Sulforaphane Increases p21 protein in Oral Cancer 63 Fig. 1. Effect of sulforaphane (SFN) on cell proliferation in KB and YD-10B cells. KB cells (A) and YD-10B cells (B) were treated with DMSO or various concentrations of SFN for 12 h. Cell proliferation was determined by a trypan blue exclusion assay. Cell viability in KB cells (C) and YD-10B cells (D) was estimated using a MTT assay. The results are reported as the mean ± SD of three independent experiments. *p<0.05 compared to the control group. (Fig. 2A). The G2/M phase arrested cells were increased from 28% and 24% in DMSO-treated KB and YD-10B cells to 54% and 35% in 40 μM of SFN-treated cells, respectively (Fig. 2B). Sulforaphane induces CDK inhibitor, p21 protein and its transactivation Because, the most of the cells were arrested at the G2/M phase, experiments were carried out to determine whether the SFN treatment also affects the expression level of G2/M phase cell cycle regulatory proteins. KB cells were treated with 20 μM of SFN for 12 h and the expression of these proteins (p21, cyclin A and cyclin B) were examined by western blot analysis. Fig. 3A and B showed a significant increase in the level of p21 protein expression and the level of cyclin B expression was significantly reduced by SFN whereas cyclin A was not changed. Furthermore, SFN induced the transactivation in KB cells transfected with pWWP (Fig. 3C). To find whether sp1 protein regulates p21 protein expression, we evaluated the level of sp1 protein expression. Unfortunately, sp1 protein was not changed by SFN compared to control (Fig. 3D). These results suggest that SFN modulates the expression level of the G2/M phase cell cycle regulatory proteins and its transactivation in KB Vol. 46, No. 1, 2010 oral carcinoma cells. Sulforaphane induces p21 protein in nude mouse xenograft model Immunohistochemistry detected the level of p21 protein from the tumor sections and they were obtained from animals in previous studies which showed that SFN inhibited oral carcinoma growth in athymic nude mice bearing KB cells as xenograft [22]. There was markedly increased immunostaining of p21 protein in oral carcinoma treated with 50 μg/kg of SFN compared to the tumors from the vehicle control (Fig. 4). Discussion Cruciferous or Brassica vegetables come from the Brassica genus, including broccoli, Brussels sprouts and cauliflower. They are a good source of glucosinolates and their hydro- lysis products, including indoles and isothiocyanates. The dietary intake of cruciferous vegetables has been associated with a lower risk of certain types of cancers including prostate, lung and colorectal cancer in some epidemiological studies [23–27]. Hence, an examination of the relationships between cancer risk in humans and phytochemicals specifi-