We have developed a simple NMR-based method to determine the turnover of nucleotides and incorporation into RNA by stable isotope resolved metabolomics (SIRM) in A549 lung cancer cells. This method requires no chemical degradation of the nucleotides or chromatography. During cell growth, the free ribonucleotide pool is rapidly replaced by de novo synthesized nucleotides. Using [U-13C]-glucose and [U-13C,15N]-glutamine as tracers, we showed that virtually all of the carbons in the nucleotide riboses were derived from glucose, whereas glutamine was preferentially utilized over glucose for pyrimidine ring biosynthesis, via the synthesis of Asp through the Krebs cycle. Incorporation of the glutamine amido nitrogen into the N3 and N9 positions of the purine rings was also demonstrated by proton-detected 15N NMR. The incorporation of 13C from glucose into total RNA was measured and shown to be a major sink for the nucleotides during cell proliferation. This method was applied to determine the metabolic action of an anti-cancer selenium agent (methylseleninic acid or MSA) on A549 cells. We found that MSA inhibited nucleotide turnover and incorporation into RNA, implicating an important role of nucleotide metabolism in the toxic action of MSA on cancer cells.
The authors conclude that Zeb1 heterozygous and null mice show features of PPCD and thus should provide an animal model for genetic dissection of pathways contributing to the disease.
BackgroundPrevious studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking.MethodsPublicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices.ResultsAnalysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture.ConclusionsOur data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.
Metabolomics provides a readout of the state of metabolism in cells or tissue and their responses to external perturbations. For this reason, the approach has great potential in clinical diagnostics. For more than two decades,, we have been using stable isotope tracer approaches to probe cellular metabolism in greater detail. The ability to enrich common compounds with rare isotopes such as carbon ( 13 C) and nitrogen ( 15 N) is the only practical means by which metabolic pathways can be traced, which entails following the fate of individual atoms from the source molecule to products via metabolic transformation. Changes in regulation of pathways are therefore captured by this approach, which leads to deeper understanding of the fundamental biochemistry of cells. Using lessons learned from pathways tracing in cells and organs, we have been applying this methodology to human cancer patients in a clinical setting. Here we review the methodologies and approaches to stable isotope tracing in cells, animal models and in humans subjects.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.