S9 Targets PI3K/mTOR/Tubulin Figure 2. S9 inhibits the kinase catalytic activity of PI3K and mTOR. A–B) Active kinases were immunoprecipitated from exponentially growing Rh30 cells and kinase assays were performed. In the presence of indicated concentrations of S9, phosphorylation PIP2 to PIP3 were measured by ELISA (A). Phosphorylation of 4E-BP1 (B) was detected by Western blot. Band intensity was quantitated by optical densitometric analysis and normalized to vehicle control. Representative images were presented and relative kinase activity was plotted as mean6SD of three independent experiments. C) S9 abrogates rapamycin-induced hyperphosphorylation of Akt (Ser 473). Rh30 cells were starved overnight and treated with 10 mM of S9 and/or rapamycin (Rapa) for 1.5 h on the next day, followed by stimulation with 50 ng/ml EGF for 10 min. Phosphorylated Akt at Serine 473 and Threonine 308 and total Akt were detected by Western blot. D) Rh30 cells were exposed to 10 mM camptothecin (CPT) for 1 h after pre-incubation with indicated concentrations of S9 or wortmannin (Wort) for 30 min. Cells were collected and c-H2AX levels were detected with Western blot analysis. E–F) The binding mode of S9 within PI3K (E), mTOR (F). The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. All of the structural diagrams were prepared using PyMOL (http://pymol.sourceforge.net). doi:10.1371/journal.pone.0004881.g002 family members [16], corresponds to Lys833 in PI3K. These results provide us a clue that S9 may directly target PI3K and mTOR. S9 perturbs microtubule skeleton via binding to colchicine-binding sites of tubulin S9 arrests tumor cells in M phase. As a common consequence of PI3K-Akt-mTOR signaling inhibition, decreased expression of cyclin D1 will blockade cell cycle transition in G0/ G1 phase [17,18]. To explore the effect of S9 on cell cycle distribution, SKOV-3 cells were treated with indicated concentrations of S9 for 24 h. As shown in Figure 3A, S9 unexpectedly arrested cells in G2/M phase in a concentration- dependent manner up to 5 mM. Cell population in G2/M phase decreased, accompanied with increase in cell population in G1 phase in the presence of 10 mM of S9, which could be a result from PI3K-Akt-mTOR inhibition. S9 also arrested SK-OV-3 cells in G2/M phase time-dependently (Fig. 3A, lower panel). To examine whether the S9-induced G2/M arrest was reversible, SKOV-3 cells were treated with S9 for 16 h (T16) and then S9 was washed out, followed by 24 h (R24) additional incubation in fresh media. We found that the cells arrested in G2/M phase could not re-enter the normal cell cycles progress (Fig. 3B), indicative of irreversible G2/M phase arrest by S9. To differentiate whether S9 induced G2 phase or M phase arrest, SKOV-3 cells were treated with S9 and stained with anti- Cyclin B1 antibody. Persistent residence of cyclin B1 in nuclei is an indication of cell mitosis block, as well as a marker to distinguish M PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4881