GROWTH-INHIBITORY EFFECTS OF THE NATURAL PHYTO-ESTROGEN GENISTEIN IN MCF-7 HUMAN BREAST-CANCER CELLS

Genistein, a natural isoflavonoid phyto-oestrogen, inhibits the tyrosi ne kinase activity of growth factor receptors and oncogene products, a s well as the in vitro growth of some tumour cell lines. The low incid ence of breast cancer in countries with a flavonoid-rich soy-based die t and the protection afforded by soy-derived products against experime ntal mammary tumours in rats suggest that genistein and other isoflavo noid compounds may exert an anti-tumour activity. We analysed the effe cts of genistein on cell number and cell cycle progression (flow cytom etric analysis of propidium iodide-stained nuclei) of human breast can cer cells (MCF-7) in vitro. Genistein produced a significant, dose-dep endent inhibition of MCF-7 cell growth with an ID50 of similar to 40 m u M after 72 h of incubation. Cell cycle analysis showed a reversible G(2)/M arrest in cell cycle progression at 10 mu M genistein concentra tions, whilst a marked fail in S-phase cell percentage associated with a persistent arrest in G(2)/M phase was observed in cultures treated with genistein doses equal to or greater than 50 mu M. These effects w ere significant at 24 h of incubation; flow cytometric analysis at lat er times (48 and 72 h) revealed a population of cells with decreased D NA content and nuclear fragmentation characteristic of apoptosis. Thus , the growth inhibitory activity of genistein in MCF-7 cells results f rom the sum of cytostatic and apoptotic effects. Since the mitogenic a ction of insulin and insulin-like growth factor (IGF)-I in MCF-7 cells is a tyrosine kinase-dependent phenomenon, we analysed the genistein impact on S-phase entry produced by insulin in cultures partially sync hronised in G(0)/G(1) phase by serum deprivation. Insulin addition aft er a 36-h culture period in serum-free medium produced a strong increa se in the percentage of S-phase cells (from 18.4 +/- 2.3 to 46.2 +/- 4 .1 after 24 h) which was almost completely blocked by 100 mu M geniste in (20.1 +/- 3.1). Immunofluorescence analysis with a fluoresceine iso thiocyanate (FITC)-conjugated anti-phosphotyrosine antibody revealed a strong increase in MCF-7 cell staining after insulin stimulation, but not when genistein was added with insulin. In conclusion, the dietary phyto-oestrogen genistein inhibits in vitro growth of MCF-7 human bre ast cancer cells through blocks in the ''critical checkpoints'' of cel l cycle control and induction of apoptosis. These effects are likely t o depend on impairment in the signal transduction pathway from tyrosin e kinase receptor(s).