br While it may seem paradoxical
While it may seem paradoxical to use a less efficient system to
* Corresponding author. E-mail address: [email protected] (K. Zhang).
generate ATP, glycolytic intermediates feed into many other important pathways. For example, the pentose phosphate pathway is used to generate ribose sugars and NADPH, both of which are important in DNA/RNA synthesis and anabolic processes. Others have argued gly-colysis allows a tumor a certain plasticity in order to rapidly respond to a changing microenvironment . Additionally the glycolytic inter-mediate, 3-phosphoglycerate, can be diverted to generate serine which can be utilized to synthesize nucleic acids that are essential for cell proliferation .
In this study we have investigated the incorporation of carbon from glucose into the amino acids glycine, serine and methionine (Fig. 1). Phosphoglycerate dehydrogenase (PHDGH) is the rate-limiting enzyme in the conversion of 3-phosphoglycerate into serine . Serine can donate a carbon atom to tetrahydrofolate by way of serine hydro-xymethyl transferase (SHMT), which can then be used for de novo purine synthesis or thymidylate synthesis [6,7]. Alternatively, the carbon unit can be transferred from serine to homocysteine to form methionine.
Methionine is not only a structural amino acid, but it is required for
Fig. 1. Glucose metabolism, energy production, and its importance in DNA replication and epigenetic homeostasis.
Glucose is metabolized to pyruvate which can serve as a substrate for oxidative phosphorylation. It can also be converted from there to lactate to maintain flux through glycolysis. This underlies the Warburg effect (red/red arrow). However, glucose metabolism is much more complex and serves many other purposes other than production of precursors for aerobic respiration alone. This includes the pentose phosphate pathway (PPP) and de novo serine synthesis which connects glycolysis to nucleic ML385 synthesis as well as one carbon metabolism (blue/blue arrow). The synthesis of serine and glycine can also branch into other pathways such as production of cystathionine, which will make cysteine, and glycine is a precursor for heme, glutathione, and purines. Glycine can also be degraded using the glycine cleavage system (GCS) to produce carbon units. An additional product of this pathway is alpha-ketoglutarate (α-KG) which can feed into the citric acid cycle. (*) Represent metabolites that show preferential accumulation in tumors by positron emission tomography scan. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
the initiation of translation of most proteins. It is also the precursor for S-adenosylmethionine (SAM), the methyl donor for numerous enzy-matic methylation reactions of an array of metabolites as well as RNA, DNA and histone proteins. The methylation of selected histone proteins, as well as cytosine bases in DNA, establishes the epigenetic landscape of both normal and cancer cells. Epigenetic patterns determine which genes are available for transcription, providing a pathway by which metabolic disturbances could result in epigenetic perturbations. For example, depleting methionine can completely alter the gene expres-sion patterns via epigenetic remodeling of chromatin due to S-adenosyl-methionine depletion . The underlying metabolic defects of cancer cells often require them to be addicted to select nutrients. Understanding these metabolic aberrations has and could lead to new and targeted chemotherapy ap-proaches . Observations of perturbed metabolism have already made possible the development of 18-Fluorodeoxyglucose positron-emission-tomography (18F-dG-PET) scanning . Since the discovery of pre-ferential glucose uptake in cancer, there have been an increasing number of other molecules discovered that also drive tumor growth and function including, glutamine, methionine, serine, and glycine [1,6,10].
Worldwide, the use of 11C methionine has been shown to have a high diagnostic performance in detecting human glioma . Ad-ditionally, there is evidence that glutamine uptake is preferential in gliomas as well [10,12]. Glutamine is rapidly taken up and converted to glutamate and ultimately alpha-ketoglutarate which feeds into the tri-carboxylic acid cycle. Interestingly, the SSP has been shown to produce as much as 50% of the intracellular alpha-ketoglutarate . This sug-gests a feature of this pathway is the production of TCA intermediates for a yet unknown purpose. Nonetheless the demand for these meta-bolites and their cellular production are intimately linked to this pathway.
The approach presented here is to study the SSP by culturing human cancer cells with stable-isotope enriched glucose and demonstrate gas chromatography-mass spectrometry can quantitatively measure the flux of carbon from glucose into serine, glycine, and methionine. Selective inhibitors of critical enzymes in this pathway including PHDGH and mitochondrial SHMT2 are currently of high interest as cancer che-motherapy agents. We demonstrate that the efficacy of selective in-hibitors of PHDGH and SHMT2 can be measured with the approach presented here.