s-adenosylhomocysteine and Carcinoma--Hepatocellular

Downregulation of liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and upregulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Being inhibited by its reaction product, MATII isoform upregulation cannot compensate for MATI/III decrease. Therefore, MAT1A:MAT2A switch contributes to decrease in SAM level in rodent and human hepatocarcinogenesis. SAM administration to carcinogen-treated rats prevents hepatocarcinogenesis, whereas MAT1A-KO mice, characterized by chronic SAM deficiency, exhibit macrovesicular steatosis, mononuclear cell infiltration in periportal areas, and HCC development. This review focuses upon the pleiotropic changes, induced by MAT1A/MAT2A switch, associated with HCC development. Epigenetic control of MATs expression occurs at transcriptional and post-transcriptional levels. In HCC cells, MAT1A/MAT2A switch is associated with global DNA hypomethylation, decrease in DNA repair, genomic instability, and signaling deregulation including c-MYC overexpression, rise in polyamine synthesis, upregulation of RAS/ERK, IKK/NF-kB, PI3K/AKT, and LKB1/AMPK axis. Furthermore, decrease in MAT1A expression and SAM levels results in increased HCC cell proliferation, cell survival, and microvascularization. All of these changes are reversed by SAM treatment in vivo or forced MAT1A overexpression or MAT2A inhibition in cultured HCC cells. In human HCC, MAT1A:MAT2A and MATI/III:MATII ratios correlate negatively with cell proliferation and genomic instability, and positively with apoptosis and global DNA methylation. This suggests that SAM decrease and MATs deregulation represent potential therapeutic targets for HCC. Finally, MATI/III:MATII ratio strongly predicts patients' survival length suggesting that MAT1A:MAT2A expression ratio is a putative prognostic marker for human HCC.

Topics: Animals; Carcinoma, Hepatocellular; Disease Progression; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Methionine Adenosyltransferase; Mice; Prognosis; Prohibitins; Rats; S-Adenosylhomocysteine; S-Adenosylmethionine; Signal Transduction

Topics: Aged; Animals; Base Sequence; Carcinoma, Hepatocellular; Cell Nucleus; Cytoplasm; Humans; Kinetics; Liver; Liver Neoplasms; Models, Biological; Neoplasms, Experimental; Ovalbumin; Oxidation-Reduction; Polyribosomes; Protein Biosynthesis; Rats; RNA, Messenger; S-Adenosylhomocysteine; S-Adenosylmethionine

The identification of viability-associated long noncoding RNAs (lncRNAs) is a means of uncovering therapeutic approaches for hepatocellular carcinoma (HCC). In addition, aberrant genome-wide hypomethylation has been implicated in HCC initiation and progression. However, the relationship between lncRNA dysregulation and genome-wide hypomethylation in hepatocarcinogenesis has not been fully elucidated. A novel lncRNA named LINC00662 was previously demonstrated to play a role in gastrointestinal cancer. In this study, we demonstrated that this lncRNA was correlated with survival and exhibited oncogenic properties, both in vitro and in vivo. Moreover, we determined that LINC00662 could lead to genome-wide hypomethylation and alter the genomic methylation profile by synchronously reducing the S-adenosylmethionine (SAM) level and enhancing the S-adenosylhomocysteine (SAH) level. Mechanistically, LINC00662 was determined to regulate the key enzymes influencing SAM and SAH levels, namely, methionine adenosyltransferase 1A (MAT1A) and S-adenosylhomocysteine hydrolase (AHCY), by RNA-RNA and RNA-protein interactions. In addition, we demonstrated that some SAM-dependent HCC-promoting genes could be regulated by LINC00662 by altering the methylation status of their promoters via the LINC00662-coupled axes of MAT1A/SAM and AHCY/SAH. Taken together, the results of this this study indicate that LINC00662 could be a potential biomarker for HCC therapy. More importantly, we proposed a new role of lncRNA in regulating genomic methylation to promote oncogene activation.

Topics: 3' Untranslated Regions; 5-Methylcytosine; Adenosylhomocysteinase; Adult; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Disease Progression; DNA Methylation; Down-Regulation; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Liver Neoplasms; Methionine Adenosyltransferase; Proteolysis; RNA, Long Noncoding; RNA, Messenger; S-Adenosylhomocysteine; S-Adenosylmethionine; Survival Analysis; Ubiquitin; Up-Regulation

The substantial rise in the prevalence of nonalcoholic steatohepatitis (NASH), an advanced form of nonalcoholic fatty liver disease, and the strong association between NASH and the development of hepatocellular carcinoma indicate the urgent need for a better understanding of the underlying mechanisms. In the present study, by using the Stelic animal model of NASH and NASH-derived liver carcinogenesis, we investigated the role of the folate-dependent 1-carbon metabolism in the pathogenesis of NASH. We demonstrated that advanced NASH and NASH-related liver carcinogenesis are characterized by a significant dysregulation of 1-carbon homeostasis, with diminished expression of key 1-carbon metabolism genes, especially a marked inhibition of the S-adenosylhomocysteine hydrolase ( Ahcy) gene and an increased level of S-adenosyl-l-homocysteine (SAH). The reduction in Ahcy expression was associated with gene-specific cytosine DNA hypermethylation and enrichment of the gene promoter by trimethylated histone H3 lysine 27 and deacetylated histone H4 lysine 16, 2 main transcription-inhibiting markers. These results indicate that epigenetically mediated inhibition of Ahcy expression may be a driving force in causing SAH elevation and subsequent downstream disturbances in transsulfuration and transmethylation pathways during the development and progression of NASH.-Pogribny, I. P., Dreval, K., Kindrat, I., Melnyk, S., Jimenez, L., de Conti, A., Tryndyak, V., Pogribna, M., Ortega, J. F., James, S. J., Rusyn, I., Beland, F. A. Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.

Topics: Adenosylhomocysteinase; Animals; Carcinoma, Hepatocellular; Epigenesis, Genetic; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; Mice; Neoplasm Proteins; Non-alcoholic Fatty Liver Disease; S-Adenosylhomocysteine

Cellular senescence is a key physiological barrier against tumor and represents an option for therapeutic intervention. One pivotal intracellular stimulus causing senescence is DNA damage response, while the senescence-associated heterochromatin in cancer limits the strength of the DNA damage response to endogenous genotoxic stress or DNA-damaging agents. Therefore, targeting the maintenance of compacted chromatin in cancer cells represents an optional intervention to improve the therapeutic efficacy in cancer treatment. Given a crosstalk between methionine cycle and histone methylation, we hypothesize that pharmacologically disrupting methylation potential, defined as the ratio of cellular S-adenosylmethionine to S-adenosylhomocysteine, could affect the chromatin structures in cancer cells and thus enhance their sensitivity to DNA damage response signaling. Our results showed that 3-deazaneplanocin A, a chemical inhibitor of S-adenosylhomocysteine hydrolase, elicited a typical cellular senescence in hepatoma cells. Therapy-induced senescence by 3-deazaneplanocin A was mediated through p53-p21 pathway and triggered by enhanced ataxia-telangiectasia mutated activation related to chromatin changes. In conclusion, our study demonstrated that metabolic perturbation of chromatin status in oncogene-activated cancers could be an optional intervention to sensitize DNA damage response signaling.

Topics: Adenosine; Ataxia Telangiectasia Mutated Proteins; Carcinoma, Hepatocellular; Cell Proliferation; Cellular Senescence; Chromatin; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Epigenomics; Hep G2 Cells; Histones; Humans; Liver Neoplasms; Metabolic Networks and Pathways; Methylation; S-Adenosylhomocysteine; S-Adenosylmethionine; Tumor Suppressor Protein p53

The etiology of liver disease remains elusive in some adults presenting with severe hepatic dysfunction.. Here we describe a woman of Pakistani descent who had elevated aminotransferases at age 23. She developed muscle weakness in her mid-20s, and was diagnosed with hepatocellular carcinoma at age 29. She died without a diagnosis at age 32 after having a liver transplant. Exome sequencing revealed that she was homozygous for a missense mutation (R49H) in AHCY, the gene encoding S-adenosylhomocysteine (SAH) hydrolase. SAH hydrolase catalyzes the final step in conversion of methionine to homocysteine and inactivating mutations in this enzyme cause a rare autosomal recessive disorder, SAH hydrolase deficiency, that typically presents in infancy. An asymptomatic 7-year old son of the proband is also homozygous for the AHCY-R49H mutation and has elevated serum aminotransferase levels, as well as markedly elevated serum levels of SAH, S-adenosylmethionine (SAM), and methionine, which are hallmarks of SAH hydrolase deficiency.. This report reveals several new aspects of SAH hydrolase deficiency. Affected women with SAH hydrolase deficiency can give birth to healthy children. SAH hydrolase deficiency can remain asymptomatic in childhood, and the disorder can be associated with early onset hepatocellular carcinoma. The measurement of serum amino acids should be considered in patients with liver disease or hepatocellular carcinoma of unknown etiology.

Topics: Adenosylhomocysteinase; Adult; Age of Onset; Amino Acid Metabolism, Inborn Errors; Amino Acid Sequence; Carcinoma, Hepatocellular; Child; Consanguinity; DNA Mutational Analysis; Exome; Female; Gene Expression; Glycine N-Methyltransferase; Heterozygote; Homozygote; Humans; Liver Neoplasms; Male; Molecular Sequence Data; Mutation, Missense; Pedigree; S-Adenosylhomocysteine; Sequence Alignment; Transaminases

Glycine N-methyltransferase (GNMT) is a folate binding protein commonly diminished in human hepatoma yet its role in tumor development remains to be established. GNMT binds to methylfolate but is also inhibited by it; how such interactions affect human carcinogenesis is unclear. We postulated that GNMT plays a role in folate-dependent methyl group homeostasis and helps maintain genome integrity by promoting nucleotide biosynthesis and DNA repair. To test the hypothesis, GNMT was over-expressed in GNMT-null cell lines cultured in conditions of folate abundance or restriction. The partitioning of folate dependent 1-carbon groups was investigated using stable isotopic tracers and GC/MS. DNA damage was assessed as uracil content in cell models, as well as in Gnmt wildtype (Gnmt(+/+)), heterozygote (Gnmt(+/-)) and knockout (Gnmt(-/-)) mice under folate deplete, replete, or supplementation conditions. Our study demonstrated that GMMT 1) supports methylene-folate dependent pyrimidine synthesis; 2) supports formylfolate dependent purine syntheses; 3) minimizes uracil incorporation into DNA when cells and animals were exposed to folate depletion; 4) translocates into nuclei during prolonged folate depletion. In conclusion, loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. To our best knowledge, the role of GNMT in folate dependent 1-carbon transfer in nucleotide biosynthesis has never been investigated. The present study gives new insights into the underlying mechanism by which GNMT can participate in tumor prevention/suppression in humans.

Topics: Adenosine; Animals; Carbon Radioisotopes; Carcinoma, Hepatocellular; Cell Nucleus; Cells, Cultured; Chromatography, High Pressure Liquid; Dietary Supplements; DNA Damage; DNA Methylation; Female; Fluorescent Antibody Technique, Indirect; Folic Acid; Glycine N-Methyltransferase; Hepatocytes; Homocysteine; Humans; Liver; Liver Neoplasms; Male; Methylenetetrahydrofolate Dehydrogenase (NADP); Mice; Mice, Knockout; Minor Histocompatibility Antigens; Protein Transport; Purines; Pyrimidines; S-Adenosylhomocysteine; S-Adenosylmethionine; Tetrahydrofolates; Uracil

Glycine-N methyltransferase (GNMT) is a potential tumor suppressor that is commonly inactivated in human hepatoma. We systematically investigated how GNMT regulates methyl group kinetics and global DNA methylation. HepG2 cells (GNMT inactive, GNMT-) and cells transfected with GNMT expressed vector (GNMT+) were cultured in low (10 μmol/L), adequate (100 μmol/L), or high (500 μmol/L) l-methionine, each with 2.27 μmol/L folate. Transmethylation kinetics were studied using stable isotopic tracers and GC-MS. Methylation status was determined by S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels, SAM:SAH ratio, DNA methyltransferase (DNMT) activity, and methylated cytidine levels in DNA. Compared with GNMT- cells, GNMT+ cells had lower homocysteine and greater cysteine concentrations. GNMT expression increased methionine clearance by inducing homocysteine transsulfuration and remethylation metabolic fluxes when cells were cultured in high or adequate l-methionine. In contrast, homocysteine remethylation flux was lower in GNMT+ cells than in GNMT- cells and homocysteine transsulfuration fluxes did not differ when cells were cultured in low methionine, suggesting that normal GNMT function helps to conserve methyl groups. Furthermore, GNMT expression decreased SAM and increased SAH levels and reduced DNMT activity in high or adequate, but not low, methionine cultures. In low methionine cultures, restoring GNMT in HepG2 cells did not lead to sarcosine synthesis, which would waste methyl groups. Methylated cytidine levels were significantly lower in GNMT- cells than in GNMT+ cells. In conclusion, we have shown that GNMT affects transmethylation kinetics and SAM synthesis and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes.

Topics: Carcinoma, Hepatocellular; Cysteine; Cytidine; DNA Methylation; DNA Modification Methylases; Gene Expression; Glycine N-Methyltransferase; Hep G2 Cells; Hepatocytes; Homeostasis; Homocysteine; Humans; Kinetics; Liver Neoplasms; Methionine; Methionine Adenosyltransferase; Methylation; Osmolar Concentration; S-Adenosylhomocysteine; S-Adenosylmethionine

Hypoxia-inducible factor 1 (HIF-1) emerges as a crucial player in tumor progression. However, its role in hepatocellular carcinoma (HCC), especially its relation with global DNA methylation patterns in HCC under hypoxic tumor microenvironment is not completely understood. Methionine adenosyltransferase 2A (MAT2A) maintains the homeostasis of S-adenosylmethionine (SAM), a critical marker of genomic methylation status. In this study, we investigated the link between HIF-1α and MAT2A as a mechanism responsible for the change in genomic DNA methylation patterns in liver cancer under hypoxia conditions. Our results showed that hypoxia induces genomic DNA demethylation in CpG islands by reducing the steady-state SAM level both in vitro and in vivo. In addition, HIF-1α and MAT2A expression is correlated with tumor size and TNM stage of liver cancer tissues. We further showed that hypoxia-induced MAT2A expression is HIF-1α dependent and requires the recruitment of p300 and HDAC1. We also identified an authentic consensus HIF-1α binding site in MAT2A promoter by site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. Taken together, we show for the first time that hypoxia induces genomic DNA demethylation through the activation of HIF-1α and transcriptional upregulation of MAT2A in hepatoma cells. These findings provide new insights into our understanding of the molecular link between genomic DNA methylation and tumor hypoxia in HCC.

Topics: Binding Sites; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Line, Transformed; Chromatin Immunoprecipitation; CpG Islands; DNA; DNA Methylation; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Histone Deacetylase 1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Methionine Adenosyltransferase; Mutagenesis, Site-Directed; p300-CBP Transcription Factors; Promoter Regions, Genetic; S-Adenosylhomocysteine; S-Adenosylmethionine; Tumor Microenvironment; Up-Regulation

Betaine-homocysteine methyltransferase (BHMT) regulates homocysteine levels in the liver. We previously reported that the alteration of BHMT is associated with alcoholic liver steatosis and injury. In this study, we tested whether BHMT protects hepatocytes from homocysteine-induced injury and lipid accumulation. Both BHMT transfectants of HepG2 cells and primary mouse hepatocytes with suppressed BHMT were generated. Comparisons were made between the cell models with respect to their response to homocysteine treatments. Homocysteine metabolism was impaired in HepG2 cells, and the expression of BHMT in HepG2 cells ameliorated the impairment and stabilized the levels of intracellular homocysteine after the addition of exogenous homocysteine. BHMT expression inhibited homocysteine-induced glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP) and homocysteine-induced cell death. A betaine treatment protected primary mouse hepatocytes from a homocysteine-induced increase in GRP78 and cell death but not a tunicamycin-induced increase. Homocysteine induced greater CHOP expression (2.7-fold) in BHMT small interfering RNA (siRNA)-transfected cells than in a control (1.9-fold). Homocysteine-induced cell death was increased by 40% in the siRNA-treated cells in comparison with the control. Apolipoprotein B (apoB) expression was higher and triglycerides and cholesterol were lower in HepG2 expressing BHMT. In primary mouse hepatocytes, homocysteine induced the accumulation of triglycerides and cholesterol, which was reduced in the presence of betaine. Betaine partially reduced homocysteine-induced sterol regulatory element binding protein 1 expression in HepG2 cells and increased S-adenosylmethionine in primary mouse hepatocytes.. The BHMT/betaine system directly protects hepatocytes from homocysteine-induced injury but not tunicamycin-induced injury, including an endoplasmic reticulum stress response, lipid accumulation, and cell death. This system also exhibits a more generalized effect on liver lipids by inducing ApoB expression and increasing S-adenosylmethionine/S-adenosylhomocysteine.

Topics: Animals; Apolipoproteins B; Betaine; Betaine-Homocysteine S-Methyltransferase; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Fatty Liver; Gene Transfer Techniques; Hepatocytes; Homocysteine; Humans; Lipid Metabolism; Liver Neoplasms; Mice; S-Adenosylhomocysteine; S-Adenosylmethionine; Sterol Regulatory Element Binding Protein 1

In alcoholic liver disease, tumor necrosis factor-alpha (TNFalpha) is a critical effector molecule, and abnormal methionine metabolism is a fundamental acquired metabolic abnormality. Although hepatocytes are resistant to TNFalpha-induced killing under normal circumstances, previous studies have shown that primary hepatocytes from rats chronically fed alcohol have increased TNFalpha cytotoxicity. Therefore, there must be mechanisms by which chronic alcohol exposure "sensitizes" to TNFalpha hepatotoxicity. S-adenosylhomocysteine (SAH) is product of methionine in transsulfuration pathway and a potent competitive inhibitor of most methyltransferases. In this study, we investigated the effects of increased SAH levels on TNFalpha hepatotoxicity. Our results demonstrated that chronic alcohol consumption in mice not only decreased hepatic S-adenosylmethionine levels but also increased hepatic SAH levels, which resulted in a significantly decreased S-adenosylmethionine-to-SAH ratio. This was associated with significant increases in hepatic TNFalpha levels, caspase-8 activity, and cell death. In vitro studies demonstrated that SAH-enhancing agents sensitized hepatocytes to TNFalpha killing, and the death was associated with increased caspase-8 activity, which was blocked by a caspase-8 inhibitor. In addition, increased intracellular SAH levels had no effect on nuclear factor kappaB activity induced by TNFalpha. In conclusion, these results provide a new link between abnormal methionine metabolism and abnormal TNFalpha metabolism in alcoholic liver disease. Increased SAH is a potent and clinically relevant sensitizer to TNFalpha hepatotoxicity. These data further support improving the S-adenosylmethionine-to-SAH ratio and removal of intracellular SAH as potential therapeutic options in alcoholic liver disease.

Topics: Adenosine; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Caspase 8; Caspases; Cell Line, Tumor; Hepatocytes; Homocysteine; Humans; Liver Diseases, Alcoholic; Methylation; Mice; Mice, Inbred C57BL; NF-kappa B; S-Adenosylhomocysteine; Tumor Necrosis Factor-alpha

S-Adenosylmethionine-homocysteine methyltransferase, which catalyzes synthesis of methionine from homocysteine, with the use of S-adenosylmethionine as the methyl donor, is absent in tumor tissue such as rat ascites hepatoma and Morris hepatoma but is present in rat liver homogenate. Absence of the enzymatic activity in tumor cells is not due to the action of an inhibitor. S-Adenosylhomocysteine hydrolase, however, is present in both rat liver and hepatoma tissue.

Topics: Animals; Carcinoma, Hepatocellular; Female; Homocysteine; Hydrolases; Liver; Liver Neoplasms; Methyltransferases; Neoplasms, Experimental; Rats; S-Adenosylhomocysteine; S-Adenosylmethionine

The analogue S-tubercidinylhomocysteine (STH) has been used to study the methylation of mRNA in vivo. Partial inhibition of cytoplasmic poly(A)-RNA methylation was observed using a level of inhibitor which still permitted cell growth. Characterization of the partially methylated mRNA indicated the presence of cap structures lacking 2'-O-methylnucleosides, m7GpppN', which are normally not found in mammalian mRNA. Inhibition of additional methylated sites in mRNA at the second 2'-O-methynucleoside, and at internal N6-methyladenosine was also observed Methylation of 7-methylguanosine was not affected under the conditions used in these experiments. The methylnucleoside composition of cap structures differed in STH-inhibited and uninhibited cells. These results indicate that a completely methylated cap is not required for transport of mRNA into the cytoplasm. Furthermore, it may now be possible to assess in vivo the sequential nature of mRNA methylation and its potential role in mRNA processing.

Topics: Carcinoma, Hepatocellular; Cell Line; Homocysteine; Liver Neoplasms; Neoplasms, Experimental; Oligoribonucleotides; Poly A; RNA, Messenger; S-Adenosylhomocysteine