p65 were investigated using specific inhibitors and activators in wild-type cell lines. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was utilized to identify the chemical constituents present in M-BJJW-medicated serum. The immunomodulatory properties and the anti-tumor activities of M-BJJW were evaluated by co-culturing with peripheral blood mononuclear cells (PBMC) and the CCK-8 assay. Additionally, we assessed M-BJJW's impact on hypoxia-induced alterations in HCC cell lines using immunofluorescence and Western blot assessments.
RESULTS
M-BJJW exhibited substantial therapeutic advantages by effectively alleviating pathological deterioration within the HCC microenvironment. In the DEN-induced rat model, M-BJJW administration notably reduced tumor growth. Flow cytometry analyses revealed an increased proportion of Cytotoxic T lymphocytes (CTLs) accompanied by a simultaneous decrease in regulatory T cells (Tregs). ELISA data supported a marked decrease in pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α). Immunohistochemistry confirmed the suppressive effect of M-BJJW on the expression of HIF-1α and PD-L1. Notably, western blotting unveiled the role of HIF-1α in regulating PD-L1 expression via the STAT3 and NF-κB signaling pathways in HCC cell lines, which was validated using activators and inhibitors of STAT3 and NF-κB. The CCK-8 assay and co-culture techniques demonstrated the anti-tumor activity of M-BJJW. Immunofluorescence and western blotting further confirmed that M-BJJW-containing serum dose-dependently inhibited HIF-1α, PD-L1, p-STAT3, and p-p65 in hypoxic HCC cell lines.
CONCLUSIONS
M-BJJW demonstrates significant therapeutic potential against HCC by influencing the hypoxic microenvironment, thereby regulating the immunosuppressive milieu. Specifically, M-BJJW modulates the HIF-1α/STAT3/NF-κB signaling pathway, leading to reduced PD-L1 expression and an elevated ratio of cytotoxic T lymphocytes (CTLs), while concurrently decreasing T regulatory cells (Tregs) and immunosuppressive factors. These synergistic effects aid in countering PD-L1-mediated immune evasion, presenting compelling pharmacological evidence supporting the clinical application of M-BJJW as a therapeutic approach for HCC.
Topics: Rats; Animals; NF-kappa B; Carcinoma, Hepatocellular; Leukocytes, Mononuclear; Liver Neoplasms; B7-H1 Antigen; Immune Evasion; Sincalide; Signal Transduction; Tumor Microenvironment
PubMed: 38104877
DOI: 10.1016/j.jep.2023.117577 -
Cancer Research Dec 2023
PubMed: 38098453
DOI: 10.1158/0008-5472.CAN-23-2367 -
Cancer Science Feb 2024Inhibition of cholesterol de novo synthesis (DNS) by statins has controversial effects on the treatment of hepatocellular carcinoma (HCC). High fatty acid conditions...
Inhibiting cholesterol de novo synthesis promotes hepatocellular carcinoma progression by upregulating prostaglandin E synthase 2-mediated arachidonic acid metabolism under high fatty acid conditions.
Inhibition of cholesterol de novo synthesis (DNS) by statins has controversial effects on the treatment of hepatocellular carcinoma (HCC). High fatty acid conditions have been reported to limit the effect of statins on metabolism diseases. Whether high fatty acid conditions interfere with the effect of statins on HCC remains unclear. Here, we reported that inhibiting cholesterol DNS with atorvastatin promoted the oncogenic capabilities of diethylnitrosamine (DEN) in mice fed high fatty acid diets (HFD). The combined analysis of metabolomics and transcriptomics revealed that arachidonic acid (AA) metabolism was the most significant changed pathway between mice with and without atorvastatin treatment. In vitro, in the presence of AA precursor linoleic acid (LA), atorvastatin promoted the proliferation and migration ability of HCC cell lines. However, in the absence of LA, these phenomena disappeared. TCGA and tissue microarray examination revealed that prostaglandin e synthase 2 (PTGES2), a key enzyme in AA metabolism, was associated with the poor outcome of HCC patients. Overexpression of PTGES2 promoted the proliferation and migration of HCC cell lines, and knockdown of PTGES2 inhibited the proliferation and migration of cells. Additionally, atorvastatin upregulated PTGES2 expression by enhancing Sterol-regulatory element binding protein 2 (SREBP2)-mediated transcription. Knockdown of PTGES2 reversed the proliferation and migration ability enhanced by atorvastatin. Overall, our study reveals that a high fatty acid background is one of the possible conditions limiting the application of statins in HCC, under which statins promote the progression of HCC by enhancing SREBP2-mediated PTGES2 transcription.
Topics: Humans; Mice; Animals; Carcinoma, Hepatocellular; Fatty Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Liver Neoplasms; Arachidonic Acid; Prostaglandin-E Synthases; Atorvastatin; Cell Line, Tumor; Cholesterol; Cell Proliferation
PubMed: 38081591
DOI: 10.1111/cas.16035 -
Scientific Reports Dec 2023Aspirin, 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...
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.
Topics: Animals; Rats; Aspirin; B7-H1 Antigen; Carcinoma, Hepatocellular; Diethylnitrosamine; Inflammation; Liver Neoplasms
PubMed: 38049630
DOI: 10.1038/s41598-023-48812-z -
Genes and Environment : the Official... Nov 2023Mutagenicity, the ability of chemical agents to cause mutations and potentially lead to cancer, is a critical aspect of substance safety assessment for protecting human...
BACKGROUND
Mutagenicity, the ability of chemical agents to cause mutations and potentially lead to cancer, is a critical aspect of substance safety assessment for protecting human health and the environment. Metabolic enzymes activate multiple mutagens in living organisms, thus in vivo animal models provide highly important information for evaluating mutagenicity in human. Rats are considered suitable models as they share a similar metabolic pathway with humans for processing toxic chemical and exhibit higher responsiveness to chemical carcinogens than mice. To assess mutagenicity in rats, transgenic rodents (TGRs) are widely used for in vivo gene mutation assays. However, such assays are labor-intensive and could only detect transgene mutations inserted into the genome. Therefore, introducing a technology to directly detect in vivo mutagenicity in rats would be necessary. The next-generation sequencing (NGS) based error-corrected sequencing technique is a promising approach for such purposes.
RESULTS
We investigated the applicability of paired-end and complementary consensus sequencing (PECC-Seq), an error-corrected sequencing technique, for detecting in vivo mutagenicity in the rat liver samples. PECC-Seq allows for the direct detection of ultra-rare somatic mutations in the genomic DNA without being constrained by the genomic locus, tissue, or organism. We tested PECC-Seq feasibility in rats treated with diethylnitrosamine (DEN), a mutagenic compound. Interestingly, the mutation and mutant frequencies between PECC-Seq and the TGR assay displayed a promising correlation. Our results also demonstrated that PECC-Seq could successfully detect the A:T > T:A mutation in rat liver samples, consistent with the TGR assay. Furthermore, we calculated the trinucleotide mutation frequency and proved that PECC-Seq accurately identified the DEN treatment-induced mutational signatures.
CONCLUSIONS
Our study provides the first evidence of using PECC-Seq for in vivo mutagenicity detection in rat liver samples. This approach could provide a valuable alternative to conventional TGR assays as it is labor- and time-efficient and eliminates the need for transgenic rodents. Error-corrected sequencing techniques, such as PECC-Seq, represent promising approaches for enhancing mutagenicity assessment and advancing regulatory science.
PubMed: 37993952
DOI: 10.1186/s41021-023-00288-z -
BioRxiv : the Preprint Server For... Nov 2023Precision-Cut Liver Slices (PCLS) are an culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the...
BACKGROUND
Precision-Cut Liver Slices (PCLS) are an culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximates pathology than methods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS.
METHODS
PCLS of 250 μm thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl). PCLS were treated with 5 μM Erlotinib for 72 hours. Histology and gene expression were then compared with murine experiments and TGF-β1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver.
RESULTS
PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genes and in PCLS from CDAHFD-induced cirrhotic mice, and and in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression of and , which was consistent with the impact of erlotinib on and expression in DEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl-induced cirrhosis caused reduced expression of and , which was consistent with the effect of erlotinib in CCl-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-β1 treatment upregulated (), while treatment with erlotinib inhibited the expression of . Similar expression results were observed in TGF-β1 treated HSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples.
CONCLUSION
The responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed and . Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).
PubMed: 37961334
DOI: 10.1101/2023.10.30.564772 -
Loss of Krüppel-like factor-10 facilitates the development of chemical-induced liver cancer in mice.Molecular Medicine (Cambridge, Mass.) Nov 2023Krüppel-like factor 10 (KLF10) is involved in a positive feedback loop that regulates transforming growth factor β (TGFβ) signaling, and TGFβ plays an important role...
BACKGROUND
Krüppel-like factor 10 (KLF10) is involved in a positive feedback loop that regulates transforming growth factor β (TGFβ) signaling, and TGFβ plays an important role in the pathogenesis of liver disease. Here, we investigated whether KLF10 deletion affects the development of liver fibrosis and hepatocellular carcinoma (HCC).
METHODS
We induced KLF10 deletion in C57BL/6 mice. Liver fibrosis was induced by feeding a diet high in fat and sucrose (high-fat diet [HFD]), whereas HCC was produced by intraperitoneal administration of N-diethylnitrosamine (DEN). An in vitro experiment was performed to evaluate the role of KLF10 in the cancer microenvironment using Hep3B and LX2 cells. An immunohistochemical study of KLF10 expression was performed using human HCC samples from 60 patients who had undergone liver resection.
RESULTS
KLF10 deletion resulted in an increased DEN-induced HCC burden with significant upregulation of SMAD2, although loss of KLF10 did not alter HFD-induced liver fibrosis. DEN-treated mice with KLF10 deletion exhibited increased levels of mesenchymal markers (N-cadherin and SNAI2) and tumor metastasis markers (matrix metalloproteinases 2 and 9). KLF10 depletion in Hep3B and LX2 cells using siRNA was associated with increased invasiveness. Compared with co-culture of KLF10-preserved Hep3B cells and KLF10-intact LX2 cells, co-culture of KLF10-preserved Hep3B cells and KLF10-depleted LX2 cells resulted in significantly enhanced invasion. Low KLF10 expression in resected human HCC specimens was associated with poor survival.
CONCLUSION
The results of this study suggest that loss of KLF10 facilitates liver cancer development with alteration in TGFβ signaling.
Topics: Animals; Humans; Mice; Carcinoma, Hepatocellular; Kruppel-Like Transcription Factors; Liver Cirrhosis; Liver Neoplasms; Mice, Inbred C57BL; Transforming Growth Factor beta; Tumor Microenvironment
PubMed: 37946098
DOI: 10.1186/s10020-023-00751-1