antibodies were obtained from Cell Signaling Technology (Danvers, MA). Rictor antibody was obtained from Novusbio (Littleton, CO). PEITC, actin antibody, TGF, SRB, MCDB105 and Medium 199 were procured from Sigma Aldrich (St. Louis, MO). RPMI and McCoy 5A were purchased from Mediatech (Manassas, VA). AnnexinV apoptosis kit was procured from BD biosciences (San Jose, CA). In vivo Tumor Xenograft Four to six week old female athymic nude mice were purchased from Charles River Laboratories (Wilmington, MA). Institutional Animal Care and Use Committee (IACUC), Texas Tech University Health Sciences Center approved the use of mice and their treatment, and all the experiments were carried out in strict compliance with regulations. Mice were fed with antioxidant-free AIN-76A special diet for a week before starting the experiment. About 56106 SKOV-3 cells were injected subcutaneously into both right and left flanks. Ten mice were assigned randomly to each group. Since each mouse was implanted two xenografts, each group had twenty tumors. Mice in the control group received PBS, whereas, mice in the treatment group received 12 mmol PEITC suspended in PBS by oral gavage every day. Tumor growth was monitored until day 42 as described by us previously [16,24]. At day 42, mice were euthanized and tumors were removed, weighed and processed for western blot analysis. Cell Cultures SKOV-3, OVCAR-3, TOV-21G cells lines were procured from American Type Culture Collection (ATCC, Manassas, VA). SKOV-3 cells were maintained in McCoy’s 5A medium supplemented with 10% Fetal Bovine Serum (FBS). OVCAR-3 cells were maintained in RPMI medium supplemented with 20% FBS, 10 mM sodium pyruvate, 10 mM HEPES, 10 mg/L bovine insulin and 4.5 g/L glucose. TOV21G cells were maintained in 1:1 mixture of MCDB105 and Medium 199 supplemented with 15% FBS. A 1% antibiotic mixture was used in all the above media. All the cell lines were maintained at 37uC in a humidified incubator circulated with 5% CO2/95% air. Sulphorhodamine B Cell Survival Assay About 5,000 cells in 0.1 ml medium were plated in 96 well plates and allowed to attach overnight. Desired concentrations of PEITC were added to the cells and incubated at 37uC for 24 h. The cells were then processed and stained with 0.4% SRB solution and the absorbance was read at 570 nm using a Biotek plate reader as described by us previously [25]. Western Blotting SKOV-3, OVCAR-3 and TOV-21G cells were exposed to varying concentrations of PEITC alone or in presence of TGF. In another experiment, cells were treated with 15 mM PEITC for different time intervals. For concentration dependent study SKOV3 cells were treated with 7.5, 15 and 30 mM PEITC for 24 hours. Cells were collected, lysed, and 20–80 mg of protein was subjected to SDS gel electrophoresis followed by immunoblotting as described by us previously [20,25]. Annexin-V Apoptosis Assay SKOV-3, OVCAR-3 or TOV-21G cells were plated at a density of 0.36106 cells per well in a six-well plate and allowed to attach overnight. Cells were then treated with or without PEITC. After 24 hours, cells were washed, suspended in binding buffer and incubated for 15 minutes with Annexin V-FITC. Fluorescence was PEITC Targets EGFR to Suppress Ovarian Cancer measured using C6 Accuri flow cytometer (Ann Arbor, MI) with a minimum of 10,000 events per sample as described by us previously [16,26]. Immunoprecipitation Assay Immunoprecipitation assay was performed to examine the effect of PEITC on the complex of mTOR with Rictor and Raptor. Briefly, SKOV3 cells were treated with 15 mM PEITC for 24 hours. The cell lysates from control and PEITC treated cells were immunoprecipitated with the mTOR antibody (Cell Signaling), as described by us earlier [27]. The proteins were separated by SDS- polyacrylamide gel and transferred to PVDF membrane. The membranes were immnobloted for Rictor, Raptor and mTOR using respective antibodies. TGF Treatment SKOV-3 or OVCAR-3 cells were treated with 50 ng/ml TGF for 1 hour followed by incubation with different concentrations of PEITC for 24 hours. Cells were then processed for western blotting as described above. AKT Overexpression SKOV-3 cells were transiently transfected with plasmid containing wil-type AKT (a generous gift from Daniel Altschuler, University of Pittsburgh, Pittsburgh, Pennsylvania) by using Fugene (Roche). Briefly, 0.36105 cells were transfected with 0.5 mg of the AKT plasmid diluted in Opti-MEM serum-free medium to which Fugene reagent was added before the mixture was added to cells. Cells were incubated with plasmid-Fugene mixture for 6 hours and then media was replaced with fresh growth medium and incubated for another 24 hours. Transfected cells were treated with 10 mM PEITC for 24 hours. Whole cell lysates were subjected to Western blot analysis and actin was used as loading control. Quantitation and Statistical Analysis All the statistical analysis was performed using Prism 5.0 (GraphPad Software Inc., San Diego, CA). The data represents mean 6 S.D. Student’s t-test was used to compare the control and treated groups. In experiments involving more than three groups, non-parametric analysis of variance followed by Bonferroni post hoc multiple comparison test was used. All statistical tests were two sided. Differences were considered statistically significant when the p value was less than 0.05. Results Phenethyl Isothiocyanate (PEITC) Suppresses Ovarian Tumor Growth in a Xenograft Model Isothiocyanates were shown to be therapeutically active against various malignancies. To test the possibility that PEITC treatment would suppress the growth of ovarian tumors, SKOV-3 tumor xenografts were established in female athymic nude mice by subcutaneously injecting 56106 cells in both right and left flanks. Mice were randomly divided into control and treatment group. Tumor bearing mice were fed 12 mmol PEITC every day and tumor growth was recorded periodically. Our results demonstrate that PEITC substantially suppressed the growth of ovarian tumors in athymic nude mice. For example, at day 42, the average tumor 3 volume in mice from treatment group was approximately 60 mm , whereas average tumor volume in mice from control group was approximately 220 mm3 (Fig. 1A). Both tumor volume and tumor PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43577