Assessment of the effect of betaine on p16 and c-myc DNA methylation and mRNA expression in a chemical induced rat liver cancer model

<p>Abstract</p> <p>Background</p> <p>The development and progression of liver cancer may involve abnormal changes in DNA methylation, which lead to the activation of certain proto-oncogenes, such as <it>c-myc</it>, as well as the inactivation of certain tumor suppressors, such as <it>p16</it>. Betaine, as an active methyl-donor, maintains normal DNA methylation patterns. However, there are few investigations on the protective effect of betaine in hepatocarcinogenesis.</p> <p>Methods</p> <p>Four groups of rats were given diethylinitrosamine (DEN) and fed with AIN-93G diets supplemented with 0, 10, 20 or 40 g betaine/kg (model, 1%, 2%, and 4% betaine, respectively), while the control group, received no DEN, fed with AIN-93G diet. Eight or 15 weeks later, the expression of <it>p16 </it>and <it>c-myc </it>mRNA was examined by Real-time PCR (Q-PCR). The DNA methylation status within the <it>p16 </it>and <it>c-myc </it>promoter was analyzed using methylation-specific PCR.</p> <p>Results</p> <p>Compared with the model group, numbers and areas of glutathione S-transferase placental form (GST-p)-positive foci were decreased in the livers of the rats treated with betaine (<it>P < 0.05</it>). Although the frequency of <it>p16 </it>promoter methylation in livers of the four DEN-fed groups appeared to increase, there is no difference among these groups after 8 or 15 weeks (<it>P > 0.05</it>). Betaine supplementation attenuated the down-regulation of <it>p16 </it>and inhibited the up-regulation of <it>c-myc </it>induced by DEN in a dose-dependent manner (<it>P </it>< 0.01). Meanwhile, increases in levels of malondialdehyde (MDA) and glutathione S-transferase (GST) in model, 2% and 4% betaine groups were observed (<it>P < 0.05</it>). Finally, enhanced antioxidative capacity (T-AOC) was observed in both the 2% and 4% betaine groups.</p> <p>Conclusion</p> <p>Our data suggest that betaine attenuates DEN-induced damage in rat liver and reverses DEN-induced changes in mRNA levels.</p

A Meta-Analysis of Array-CGH Studies Implicates Antiviral Immunity Pathways in the Development of Hepatocellular Carcinoma

BACKGROUND: The development and progression of hepatocellular carcinoma (HCC) is significantly correlated to the accumulation of genomic alterations. Array-based comparative genomic hybridization (array CGH) has been applied to a wide range of tumors including HCCs for the genome-wide high resolution screening of DNA copy number changes. However, the relevant chromosomal variations that play a central role in the development of HCC still are not fully elucidated. METHODS: In present study, in order to further characterize the copy number alterations (CNAs) important to HCC development, we conducted a meta-analysis of four published independent array-CGH datasets including total 159 samples. RESULTS: Eighty five significant gains (frequency ≥ 25%) were mostly mapped to five broad chromosomal regions including 1q, 6p, 8q, 17q and 20p, as well as two narrow regions 5p15.33 and 9q34.2-34.3. Eighty eight significant losses (frequency ≥ 25%) were most frequently present in 4q, 6q, 8p, 9p, 13q, 14q, 16q, and 17p. Significant correlations existed between chromosomal aberrations either located on the same chromosome or the different chromosomes. HCCs with different etiologies largely exhibited surprisingly similar profiles of chromosomal aberrations with only a few exceptions. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the genes affected by these chromosomal aberrations were significantly enriched in 31 canonical pathways with the highest enrichment observed for antiviral immunity pathways. CONCLUSIONS: Taken together, our findings provide novel and important clues for the implications of antiviral immunity-related gene pathways in the pathogenesis and progression of HCC

Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (gt;99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMOdependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness

Loss of runt-related transcription factor 3 expression leads hepatocellular carcinoma cells to escape apoptosis

Background: Runt-related transcription factor 3 (RUNX3) is known as a tumor suppressor gene for gastric cancer and other cancers, this gene may be involved in the development of hepatocellular carcinoma (HCC). Methods: RUNX3 expression was analyzed by immunoblot and immunohistochemistry in HCC cells and tissues, respectively. Hep3B cells, lacking endogenous RUNX3, were introduced with RUNX3 constructs. Cell proliferation was measured using the MTT assay and apoptosis was evaluated using DAPI staining. Apoptosis signaling was assessed by immunoblot analysis. Results: RUNX3 protein expression was frequently inactivated in the HCC cell lines (91%) and tissues (90%). RUNX3 expression inhibited 90 +/- 8% of cell growth at 72 h in serum starved Hep3B cells. Forty-eight hour serum starvation-induced apoptosis and the percentage of apoptotic cells reached 31 +/- 4% and 4 +/- 1% in RUNX3-expressing Hep3B and control cells, respectively. Apoptotic activity was increased by Bim expression and caspase-3 and caspase-9 activation. Conclusion: RUNX3 expression enhanced serum starvation-induced apoptosis in HCC cell lines. RUNX3 is deleted or weakly expressed in HCC, which leads to tumorigenesis by escaping apoptosis

FGFR1 and NTRK3 actionable alterations in “Wild-Type” gastrointestinal stromal tumors

BACKGROUND: About 10–15% of adult, and most pediatric, gastrointestinal stromal tumors (GIST) lack mutations in KIT, PDGFRA, SDHx, or RAS pathway components (KRAS, BRAF, NF1). The identification of additional mutated genes in this rare subset of tumors can have important clinical benefit to identify altered biological pathways and select targeted therapies. METHODS: We performed comprehensive genomic profiling (CGP) for coding regions in more than 300 cancer-related genes of 186 GISTs to assess for their somatic alterations. RESULTS: We identified 24 GIST lacking alterations in the canonical KIT/PDGFRA/RAS pathways, including 12 without SDHx alterations. These 24 patients were mostly adults (96%). The tumors had a 46% rate of nodal metastases. These 24 GIST were more commonly mutated at 7 genes: ARID1B, ATR, FGFR1, LTK, SUFU, PARK2 and ZNF217. Two tumors harbored FGFR1 gene fusions (FGFR1–HOOK3, FGFR1–TACC1) and one harbored an ETV6–NTRK3 fusion that responded to TRK inhibition. In an independent sample set, we identified 5 GIST cases lacking alterations in the KIT/PDGFRA/SDHx/RAS pathways, including two additional cases with FGFR1–TACC1 and ETV6–NTRK3 fusions. CONCLUSIONS: Using patient demographics, tumor characteristics, and CGP, we show that GIST lacking alterations in canonical genes occur in younger patients, frequently metastasize to lymph nodes, and most contain deleterious genomic alterations, including gene fusions involving FGFR1 and NTRK3. If confirmed in larger series, routine testing for these translocations may be indicated for this subset of GIST. Moreover, these findings can be used to guide personalized treatments for patients with GIST. Trial registration NCT 02576431. Registered October 12, 2015 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12967-016-1075-6) contains supplementary material, which is available to authorized users

Primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic and slowly progressive cholestatic liver disease of autoimmune etiology characterized by injury of the intrahepatic bile ducts that may eventually lead to liver failure. Affected individuals are usually in their fifth to seventh decades of life at time of diagnosis, and 90% are women. Annual incidence is estimated between 0.7 and 49 cases per million-population and prevalence between 6.7 and 940 cases per million-population (depending on age and sex). The majority of patients are asymptomatic at diagnosis, however, some patients present with symptoms of fatigue and/or pruritus. Patients may even present with ascites, hepatic encephalopathy and/or esophageal variceal hemorrhage. PBC is associated with other autoimmune diseases such as Sjogren's syndrome, scleroderma, Raynaud's phenomenon and CREST syndrome and is regarded as an organ specific autoimmune disease. Genetic susceptibility as a predisposing factor for PBC has been suggested. Environmental factors may have potential causative role (infection, chemicals, smoking). Diagnosis is based on a combination of clinical features, abnormal liver biochemical pattern in a cholestatic picture persisting for more than six months and presence of detectable antimitochondrial antibodies (AMA) in serum. All AMA negative patients with cholestatic liver disease should be carefully evaluated with cholangiography and liver biopsy. Ursodeoxycholic acid (UDCA) is the only currently known medication that can slow the disease progression. Patients, particularly those who start UDCA treatment at early-stage disease and who respond in terms of improvement of the liver biochemistry, have a good prognosis. Liver transplantation is usually an option for patients with liver failure and the outcome is 70% survival at 7 years. Recently, animal models have been discovered that may provide a new insight into the pathogenesis of this disease and facilitate appreciation for novel treatment in PBC