s-adenosylhomocysteine and Breast-Neoplasms

BCDIN3 domain containing RNA methyltransferase, BCDIN3D, monomethylates the 5'-monophosphate of cytoplasmic tRNAHis with a G-1:A73 mispair at the top of an eight-nucleotide-long acceptor helix, using S-adenosyl-l-methionine (SAM) as a methyl group donor. In humans, BCDIN3D overexpression is associated with the tumorigenic phenotype and poor prognosis in breast cancer. Here, we present the crystal structure of human BCDIN3D complexed with S-adenosyl-l-homocysteine. BCDIN3D adopts a classical Rossmann-fold methyltransferase structure. A comparison of the structure with that of the closely related methylphosphate capping enzyme, MePCE, which monomethylates the 5'-γ-phosphate of 7SK RNA, revealed the important residues for monomethyl transfer from SAM onto the 5'-monophosphate of tRNAHis and for tRNAHis recognition by BCDIN3D. A structural model of tRNAHis docking onto BCDIN3D suggested the molecular mechanism underlying the different activities between BCDIN3D and MePCE. A loop in BCDIN3D is shorter, as compared to the corresponding region that forms an α-helix to recognize the 5'-end of RNA in MePCE, and the G-1:A73 mispair in tRNAHis allows the N-terminal α-helix of BCDIN3D to wedge the G-1:A73 mispair of tRNAHis. As a result, the 5'-monophosphate of G-1 of tRNAHis is deep in the catalytic pocket for 5'-phosphate methylation. Thus, BCDIN3D is a tRNAHis-specific 5'-monomethylphosphate capping enzyme that discriminates tRNAHis from other tRNA species, and the structural information presented in this study also provides the molecular basis for the development of drugs against breast cancers.

Topics: Antineoplastic Agents; Breast Neoplasms; Crystallography, X-Ray; Cytoplasm; Female; Gene Expression Regulation, Enzymologic; Humans; Methylation; Methyltransferases; Protein Conformation, alpha-Helical; Protein Folding; RNA, Transfer; RNA, Transfer, His; S-Adenosylhomocysteine

The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

Topics: AMP-Activated Protein Kinases; Animals; Biomarkers, Tumor; Breast Neoplasms; Carbon; Colonic Neoplasms; DNA Methylation; Electron Transport Complex I; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Hypoglycemic Agents; Metformin; Mice; Mitochondria; S-Adenosylhomocysteine; S-Adenosylmethionine; Tumor Cells, Cultured

Tamoxifen (TAM) resistance is a main cause of therapeutic failure in breast cancers. Although methionine dependency is a phenotypic characteristic of tumor cells, the role of sulfur amino acid metabolism in chemotherapy resistance remains to be elucidated. This study compared metabolite profiles of sulfur amino acid metabolism from methionine to taurine or glutathione (GSH) between normal MCF-7 and TAM-resistant MCF-7 (TAMR-MCF-7) cells. TAMR-MCF-7 cells showed elevated levels and activities of enzymes involved in both transsulfuration from methionine to cysteine and metabolism of cysteine to GSH and taurine. Cysteine concentrations in TAMR-MCF-7 cells and medium conditioned by cell culture for 42h were markedly decreased, while GSH, hypotaurine, and taurine concentrations in the medium were increased. These results show that TAMR-MCF-7 cells display enhanced cysteine utilization. The addition of propargylglycine, a specific cystathionine γ-lyase inhibitor, and buthionine sulfoximine, a specific γ-glutamylcysteine ligase inhibitor, to TAMR-MCF-7 cells, but not to MCF-7 cells, resulted in cytotoxicity after sulfur amino acid deprivation. These results suggest that cell viability of TAMR-MCF-7 cells is affected by inhibition of sulfur amino acid metabolism, particularly cysteine synthesis from homocysteine and GSH synthesis from cysteine. Additionally, the S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in TAMR-MCF-7 cells increased to ~3.6-fold relative to that in MCF-7 cells, a finding that may result from upregulation of methionine adenosyltransferase IIa and S-adenosylhomocysteine hydrolase. In conclusion, this study suggests that TAMR-MCF-7 cells display enhanced cysteine utilization for synthesis of GSH and taurine, and are sensitive to inhibition of cysteine metabolism.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cystathionine gamma-Lyase; Cysteine; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Glutamate-Cysteine Ligase; Glutathione; Humans; Membrane Transport Modulators; Methionine; Methylation; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; S-Adenosylhomocysteine; S-Adenosylmethionine; Tamoxifen; Taurine; Up-Regulation

We studied the modulating effects of caffeic acid and chlorogenic acid (two common coffee polyphenols) on the in vitro methylation of synthetic DNA substrates and also on the methylation status of the promoter region of a representative gene in two human cancer cells lines. Under conditions that were suitable for the in vitro enzymatic methylation of DNA and dietary catechols, we found that the presence of caffeic acid or chlorogenic acid inhibited in a concentration-dependent manner the DNA methylation catalyzed by prokaryotic M.SssI DNA methyltransferase (DNMT) and human DNMT1. The IC50 values of caffeic acid and chlorogenic acid were 3.0 and 0.75 microM, respectively, for the inhibition of M.SssI DNMT-mediated DNA methylation, and were 2.3 and 0.9 microM, respectively, for the inhibition of human DNMT1-mediated DNA methylation. The maximal in vitro inhibition of DNA methylation was approximately 80% when the highest concentration (20 microM) of caffeic acid or chlorogenic acid was tested. Kinetic analyses showed that DNA methylation catalyzed by M.SssI DNMT or human DNMT1 followed the Michaelis-Menten curve patterns. The presence of caffeic acid or chlorogenic acid inhibited DNA methylation predominantly through a non-competitive mechanism, and this inhibition was largely due to the increased formation of S-adenosyl-L-homocysteine (SAH, a potent inhibitor of DNA methylation), resulting from the catechol-O-methyltransferase (COMT)-mediated O-methylation of these dietary catechols. Using cultured MCF-7 and MAD-MB-231 human breast cancer cells, we also demonstrated that treatment of these cells with caffeic acid or chlorogenic acid partially inhibited the methylation of the promoter region of the RARbeta gene. The findings of our present study provide a general mechanistic basis for the notion that a variety of dietary catechols can function as inhibitors of DNA methylation through increased formation of SAH during the COMT-mediated O-methylation of these dietary chemicals.

Topics: Antioxidants; Breast Neoplasms; Caffeic Acids; Catechol O-Methyltransferase; Cell Line, Tumor; Chlorogenic Acid; Coffee; Diet; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Female; Humans; Promoter Regions, Genetic; S-Adenosylhomocysteine

A novel mechanistic hypothesis is proposed which suggests that hyperhomocysteinemia is a risk factor for the development of estrogen-induced hormonal cancer in humans. Mechanistically, hyperhomocysteinemia may exert its pathogenic effects largely through metabolic accumulation of intracellular S-adenosyl-L-homocysteine, a strong non-competitive inhibitor of the catechol-O-methyltransferase-mediated methylation metabolism of endogenous and exogenous catechol estrogens (mainly 2-hydroxyestradiol and 4-hydroxyestradiol). While a strong inhibition of the methylation metabolism of 2-hydroxyestradiol would decrease the formation of 2-methoxyestradiol (an antitumorigenic endogenous metabolite of 17beta-estradiol), an inhibition of the methylation of 4-hydroxyestradiol would lead to accumulation of this hormonally-active and strongly procarcinogenic catechol estrogen metabolite. Both of these effects resulting from inhibition of the methylation metabolism of catechol estrogens would facilitate the development of estrogen-induced hormonal cancer in the target organs. This hypothesis also predicts that adequate dietary intake of folate, vitamin B6, and vitamin B12 may reduce hyperhomocysteinemia-associated risk for hormonal cancer. Experimental studies are warranted to determine the relations of hyperhomocysteinemia with the altered circulating or tissue levels of 4-hydroxyestradiol and 2-methoxyestradiol and also with the altered risk for estrogen-induced hormonal cancer.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Estradiol; Estrogens; Estrogens, Catechol; Female; Folic Acid; Humans; Hyperhomocysteinemia; Kinetics; Methylation; Models, Biological; Mutagenicity Tests; Neoplasms, Experimental; Neoplasms, Hormone-Dependent; Risk Factors; S-Adenosylhomocysteine; Uterine Neoplasms; Vitamin B 12; Vitamin B 6

In this study we investigated the signalling requirements for TNF-induced cytotoxicity modulated by the methyltransferase inhibitor S-adenosyl-L-homocysteine (AdoHcy) using the TNF-sensitive human breast carcinoma MCF7 cells and its established TNF-resistant clones (R-A1 and clone 1001). Our data indicate that inhibition of methylation reactions by adenosine plus homocysteine, which are known to condense within cells to AdoHcy, markedly potentiated TNF-induced cytotoxicity in MCF7 cells and rendered related TNF-resistant variants, TNF-sensitive by a mechanism independent from the ceramide pathway. We demonstrated that the dominant-negative derivative of FADD (FADD-DN) blocked methylation inhibition/TNF-induced cell death. Moreover, TNF-mediated cytotoxicity modulated by AdoHcy was blocked by the ICE-inhibiting peptide z-VAD-fmk, suggesting that an ICE-like protease is required for the methylation inhibition/TNF-inducible death pathway. In conclusion, these results suggest that the methyltransferase inhibitor AdoHcy potentiates TNF-induced cytotoxicity in MCF7 cells and renders TNF-resistant MCF7 clones, TNF-sensitive via the ceramide independent pathway and that FADD and the ICE-like protease are likely necessary components in transducing methylation inhibition/TNF signals for cell death.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Apoptosis; Arabidopsis Proteins; Breast Neoplasms; Caspase Inhibitors; Ceramides; Cysteine Proteinase Inhibitors; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Drug Resistance; Fatty Acid Desaturases; Female; Humans; Recombinant Fusion Proteins; S-Adenosylhomocysteine; Tumor Necrosis Factor-alpha