214 TRICLOSAN-INDUCED CELL CYCLE RELATED AND APOPTOTIC RELATED GENES WERE REVERSED BY KAEMPFEROL IN IN VITRO BREAST CANCER AND XENOGRATED MOUSE MODELS

Abstract

Triclosan (Tri) is one of many endocrine-disrupting chemicals (EDCs) that are scattered with environment agents, such as toothpastes, deodorants, and cleaning supplies. As a phytoestrogen, kaempferol (Kae) is one of bioflavonoids, which has been found in a variety of vegetables including broccoli, tea, and tomatoes. Although Kae may have anti-cancer activity, its exact mechanism is under investigation, and might be the induction of apoptosis and inhibition of cell proliferation or angiogenesis. In this study, we examined the anti-proliferative effects of Kae in Tri-induced cell growth in MCF-7 breast cancer cells. A proper concentration and co-treatment effect of Tri and Kae were determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay to measure cell viability in vitro. MCF-7 cells were cultured with a negative control (0.1% DMSO), E2 (1 × 10–9 M), Tri (10–5–10–8 M) and Kae (50, 70, and 90 mM). In this study, treatment with Tri (10–6 M) increased the cell viability of MCF-7 cells, while Kae (50 mM) significantly reduced the cell viability compared to the negative control (P < 0.05). In addition, Kae significantly reversed Tri-induced MCF-7 cell growth at 50 mM compared with a higher concentration (100 mM; P < 0.05). To confirm that Kae inhibited Tri-induced cell growth, we examined the transcriptional levels of cell growth and apoptosis-related markers, i.e. cyclin D, p21, cyclin E, p27 and bcl-2, and bax genes, using reverse transcription (RT)-PCR. The expression levels of cyclin D, cyclin E, and bax/bcl-2 ratio were increased, while those of p21 and p27 mRNAs were decreased by Tri in MCF-7 cells. In addition, Kae treatment significantly reversed Tri-induced gene expressions in an opposite manner. In parallel with its mRNA level, the protein level of cyclin E, p-ERK and p-MEK1/2 were induced by Tri while it was reversed by Kae as shown by Western blot analysis. The expression levels of p21 and bax genes were altered by Tri and reversed by Kae treatment in this study. As an in vivo model, a xenografted mouse model was generated following injection with MCF-7 breast cancer cells in 6 weeks. In parallel with in vitro results, tumour volumes following treatment with E2 and Tri were continually increased compared to a vehicle (corn oil). It was of interest that treatment of the mice with combination of E2 plus Kae or Tri plus Kae showed less tumour formation rather than that of singly treated mice with E2 or Tri. Taken together, these results indicate that Kae may inhibit the growth of MCF-7 cells via regulating of cell cycle and apoptosis-related genes. In addition, EDC-induced progression of breast cancer may be suppressed by a phytoestrogen, i.e. Kae, in a specific manner.