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Exometabolom analysis of breast cancer cell lines: Metabolic signature.

Willmann L, Erbes T, Halbach S, Brummer T, Jäger M, Hirschfeld M, Fehm T, Neubauer H, Stickeler E, Kammerer B - Sci Rep (2015)

Bottom Line: Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer.Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism.Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level.

View Article: PubMed Central - PubMed

Affiliation: Center for Biological Systems Analysis ZBSA, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany.

ABSTRACT
Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

No MeSH data available.


Related in: MedlinePlus

Polyamine/Methionine cycle (red: analyzed compound; involved enzymes: 1) S-adenosylmethionine synthetase [EC:2.5.1.6]; 2) S-adenosylmethionine decarboxylase [EC:4.1.1.50]; 3) spermidine synthase [EC:2.5.1.16]/spermine synthase [2.5.1.22]; 4) 5’-methylthioadenosine phosphorylase [EC:2.4.2.28]; 5) methylthioribose-1-phosphate isomerase [EC:5.3.1.23]; 6) betaine-homocysteine S-methyltransferase [EC:2.1.1.5]/5-methyltetrahydrofolate-homocysteine methyltransferase [2.1.1.13]; 7) adenosylhomocysteinase [EC:3.3.1.1]; 8) DNA (cytosine-5)-methyltransferase 1 [EC:2.1.1.37]; 9) tRNA-uridine aminocarboxy-propyltransferase [EC:2.5.1.25]; 10) adenine phosphoribosyltransferase [EC:2.4.2.7]; 11) adenosine kinase [2.7.1.20]).
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f6: Polyamine/Methionine cycle (red: analyzed compound; involved enzymes: 1) S-adenosylmethionine synthetase [EC:2.5.1.6]; 2) S-adenosylmethionine decarboxylase [EC:4.1.1.50]; 3) spermidine synthase [EC:2.5.1.16]/spermine synthase [2.5.1.22]; 4) 5’-methylthioadenosine phosphorylase [EC:2.4.2.28]; 5) methylthioribose-1-phosphate isomerase [EC:5.3.1.23]; 6) betaine-homocysteine S-methyltransferase [EC:2.1.1.5]/5-methyltetrahydrofolate-homocysteine methyltransferase [2.1.1.13]; 7) adenosylhomocysteinase [EC:3.3.1.1]; 8) DNA (cytosine-5)-methyltransferase 1 [EC:2.1.1.37]; 9) tRNA-uridine aminocarboxy-propyltransferase [EC:2.5.1.25]; 10) adenine phosphoribosyltransferase [EC:2.4.2.7]; 11) adenosine kinase [2.7.1.20]).

Mentions: In our LC-MS analysis of the different breast cancer cell lines and the breast epithelial cell line MCF-10A we detected the following substances derived from the polyamine/methionine cycle: SAM, SAH, MTA, MTA-SO and 3-(3-Amino-carboxypropyl)-uridine (acp3U) (Fig. 6). The elevated methylation of nucleosides in cancer cells may be interrelated with the most popular methyldonor SAM. SAM-synthetase catalyzes the synthesis of SAM from L-Methionine. SAM has found to be elevated in supernatants of MDA-MB-453, in all other cell lines SAM was not detectable. The low excretion patterns of SAM may be explained by the high intracellular utilization of SAM as a methyl donor, e.g. for intracellular nucleoside methylation. In contrast, SAH was elevated in supernatants of all breast cancer cell lines, except BT-474. As SAH is a byproduct of SAM triggered methylation, the elevated excretion might be related to elevated nucleoside methylation in breast cancer cell lines. MTA and MTA-SO excretion levels were decreased in supernatants of all breast cancer cell lines. In the breast cancer cell line MDA-MB-453 the MTA levels of the cell culture medium was higher than the levels of cell culture supernatants. Consequently, MTA was assimilated by the MDA-MB-453 cells. MTA-SO has been found in urine of immunodeficient children and it has been suggested, that MTA-SO results from in vivo MTA oxidation by oxides and superoxides or enzymatically by enzymes53. The modified nucleoside MTA is part of the methionine metabolism and results from SAM or S-Adenosylmethioninamine. MTA can be converted into S-Methyl-5-thio-D-ribose 1-phosphate catalyzed by 5-Methylthioadenosine phosphorylase (MTAP), thereby Adenine is produced as a byproduct. MTAP is ubiquitously expressed in mammalian tissue and catalyses the first and rate-limiting step of the methionine salvage pathway, resulting in methionine and A. MTAP has been reported to be deficient in breast cancer54. Methionine-dependant growth has been discussed in tumor cell lines55. Cell lines, that are considered to be methionine-dependant, are unable to grow in vitro when methionine is replaced with homocysteine56. A relative survival advantage, that would be characteristic for cancer cells, has been suggested for methionine-dependant cell lines57. As a result of MTAP deficiency adenine, as well as AMP and A, is solely synthesized in the de novo purine biosynthesis pathway. The decreased excretion of A in BT-474 and MDA-MB-231 might be related with this phenomenon. The depletion of the rate-limiting enzyme MTAP is not responsible for the methionine-dependant growth of human tumor cell lines56. It has been observed, that MTA has inhibitory effects on spermidine and spermine synthase and on ornithine decarboxylase. Additionally, MTA influences critical responses of the cell, e.g. regulation of gene expression, proliferation, differentiation and apoptosis58. The methyldonor SAM can also be transformed into MTA catalyzed by tRNA-uridine aminocarboxypropyltransferase. Thereby Uridine is transformed into acp3U. acp3U has been found in phenylalanine tRNA of Escherichia coli59. As there is no other enzyme reported, we suggest tRNA-uridine aminocarboxypropyltransferase to catalyze acp3U synthesis in breast cancer cell lines MDA-MB-231 and -453. Although acp3U was slightly elevated in MDA-MB-231 and -453, the MTA levels were decreased in all breast cancer cell lines. Consequently, MTA is further degradaded by MTAP or acp3U results from other sources. Another explanation for this phenomenon would be a differential exocytosis between MTA and acp3U.


Exometabolom analysis of breast cancer cell lines: Metabolic signature.

Willmann L, Erbes T, Halbach S, Brummer T, Jäger M, Hirschfeld M, Fehm T, Neubauer H, Stickeler E, Kammerer B - Sci Rep (2015)

Polyamine/Methionine cycle (red: analyzed compound; involved enzymes: 1) S-adenosylmethionine synthetase [EC:2.5.1.6]; 2) S-adenosylmethionine decarboxylase [EC:4.1.1.50]; 3) spermidine synthase [EC:2.5.1.16]/spermine synthase [2.5.1.22]; 4) 5’-methylthioadenosine phosphorylase [EC:2.4.2.28]; 5) methylthioribose-1-phosphate isomerase [EC:5.3.1.23]; 6) betaine-homocysteine S-methyltransferase [EC:2.1.1.5]/5-methyltetrahydrofolate-homocysteine methyltransferase [2.1.1.13]; 7) adenosylhomocysteinase [EC:3.3.1.1]; 8) DNA (cytosine-5)-methyltransferase 1 [EC:2.1.1.37]; 9) tRNA-uridine aminocarboxy-propyltransferase [EC:2.5.1.25]; 10) adenine phosphoribosyltransferase [EC:2.4.2.7]; 11) adenosine kinase [2.7.1.20]).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4544000&req=5

f6: Polyamine/Methionine cycle (red: analyzed compound; involved enzymes: 1) S-adenosylmethionine synthetase [EC:2.5.1.6]; 2) S-adenosylmethionine decarboxylase [EC:4.1.1.50]; 3) spermidine synthase [EC:2.5.1.16]/spermine synthase [2.5.1.22]; 4) 5’-methylthioadenosine phosphorylase [EC:2.4.2.28]; 5) methylthioribose-1-phosphate isomerase [EC:5.3.1.23]; 6) betaine-homocysteine S-methyltransferase [EC:2.1.1.5]/5-methyltetrahydrofolate-homocysteine methyltransferase [2.1.1.13]; 7) adenosylhomocysteinase [EC:3.3.1.1]; 8) DNA (cytosine-5)-methyltransferase 1 [EC:2.1.1.37]; 9) tRNA-uridine aminocarboxy-propyltransferase [EC:2.5.1.25]; 10) adenine phosphoribosyltransferase [EC:2.4.2.7]; 11) adenosine kinase [2.7.1.20]).
Mentions: In our LC-MS analysis of the different breast cancer cell lines and the breast epithelial cell line MCF-10A we detected the following substances derived from the polyamine/methionine cycle: SAM, SAH, MTA, MTA-SO and 3-(3-Amino-carboxypropyl)-uridine (acp3U) (Fig. 6). The elevated methylation of nucleosides in cancer cells may be interrelated with the most popular methyldonor SAM. SAM-synthetase catalyzes the synthesis of SAM from L-Methionine. SAM has found to be elevated in supernatants of MDA-MB-453, in all other cell lines SAM was not detectable. The low excretion patterns of SAM may be explained by the high intracellular utilization of SAM as a methyl donor, e.g. for intracellular nucleoside methylation. In contrast, SAH was elevated in supernatants of all breast cancer cell lines, except BT-474. As SAH is a byproduct of SAM triggered methylation, the elevated excretion might be related to elevated nucleoside methylation in breast cancer cell lines. MTA and MTA-SO excretion levels were decreased in supernatants of all breast cancer cell lines. In the breast cancer cell line MDA-MB-453 the MTA levels of the cell culture medium was higher than the levels of cell culture supernatants. Consequently, MTA was assimilated by the MDA-MB-453 cells. MTA-SO has been found in urine of immunodeficient children and it has been suggested, that MTA-SO results from in vivo MTA oxidation by oxides and superoxides or enzymatically by enzymes53. The modified nucleoside MTA is part of the methionine metabolism and results from SAM or S-Adenosylmethioninamine. MTA can be converted into S-Methyl-5-thio-D-ribose 1-phosphate catalyzed by 5-Methylthioadenosine phosphorylase (MTAP), thereby Adenine is produced as a byproduct. MTAP is ubiquitously expressed in mammalian tissue and catalyses the first and rate-limiting step of the methionine salvage pathway, resulting in methionine and A. MTAP has been reported to be deficient in breast cancer54. Methionine-dependant growth has been discussed in tumor cell lines55. Cell lines, that are considered to be methionine-dependant, are unable to grow in vitro when methionine is replaced with homocysteine56. A relative survival advantage, that would be characteristic for cancer cells, has been suggested for methionine-dependant cell lines57. As a result of MTAP deficiency adenine, as well as AMP and A, is solely synthesized in the de novo purine biosynthesis pathway. The decreased excretion of A in BT-474 and MDA-MB-231 might be related with this phenomenon. The depletion of the rate-limiting enzyme MTAP is not responsible for the methionine-dependant growth of human tumor cell lines56. It has been observed, that MTA has inhibitory effects on spermidine and spermine synthase and on ornithine decarboxylase. Additionally, MTA influences critical responses of the cell, e.g. regulation of gene expression, proliferation, differentiation and apoptosis58. The methyldonor SAM can also be transformed into MTA catalyzed by tRNA-uridine aminocarboxypropyltransferase. Thereby Uridine is transformed into acp3U. acp3U has been found in phenylalanine tRNA of Escherichia coli59. As there is no other enzyme reported, we suggest tRNA-uridine aminocarboxypropyltransferase to catalyze acp3U synthesis in breast cancer cell lines MDA-MB-231 and -453. Although acp3U was slightly elevated in MDA-MB-231 and -453, the MTA levels were decreased in all breast cancer cell lines. Consequently, MTA is further degradaded by MTAP or acp3U results from other sources. Another explanation for this phenomenon would be a differential exocytosis between MTA and acp3U.

Bottom Line: Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer.Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism.Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level.

View Article: PubMed Central - PubMed

Affiliation: Center for Biological Systems Analysis ZBSA, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany.

ABSTRACT
Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

No MeSH data available.


Related in: MedlinePlus