S9 Targets PI3K/mTOR/Tubulin Figure 3. S9 arrests tumor cells in M phase. A) S9 induces does-dependent and time-dependent G2/M phase arrest. SK-OV-3 cells grown in serum-supplemented medium were treated with S9 for 24 h. Cells were collected and subjected to FACS analysis. Data shown are percentage of cell population in G2/M phase. B) S9-induced G2/M phase arrest is irreversible. SK-OV-3 cells were treated with S9 for 16 h (T16), and then S9 was washed out. Cells were further incubated in fresh medium for 24 h (R24). Cells were collected and DNA content was analyzed by FACS analysis. C) Aggregation of cyclin B1 in nucleus after S9 treatment. SK-OV-3 cells were exposed to 5 mM S9 for 24 h. Cells were fixed and stained with anti- cyclinB1 antibody (green) and DAPI (blue). D) S9 decreases the level of phosphorylated cyclin B1 and increases phosphorylation of cdc25C. SK-OV-3 cells were exposed to 5 mM S9 for 24 h and western blot analyses for p-cyclinB1 (Ser 147), total cyclinB1, p-cdc25C (Ser 216) and cdc25C were performed. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a loading control. doi:10.1371/journal.pone.0004881.g003 phase arrest from G2 phase arrest [19]. Most of cylclin B1 aggregated in nuclei after S9 treatment, indicating that S9 induced M phase arrest (Fig 3C). S9 treatment also resulted in decreased phosphorylated cyclin B1 at Ser 147 and increased phosphorylated cdc25c at Ser 216 and total cyclin B1 (Fig 3C), which is in accordance with those agents that induce M phase arrest [20,21]. S9 disturbs cytoskeleton. The compounds which arrest cells in M phase are mostly tubulin-targeting agents. We tested whether S9 disrupted the organization of cytoskeleton. The microtubule structure of S9-treated cells was visualized via immunocytochemistry. As shown in Figure 4A, microtubule formed intact network with fine mesh in untreated cells, while in S9-treated cells, tubulin was dispersed as dot, and cells appeared as weaker staining of anti-tubulin antibody which was presumably due to reduction of microtubular materials by depolymerization resulted from S9 treatment (Fig. 4A). S9 inhibits tubulin polymerization in vitro and causes a conformational change in tubulin. As S9 has been shown to disturb microtubule cytoskeleton in SKOV-3 cells, we tested the effects of S9 on tubulin polymerization in vitro. The polymerization of purified tubulin at 37uC in the presence of tested compounds or vehicle control was monitored spectrophotometrically. In the absence of compounds, purified tubulin (15 mM) was polymerized in a time-dependent manner. S9 treatment inhibited tubulin polymerization in a concentration-dependent manner (Fig. 4B). The effect of S9 on the second structure of tubulin was monitored by far-UV CD spectroscopy. As shown in Figure 4C, 25 mM S9 induced a secondary structure change of tubulin, characterized by the alteration of a-helix and b-sheet content. S9 directly bind to colchicine-binding site on tubulin. Because S9 inhibits tubulin polymerization and causes conformational change of tubulin, direct binding of S9 to tubulin was next determined using SPR biosensor technology, with a well-characterized tubulin destabilizer colchicine as a positive control. Various concentrations (50.0, 35.0, 24.5, 17.2, 12.0, 8.40, 5.88 mM) of S9 and colchicine were injected for binding with tubulin immobilized on the CM-5 sensor chips. Real time sensor grams were recorded. As shown in Figure 4D, injection of S9 or colchicine caused a significant increase in SPR response in a concentration-dependent manner. General fitting and steady-state binding affinity calculated with Biacore evaluation Version 3.0 software helped release a relative high binding affinity for tubulin with the dissociation constant (Kd) value of 11.1 mM, which was two-fold lower than that of colchicine (24.6 mM). Most compounds that inhibit tubulin polymerization bind to either colchicine- or vinblastine-binding sites[21]. We investigated whether S9 binds to either of these two sites via competitive binding assays. The intrinsic fluorescence of colchicine increases upon binding to tubulin [22], which could be used as an index for S9 competition with colchicine in tubulin binding. As shown in Figure 4E, left panel, the fluorescence of colchicine-tubulin complex reduced in the presence of S9 in a dose-dependent manner, suggesting that colchicine failed to bind to tubulin-S9 complex. In contrast, the fluorescence remained unchanged when vincristine, a typical vinblastine-binding site drug, was incubated with colchicine and tubulin. A fluorescent analogue of vinblastine (BODIPY FL-vinblastine) was employed to detect whether S9 bound to vinblastine-binding site. S9 had no effect on the PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4881