Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.
BACKGROUND
The chemopreventive effect of green tea polyphenols, such as (-)-epigallocatechin-3-gallate (EGCG), has been well demonstrated in cell culture studies. However, a wide range of IC(50) concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature.


RESULTS
We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP) and deoxynucleic acid (DNA) showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95). However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC(50) concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays.


CONCLUSIONS
These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.
Wang
Piwen
P
Center for Human Nutrition, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
Henning
Susanne M
SM
Heber
David
D
D016588:Anticarcinogenic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D010636:Phenols / Q000494:pharmacology*; D059808:Polyphenols; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D013662:Tea; D013778:Tetrazolium Salts; D013844:Thiazoles
D016588:Anticarcinogenic Agents; D004396:Coloring Agents; D005419:Flavonoids; D010636:Phenols; D059808:Polyphenols; D011933:Reagent Kits, Diagnostic; D013662:Tea; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; C022616:thiazolyl blue