Evaluation of in vitro toxicity and efficacy of ferutinin, a natural promising chemopreventive compound

The biological activity of the natural phytoestrogen ferutinin have not been extensively examined as yet, in spite of the interest about plant-derived products as possible chemopreventives. In this study, the efficacy of ferutinin on several in vitro experimental endpoints correlated with tumour onset and progression has been compared to that of the well characterized soy isoflavone genistein. Effects of ferutinin and genistein have been examined on cell proliferation and growth inhibition, anchorage-independent growth and Matrigel invasion, cell growth in estrogen depleted media, programmed cell death. Like genistein, ferutinin acts as an estrogen agonist in the E-screen assay and exerts a biphasic effect on cell growth and proliferation in ER-positive MCF-7 cells, with an induction of proliferation at lower concentration (1µM) and an antiproliferative dose-response effect at higher concentrations (10-100 µM). In MDA-MB-231 ER-negative cells, the dose-related inhibition of cell growth induced by ferutinin or genistein was evident, even if no biphasic effect was shown. In the agar clonogenic assay, ferutinin did not induce any significant increase in colony growth of MCF-7 cells at the assayed doses, while it showed a strong dose-related antiproliferative activity at high concentrations (10-100 µM). The biphasic effect of genistein on anchorage-independent growth was evident. The effect of the phytoestrogens on the malignant phenotype was evaluated in the in vitro Matrigel invasion assay. Ferutinin (1-100 µM) induced a dose-dependent inhibition of the invasive ability of MDA-MB-231 cells. The effect of ferutinin on cell death was also evaluated. The morphology of dying cells suggested the induction of a different mechanism of cell death induced by ferutinin, possibly alternative to apoptosis. The monodansylcadaverine (MDC) assay for autophagic cell death revealed some MDC-positive structures, which could be classified as autophagic vacuoles, in cells treated with high ferutinin concentrations (80 and 40 µM). The results show that ferutinin is more effective than genistein in the assayed in vitro endpoints in human breast cancer cells, suggesting a possible use of this natural compound as a chemopreventive or chemoterapeutic drug, even if the mechanisms of action and the benefit/risk ratio need to be further evaluated.