have been manifested to sensitize tumor cells to apoptosis induced by cytotoxic drugs including DNA damaging agents and tubulin destabilizer [25]. This principle of concept appeals a surge of interests in discovery of dual cytostatic and cytotoxic identities. In current study, S9 induces rapid apoptosis in a number of tumor cells. S9 also displays potent antiproliferative activity in MDR cells, which is distinct in tubulin interfering drugs such as colchicine and vincristine (Fig.6B). Moreover, S9 overcomes drug resistance caused by low expression of 4E-BP1 or overexpression of Bcr-Abl (Fig 6B) and circumvents Akt activation triggered by mTOR inhibition. In contrast, S7, which failed to interfere the structure of tubulin but with similar potency in impairing PI3K- Akt-mTOR signaling, was less active against the proliferation of Rh30 cells compared to S9 (data not shown). All these favor S9 to possess synergistic cytostatic and cytotoxic efficacy and stand out distinctly from the established PI3K-Akt-mTOR inhibitors or known tubulin-interfering drugs. In summary, we herein show for the first time that S9 functions as a multi-inhibitor simultaneously targeting both PI3K-Akt- mTOR axis and microtubule cytoskeleton. The converged outcome of both cytostatic and cytotoxic paradigms of S9 in vitro and in vivo, together with its appreciable anti-MDR profiles, help prove to be a promising alternative template for rational designing of anticancer drugs. Given the family of PI3K inhibitors could impact such general processes as insulin signaling, diabetes, obesity, and aging, unwanted side-effects should be carefully considered. In regards to these potential toxicities, we believe that optimal drug candidates that is more potent with improved water- solubility derived from S9 must be evaluated extensively for both efficacy and safety in cellular and animal systems. Materials and Methods Chemicals Wortmannin, colchicine, vinblastine and vincristine were purchased from Sigma (St. Louis, MO), triciribine from Biomol (Plymouth Meeting, PA) and rapamycin from LclLab (Woburn, MA), fluorescent vinblastine (BODIPY FL-vinblastine) from Invitrogen (Eugene, OR). S9 (Fig. 1) was prepared as described previously [8] with a purity .99.5%. Each compound was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 10 mM. Aliquots were stored at 220uC. All the compounds were diluted to desired concentrations immediately before each experiment. Cell Lines and Culture Conditions Human colorectal carcinoma LOVO, human breast carcinoma MDA-MB-468, human prostate carcinoma PC3, human osteo- sarcoma U2OS cells were from American Type Culture Collection (ATCC, Manassas, VA); human lung adenocarcinoma NCI-H23 cells were obtained from the National Cancer Institute (NCI); rhabdomyosarcoma (RMS) Rh30 cells were generous gift from Dr. P.J. Houghton (St. Jude Children’s Research Hospital, Memphis, TN). MDA-MB-468, Rh30, LOVO, NCI-H23 and U2OS cells were grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS; GIBCO, Grand Island, NY), 2 mM L-glutamine, 100 U/ml benzylpenicillin, 100 mg/ml streptomycin and 10 mM HEPES (pH 7.4). PC3 cells were grown in Ham’s F12 (GIBCO, Grand Island, NY) with 10% FBS. CHO (pCORON1000-EGFP-Akt) was purchased from GE Healthcare (EGFP: Enhanced Green Fluorescence Protein) and propagated in F-12 Ham medium with Glutamax supplemented with 10% FBS, penicillin-streptomycin (50 U/ml) and geneticin (0.5 mg/ml, Sigma-Aldrich, St. Louis, MO). Rest of cells used in this study S9 Targets PI3K/mTOR/Tubulin were grown as described previously [26]. All cells were cultured in a highly humidified atmosphere containing 5% CO2 at 37uC. Western Blot analysis Rh30 and SK-OV-3 cells were seeded at density of 56105 cells/ well in six-well plates. Cells were washed twice with serum-free medium on the next day and further incubated in serum-free medium for 24 h. Cells were exposed to S9 or wortmannin for 1 h before stimulation with 50 ng/ml epithelial growth factor (EGF, R&D system, Minneapolis, MN) for 10 min. Cells were collected and subjected to western analysis as described [26], using antibodies against phosphorylated PDK1 at serine 241, phosphorylated Akt at threonine 308 (#9275) or serine 473 (#9271), phosphorylated mTOR at serine 2441 (#2971), phosphorylated p70S6K1 at threonine 389 (#9209) and phosphorylated 4E-BP1 at threonine 37/46 (#9459)or 70 (#9455) (Cell Signaling Technology, Wal- tham, MA). Membranes detected phosphorylated proteins were stripped with Re-blot plus mild solution (Chemicon International, CA) and re-blotted with antibodies against corresponding total PDK1 (#3062), Akt (#9272), mTOR (#2972), p70S6K1(#9202) and 4E-BP1 (#9452). For detection cH2AX, Rh30 cells were treated with DMSO or 10 mM camptothecin for 1 h following incubated with indicated concentrations of S9 or wortmannin for 30 min. Cells were collected and subjected to Western blot analysis using antibodies against c-H2AX (#9718). b-actin (#4967) or GAPDH (Kangchen, China) were used as loading control. Immunoprecipitation and Kinase Assays Rh30 cells were treated with EGF 50 ng/ml for 10 min after incubated in serum-free medium for 24 h. Cells were then lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 50 mM b-glycerophosphate, 10% glycerol (w/v), 1% Tween-20 (w/v), 1 mM EDTA, 10 mg/mL aprotinin, 1 mg/ml pepstatin A, 10 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, 20 nM microcystin-LR and 25 mM NaF. Cell lysates were clarified by centrifugation at 17,500 g for 15 min at 4uC. One mg of the cell protein was used for immunoprecipitation. Immuno-complexes were suspended in kinase buffer [10 mM HEPES (pH 7.4), 50 mM NaCl, 50 mM b-glycerophosphate, 10% glycerol (w/v), 10 mM MnCl2, 1 mM DTT]. Protein A/G agarose beads slurry was equally divided into 5 aliquots, mixed with vehicle or different concentrations of S9. mTOR kinase assay was performed with 50 mM ATP and 1 mg recombinant 4E-BP1 (A.G. Scientific, San Diego, CA). Reactions were terminated by adding 56SDS loading buffer and samples were separated on SDS PAGEs. Phosphory- lation of 4E-BP1 was visualized with Western blot analysis. Band optical densitometric (OD) analysis was performed using ImageJ (http://rsb.info.nih.gov//ij/) and relative kinase activity was calculated with the formula: ODdrugs/ODEGF6100%. The mean values were obtained from at least 3 independent tests. Enzyme Linked Immunosorbent Assays (ELISAs) Effect of S9 on PI3K activity was measured by PI3K ELISA kit (Echelon Biosciences Inc., Salt Lake City, UT) according to the manufacturer’s instructions. PI3K was immunoprecipitated using an anti-human PI3K p85 subunit antibody (Santa Cruz Biotech- nology, Inc., CA) and kinase assay was initiated in the presence of PtdIns(4,5)P2 substrate and ATP (25 mM). The relative PI3K activity was computed as (PIP3S9/PIP3control)6100%. Akt translocation Assay The CHO (pCORON1000-EGFP-Akt) cells were seeded in a 96-well black plate (Corning, NY 14831). On the next day, cells PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4881