64 J.-H. Kim et al. Fig. 2. Effect of SFN on the cell cycle distribution in KB and YD-10B cells. KB and YD-10B cells were treated with DMSO or various concentrations of SFN for 12 h. Both floating and attached cells were collected and processed for an analysis of cell cycle reg- ulation as described in “Materials and Methods” (A) Representative histograms of cell cycle distribution in the control and SFN-treated cells are shown. The data points in the graph are the mean ± SD of three independent experiments in KB cells (B) and YD-10B cells (C). *p<0.05 compared to control group. cally rich in cruciferous vegetables is expected to help pre- vent cancer. Among the phytochemicalsin these vegetables, SFN is the most characterized isothiocyanate compound. Many studies have reported that SFN is an effective agent against the proliferation of a variety of cancer cells [22, 28– 30]. It was previously reported that SFN exhibited a signifi- cant inhibition on the growth of KB and YD-10B cells with caspase-dependent apoptosis [22]. In this study, it was found that early-time exposure (12 h) to SFN significantly decreases the number of viable KB and YD-10B cell growth in a dose-dependent manner (Fig. 1). On the other hand, there were no apoptotic events observed in SFN-treated cell for 12 h. Therefore, other possible mechanisms for how SFN inhibits the proliferation of oral squamous carcinoma cells were examined. Several studies on SFN reported that SFN induces G2/M cell cycle arrest in cancer cells [18, 31, 32]. Therefore, this study examined cell cycle regulation in KB and YD-10B cells and SFN induced a profound arrest in the G2/M phase (Fig. 2). These results suggest that the G2/M cell cycle arrest induced by SFN is a prerequisite for caspase- dependent apoptosis in these cell lines. However, more critical experiments will be needed to test this hypothesis. CDK inhibitors have grown in importance and number since they were first described and they appear to be universal inhibitors of cyclin/CDK activity which functions by forming a complex with the cyclin/CDK to inhibit the growth of cancer [2, 33, 34]. p21, a CDK inhibitor also inhibits CDKs. The induction of p21 molecule causes a number of different cell lines to arrest in both G1 and G2 checkpoint. SFN was reported to induce G2/M arrest in a variety of cancer cell lines and p21 expression is associated with G2/M phase cell cycle arrest [31, 35, 36]. Our study showed that SFN induces the p21 expression and its trans- activation to induce G2/M phase arrest (Fig. 3). There results are fully consistent with other studies. We also previously reported that SFN decreased tumor growth in athymic nude mice bearing KB cells as xenograft. Fig. 4 shows that p21 was also induced in the tumor sections from the same animals demonstrating comparable induction of this pro- apoptotic protein in both in vivo and in vitro tumor models. The specificity protein (Sp) family recognizes GC-rich or GT-rich DNA and regulates the promoters of several genes [37, 38]. The human p21 gene contains six Sp1 binding sites [39]. Due to close relationship between Sp1 and p21, we J. Clin. Biochem. Nutr.