Stable isotope resolved metabolomics analysis of ribonucleotide and RNA metabolism in human lung cancer cells

Fan, Teresa W.‐M.; Tan, Jinlian; McKinney, Martin M.; Lane, Andrew N.

doi:10.1007/s11306-011-0337-9

Cited by 40 publications

(68 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We hypothesized that glutaminase inhibition would affect DNA replication, because glutamine is the preferred carbon source for pyrimidine synthesis and is the nitrogen donor for N1 and N9 of purines (38). Indeed, our data showed that the cycling cell population decreased upon glutaminase inhibition.…”

Section: Combined Bptes-np and Metformin Treatment Provides Enhancedmentioning

confidence: 72%

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer

Elgogary

Poore

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

192

159

View full text Add to dashboard Cite

Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.pancreatic ductal adenocarcinoma | glutaminolysis | glucose metabolism | KRAS mutation | intratumoral hypoxia P atients with pancreatic ductal adenocarcinoma (PDAC) have among the highest fatality rates of all cancers (1). Pancreatic cancer is predicted to become the second-leading cause of cancer death in the United States by the year 2030 (2). Over 90% of PDACs display mutations in oncogenic KRAS (Kirsten rat sarcoma viral oncogene homolog) (3, 4), a known regulator of glutamine metabolism that can render cancer cells dependent on glutamine for survival and proliferation (5, 6)-a state known as "glutamine addiction" (7, 8)-suggesting that dependency on glutamine could be exploited to develop new therapies for KRAS-mutated PDAC. The first step of glutamine metabolism is the conversion of glutamine to glutamate and ammonia, which is catalyzed by glutaminase (GLS). Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), which is an allosteric, time-dependent (9), and specific inhibitor of GLS1, exhibits unique binding at the oligomerization interface of the glutaminase tetramer (10, 11). Although BPTES is more selective than other prototype glutaminase inhibitors, such as 6-diazo-5-oxo-L-norleucine (12) or ebselen (9), and can effectively inhibit GLS1 (13) and tumor growth (13-15), poor solubility (0.144 μg/mL) (16) has limited its clinical development. Recently, CB-839 (17) was tested in a phase I clinical trial. Abnormal liver and kidney function tests, lymphop...

show abstract

Section: Combined Bptes-np and Metformin Treatment Provides Enhancedmentioning

confidence: 72%

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer

Elgogary

Poore

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

192

159

View full text Add to dashboard Cite

show abstract

“…The amination of U to C consumes an additional ATP; de novo synthesis of 5′-UMP and 5′-CMP therefore require four and five molecules of ATP respectively. Aspartate provides three of the four carbon atoms of pyrimidines, which derive largely from glutamine and to a lesser extent from glucose (92) (and see below).…”

Section: Nucleic Acid Synthesis: Energetics and Nutrient Requirementsmentioning

confidence: 99%

Regulation of mammalian nucleotide metabolism and biosynthesis

Lane

Fan

2015

Nucleic Acids Research

692

631

View full text Add to dashboard Cite

Nucleotides are required for a wide variety of biological processes and are constantly synthesized de novo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer.

show abstract

“…

Lyophilize the extracted RNA

Redissolve in 0.1 M sodium acetate buffer containing 1 mM ZnCl 2 , pH 5.3.

The conditions for digestion are determined using unlabeled Sigma RNA. Two units of P1 micrococcal nuclease (US Biological) are added to 1 ml RNA solution in the assay buffer (A 260 = 0.5 to 1 unit) in a capped quartz cuvette

Incubate at 50 °C.Periodically measure the absorbance at 260 nm (or closest available wavelength) as a function of time for the progress of hydrolysis in a spectrophotometer.After the reaction stops (absorbance levels off), add another aliquot of the enzyme to ensure complete digestion.Divide the solution is into two parts, and lyophilize each.One freeze dried powder sample is redissolved in 350 μL D 2 O containing 30 nmol DSS-d 6 for analysis by NMR (75). The NMR analysis provides quality control, and a check on the atom labeling in the ribose subunit of the free nucleotides.…”

Section: Methodsmentioning

confidence: 99%

“…One freeze dried powder sample is redissolved in 350 μL D 2 O containing 30 nmol DSS-d 6 for analysis by NMR (75). The NMR analysis provides quality control, and a check on the atom labeling in the ribose subunit of the free nucleotides.…”

Section: Methodsmentioning

confidence: 99%

Stable Isotope-Labeled Tracers for Metabolic Pathway Elucidation by GC-MS and FT-MS

Higashi

Fan

Lorkiewicz

et al. 2014

Methods in Molecular Biology

Self Cite

View full text Add to dashboard Cite

Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics is poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, Stable Isotope Resolved Metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks, in a wide range of experimental systems, including human subjects. MS offers a wide range of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS affordable by many individual laboratories, to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter will focus on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM.

show abstract

Stable isotope resolved metabolomics analysis of ribonucleotide and RNA metabolism in human lung cancer cells

Cited by 40 publications

References 39 publications

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer

Regulation of mammalian nucleotide metabolism and biosynthesis

Stable Isotope-Labeled Tracers for Metabolic Pathway Elucidation by GC-MS and FT-MS

Contact Info

Product

Resources

About