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Scientific Reports May 2020The gloomy outcome of liver cancer is mainly due to the high rates of metastasis and recurrence, even after curative resection for early stage liver cancer. Our study...
The gloomy outcome of liver cancer is mainly due to the high rates of metastasis and recurrence, even after curative resection for early stage liver cancer. Our study was conducted to find the animal model suitable for the study of liver cancer metastasis. In our study, two liver cancer cells were obtained from N-nitrosodiethylamine (DEN) and N-nitrosomorpholine (NMOR) induced rats, and they were cultivated, screened and cloning cultivated. Bionomics of cells was analyzed. The results show that 2 cells had different metastatic potentiality. They were named Wrh-f2 and Wrh-s2, and they have the characteristics of Hepatocellular carcinoma cells. The bionomics of 2 cells showed: (1) The chromosome karyotype analysis showed that the mode of Wrh-f2 was 80-83 and Wrh-s2 was 55-57; (2) AFP positive cytoplasmic staining was observed in Wrh-f2 and Wrh-s2. Cytokeratin (CK) 7 and CK8 positive staining was present in Wrh-f2. CK8 positive staining was present in Wrh-s2; (3) The numbers of Wrh-f2 and Wrh-s2 that passed through the Transwells were 98 ± 12 and 55 ± 15;(4) Wrh-f2 had the significant higher colony formation (78%) than Wrh-s2(8%) (P < 0.01). (5) The animal models generated solid tumours when 2 cells were inoculated to nude mouse and rat. And Wrh-f2 developed stable pulmonary metastasis. The established cell lines with different metastatic potential showed obvious advantages over liver cancer in mimicking the biological properties of malignant liver cancer tumors. It provided a suitable model for the mechanism of liver cancer metastasis in vivo and in vitro.
Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Diethylnitrosamine; Disease Models, Animal; Karyotyping; Liver Neoplasms, Experimental; Male; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Nitrosamines; Rats
PubMed: 32433581
DOI: 10.1038/s41598-020-65338-w -
Scientific Reports Apr 2024Hepatocellular carcinoma (HCC) seriously threatens human health, mostly developed from liver fibrosis or cirrhosis. Since diethylnitrosamine (DEN) and carbon...
Hepatocellular carcinoma (HCC) seriously threatens human health, mostly developed from liver fibrosis or cirrhosis. Since diethylnitrosamine (DEN) and carbon tetrachloride (CCl)-induced HCC mouse model almost recapitulates the characteristic of HCC with fibrosis and inflammation, it is taken as an essential tool to investigate the pathogenesis of HCC. However, a comprehensive understanding of the protein expression profile of this model is little. In this study, we performed proteomic analysis of this model to elucidate its proteomic characteristics. Compared with normal liver tissues, 432 differentially expressed proteins (DEPs) were identified in tumor tissues, among which 365 were up-regulated and 67 were down-regulated. Through Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), protein-protein interaction networks (PPI) analysis and Gene-set enrichment analysis (GSEA) analysis of DEPs, we identified two distinguishing features of DEN and CCl-induced HCC mouse model in protein expression, the upregulation of actin cytoskeleton and branched-chain amino acids metabolic reprogramming. In addition, matching DEPs from the mouse model to homologous proteins in the human HCC cohort revealed that the DEN and CCl-induced HCC mouse model was relatively similar to the subtype of HCC with poor prognosis. Finally, combining clinical information from the HCC cohort, we screened seven proteins with prognostic significance, SMAD2, PTPN1, PCNA, MTHFD1L, MBOAT7, FABP5, and AGRN. Overall, we provided proteomic data of the DEN and CCl-induced HCC mouse model and highlighted the important proteins and pathways in it, contributing to the rational application of this model in HCC research.
Topics: Mice; Animals; Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Proteomics; Liver Neoplasms, Experimental; Diethylnitrosamine; Liver Cirrhosis; Disease Models, Animal; Fatty Acid-Binding Proteins
PubMed: 38580754
DOI: 10.1038/s41598-024-58587-6 -
Biological & Pharmaceutical Bulletin 2023This study was designed to evaluate the potential protective impact of estrogen and estrogen receptor against diethylnitrosamine (DEN)-induced hepatocellular carcinoma...
This study was designed to evaluate the potential protective impact of estrogen and estrogen receptor against diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The levels of liver injury serum biomarkers, liver content of interleukin-6 (IL-6), relative liver weight and distortion of liver histological pictures were significantly increased in ovariectomized (OVX) rats and SHAM rats that received DEN alone and were further exaggerated when DEN was combined with fulvestrant (F) compared to non-DEN treated rats. The OVX rats showed higher insults than SHAM rats. The tapering impact on these parameters was clear in OVX rats that received estradiol benzoate (EB), silymarin (S) or orlistat (ORS). The immunohistochemistry and/or Western blot analysis of liver tissues showed a prominent increase in fatty acid synthase (FASN) and cluster of differentiation 36 (CD36) expressions in OVX and SHAM rats who received DEN and/ or F compared to SHAM rats. In contrast to S, treatment of OVX rats with EB mitigated DEN-induced expression of FASN and CD36 in liver tissue, while ORS improved DEN-induced expression of FASN. In conclusion, the protective effect against HCC was mediated via estrogen receptor alpha (ER-α) which abrogates its downstream genes involved in lipid metabolism namely FASN and CD36 depriving the tumor from survival vital energy source. In addition, ORS induced similar mitigating effect against DEN-induced HCC which could be attributed to FASN inhibition and anti-inflammatory effect. Furthermore, S alleviated DEN-induced HCC, independent of its estrogenic effect.
Topics: Animals; Female; Rats; Carcinoma, Hepatocellular; Diethylnitrosamine; Estrogens; Fatty Acid Synthases; Interleukin-6; Liver; Liver Neoplasms; Receptors, Estrogen
PubMed: 37914358
DOI: 10.1248/bpb.b23-00342 -
Intestinal microecology in mice bearing diethylnitrosamine-induced primary hepatocellular carcinoma.Zhejiang Da Xue Xue Bao. Yi Xue Ban =... Aug 2022To explore the characteristics of intestinal microecology in hepatocellular carcinoma (HCC) model mice.
OBJECTIVE
To explore the characteristics of intestinal microecology in hepatocellular carcinoma (HCC) model mice.
METHODS
C57BL/6 male mice aged 2 weeks were divided into normal control group and HCC model group. Mice in HCC model group were exposed to a single intraperitoneal injection of diethylnitrosamine (DEN) 2 weeks after birth; the surviving mice were intraperitoneally injected with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), once every 2 weeks for 8 times starting from the 4 week after birth. Mice in each group were randomly selected and sacrificed at 10 , 18 and 32 weeks after birth, respectively, the liver tissue samples were obtained for histopathological examination. At the 32 week, all mice in both groups were sacrificed and the feces samples were collected under sterile conditions right before the sacrifice. The feces samples were sequenced for the V3-V4 hypervariable regions of the 16S rRNA gene, and the species abundance, flora diversity and phenotype, as well as flora correlation and functional prediction were analyzed.
RESULTS
Alpha diversity analysis showed that all Good's coverage reached the maximum value of 1.00, and the differences in the Observed features, Chao1 index, Shannon index and Simpson index of the intestinal flora of mice between normal control group and HCC model group were all statistically significant (all <0.05). Beta diversity analysis showed that PCoA based on weighted or unweighted Unifrac distances all yielded >0, confirming that the intra-group differences of the samples were less than the inter-group differences; the trend of separation between the two groups was significant ( <0.05). Bacteroidetes, Firmicutes, Actinobacteria and Patescibacteria were the dominant taxa at the phylum level in both normal control group and HCC model group. However, compared with normal control group, the abundance of Bacteroidetes in HCC model group was significantly decreased ( <0.01), while the abundance of Patescibacteria was significantly increased ( <0.05). Moreover, the dominant taxa at the genus level in normal control group mainly included , , , , . The dominant taxa at the genus level in HCC model group mainly included , , , , . There were 30 genera with statistically significant differences in relative abundance at the genus level between the two groups (all <0.05). LEfSe analysis of the intestinal flora of mice in the two groups revealed a total of 14 multi-level differential taxa (all <0.05, LDA score>4.0), which were mainly enriched in Bacteroidetes. The enrichment of 10 differential taxa including Bacteroidetes, Bacteroidia, Bacteroidales, Muribaculaceae, etc. were found in normal control group, and the enrichment of 4 differential taxa including , , etc. were found in HCC model group. There were both positive and negative correlations between the dominant intestinal genera in normal control group (|rho|>0.5, <0.05), while the correlations of the dominant intestinal genera in HCC model group, being less complex than that in normal control group, were all positive. The relative abundance of gram positive and mobile element containing in the intestinal flora of mice in HCC model group was significantly up-regulated compared with normal control group (both <0.05), while that of gram negative ( <0.05) and pathogenic potential ( <0.05) was significantly down-regulated. The metabolic pathways of the intestinal flora in the two groups were significantly different. For instance, 18 metabolic pathways were enriched in normal control group (all <0.005), including those related to energy metabolism, cell division, nucleotide metabolism, etc., while 12 metabolic pathways were enriched in HCC model group (all <0.005), including those related to energy metabolism, amino acid metabolism, carbohydrate metabolism, etc. Conclusions: The amount of intestinal flora in DEN-induced primary HCC model mice decreased, and the composition, correlation, phenotype and function of the intestinal flora in mice were significantly altered. Bacteroidetes at the phylum level, as well as several microbial taxa at the genus level such as , , and could be closely associated with DEN-induced primary HCC in mice.
Topics: Male; Animals; Mice; Carcinoma, Hepatocellular; Diethylnitrosamine; RNA, Ribosomal, 16S; Mice, Inbred C57BL; Liver Neoplasms; Bacteria
PubMed: 37202098
DOI: 10.3724/zdxbyxb-2022-0283 -
Journal of Experimental & Clinical... Oct 2019Glutathione S-transferase zeta 1 (GSTZ1) is the penultimate enzyme in phenylalanine/tyrosine catabolism. GSTZ1 is dysregulated in cancers; however, its role in...
BACKGROUND
Glutathione S-transferase zeta 1 (GSTZ1) is the penultimate enzyme in phenylalanine/tyrosine catabolism. GSTZ1 is dysregulated in cancers; however, its role in tumorigenesis and progression of hepatocellular carcinoma (HCC) is largely unknown. We aimed to assess the role of GSTZ1 in HCC and to reveal the underlying mechanisms, which may contribute to finding a potential therapeutic strategy against HCC.
METHODS
We first analyzed GSTZ1 expression levels in paired human HCC and adjacent normal tissue specimens and the prognostic effect of GSTZ1 on HCC patients. Thereafter, we evaluated the role of GSTZ1 in aerobic glycolysis in HCC cells on the basis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Furthermore, we assessed the effect of GSTZ1 on HCC proliferation, glutathione (GSH) concentration, levels of reactive oxygen species (ROS), and nuclear factor erythroid 2-related factor 2 (NRF2) signaling via gain- and loss- of GSTZ1 function in vitro. Moreover, we investigated the effect of GSTZ1 on diethylnitrosamine (DEN) and carbon tetrachloride (CCl) induced hepatocarcinogenesis in a mouse model of HCC.
RESULTS
GSTZ1 was downregulated in HCC, thus indicating a poor prognosis. GSTZ1 deficiency significantly promoted hepatoma cell proliferation and aerobic glycolysis in HCC cells. Moreover, loss of GSTZ1 function depleted GSH, increased ROS levels, and enhanced lipid peroxidation, thus activating the NRF2-mediated antioxidant pathway. Furthermore, Gstz1 knockout in mice promoted DEN/CCl-induced hepatocarcinogenesis via activation of the NRF2 signaling pathway. Furthermore, the antioxidant agent N-acetylcysteine and NRF2 inhibitor brusatol effectively suppressed the growth of Gstz1-knockout HepG2 cells and HCC progression in Gstz1 mice.
CONCLUSIONS
GSTZ1 serves as a tumor suppressor in HCC. GSH depletion caused by GSTZ1 deficiency elevates oxidative stress, thus constitutively activating the NRF2 antioxidant response pathway and accelerating HCC progression. Targeting the NRF2 signaling pathway may be a promising therapeutic approach for this subset of HCC.
Topics: Animals; Carbon Tetrachloride; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Diethylnitrosamine; Down-Regulation; Gene Expression Regulation, Neoplastic; Glutathione Transferase; Hep G2 Cells; Humans; Kelch-Like ECH-Associated Protein 1; Lipid Peroxidation; Liver Neoplasms; Mice; NF-E2-Related Factor 2; Neoplasm Transplantation; Oxidative Stress; Prognosis; Signal Transduction
PubMed: 31666108
DOI: 10.1186/s13046-019-1459-6 -
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 -
Molecules (Basel, Switzerland) Jan 2021This study investigated the cancer chemopreventive effects of an acidic methanol extract of purple rice husk on chemically induced carcinogenesis in rats. This purple...
This study investigated the cancer chemopreventive effects of an acidic methanol extract of purple rice husk on chemically induced carcinogenesis in rats. This purple rice husk extract (PRHE) had high polyphenol contents. Vanillic acid was a major phenolic compound in PRHE. Three major anthocyanins found in PRHE were malvidin-3-glucoside, peonidin-3-glucoside and cyanidin-3-glucoside. PRHE was not toxic and clastogenic in rats. The LD of PRHE was greater than 2000 mg kg body weight (BW). The oral administration of 300 or 1000 mg kg BW of PRHE for 28 days significantly decreased the number of micronucleated hepatocytes in diethylnitrosamine-initiated rats. The inhibitory mechanisms were associated with the reduction of cytochrome P450 2E1 expression and induction of some detoxifying enzymes in the liver. In addition, treatment with 500 mg kg BW of PRHE for eight weeks did not induce preneoplastic lesions in the liver and colon. It significantly inhibited hepatic glutathione--transferase positive foci formation induced by diethylnitrosamine and 1,2-dimethylhydrazine by suppression of hepatocyte proliferation and induction of apoptosis. In conclusion, PRHE did not present toxicity, clastogenicity or carcinogenicity in rats. It exhibited cancer chemopreventive properties against chemically induced early stages rat hepatocarcinogenesis. Anthocyanins and vanillic acid might be candidate anticarcinogenic compounds in purple rice husk.
Topics: 3,3'-Diaminobenzidine; Animals; Apoptosis; Carcinogenesis; Diethylnitrosamine; Liver; Male; Oryza; Plant Extracts; Proliferating Cell Nuclear Antigen; Rats, Wistar; Toxicity Tests, Acute; Xenobiotics; Rats
PubMed: 33445792
DOI: 10.3390/molecules26020360 -
European Review For Medical and... Aug 2018Immune therapy has recently become a novel strategy for treating liver cancer, making it of critical importance to identify novel targets for treatment. Programmed...
OBJECTIVE
Immune therapy has recently become a novel strategy for treating liver cancer, making it of critical importance to identify novel targets for treatment. Programmed death-1 homology (PD-1H) is one newly discovered negative co-stimulating molecule, and plays important regulatory roles in suppressing T cell activation. However, the expression or function of PD-1H in liver tumors has not been reported.
MATERIALS AND METHODS
Liver cancer tissues were collected from The Cancer Genome Atlas (TCGA) (http://tcga-data.nci-nih.gov). This study then utilized diethylnitrosamine (DEN) induced liver cancer mice, on which PD-1H monoclonal antibody and PD-1H extra-cellular Fc domain fusion protein were injected intraperitoneal. General status, gross morphology of liver tissues was examined, followed by hematoxylin-eosin (HE) staining and plotting survival curve.
RESULTS
Among TCGA samples, PD-1H expression was significantly elevated. Induced liver cancer mice showed depressed mental status, early onset of hepatitis and liver cirrhosis. Five mice dead in model group (mortality=33.33%). No natural death occurred in control group. Injection of PD-1H-Fc-Ig fusion and PD-1H monoclonal antibody improved the condition to certain extents, with morality at about 20%. Comparing to DEN group, combined treatment group showed significantly fewer tumor lesion on liver surface, with increased body weight and lower liver-body weight ratio. HE staining showed significantly elevated ratio of normal cells in combined treatment group, although large amounts of cancer cells still existed.
CONCLUSIONS
Blocking of PD-1H signal pathway could suppress liver cancer cell growth, decrease mouse mortality, indicating promising application of PD-1H in tumor immune therapy.
Topics: Alkylating Agents; Animals; Diethylnitrosamine; Disease Models, Animal; Humans; Liver Neoplasms; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Random Allocation
PubMed: 30178841
DOI: 10.26355/eurrev_201808_15716 -
Mutation Research. Genetic Toxicology... Apr 2018Regulatory guidance documents stress the value of assessing multiple tissues and the most appropriate endpoints when evaluating chemicals for in vivo genotoxic...
Regulatory guidance documents stress the value of assessing multiple tissues and the most appropriate endpoints when evaluating chemicals for in vivo genotoxic potential. However, conducting several independent studies to consider multiple endpoints and/or tissue compartments is resource intensive. Furthermore, conventional approaches for scoring genotoxicity endpoints are slow, tedious, and less objective than what would be considered ideal. In an effort to address these issues with current practices, we attempted to i) employ flow cytometry-based methods to score liver micronuclei, blood micronuclei, and blood Pig-a gene mutation, and ii) integrate the endpoints into a common general toxicology study design-the rat 28-day repeat dose study. A proof-of-principle experiment was performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) for 28 consecutive days. One day later blood was collected for micronucleated reticulocyte (MN-RET) and Pig-a mutation assays, and liver tissue was obtained for micronucleated hepatocyte (MNHEP) scoring. MN-RET frequencies were not affected by DEN exposure, and mean Pig-a mutant cell frequencies were only slightly elevated. On the other hand, % MNHEP showed marked, dose-related increases (2.2, 7.2, and 9.1 mean fold-increase for 5, 10, 15 mg DEN/kg/day, respectively). Concurrent with MNHEP analyses, assessments of Ki-67-positive events and the proportion of 8n nuclei provided evidence for treatment-related changes to hepatocyte proliferation. Collectively, these results reinforce the importance of evaluating chemicals' genotoxic potential in liver in addition to hematopoietic cells, and suggest that several automated measurements can be successfully integrated into repeat-dose studies for higher efficiencies and better utilization of fewer animals.
Topics: Animals; Diethylnitrosamine; Dose-Response Relationship, Drug; Flow Cytometry; Hepatocytes; Male; Membrane Proteins; Micronucleus Tests; Mutagenicity Tests; Mutation; Rats, Sprague-Dawley; Reticulocytes
PubMed: 29555062
DOI: 10.1016/j.mrgentox.2018.02.005 -
Molecules (Basel, Switzerland) Mar 2015Caudatin is a potential antitumor agent isolated from the traditional Chinese medicine "baishouwu", which was the root tuber of Cynanchum auriculatum Royle ex Wight. In...
Caudatin is a potential antitumor agent isolated from the traditional Chinese medicine "baishouwu", which was the root tuber of Cynanchum auriculatum Royle ex Wight. In our previous studies, caudatin showed selectivity on human hepatoma cell line SMMC7721 among several different tumor cell lines, and further in vivo tests validated the inhibitory action of caudatin against hepatic cancer using an H22 solid tumor model in mice, but to our knowledge, the biopharmaceutical properties of caudatin are largely unknown. In this study, a simple, rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of caudatin in rat plasma and tissues, which kept the run time to detect one sample within 4 min, was developed and validated. Pharmacokinetics and tissue distribution studies of caudatin in conventional rats and hepatocellular carcinoma (HCC) model rats were then conducted for the first time. Statistically significant differences were observed between conventional rats and diethylnitrosamine (DEN)-induced HCC rats with respect to pharmacokinetic parameters, including maximum concentration (Cmax), time to reach Cmax (Tmax), half-life (t1/2), area under the concentration-time curve (AUC0-t, AUC0-∞), mean residence time (MRT0-t and MRT0-∞), and oral clearance (CL/F). Increased exposures of caudatin were found in the plasma and livers of HCC model rats, which would be helpful for a better understanding of pharmacological effect of caudatin in treating HCC disease.
Topics: Alkylating Agents; Animals; Carcinoma, Hepatocellular; Chromatography, Liquid; Diethylnitrosamine; Disease Models, Animal; Drugs, Chinese Herbal; Glycosides; Humans; Liver Neoplasms; Male; Mice; Rats; Rats, Sprague-Dawley; Steroids; Tandem Mass Spectrometry; Tissue Distribution
PubMed: 25751784
DOI: 10.3390/molecules20034225