Sulforaphane Increases p21 protein in Oral Cancer 65 Fig. 3. Effects of SFN on the levels of the proteins involved in the regulation of G2/M transition. (A) immunoblotting for p21, cyclin B and cyclin A using lysates from control and SFN-treated KB cells. (B) The data points in the graph are the mean ± SD of three independent experiments. *p<0.05 compared to the control group. (C) Transactivation activity of pWWP (p21 promoter construct) induced by SFN in KB cells. (D) immunobloting for sp1 protein using lysates from control and SFN-treated KB cells. Fig. 4. Effects of SFN on the expression level of p21 protein expression in an athymic nude mouse xenograft model. (A) Immunostain- ing of p21 in tumor tissue samples. Tumor tissue samples from mouse xenografts bearing KB cells were deparaffinized and immunostained with the p21 antibody as described in Materials and Methods. (B) Immunoreactivity score values of p21 protein in tumor tissues. 0; negative, 1; weakly positive, 2; moderately positive, 3; strongly positive. *p<0.05 compared to control group. evaluated Sp1 protein expression level in this system. Unfortunately, Sp1 protein was not affected by SFN indi- cating that Sp1 is not an upstream protein of p21 (Fig. 3D). Of several CDK inhibitors, p21 is an important mediator of cell cycle arrest regulated by tumor suppressor p53 in response to DNA damage [40]. In addition to being induced by p53, p21 is also induced by other factors in a p53- independent pathway [41, 42]. Recently, Traka et al. (2009) reported that the induction of p21 protein by SFN can be mediated by Kruppel-like factor 4 (KLF4) in colon cancer cells and tissues suggesting that KLF4 might be a possible mediator for p21 protein in this study [43]. Therefore, current studies are focused on identifying these critical proteins induced by SFN in oral squamous carcinoma cell lines to induce p21 protein. In summary, this study demonstrates for the first time that SFN inhibit growth and induce G2/M cell cycle arrest in oral squamous carcinoma cell lines. Moreover, p21 protein plays an important role in SFN-induced cell cycle arrest. Therefore, these findings confirm that SFN is a promising Vol. 46, No. 1, 2010