GENISTEIN INHIBITS TUMOR-CELL GROWTH IN-VITRO BUT ENHANCES MITOCHONDRIAL REDUCTION OF TETRAZOLIUM SALTS - A FURTHER PITFALL IN THE USE OF THE MTT ASSAY FOR EVALUATING CELL-GROWTH AND SURVIVAL

The natural isoflavone genistein inhibits the growth of a number of tu mour cell lines in vitro. During investigations on the antiproliferati ve effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underest imated the growth inhibitory activity of the substance. Cell prolifera tion was markedly inhibited by genistein in three tumour cell lines (M CF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929 , mouse transformed fibroblasts) when cell number was evaluated by dir ect counting, whereas a 72-h MTT assay failed to reveal any growth-inh ibitory effect. Cell cycle analysis by propidium iodide staining and f low-cytometry revealed a G2/M cell cycle arrest after genistein treatm ent. Genistein-treated cells displayed an increase in cell volume and in mitochondrial number and/or activity, as revealed by enhanced forma zan generation and increased uptake of the vital mitochondrial dye rho damine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly influen ce mitochondrial number and/or function and, consequently, MTT reducti on to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M tr ansition, on growth and viability of cancer cells in vitro by MTT assa y.