Sulforaphane Increases p21 protein in Oral Cancer 61 and/or inactivation of CDK inhibitors resulting in un- controlled proliferation which is one of the causes of cancer [1]. p21 is a CDK inhibitor protein that is essential for cellular growth, differentiation and apoptosis [2]. Although p21 is rarely mutated in human tumors, a decrease in p21 protein expression contributes to the neoplastic progression of many tumors [3–11]. A number of scientific reports show that p21 is induced by DNA damage or oncogene expression, triggering irreversible cell cycle arrest, which facilitates tumor growth arrest [12–15]. For this reason, induction of the p21 protein is considered a good strategy for molecular target therapy. The use of natural products as chemopreventive agents has been a major focus for a long time due to their possible cancer chemotherapeutic activity. Among the many natu- rally occurring anti-cancer agents, sulforaphane (SFN) has attracted the attention of cancer investigators. There is accu- mulating evidences that SFN inhibits the proliferation of cancer cells both in vitro and in vivo by causing apoptosis and/or cell cycle arrest [16–21]. Our previous study also found that SFN induced caspase-dependent apoptosis in human oral squamous carcinoma cells and a nude mouse xenograft model [22]. However, the mechanism of the cell cycle arrest induced by SFN to inhibit the proliferation of oral cancer cells is not completely understood. This study confirmed that the treatment with SFN causes cell cycle arrest of KB and YD-10B human oral squamous carcinoma cell at the G2/M phase. In addition, it is associated with a significant increase in p21 protein expression. More- over, SFN induced p21 protein expression in a nude mouse xenograft model confirming that SFN activates p21 protein which contributes to the potent anticancer activity of this compound. Materials and Methods Reagents Sulforaphane was purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies for sp1, cyclin A, cyclin B and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The p21 antibody was purchased from BD Pharmingen (San Diego, CA). Propidium Iodide was obtained by Calbiochem (San Diego, CA), and RNase A was from Sigma-Aldrich. p21 promoter reporter constructs, pWWP was provided by Dr. Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan). Lipofectamine 2000 transfection reagent was purchased from Invitrogen (Carlsbad, CA). Reporter lysis buffer and luciferase reagent for dual luciferase assays were purchased from Promega (Madison, WI). Vol. 46, No. 1, 2010 Cell culture and drug treatment The human oral squamous carcinoma cell line KB was obtained from American Tissue Culture Collection (Manassas, VA). Human oral squamous carcinoma cell line YD-10B was obtained from College of Dentistry, Yonsei University (Seoul, Korea). KB Cells were maintained in Dulbecco’s modified essential medium (DMEM; Welgene, Dae-Ku, Korea) without phenol red and YD-10B cells were cultured in DMEM/F-12 media (Welgene) containing 5% fetal bovine serum (FBS) and 10 ml/L of 100X antibiotics antimycotic solution (Welgene) at 37°C in a humidified atmosphere of 5% CO2 incubator. For the treatment of cells with SFN, a stock solution of 40 mM was prepared in dimethyl sulfoxide (DMSO). An equal number of cells were seeded in 96-well, 12-well, or 6-well plates and allowed to attach until plates reached 50–60% confluence, which was usually observed 24 h after seeding. Cells were treated with vehicle (DMSO) or compounds of various concentrations diluted in DMEM without phenol red and supplemented with 2.5% FBS. Cell proliferation assay KB and YD-10B cells were seeded in 12-well plates and were treated with DMSO and two concentrations of the test compounds in DMEM containing 2.5% FBS for 12 h. KB and YD-10B cells were counted to evaluate the effects of SFN and the vehicle control on the number of viable cells. A cell count was carried out with a hematocytometer using equal volumes of cell suspension and 0.4% trypan blue solution (Sigma Chemical Co.). Each experiment was carried out in triplicate and results were expressed as means ± SD for each treatment group. MTT assay The MTT assay kit purchased from AMRESCO (Solon, OH) was used to evaluate the effect of SFN on cell pro- liferation in KB and YD-10B cells. Cells were seeded in 96-well plates and incubated for 24 h before the treatment with SFN. After SFN treatment for 12 h, 20 μl of the MTT solution (2 mg/ml) was added to each well and cells were incubated for 2 h at 37°C in 5% CO2. The formazan absor- bance was measured at 560 nm using a microtiter plate reader (Molecular Devices). FACS analysis for cell cycle regulation After treatment with SFN, detached cells (floaters) were collected by centrifugation and combined with adherent cells that were released by trypsinization. Cell cycle analysis was carried out by flow cytometry; cells were fixed in 70% ethanol overnight at −20°C. After centrifugation, ethanol was removed and the cells were subsequently stained with 0.02 mg/ml propidium iodide (PI) and subjected to DNA content analysis using a FACScan cytometer (Becton