Cellular glucose metabolism is essential for the reduction of cell-impermeable water-soluble tetrazolium (WST) dyes

• Published in: International journal of biological sciences 2018-01, Vol.14 (11), p.1535-1544
• Main Authors: Xie, Linna, Dai, Zichan, Pang, Chunxiu, Lin, Dexin, Zheng, Min
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; AKT protein; Assaying; ATP; Cell death; Cell number; Cell proliferation; Drug metabolism; Drug screening; Dyes; Epidermal growth factor receptors; Fructose; Galactose; Glucose; Glucose metabolism; Glucose transport; Glutamine; HIV; Human immunodeficiency virus; Hyperglycemia; In vivo methods and tests; Intracellular; Intracellular signalling; Metabolism; Protease inhibitors; Proteinase inhibitors; Reduction; Research Paper; Ritonavir; Water chemistry
• ISSN: 1449-2288; 1449-2288
• DOI: 10.7150/ijbs.25629

Water-soluble tetrazolium (WST) dyes, such as WST-1 and WST-8, are widely used in cell proliferation and anti-cell-growth drug screen assays. However, the underlying determinants for WST reduction are still largely unknown. In addition, application of tetrazolium-based assays to cellular glucose metabolism studies has not been fully explored. In the present study, we show here that WST-8 reduction is dependent on cellular glucose metabolism. In order to minimize the variance of live cell number during stimulation, we treated cells with different stimuli and performed tetrazolium-based assays within 6 hours. Withdrawal of medium glucose supply greatly attenuated WST-8 reduction but not intracellular ATP levels, while re-adding glucose reconstituted WST-8 reduction, indicating the effect was not due to cell death. The role of glucose on WST-8 reduction is specific since glutamine, fructose or galactose did not substitute for the effect of glucose on WST-8 reduction. Furthermore, inhibition of glucose transporters, intracellular glucose metabolic enzymes or EGFR-PI3K-Akt signaling also attenuated WST-8 reduction. In an attempt to screen inhibitors targeting cellular glucose metabolism from hyperglycemia-associated drugs, it turned out that HIV protease inhibitor, ritonavir, could largely block WST-8 reduction, but not cellular ATP level. Interestingly, ritonavir has been shown to acutely block glucose transport and . Taken together, our studies not only demonstrate an essential role of cellular glucose metabolism on WST-8 reduction, but also propose a novel application of tetrazolium-based assays in screening for inhibitors of cellular glucose metabolism when used in combination with ATP assay.