Growth inhibitory and cell cycle arresting properties of the brown rice constituent tricin in MDA-MB-468 breast cancer cells in vitro and in vivo: Relationship with tricin concentrations Free

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The flavone tricin (4’,5,7-trihydroxy-3’,5’-dimethoxyflavone) occurs as a glucoside in brown rice and rice bran as well as in other grass species, such as wheat, barley and maize. Little is known about the effect of this flavone on human health. In order to explore the potential of tricin to retard or prevent breast cancer, we investigated its ability to interfere with the growth of human breast-derived cell lines in vitro and after implantation in mice in vivo. The concentration of tricin in cells and tissues was analyzed by HPLC using a Hypersil BDS C18 column by isocratic elution with 52% methanol in 0.1 M ammonium acetate buffer pH 5.1. Malignant MDA-MB-468 cells (MDA) and transformed, but non-malignant, HBL-100 cells (HBL) were incubated with tricin for up to 168 h. Cells were counted and subjected to flow cytometric evaluation using propidium iodide and annexin-V for cell cycle analysis and quantitation of apoptosis, respectively. Tricin inhibited the growth of MDA and HBL cells with IC₅₀ values of approximately 0.7 and 21 μM, respectively. Tricin did not induce apoptosis in either cell line within the concentration range 1 to 40 μM. In MDA cells tricin (5 to 40 μM) arrested cell cycle progression at G2/M in a concentration- and time-dependent manner. G2/M arrest was not observed in HBL cells. HPLC analysis of cells and supernatant suggests that the agent was stable during the incubation period and that tricin did not undergo metabolism. MDA cells (10⁷) were implanted sc into the flanks of female nude MF1 mice. Tricin was added to their diet at 0.2% (∼300 mg/kg body weight per day) from the day of tumor implantation. Tumors developed, which after 8 weeks reached a volume of 215.5 ± 279.9 mm³ in untreated mice. Tumors in mice, which had received tricin, were not significantly smaller than those in untreated mice. Levels of tricin in tumors at termination were 0.11 ± 0.13 nmol/g (mean ± SD, n=5), thus probably too low to exert growth inhibition. MDA cells were incubated for 72 h with tricin (0, 0.11, 1.1 or 11 μM) and subsequently implanted into mice. Whilst tumors in mice, which had received cells incubated with 0.1 or 1.1 μM tricin, were indistinguishable from tumors with no pre exposure to tricin, the size of tumors in animals bearing cells incubated with 11 μM tricin was less than one third of that in mice, which had received control cells. These results suggest that i) tricin is a potent cytostatic agent in MDA cells in vitro involving G2/M arrest but not induction of apoptosis, ii) target tissue levels of 5 μM or higher are required to elicit growth inhibition in vivo, and iii) tricin may have limited bioavailability. We thank the NCI RAPID initiative for the synthesis of tricin.