S9 Targets PI3K/mTOR/Tubulin Figure 6. S9 displayed anti-tumor activity in vitro and in vivo and induces rapid apoptosis in human cancer cells. A) Antiproliferative activity of S9 and wortmannin against a wide panel of tumor cell lines. IC50s plotted as mean6SD (mM) were from three separate experiments. B) Resistance factor (RF) of S9 and reference compounds in drug-resistant cancer cells. RF was calculated as the ratio of the IC50 value of the drug- resistant cells to that of the corresponding parental cells. C) S9 induces rapid apoptosis in human cancer cells. Rh30, A375, MKN-28 and U2OS cells were treated with S9 for 8 h. Apoptosis was measured by Annexin V-PI double staining. Apoptosis measured as Annexin-V positive cells were plotted. Data shown were mean6SD from three independent experiments. D) Rh30 (left panel) and SMMC7721 (right panel) xenograft growth inhibition after administration of S9. Rh30 and SMMC7721 nude mice were subcutaneously inoculated with a tumor fragment. The animals were randomly divided in groups when tumor volume reached 100 to 200 mm3 and received i.p. injection of normal saline or S9 five times a week for two weeks. Tumor volumes were measured and relative tumor volumes were calculated as tumor volumeDn/tumor volumeD0. Data shown are the mean of six mice. doi:10.1371/journal.pone.0004881.g006 altered a-helix and b-sheet contents in tubulin induced by S9 further confirmed its activity of microtubule perturbation. It has been reported that 2-phnylindole derivatives inhibited tubulin polymerization by binding to the colchicines site on tubulin [24]. Consistently, the 2-phenylindole moiety interacted with tubulin in our computational exercise, which occupies similar Cartesian space in the colchicine site of tubulin, releasing similar hydrophobic interactions to that of colchicine. Moreover, when the MOMO group on the side chain of 2-phnylindole was replaced by H in compound S7, no tubulin disturbance was observed even though S7 possesses similar potency with S9 against PI3K-Akt-mTOR signaling [8]. Thus, the hydrogen bond formed between Lys 254 and the MOMO group is essential for S9 to interact with tubulin. This scenario, together with the aforemen- tioned findings, helps understand that a smart manipulation of 2- phnylindole together with a-methylene-c-lactone might offer the appreciable selectivity and targetability for S9. The dramatic clinical efficacy of targeted inhibitors combined with the cytotoxic drugs has long been hoped to revolutionize the cancer therapy. In fact, inhibitors of PI3K-Akt-mTOR pathway PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4881