cyclin-d1 and Liver-Cirrhosis

Hastatoside is an iridoid glycoside extracted from the herb, Verbena officinalis, that exerts various pharmacological effects, including anti-inflammatory, sleep-promoting, and analgesic effects. However, only a few studies have reported the efficacy of hastatoside in liver fibrosis. Liver fibrosis is a pathophysiological process, and its persistence can seriously affect the quality of life and well-being of the patients.. This study aimed to investigate the role of hastatoside on liver fibrosis and its possible underlying mechanisms.. C57BL/6 J mice with carbon tetrachloride (CCl. These findings suggest that hastatoside can bind to GSK-3β and promote its activity, while inhibiting the GSK-3β downstream effector expression of β-catenin, thereby inhibiting the activation and proliferation of HSCs, which further prevents the development of liver fibrosis. These results provide innovative insights into the underlying liver fibrosis. Moreover, hastatoside is a potential anti-fibrosis monomer that can potentially be used for the treatment of liver fibrosis.

Topics: Animals; beta Catenin; Chemical and Drug Induced Liver Injury; Cyclin D1; Disease Models, Animal; Glycogen Synthase Kinase 3 beta; Hepatic Stellate Cells; Humans; Iridoid Glycosides; Liver; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Quality of Life; Signal Transduction

Liver fibrosis is a chronic wound-healing response to liver injury of various origins and represents a major health problem.. The current study endeavored to investigate the repressing effect of fisetin on hepatic fibrosis induced by thioacetamide (TAA) in rats.. Rats were injected with TAA (200 mg/kg) intraperitoneally twice per week for 6 weeks to induce liver fibrosis. Fisetin (50 and 100 mg/kg/day) or silymarin (50 mg/kg/day) were given orally on a daily basis along with TAA. Liver function parameters, oxidative stress, inflammatory and fibrogenic biomarkers as well as wnt3a, β-catenin, glycogen synthase kinase 3 (GSK-3β) and cyclin D1 were estimated. Histoapthological and immunohistochemical examinations were performed.. Fisetin restored normal liver functions, increased reduced glutathione (GSH) level and decreased malondialdehyde (MDA), as well as inflammatory biomarkers including; tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). Additionally, it lessened transforming growth factor β1 (TGF-β1), collagen I and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels as well as elevated matrix metalloproteinase-9 (MMP-9) hepatic content. Furthermore, fisetin significantly suppressed wnt3a gene expression associated with decreased β-catenin and increased GSK-3β levels. Moreover, fisetin decreased the progress of histologic hepatic fibroplasia and diminished hepatic expression of α-SMA and cyclin D1.

Topics: Animals; beta Catenin; Biomarkers; Cyclin D1; Flavonols; Glycogen Synthase Kinase 3 beta; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Matrix Metalloproteinase 9; Rats; Silymarin; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Wnt Signaling Pathway

This study has been designed to investigate the role of vanillin either as prophylaxis or treatment in liver regeneration augmentation and liver fibrosis regression in thioacetamide (TAA) induced liver damage.. Animals were injected with TAA to induce liver injury (200mg/kg twice weekly) for 8 weeks. In vanillin prophylaxis group; rats were administered vanillin (100 mg/Kg; IP, daily) from day 1 of TAA injection for 8 weeks. In vanillin treatment group; rats were confronted with the same dose of TAA injection for 8 weeks then treated with vanillin (100 mg/Kg, IP, daily) for 4 weeks. ALT, AST activities, serum albumin, hepatic GSH, MDA, HGF, VEGF, IL-6 and TNF-α levels were measured and also, MMP-2, TIMP-1 and cyclin D gene expression were determined. Liver sections were stained with H&E and Sirius red and immunostained for Ki-67 and α-SMA for histological and immunohistological changes analysis.. Vanillin improved liver function and histology. Also, showed a remarkable increase in hepatic HGF and VEGF level, and up-regulation of cyclin D1 expression accompanied by a significant up-regulation of MMP-2 and down- regulation of TIMP-1. All these effects were accompanied by TNF-α, IL-6 and oxidative stress significant attenuation.. In conclusion, vanillin enhanced liver regeneration in TAA induced liver damage model; targeting growth factors (HGF, VEGF) and cellular proliferation marker cyclin D1. As well as stimulating fibrosis regression by inhibition of ECM accumulation and enhancing its degradation.

Topics: Animals; Benzaldehydes; Cell Proliferation; Cyclin D1; Intercellular Signaling Peptides and Proteins; Liver; Liver Cirrhosis; Liver Regeneration; Male; Oxidative Stress; Rats; Rats, Wistar; Thioacetamide

Though honokiol, derived from the Magnolia tree, was known to suppress renal fibrosis, pulmonary fibrosis, non-alcoholic steatoheptitis, inflammation and cancers, the underlying antifibrotic mechanisms of honokiol are not fully understood in hepatic stellate cells until now. Thus, in the present study, inhibitory mechanism of honokiol on liver fibrosis was elucidated mainly in hepatic stellate cells (HSCs) by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle analysis and western-blotting. Honokiol exerted cytotoxicity in LX-2, HSC-T6 and Hep-G2 cells. Honokiol increased sub G1 population and activated caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) in HSCs. Moreover, honokiol attenuated the expression of alpha smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β1), phospho-Smad3, phospho-AKT, cyclin D1, c-Myc, Wnt3a, β-catenin, and activated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) in HSCs. Conversely, GSK3β inhibitor SB216763 reversed the effect of honokiol on PARP, α-SMA, phospho-GSK3β, β-catenin and sub G1 population in LX-2 cells. Overall, honokiol exerts apoptotic and antifibrotic effects via activation of GSK3β and inhibition of Wnt3a/β-catenin signalling pathway.

Topics: Actins; beta Catenin; Biphenyl Compounds; Caspase 3; Cell Line; Cyclin D1; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Hepatic Stellate Cells; Humans; Lignans; Liver Cirrhosis; Phosphorylation; Smad3 Protein; Transforming Growth Factor beta1; Wnt Signaling Pathway

Liver fibrosis is an increasing health problem worldwide, for which no effective antifibrosis drugs are available. Although the involvement of aerobic glycolysis in hepatic stellate cell (HSC) activation has been reported, the role of pyruvate kinase M2 (PKM2) in liver fibrogenesis still remains unknown. We examined PKM2 expression and location in liver tissues and primary hepatic cells. The in vitro and in vivo effects of a PKM2 antagonist (shikonin) and its allosteric agent (TEPP-46) on liver fibrosis were investigated in HSCs and liver fibrosis mouse model. Chromatin immunoprecipitation sequencing and immunoprecipitation were performed to identify the relevant molecular mechanisms. PKM2 expression was significantly up-regulated in both mouse and human fibrotic livers compared with normal livers, and mainly detected in activated, rather than quiescent, HSCs. PKM2 knockdown markedly inhibited the activation and proliferation of HSCs in vitro. Interestingly, the PKM2 dimer, rather than the tetramer, induced HSC activation. PKM2 tetramerization induced by TEPP-46 effectively inhibited HSC activation, reduced aerobic glycolysis, and decreased MYC and CCND1 expression via regulating histone H3K9 acetylation in activated HSCs. TEPP-46 and shikonin dramatically attenuated liver fibrosis in vivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.

Topics: Acetylation; Animals; Cyclin D1; Female; Hepatic Stellate Cells; Histones; Humans; Liver Cirrhosis; Male; Mice; Organic Chemicals; Protein Multimerization; Proto-Oncogene Proteins c-myc; Pyridazines; Pyrroles; Pyruvate Kinase

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl

Topics: Actins; Animals; Cell Line; Cell Proliferation; Collagen Type I; Collagen Type I, alpha 1 Chain; Cyclin D1; Cyclin-Dependent Kinases; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Extracellular Signal-Regulated MAP Kinases; Hepatic Stellate Cells; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Protein Binding; PTEN Phosphohydrolase; Sennosides; Signal Transduction; Transforming Growth Factor beta

The associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure promotes the proliferation of the future liver remnant, but evidence to support the feasibility of ALPPS in livers with fibrosis is needed. Therefore the aim of this study was to establish a fibrotic ALPPS model in the rat to compare the capacity of regeneration in the remnant liver with or without fibrosis.. In our study we first established a thioacetamide-induced fibrotic ALPPS model in rats. Then the ALPPS-induced regenerative capacities of normal and fibrotic liver were compared in this animal model. In addition, markers of regeneration, including the proliferative index and cyclin D1 and proliferating cell nuclear antigen levels, as well as various indicators of liver function were determined to evaluate the quality of the hepatic regeneration.. Compared with that of the sham group (opening of the peritoneal cavity with no further operative manipulation), the proliferation of the future liver remnant in fibrotic rat liver after the ALPPS procedure was increased on postoperative days 1, 2, and 5 (P < .039 each). In addition, the proliferative response was greater in the ALPPS group than in the ligation group subjected only to portal vein ligation of the left lateral, left middle, right, and caudate lobes (P = .099, P = .006, and P = .020 on postoperative days 1, 2, and 5, respectively). In contrast, the ALPPS-induced regenerative capacity in the fibrotic rat livers was attenuated compared with that in the normal liver on postoperative days 1, 2, and 5 (P < .031 for each) after stage I and on postoperative day 5 after stage II of the ALPPS procedure (P < .005). This attenuated the recovery of liver function, and the greater mortality rate indicated that functional proliferation was either delayed or not as extensive in the fibrotic rat livers.. Through establishing a rat model of thioacetamide-induced liver fibrosis, we found that ALPPS-derived liver regeneration was present and feasible in fibrotic livers, but this effect was attenuated compared with that in normal liver.

Topics: Animals; Biomarkers; Cyclin D1; Disease Models, Animal; Feasibility Studies; Hepatectomy; Ki-67 Antigen; Ligation; Liver; Liver Cirrhosis; Liver Regeneration; Portal Vein; Proliferating Cell Nuclear Antigen; Random Allocation; Rats, Sprague-Dawley; Thioacetamide

Chemokine-like factor (CKLF)-like, MAL and related proteins for vesicle trafficking and membrane link (MARVEL) transmembrane domain-containing family proteins (CMTMs) have significant roles in the immune system, in male reproduction, as well as in tumorigenesis. Previous studies have shown that CMTM family member 7 (CMTM7) was broadly expressed in various normal tissues, but not in lung, gastric, esophageal, pancreas, and cervix cancers. To explore its relationship with liver cancer, we examined the expression of CMTM7 in liver cancers and its correlation with clinical and pathological conditions. We found that CMTM7 expression was markedly reduced in liver cancer tissues, and negatively correlated with TNM staging and tumor metastasis. In vitro studies showed that enforced expression of CMTM7 inhibited the cell growth and migration of liver cancer cells. Further analysis revealed that CMTM7 suppressed AKT signaling and induced cell cycle arrest at the G0/G1 phase in the liver cancer cells, likely as the consequent of decreased levels of cyclin D1, cyclin-dependent kinase 4 (CDK4), and CDK6, and increased p27 expression. Thus, CMTM7 functions as a tumor suppressor in liver cancer through suppressing cell cycle progression.

Topics: Aged; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chemokines; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Cirrhosis; Liver Neoplasms; Lymphatic Metastasis; Male; MARVEL Domain-Containing Proteins; Middle Aged; Neoplasm Staging; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; Signal Transduction

Topics: Animals; Carbon Tetrachloride; Cell Line; Cell Proliferation; Cyclin D1; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Octreotide; Proto-Oncogene Proteins c-myc; Rats, Sprague-Dawley; Wnt Signaling Pathway

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with highest incidence in Asia and Africa. MicroRNAs (miRNAs), a class of non-coding single stranded RNA, which not only post transcriptionally regulate gene expression but also respond to signaling molecules to affect cell functions such as Wnt/β-catenin signaling specifically in HCC. The goal of this study is to investigate the crosstalk between Wnt/β-catenin signaling proteins and microRNAs expression in HCC patients.. Fresh tissue samples of 30 primary HCC patients and 10 control subjects were included. Expression level of 13 different miRNAs (miR-10a- miR-106b- miR-99a- miR-148a- miR-125b- miR-30e- miR-183- miR-155- miR-199a- miR-199a3p- miR-24- miR-122 and miR-215) were examined using real-time PCR assay. Five proteins involved in the Wnt/β-catenin pathway (β-catenin, APC, c-myc, survivin and cyclin D1) were analysed by immunohistochemistry technique. The correlation between miRNAs expression levels with protein expressions was assessed.. Up-regulation of miR-155 and miR-183 was reported in HCC patients compared to normal controls and this up-regulation was significantly correlated with liver cirrhosis in the case of miR-155 (p<0.05) referring to their oncogenic activity. Down-regulation was observed for 11 miRNAs in HCC indicating their tumour suppression activity. MiRNA-10a, miR-30e, miR-215, miR-125b and miR-148a were significantly correlated with the expression of important players in Wnt/β-catenin pathway including β-catenin, APC and c-myc (p<0.05). Detailed analysis revealed that miR-215 is associated with the grade of the disease and miR-125b is associated with HCV infection.. Collectively, our data showed potential role of miR-10a, miR-30e, miR-215, miR-125b and miR-148a as important mediators in HCC progression. Furthermore, their association with Wnt/β-catenin cascade proteins could be exploited to develop new therapeutic target strategies in HCC.

Topics: Adenomatous Polyposis Coli Protein; Aged; beta Catenin; Carcinoma, Hepatocellular; Case-Control Studies; Cyclin D1; Down-Regulation; Female; Gene Expression; Hepatitis C, Chronic; Humans; Inhibitor of Apoptosis Proteins; Liver Cirrhosis; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Grading; Proto-Oncogene Proteins c-myc; Survivin; Up-Regulation; Wnt Signaling Pathway

Varied pathogenetic elements have been touched upon the liver fibrosis, including inflammatory, stress, apoptosis and unfolded proteins aggregation. Magnesium Isoglycyrrhizinate (MgIG) has been accepted to be a neuroprotective effect, hepatoprotective and anti-inflammatory molecule. In our vitro researches, MgIG was considered to activate hepatic stellate cells (HSCs) apoptosis by promoting endoplasmic reticulum stress (ERS) detrimental response to a certain extent. Consequently, MgIG showed its potential therapeutic capacity in fibrogenesis and counteracted the pathogenetic aspects, which were involved in integrating current treatments correcting liver fibrosis. In addition, we further verificated the behavior and pathogenic mechanisms in the CCl

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Carbon Tetrachloride; Cell Line; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; eIF-2 Kinase; Endoplasmic Reticulum Stress; Gene Expression Regulation; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred ICR; Oncogene Proteins; Saponins; Signal Transduction; Triterpenes; Unfolded Protein Response

Matrine (MT), the effective component of Sophora flavescens Ait, has been shown to have anti-inflammation, immune-suppressive, anti-tumor, and anti-hepatic fibrosis activities. However, the pharmacological effects of MT still need to be strengthened due to its relatively low efficacy and short half-life. In the present study, we report a more effective thio derivative of MT, MD-1, and its inhibitory effects on the activation of hepatic stellate cells (HSCs) in both cell culture and animal models. Cytological experiments showed that MD-1 can inhibit the proliferation of HSC-T6 cells with a half-maximal inhibitory concentration (IC50) of 62 μmol/L. In addition, MD-1 more strongly inhibits the migration of HSC-T6 cells compared to MT and can more effectively induce G0/G1 arrest and apoptosis. Investigating the biological mechanisms underlying anti-hepatic fibrosis in the presence of MD-1, we found that MD-1 can bind the epidermal growth factor receptor (EGFR) on the surface of HSC-T6 cells, which can further inhibit the phosphorylation of EGFR and its downstream protein kinase B (Akt), resulting in decreased expression of cyclin D1 and eventual inhibition of the activation of HSC-T6 cells. Furthermore, in rats with dimethylnitrosamine (DMN)-induced hepatic fibrosis, MD-1 slowed the development and progression of hepatic fibrosis, protecting hepatic parenchymal cells and improving hepatic functions. Therefore, MD-1 is a potential drug for anti-hepatic fibrosis.

Topics: Alkaloids; Animals; Cell Line; Cyclin D1; Dimethylnitrosamine; Enzyme Activation; ErbB Receptors; G1 Phase Cell Cycle Checkpoints; Hepatic Stellate Cells; Liver Cirrhosis; Matrines; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinolizines; Rats

To assess the distribution of proteins coded by genes reported as relevant for the molecular classification of hepatocellular carcinoma (HCC).. In this retrospective cross-sectional study, the following clinicopathological data were analyzed in 80 autopsied HCC patients: sex, age, ethnicity, alcohol intake, infection with hepatitis B and/or C virus, infection with human immunodeficiency virus, prior treatment, basic and immediate causes of death, liver weight, presence of cirrhosis, number and size of nodules, gross pattern, histological grade and variants, architectural pattern, invasion of large veins, and presence and location of extrahepatic metastases. The protein products of genes known to be involved in molecular pathogenesis of HCC, including epidermal growth factor receptor (EGFR), MET, keratin 19 (K19), vimentin, beta-catenin, mechanistic target of rapamycin (mTOR), extracellular signaling-related kinase (ERK)1, ERK2, Ki67, cyclin D1, caspase 3 and p53, were detected by immunohistochemistry on tissue microarrays. The expression levels were scored and statistically assessed for correlation with HCC parameters.. Infection with hepatitis C virus was identified in 49% of the 80 autopsy patients, cirrhosis in 90%, advanced tumors in 95%, and extrahepatic metastases in 38%. Expression of K19, p53 and ERK1 correlated to high-grade lesions. Expression of ERK1, nuclear beta-catenin, cyclin D1 and ERK2 correlated to higher rates of cell proliferation as determined by Ki67. Expression of MET, EGFR (> 0) and caspase 3 correlated with lower histological grades. Expression of EGFR correlated to that of caspase 3, and overexpression of EGFR (≥ 200/300) was observed in low-grade tumors more frequently (grades 1 and 2: 67% vs grade 3: 27% and grade 4: 30%). Expression of ERK1 was associated with that of K19 and vimentin, whereas expression of ERK2 was associated with that of cyclin D1, MET and membrane beta-catenin. Expression of vimentin was strongly correlated with that of K19.. Expression of K19, p53, ERK1, ERK2, vimentin and nuclear beta-catenin was related to higher-grade markers, as opposed to expression/overexpression of EGFR, MET and caspase 3.

Topics: Adult; Aged; Aged, 80 and over; Autopsy; beta Catenin; Brazil; Carcinoma, Hepatocellular; Case-Control Studies; Caspase 3; Cross-Sectional Studies; Cyclin D1; ErbB Receptors; Female; Hepatitis B, Chronic; Hepatitis C, Chronic; HIV Infections; Humans; Immunohistochemistry; Keratin-19; Ki-67 Antigen; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-met; Retrospective Studies; Tissue Array Analysis; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53; Vimentin

To investigate the effect of Ganfukang (GFK) on connective tissue growth factor (CTGF) and focal adhesion kinase (FAK)/protein kinase B (PKB or Akt) signal pathway in a hepatic fibrosis rat model and to explore the underlying therapeutic molecular mechanisms of GFK.. Fifty SD rats were randomly divided into five groups as follows: the control group, the model group (repeated subcutaneous injection of CCl4), and the three GFK treatment groups (31.25, 312.5, and 3125 mg/kg, intragastric administration). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to examine the expression of CTGF, integrin α5, integrin β1, FAK/Akt signal pathway, cyclinD1, and collagen in the different-treated rats.. GFK attenuated the up-regulation of CTGF, integrin α5, and integrin β1 in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the phosphorylation of Akt simultaneously (P<0.01). At the same time, the expression of cyclinD1, collagen I, and collagen III was decreased by GFK significantly (P<0.01).. CTGF and FAK/Akt signal pathway were activated in the CCl4-induced hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due to its effects against CTGF and FAk/Akt signal pathway.

Topics: Animals; Collagen; Connective Tissue Growth Factor; Cyclin D1; Drugs, Chinese Herbal; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Integrin alpha5; Integrin beta1; Liver; Liver Cirrhosis; Male; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Signal Transduction

Metabolic syndrome (MS) is an emerging risk factor in hepatocellular carcinoma (HCC). HCC related to MS may occur either in advanced fibrosis or before the development of cirrhosis, suggesting involvement of different molecular pathways according to the features of background liver.. To investigate genomic aberrations in HCC related to MS in order to identify new target genes involved in liver carcinogenesis.. Chromosomal aberrations of HCC obtained from 20 patients with MS (HCC/MS) were studied by comparative genomic hybridisation and compared with HCC related to hepatitis C virus (HCV) infection (HCC/HCV, n=10) and, within the group of HCC with MS, according to the condition of the background liver (presence or absence of significant fibrosis).. Among the most frequent chromosomal alterations observed in HCC, 6p21.1 amplification had a higher incidence in HCC/MS than in HCC/HCV (60% vs 20%, p<0.01). Advanced fibrosis/cirrhosis in the peritumoral liver was the only clinicopathological factor associated with the 6p21.1 amplicon in HCC/MS. Increased expression of cullin7 (CUL7), a gene located at the 6p21.1 locus, was demonstrated in HCC with the 6p21.1 amplicon, in parallel with a decrease in cyclin D1 expression. CUL7 downregulation using siRNA transfection in hepatoma cell lines induced significant cyclin D1 expression (by promoting its degradation), decreased cell proliferation and increased apoptosis.. This study demonstrates specific genomic alterations in HCC/MS and points to CUL7 as a novel gene potentially involved in liver carcinogenesis associated with MS, the amplification of which might influence cell proliferation.

Topics: Aged; Aged, 80 and over; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosomes, Human, Pair 6; Cullin Proteins; Cyclin D1; Female; Gene Expression; Hepatitis C; Humans; Immunohistochemistry; Liver Cirrhosis; Liver Neoplasms; Male; Metabolic Syndrome; Middle Aged; Nucleic Acid Hybridization; Real-Time Polymerase Chain Reaction

Hepatic stellate cells (HSCs), activated during liver injury, are defined as the most important target in the therapy of hepatic fibrosis. In the present study, we evaluated the effect of Rosmarinic acid (RosA) on the proliferation and apoptosis in activated hepatic stellate cells (HSC-T6), which is useful to decrease this cell population. The proliferation of HSC-T6 was significantly inhibited after treated with various concentrations of RosA for different times. Flow cytometric analyses and transmission electron microscope (TEM) observations revealed that HSC-T6 treated with RosA underwent apoptosis in a time dependent manner and displayed typical apoptotic features in the cells. The phosphorylation in signal transducer and activator of transcription protein-3 (STAT3), which regulates cell survival, proliferation and differentiation in a variety of tissues, was markedly decreased as the result of Western blot assay and correlated with downregulation of CyclinD1 and B cell lymphoma/leukemia-2 (Bcl-2). In conclusion, these results suggested that RosA was able to inhibit proliferation and induce apoptosis in HSC-T6, partly due to the inhibition of phosphorylation in STAT3, which contributed to the reversal of hepatic fibrosis.

Topics: Animals; Apoptosis; bcl-Associated Death Protein; Cell Line; Cell Proliferation; Cinnamates; Cyclin D1; Depsides; Down-Regulation; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Phosphorylation; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rats; Rosmarinic Acid; Signal Transduction; STAT3 Transcription Factor

Plasminogen activator inhibitor-1 (PAI-1) is an acute phase protein that has been shown to play a role in experimental fibrosis caused by bile duct ligation (BDL) in mice. However, its role in more severe models of hepatic fibrosis (e.g., carbon tetrachloride; CCl(4)) has not been determined and is important for extrapolation to human disease. Wild-type or PAI-1 knockout mice were administered CCl(4) (1 ml/kg body wt ip) 2x/wk for 4 wk. Plasma (e.g., transaminase activity) and histological (e.g., Sirius red staining) indexes of liver damage and fibrosis were evaluated. Proliferation and apoptosis were assessed by PCNA and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively, as well as by indexes of cell cycle (e.g., p53, cyclin D1). In contrast to previous studies with BDL, hepatic fibrosis was enhanced in PAI-1(-/-) mice after chronic CCl(4) administration. Indeed, all indexes of liver damage were elevated in PAI-1(-/-) mice compared with wild-type mice. This enhanced liver damage correlated with impaired hepatocyte proliferation. A similar effect on proliferation was observed after one bolus dose of CCl(4), without concomitant increases in liver damage. Under these conditions, a decrease in phospho-p38, coupled with elevated p53 protein, was observed; these results suggest impaired proliferation and a potential G(1)/S cell cycle arrest in PAI-1(-/-) mice. These data suggest that PAI-1 may play multiple roles in chronic liver diseases, both protective and damaging, the latter mediated by its influence on inflammation and fibrosis and the former via helping maintain hepatocyte division after an injury.

Topics: Animals; Apoptosis; Carbon Tetrachloride Poisoning; Cell Proliferation; Cyclin D1; Hepatocytes; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Plasminogen Activator Inhibitor 1; Tissue Inhibitor of Metalloproteinase-1; Tumor Suppressor Protein p53

The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle alpha-actin (alphaSMA) promoter was generated (Ad-SMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced.. Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis.

Topics: Actins; Adenoviridae; Animals; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen Type I; Cyclin D1; Disease Models, Animal; Disease Progression; Extracellular Matrix; Hepatic Stellate Cells; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Mice, Transgenic; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction

Topics: Carcinoma, Hepatocellular; Cyclin D1; Hepatitis B virus; Humans; Liver; Liver Cirrhosis; NF-kappa B; Trans-Activators; Viral Regulatory and Accessory Proteins

The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type I collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-beta1 (TGF-beta1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde.. Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-beta1 was examined using the reverse transcriptase-polymerase chain reaction. Type I collagen in the culture medium was detected by enzyme-linked immunosorbent assay.. 20, 50 and 100 micromol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1-phase arrest of acetaldehyde-induced HSCs in a dose-dependent manner. The secretion of type I collagen and the expression of cyclin D1, CDK4 and TGF-beta1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 micromol/L PD98059, respectively.. The ERK pathway regulates the cell proliferation, secretion of type I collagen and the expression of TGF-beta1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.

Topics: Acetaldehyde; Animals; Cells, Cultured; Collagen Type I; Cyclin D1; Cyclin-Dependent Kinase 4; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; G1 Phase; Hepatocytes; Liver Cirrhosis; MAP Kinase Signaling System; Rats; Rats, Inbred Strains; Resting Phase, Cell Cycle; RNA, Messenger; Transforming Growth Factor beta1

The function of peroxisome proliferator-activated receptor gamma (PPARgamma) in hepatic fibrogenesis remains largely unknown. Curcumin is a natural substance extracted form Curcuma Longa Linn and has a variety of pharmacological effects. In this study, the effects of curcumin on the proliferation, activation and apoptosis of rat hepatic stellate cells (HSCs) through PPARgamma signaling were investigated.. HSCs were isolated from the normal Sprague Dawley rats through in situ perfusion of the liver with Pronase E and density-gradient centrifugation with Nycodenz. Cells were treated with curcumin, troglitazone, salvianolic acid B or GW9662. The effect on HSCs proliferation was determined by MTT colorimetry. Total RNA was extracted by TRizol reagent and gene levels were determined by semi-quantitative RT-PCR. Total cellular and nuclear protein were isolated and separated by 10% sodium dodecy lsulfate polyacrylamide gel electrophoresis. Protein levels were determined by Western blot. Cell apoptosis was detected by Hoechst 33258 staining. PPARgamma subcellular distribution was detected by immunofluorescent staining. The activities of MMP-2 and 9 were measured by Gelatin zymograph assay.. Curcumin suppressed HSCs proliferation in a dose-dependent manner. As HSCs underwent gradual activation with culture prolongation the PPARgamma nuclear expression level decreased. Curcumin up-regulated PPARgamma expression and significantly inhibited the production of alpha-SMA and collagen I. PPARgamma is expressed in the cytoplasm and nucleus and is evenly distributed in HSCs, but accumulated in the nucleus of HSCs and disappeared from cytoplasm after curcumin treatment. Hoechst 33258 staining showed that curcumin induced the apoptosis of culture-activated HSCs and significantly increased pro-apoptotic Bax expression and reduced anti-apoptotic Bcl-2 expression. Cyclin D1 gene, activated NFkappaB p65 protein and TGFbetaR-I protein expression were down-regulated significantly by curcumin. The activities of MMP-2 and MMP-9 were enhanced significantly by curcumin.. Curcumin can inhibit the proliferation and activation of HSCs, induce the apoptosis of activated HSCs and enhance the activities of MMP-2 and MMP-9. The effects of curcumin are mediated through activating the PPARgamma signal transduction pathway and associated with PPARgamma nuclear translocation/redistribution.

Topics: Active Transport, Cell Nucleus; Activin Receptors, Type I; Animals; Apoptosis; bcl-2-Associated X Protein; Cell Nucleus; Cell Proliferation; Cells, Cultured; Curcumin; Cyclin D1; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; PPAR gamma; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Transcription Factor RelA

The mechanisms responsible for impaired regenerative ability after hepatic resection observed in chronic liver disease are not fully understood. We have examined the relationships between an altered expression of cell cycle-related proteins in regenerating liver after partial hepatectomy and the impaired regenerative process observed in fibrotic and cirrhotic rats.. We performed 70% partial hepatectomy in both control and porcine serum-induced fibrotic rats, and 45% partial hepatectomy in thioacetamide-induced cirrhotic rats because of the high mortality associated with 70% partial hepatectomy. Liver regeneration was monitored by proliferating cell nuclear antigen labeling index and the expression of G1 regulatory cell cycle-related proteins was determined by immunoblot analysis.. Compared with controls, hepatocyte DNA synthesis, and induction of cyclin D1 and p21(CIP1) proteins were delayed but not suppressed in porcine serum-induced fibrotic rats and markedly inhibited in thioacetamide-induced cirrhotic rats. p27(KIP1) protein levels were unaffected by partial hepatectomy and did not differ among all three groups.. Two distinct rat models of liver fibrosis and cirrhosis showed markedly different proliferative responses after partial hepatectomy. The delay or failure of cyclin D1 induction, but not the increase of p21(CIP1) or p27(KIP1) might be responsible for their impaired liver regeneration.

Topics: Animals; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; DNA; Hepatectomy; Liver; Liver Cirrhosis; Liver Regeneration; Male; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; Serum; Thioacetamide

Nicotinamide has been shown to inhibit proliferation and induce apoptosis in a variety of cells. Moreover, nicotinamide treatment attenuates collagen accumulation and fibrogenesis in the bleomycin model of lung fibrosis. We hypothesized that nicotinamide may be useful as an antifibrotic agent in liver fibrosis and we investigated the in vitro effect of nicotinamide on hepatic stellate cells proliferation, apoptosis and collagen I expression.. Transforming growth factor beta1 (TGF-beta1) was used for activation of the rat HSC-T6 cell line. Apoptosis was determined by fluorescence activated cell sorter (FACS) analysis after propidium iodide staining and by immunohistochemistry showing presence of the active form of caspase 3. Expression of activation marker alpha-smooth muscle actin (alpha-SMA), apoptotic and cell cycle markers cyclin D1, P53 and caspase 3 was determined by Western blotting. Collagen I expression was assessed by Northern blotting.. Nicotinamide inhibits hepatic stellate cell proliferation and induces apoptosis with caspase-3 activation. There is no effect of nicotinamide on the levels of cell cycle stimulator cyclin D1. Expression of p53 is induced in the presence of nicotinamide. Nicotinamide reduces activation marker alpha-SMA and decreases both basal and TGFbetaepsilon-induced collagen I expression. Moreover, in TGFbeta-activated cells, nicotinamide reduces expression of pro-inflammatory and pro-fibrotic cytokines TGFbeta2, IL-1beta, TNFalpha and macrophage chemotactic protein-1.. The in vitro effect of nicotinamide on activation and proliferation of hepatic stellate cells suggests that nicotinamide may have a potential beneficial role in attenuation of liver fibrogenesis.

Topics: Actins; Animals; Apoptosis; Blotting, Northern; Blotting, Western; Caspase 3; Caspases; Cell Cycle; Cell Line; Cell Proliferation; Collagen Type I; Cyclin D1; Cytokines; Enzyme Activation; Flow Cytometry; Glyceraldehyde-3-Phosphate Dehydrogenases; Hepatocytes; Immunohistochemistry; Inflammation Mediators; Liver Cirrhosis; Niacinamide; Rats; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinases; Tumor Suppressor Protein p53; Vitamin B Complex

It has been shown that a variety of cell cycle-related proteins play important roles in the process of carcinogenesis including hepatocarcinogenesis. In the present study, we evaluated mRNA and protein expression of G1 phase-related cell cycle molecules in the process of hepatocarcinogenesis, using Long-Evans Cinnamon (LEC) rats, an animal model of hepatocellular carcinoma (HCC). The expression of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6 was measured quantitatively by real-time polymerase chain reaction. Cyclin D1 mRNA expression was increased significantly in chronic hepatitis liver compared with normal liver, and then decreased in HCC and the surrounding precancerous liver of LEC rats. Levels of Cdk4 mRNA were increased significantly in HCC compared to precancerous and chronic hepatitis livers. In contrast, mRNA levels of Cdk6 did not change significantly during hepatocarcinogenesis. We also evaluated the protein levels of these G1 phase-related cell cycle molecules by Western blot analyses and confirmed similar results. Total amounts of retinoblastoma protein (pRb) in the liver did not change significantly in the process of hepatocarcinogenesis in LEC rats. However, levels of phosphorylated pRb were increased markedly in the process of hepatocarcinogenesis, and the highest in HCC compared to precancerous, chronic hepatitis and normal livers. These results indicate that cyclin D1 may be involved in the regeneration of hepatocytes rather than hepatocarcinogenesis, while Cdk4 but not Cdk6 may play an important role in the development of HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; G1 Phase; Hepatitis, Animal; Hepatitis, Chronic; Liver Cirrhosis; Liver Neoplasms, Experimental; Phosphorylation; Proto-Oncogene Proteins; Rats; Rats, Inbred LEC; Retinoblastoma Protein; RNA, Messenger

Liver cirrhosis is the end stage of various chronic liver diseases and its prognosis is very poor. One of the most important causes of liver cirrhosis appears to be impaired proliferative capability of hepatocytes caused by continuous hepatic damage. Cell cycle-related molecules have been shown to play essential roles in cell proliferation. Specifically, G1-related cell cycle molecules are important, because they are requisite for the entry into the cell cycle from the quiescent state. However, the role of these cell cycle molecules during the development of liver cirrhosis remains to be examined. In the present study, liver cirrhosis was produced in rats by intraperitoneally administering dimethylnitrosamine (DMN). Proliferative capability of hepatocytes estimated immunohistochemically by proliferating cell nuclear antigen staining was markedly increased at an early stage of cirrhosis development. However, it was gradually decreased thereafter and suppressed substantially at the time of cirrhosis manifestation. Cyclin D1 expression estimated by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method was also increased markedly at an early stage of cirrhosis development but decreased substantially thereafter. mRNA levels of catalytic subunits of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6, did not show significant changes during the development of liver cirrhosis. Among G1-specific Cdk inhibitors, expression of p15INK4b and p16INK4a estimated by an RT-PCR method was increased according to the progression of cirrhosis and reached a peak at the time of cirrhosis manifestation. Conversely, p18INK4c expression did not change significantly during the development of liver cirrhosis. These results suggest that cyclin D1 plays an essential role in hepatocyte proliferation in response to hepatic damage. However, with the decrease of cyclin D1 expression and increase of p15INK4b and p16INK4a expression, proliferative capability of hepatocytes is severely impaired and extracellular matrix components are deposited to retrieve space lost by the destruction of hepatic parenchyma, resulting in establishment of liver cirrhosis.

Topics: Animals; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinases; Dimethylnitrosamine; Extracellular Matrix; G1 Phase; Hepatocytes; Liver; Liver Cirrhosis; Male; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Tumor Suppressor Proteins

Increasing evidence has indicated that perturbation of cyclins is one of the major factors leading to cancer. The aim of this study was not only to investigate various cell cycle-related kinase activities in hepatocellular carcinoma (HCC), but also to analyze the difference of cell cycle-related kinase activity levels between hepatitis C virus (HCV)-induced HCC and HCV-induced cirrhosis. The protein levels of cyclins D1, E, A, and H, and of cyclin dependent kinase 1 (Cdk1), Cdk2, Cdk4, Cdk6, and Cdk7 in HCC and in surrounding nontumorous cirrhosis were determined by Western blot. The enzymatic activities of cyclins D1, E, A, Cdk1, Cdk4, Cdk6, Cdk7, and Wee1 were measured using in vitro kinase assays. Protein levels and kinase activities of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 were significantly elevated in HCC compared with surrounding cirrhotic tissues. The enhanced cyclin D1-related kinase activity in HCC was accompanied by the up-regulation of Cdk4 activity, but not Cdk6 activity. The kinase activities of Cdk6, Cdk7, and Cdk1 did not differ between HCC and surrounding cirrhotic tissues. In addition, the protein levels and kinase activities of cyclin D1, Cdk4, and cyclin E were higher in poorly differentiated HCC and advanced HCC. In conclusion, the increases of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 play an important role in the development of HCC from cirrhosis. Cyclin D1, Cdk4, and cyclin E activation may be closely related to the histopathologic grade and progression of HCC.

Topics: Aged; Blotting, Western; Carcinoma, Hepatocellular; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin E; Cyclin H; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Cyclins; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein