Figure 1.
S9 inhibits PI3K-Akt-mTOR signaling.
A–B), S9 depresses EGF-triggered activation of PI3K-Akt-mTOR signaling pathway. Serum-deprived Rh30 cells (A) and SK-OV-3 cells (B) were treated with indicated concentrations of S9 for 1 h followed by EGF (50 ng/ml) stimulation for 10 min. Cells were harvested for Western blot analysis with antibodies specific for p-PDK1 (S241), PDK, p-Akt (T308), p-Akt (S473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP1(T37/64), p-4E-BP1 (T70), 4E-BP1 and actin. Arrows indicate p70 isoform of S6 kinase protein. C) S9 blocks Akt membrane translocation and membrane ruffling. CHO (pCORON1000-EGFP-Akt) cells seeded on chamber were starved for 2 h then treated with 5 or 10 µM S9 for 1 h followed by IGF stimulation for 5 min. Fluorescent pictures were captured with confocal fluorescent microscopy. White arrows indicate cell membrane ruffling. Blue arrows indicate fluorescent foci. D) Total Akt granule intensity in each CHO (pCORON1000-EGFP-Akt) cell was counted with statistic module by IN Cell Analyzer 1000. Data shown are representative from two independent experiments.
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 mean±SD of three independent experiments. C) S9 abrogates rapamycin-induced hyperphosphorylation of Akt (Ser 473). Rh30 cells were starved overnight and treated with 10 µM 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 µM camptothecin (CPT) for 1 h after pre-incubation with indicated concentrations of S9 or wortmannin (Wort) for 30 min. Cells were collected and γ-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).
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 µM 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 µM 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.
Figure 4.
S9 disturbs the microtubular structure by binding to tubulin directly and inhibiting tubulin polymerization.
A) S9 disturbs the cell skeleton structure. SK-OV-3 cells were exposed to 5 µM S9 for 24 h and stained with anti-tubulin antibody (green) and DAPI (blue). Scale bar: 30 µm. Data shown are representative of at least three independent experiments. B) Inhibition on purified tubulin polymerization by S9. Purified pork tubulin was incubated with S9, vincristine (VCR) or colchicine (COL) or vehicle in reaction buffer. The optical density (OD) at 340 nm was measured with a spectrophotometer every 1 min till the OD value became stable. C). Disruption secondary structure of tubulin by S9. Purified tubulin and 25 µM S9 were co-incubated in reaction buffer for 1 h. The circular dichroism (CD) spectra were recorded. The characterized absorbance wave lengths were 209/222 nm for α–helix and 196 nm for β–sheet. D) S9 (left panel) and colchicines (right panel) bind to tubulin directly. Purified tubulin was immobilized on sensor chip. Association and dissociation rate constants and real-time binding capacity were recorded by Surface Plasmon Resonance (SPR). The Kd values were computed by Biacore evaluation Version 3.0 software. E) Competitive binding of S9 to colchicine-binding sites on tubulin but not vinblastine-binding sites. Tubulin was co-incubated with indicated concentrations of S9 or vincristine for 1 h, then colchicine was added (left panel). Right panel, tubulin was co-incubated with S9 or vinblastine (VLB) for 1 h followed by addition of BODIPY-FL-vinblastine. The fluorescence was measured by spectrofluorometer. All assays were repeated twice and representative data were shown.
Figure 5.
The binding mode of S9 within tubulin.
The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. S9 and colchicine are also represented by sticks and colored in cyan and yellow, respectively. All of the structural diagrams were prepared using PyMOL (http://pymol.sourceforge.net).
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 mean±SD (µM) 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 mean±SD 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.