title: Genistein inhibits tumour 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.
abstract: The natural isoflavone genistein inhibits the growth of a number of tumour cell lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour cell lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect. Cell cycle analysis by propidium iodide staining and flow-cytometry revealed a G2/M cell cycle arrest after genistein treatment. Genistein-treated cells displayed an increase in cell volume and in mitochondrial number and/or activity, as revealed by enhanced formazan generation and increased uptake of the vital mitochondrial dye rhodamine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly influence mitochondrial number and/or function and, consequently, MTT reduction to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M transition, on growth and viability of cancer cells in vitro by MTT assay.
authors.affiliation: Istituto di Clinica Medica 1, Perugia University School of Medicine, Italy.
authors.forename: M C
authors.initials: MC
authors.lastname: Pagliacci
authors.forename: F
authors.initials: F
authors.lastname: Spinozzi
authors.forename: G
authors.initials: G
authors.lastname: Migliorati
authors.forename: G
authors.initials: G
authors.lastname: Fumi
authors.forename: M
authors.initials: M
authors.lastname: Smacchia
authors.forename: F
authors.initials: F
authors.lastname: Grignani
authors.forename: C
authors.initials: C
authors.lastname: Riccardi
authors.forename: I
authors.initials: I
authors.lastname: Nicoletti
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D002452:Cell Count; D005562:Formazans / Q000378:metabolism; D019833:Genistein; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology*; D008928:Mitochondria / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000494:pharmacology*; D012235:Rhodamines / Q000378:metabolism*; D013268:Stimulation, Chemical; D013778:Tetrazolium Salts / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects*
chemical_list: D005562:Formazans; D006131:Growth Inhibitors; D007529:Isoflavones; D012235:Rhodamines; D013778:Tetrazolium Salts; D013844:Thiazoles; D019833:Genistein; D011505:Protein-Tyrosine Kinases; C022616:thiazolyl blue