were incubated in FBS-free medium for 2 h then treated with different concentrations of S9 dissolved in assay buffer containing 2 mM Hoechst 33342 for 1 h. Insulin-like growth factor 1 (IGF-1 10 ng.ml21) was added to trigger Akt membrane translocation. Images were captured by IN Cell Analyzer 1000 (GE Healthcare) with 475/535 nm excitation/emission filters 5 min later. Each tested concentration was repeated in 12 wells and total granule intensity of EGFP-Akt foci in each cell were measured and quantitated by statistic software module. Fluorescent Microscopy SKOV-3 cells were grown on 1.561.5 cm2 glass chamber slides and treated with 5 mM S9 for 24 h. Immunofluorescent staining with anti-cyclin B1 antibody (Santa Cruz Biotechnology, Inc., CA) or anti-tubulin antibody (Invitrogen, Eugene, OR) was visualized as described [26]. Fluorescence Activated Cell Sorter (FACS) Assay DNA content was measured with a FACS-Calibur cytometer (Becton Dickinson, San Jose, CA) and cell cycle distribution were calculated as described previously [27]. Apoptosis were detected with Annexin V-FITC apoptosis detection kit (Beckon Dickinson Labware, Bedford, MA) and fraction of Annexin V positive cells was measured with Cell Quest Software. Each experiment was repeated thrice. Purified Tubulin Assembly Assay Pork tubulin was isolated, purified and stored as reported [28]. Prepared tubulin (15 mM) was mixed with different concentrations of S9, colchicine or vincristine in PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA) containing 1 mM GTP and 5% glycerol. Microtubule polymerization was monitored at 37uC by light scattering at 340 nm using a VERSAmax multiwell spectrophotometer [27]. Circular dichroism (CD) spectroscopy CD spectroscopy was performed as described [27]. Briefly, 3 mM tubulin was incubated with 25 mM S9 in PEM buffer at 37uC for 1 h. CD spectra were recorded on a Jasco-810 spectropolarimeter (Jasco, Tokyo, Japan) at 25uC. Competitive tubulin-binding Assay For colchicine competitive binding assay, tubulin was co- incubated with indicated concentrations of S9 or vincristine at 37uC for 1 h. Then colchicine was added to a final concentration of 5 mM. For BODIPY-FL-vinblastine competitive binding assay, BODIPY FL-vinblastine was added to S9-tubulin or vinblastine- tubulin binding equilibria. Fluorescence was determined using a Hitachi F-2500 spectrofluorometer (Tokyo, Japan) at excitation wavelengths of 365 nm and emission wavelengths of 435 nm or 502–528 nm for BODIPY-FL vinblastine. Blank values (buffer alone) as background were subtracted from all samples. Surface Plasmon Resonance (SPR) Assay The binding affinities of S9 or colchicine to the tubulin were determined using the SPR biosensor technology. The purified tubulin (Cytoskeleton, Denver) were immobilized to CM5 chip surface by standard Biacore procedure using HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% (v/v) surfactant P20, pH 7.4) as running buffer. The specific binding profiles of S9 and colchicine to the immobilized tubulin were obtained after subtracting the response signal from the control flow cell. All the data were collected at 25uC with running S9 Targets PI3K/mTOR/Tubulin buffer HBS-EP at a constant flow of 30 ml/min. Binding affinities of the compounds to tubulin were evaluated with Biacore evaluation Version 3.0 software (GE Healthcare, Wisconsin, USA). Molecular Modeling of the S9-protein Complex The crystal structures of the PI3K-a [Protein Data Bank (PDB) Code: 2RD0][29], tubulin-colchicine complex (PDB Code: 1SAO)[30] were obtained as the target structures in the molecular docking. The sequence of mTOR was retrieved from the Swiss- Prot database and the crystal structure of the PI3K-a [29] was used as the template structure for homology modeling of mTOR structure. The sequence identity in catalytic domain between mTOR and PI3K-a is about 29%. The ClustalW algorithm [31] and the Homology module of InsightII (version 2000 Accelrys San Diego CA) were applied in sequence alignment, and the best-fit alignment was obtained based on the Blosum scoring matrix [32] and previous alignments [16]. Model based on the alignment was constructed using the Modeller module of InsightII. Based on these structures, S9 was docked into the binding site of PI3K-a, mTOR, and tubulin with AutoDock 3.0 program [33], respec- tively. In each ligand-receptor docking procedure, 20 different docking solutions were obtained and the most possible binding conformations of S9 with enzymes were determined based on the combination of experimental information and free energy scores. Sulforhodamine B (SRB) Assay The anti-proliferative effect of S9 in a panel of tumor cells was examined with the SRB assay as described [26]. In vivo Antitumor Activity Assay Animal experiments were performed according to institutional ethical guidelines of animal care. Well-developed tumors were cut into 1-mm3 fragments and transplanted s.c. into the right flank of nude mice using a trocar. When the tumor volume reached 100 to 150 mm3, the mice were randomly assigned into control and treatment groups. Control groups were given vehicle alone, and treatment groups received S9 as indicated doses via i.p. administration five days per week for 2 weeks. The sizes of the tumors were measured twice per week using microcaliper. The tumor volume (TV) was calculated as: TV = (length6width2)/2. The tumor volume on day n was expressed as relative tumor volume (RTV) calculated with formula RTV = TVn/TV0, where TVn is the volume on day n, and TV0 is the volume at the beginning of the treatment. The therapeutic effect of the compounds on day 14 was presented as T/C values, which were calculated as: T/C = mean RTV of the treated group / mean RTV of the control group. Data Analysis Statistics Data were presented as X6SD, and differences were considered significant when P,0.05 as determined by Student’s t test. Supporting Information Table S1 Found at: doi:10.1371/journal.pone.0004881.s001 (0.05 DOC) Table S2 Found at: doi:10.1371/journal.pone.0004881.s002 (0.06 DOC) MB MB Figure S1 Found at: doi:10.1371/journal.pone.0004881.s003 (2.38 MB EPS) PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4881