Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling

Sellick, Christopher A.; Hansen, Rasmus; Stephens, Gill; Goodacre, Royston; Dickson, Alan J.

doi:10.1038/nprot.2011.366

Cited by 199 publications

(115 citation statements)

References 41 publications

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“…Metabolite extraction generally has two parts: quenching and extraction. Quenching first limits further metabolic activity and removes contaminants, which is then followed by a metabolite extraction step (Sellick, Hansen, Stephens, Goodacre, & Dickson, 2011). Metabolites with a high turnover rate, such as ATP, depend on complete and quick quenching methods for accurate detection.…”

Section: Metabolite Measurements and Glucose Tracingmentioning

confidence: 99%

Techniques to Monitor Glycolysis

TeSlaa¹,

Teitell

2014

Methods in Enzymology

232

190

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An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation.

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Section: Metabolite Measurements and Glucose Tracingmentioning

confidence: 99%

Techniques to Monitor Glycolysis

TeSlaa¹,

Teitell

2014

Methods in Enzymology

232

190

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“…Then, metabolites were extracted from the quenched cells by two 100% methanol extractions followed by a single water extraction. The metabolite samples generated using this protocol were amenable to analysis by GC-MS as previously described [23].…”

Section: Metabolic Assaysmentioning

confidence: 99%

Metabolic Shift Induced by ω -3 PUFAs and Rapamycin Lead to Cancer Cell Death

Zhu

Feng

Lin

et al. 2018

Cell Physiol Biochem

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Background/Aims: Rapamycin (Rp), the main mammalian target of rapamycin complex inhibitor, is a promising therapeutic agent for breast cancer. However, metabolic disorders and drug resistance reduce its efficacy. Epidemiological, clinical, and experimental studies have demonstrated that omega-3 polyunsaturated fatty acids (ω-3 PUFAs) significantly reduce the incidence and mortality of breast cancer and improve metabolic disorders. Methods: Three breast cancer cell lines and immunocompetent and immunodeficient mice were used to evaluate the therapeutic effects of Rp plus ω-3 PUFA treatment. The production of reactive oxygen species (ROS) and glucose uptake were examined by flow cytometry. Metabolic shift was examined by metabonomics, seahorse experiments, and western blot analysis. Results: We found that ω-3 PUFAs and Rp synergistically induced cell cycle arrest and apoptosis in vitro and in vivo, accompanied by autophagy blockage. In addition, Rp-induced hypertriglyceridemia and hypercholesterolemia were completely abolished by ω-3 PUFA supplementation. Moreover, the combined treatment of ω-3 PUFA and Rp significantly inhibited glycolysis and glutamine metabolism. The anti-tumor effects of this combination treatment were dependent on ROS production, which was increased by β-oxidation and oxidative phosphorylation. Conclusion: Our study revealed that ω-3 PUFA enhanced the anti-tumor activity of Rp while minimizing its side effects in vitro and in vivo. These results provide novel insights into the mechanisms underlying the potential beneficial effects of Rp combined with ω-3 PUFAs on the prevention of breast cancer.

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“…Cell suspensions for metabolite extractions were collected from the same flasks at 0, 24, 48 & 72 h after the treatments and subjected to fast filtration and quenching. Briefly, vacuum assisted-fast filtration 20, 21 on a vacuum manifold (Pall, Port Washington, NY, USA) was used to quickly remove media, followed by a 10 ml PBS wash. Filters (47 mm, 0.45 μM, PES; Millipore, Billerica, MA, US; catalog #HPWP04700) capturing the cells were immediately transferred to 1.5ml of −80°C methanol in a petri dish (60 mm × 15 mm) on dry ice and stored at −80°C for 24 h for thorough extraction. The filters were rinsed with the methanol cell suspension, and the suspension was transferred to microcentrifuge tubes, vigorously mixed for 30 sec and centrifuged at 5000 g for 5 min at −5°C.…”

Section: Experimental Methodsmentioning

confidence: 99%

Metabolomics identifies the intersection of phosphoethanolamine with menaquinone-triggered apoptosis in an in vitro model of leukemia

et al. 2015

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Altered metabolism is increasingly acknowledged as an important aspect of cancer, and thus serves as a potentially fertile area for the identification of therapeutic targets or leads. Our recent work using transcriptional data to predict metabolite levels in cancer cells led to preliminary evidence of the antiproliferative role of menaquinone (Vitamin K2) in the Jurkat cell line model of acute lymphoblastic leukemia. However, nothing is known about the direct metabolic impacts of menaquinone in cancer, which could provide insights into its mechanism of action. Here, we used metabolomics to investigate the process by which menaquinone exerts antiproliferative activity on Jurkat cells. We first validated the dose-dependent, semi-selective, pro-apoptotic activity of menaquinone treatment on Jurkat cells relative to non-cancerous lymphoblasts. We then used mass spectrometry-based metabolomics to identify systems-scale changes in metabolic dynamics that are distinct from changes induced in non-cancerous cells or by other chemotherapeutics. One of the most significantly affected metabolites was phosphoethanolamine, which exhibited a two-fold increase in menaquinone-treated Jurkat cells compared to vehicle-treated cells at 24 h, growing to a five-fold increase at 72 h. Phosphoethanolamine elevation was observed prior to the induction of apoptosis, and was not observed in menaquinone-treated lymphoblasts or chemotherapeutic-treated Jurkat cells. We also validated the link between menaquinone and phosphoethanolamine in an ovarian cancer cell line, suggesting potentially broad applicability of their relationship. This metabolomics-based work is the first detailed characterization of the metabolic impacts of menaquinone treatment and the first identified link between phosphoethanolamine and menaquinone-induced apoptosis.

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Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling

Cited by 199 publications

References 41 publications

Techniques to Monitor Glycolysis

Techniques to Monitor Glycolysis

Metabolic Shift Induced by ω -3 PUFAs and Rapamycin Lead to Cancer Cell Death

Metabolomics identifies the intersection of phosphoethanolamine with menaquinone-triggered apoptosis in an in vitro model of leukemia

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