Ganapathy-Kanniappan Shanmugasundaram, Geschwind Jean-Francois H, Kunjithapatham Rani, Buijs Manon, Syed Labiq H, Rao Pramod P, Ota Shinichi, Vali Mustafa
Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Assay Drug Dev Technol. 2010 Apr;8(2):258-62. doi: 10.1089/adt.2009.0226.
3-Bromopyruvate (3BrPA) is a pyruvate analog known for its alkylating property. Recently, several reports have documented the antiglycolytic and anticancer effects of 3BrPA and its potential for therapeutic applications. 3BrPA-mediated cytotoxicity has been evaluated in vitro by various methods including tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)-based assays such as MTT, MTS, and so on. However, growing body of evidences has shown that tetrazolium reagent may interfere with the test compounds. In this study, we investigated whether the tetrazolium reagent interferes with the assessment of 3BrPA cytotoxicity. The results of the tetrazolium-based MTS assay were compared with 3 distinct cell viability detection methods, that is, Trypan Blue staining, ATP depletion, and Annexin V staining in 2 different cell lines, Vx-2 and HepG2. The MTS assay data showed false positive results by indicating increased cell viability at 1 mM and 2 mM 3BrPA whereas the other cell viability assays demonstrated that both Vx-2 and HepG2 cells are not viable at the same treatment conditions. In order to validate the direct interaction of 3BrPA with MTS reagent, we tested cell-free media incubated with different concentrations of 3BrPA. The results of cell-free media showed an increase in absorbance in a dose-dependent manner confirming the interaction of MTS with 3BrPA. Thus, our data clearly demonstrate that 3BrPA interferes with the accuracy of MTS-based cytotoxicity evaluation. Hence, we suggest that employing multiple methods of biochemical as well as morphological cytotoxicity assays is critical to evaluate 3BrPA-mediated cell death.
3-溴丙酮酸（3BrPA）是一种因其烷基化特性而闻名的丙酮酸类似物。最近，有几份报告记录了3BrPA的抗糖酵解和抗癌作用及其治疗应用潜力。3BrPA介导的细胞毒性已通过多种体外方法进行评估，包括基于四唑盐（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐）的检测方法，如MTT、MTS等。然而，越来越多的证据表明四唑试剂可能会干扰测试化合物。在本研究中，我们调查了四唑试剂是否会干扰3BrPA细胞毒性的评估。将基于四唑的MTS检测结果与3种不同的细胞活力检测方法进行比较，即锥虫蓝染色、ATP消耗和膜联蛋白V染色，使用2种不同的细胞系Vx-2和HepG2。MTS检测数据显示出假阳性结果，表明在1 mM和2 mM 3BrPA时细胞活力增加，而其他细胞活力检测表明在相同处理条件下Vx-2和HepG2细胞均无活力。为了验证3BrPA与MTS试剂的直接相互作用，我们测试了与不同浓度3BrPA孵育的无细胞培养基。无细胞培养基的结果显示吸光度呈剂量依赖性增加，证实了MTS与3BrPA的相互作用。因此，我们的数据清楚地表明3BrPA会干扰基于MTS的细胞毒性评估的准确性。因此，我们建议采用多种生化以及形态学细胞毒性检测方法对于评估3BrPA介导的细胞死亡至关重要。
