I3C (purity: 95%) was purchased from Sigma-Aldrich (St Louis, MO). 3CAI (purity: 95%), 5-methoxy-3CAI (purity: 95%), 5-fluoro-3CAI (purity: 95%) and 2-(4-(2-hydroxyethyl)piperazin-1-yl)-1-(5-methoxy-1H-indol-3-yl)ethanone) (purity: 95%) were purchased from InterBioScreen (Moscow, Russia). CNBr-Sepharose 4B beads were purchased from GE Healthcare (Piscataway, NJ). The active AKTs, active MEK1, active JNK1, active ERK1 human recombinant protein, histone H2B and H2AX for kinase assays were purchased from Millipore (Temecula, CA). The active TOPK human recombinant protein for the kinase assay was purchased from SignalChem (Richmond, BC). PI3K was obtained from Upstate Biotechnology (Lake placid, NY). AKT, p-AKT (Thr308), mTOR, p-mTOR (Ser2448), GSK3β, p-GSK3β (Ser9), Bad, Bcl2 and p-ASK1 (Ser83) and CDKN1A antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to detect p53 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LY294002 was purchased from Gibco BRL (Grand Island, NY). AKT inhibitor VIII was purchased from Merck KGaA (Darmstadt, Germany).
Synthesis of 3CAI
3CAI (purity: 95%) was synthesized as described (21
) and purity and structure were analyzed using HPLC and NMR.
All cell lines were purchased from American Type Culture Collection (ATCC) and were cytogenetically tested and authenticated before the cells were frozen. Each vial of frozen cells was thawed and maintained in culture for a maximum of 8 weeks. Enough frozen vials were available for each cell line to ensure that all cell-based experiments were conducted on cells that had been tested and in culture for 8 weeks or less. HCT116 and HT29 human colon cancer cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic.
The crystal structure of the pleckstrin homology (PH) domain of AKT1 was obtained from the RCSB Protein Data Bank, PDB entry 1UNQ (22
), which is a complex structure of the AKT1 PH domain and Ins(1,3,4,5)P4
and has an atomic resolution of 0.98 Å. The crystal structure was prepared using the Protein Preparation Wizard in Maestro v.9.2. Hydrogens were added to the protein structure consistent with a pH of 7. All water molecules in the crystal structure were removed. Then the crystal structure was minimized with an RMSD cutoff value of 0.3 Å. The structure of the AKT2 PH domain used in this study was modeled with the template structure of 1UNQ using Prime v.3.0. Energy grids for docking were computed for each protein structure using default settings in Glide v.5.7. 3CAI was prepared using LigPrep v.2.5 and then was docked into the PH domains of AKT1 and 2 with Glide extra precision (XP) mode.
Anchorage independent cell growth
Cells (8 × 103 per well) suspended in complete growth medium (McCoy's 5A supplemented with 10% FBS and 1% antibiotics) were added to 0.6% agar with different doses of each compound in a base layer and a top layer of 0.3% agar. The cultures were maintained at 37°C in a 5% CO2 incubator for 3 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v. 4) program (Media Cybernetics).
Western blot analysis
Cell lysates were prepared with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 × protease inhibitor tablet). Equal amounts of protein were determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Proteins were separated by SDS/PAGE and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with appropriate primary antibodies overnight at 4°C. After washing with PBS containing 0.1% Tween 20, the membrane was incubated with a horseradish peroxidase–conjugated secondary antibody at 1:5,000 dilution and the signal detected with a chemiluminescence reagent (Amersham Biosciences Corp).
In vitro pull-down assay
Recombinant human AKTs (200 ng) were incubated with 3CAI-Sepharose 4B (or Sepharose 4B only as a control) beads (50 μl, 50% slurry) in reaction buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 2 μg/mL bovine serum albumin). After incubation with gentle rocking overnight at 4°C, the beads were washed 5 times with buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40) and binding was visualized by Western blotting.
Cell proliferation assay
Cells were seeded (1 × 103 cells per well) in 96-well plates and incubated for 24 h and then treated with different doses of each compound. After incubation for 48 h, 20 μl of CellTiter96 Aqueous One Solution (Promega) were added and then cells were incubated for 1 h at 37°C in a 5% CO2 incubator. Absorbance was measured at 492 nm.
Colon cancer cells were plated into 60-mm culture dishes (1 × 105 cells/dish) and incubated for 1 day in medium containing 10% FBS. The culture medium was then replaced with a 1% serum medium and cultured for 4 days with 3CAI, I3C or a commercial AKT inhibitor. The cells were collected by trypsinization and washed with phosphate buffered saline (PBS). The cells were resuspended in 200 μl of binding buffer. Annexin V staining was accomplished following the product instructions (Clontech, Palo Alto, CA). The cells were observed under a fluorescence microscope using a dual filter set for FITC and propidium iodide and then analyzed by flow cytometry.
In vitro kinase assay
The kinase assay was performed in accordance with instructions provided by Upstate Biotechnology (Billerica, MA). Briefly, the reaction was carried out in the presence of 10 μCi of [γ-32P]ATP with each compound in 40 μl of reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, and 1 mM dithiothreitol. After incubation at room temperature for 30 min, the reaction was stopped by adding 10 μl protein loading buffer and the mixture was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The relative amounts of incorporated radioactivity were assessed by autoradiography.
Hematoxylin-eosin staining and immunohistochemistry
Tumor tissues from mice were embedded in a paraffin block and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry. Tumor tissues were de-paraffinized and hydrated then permeabilized with 0.5% Triton X-100/1 × PBS for 10 min, hybridized with Ki-67 (1:500) as the primary antibody and horse-radish peroxidase (HRP)-conjugated goat anti-rabbit or mouse IgG antibody was used as secondary antibody. After developing with 3, 3′-diaminobenzidine, the sections were counterstained with H&E. All sections were observed by microscope and the Image-Pro Plus software (v. 4) program (Media Cybernetics).
Xenograft mouse model
Athymic mice [Cr:NIH(S), NIH Swiss nude, 6–9 wk old] were obtained from Charles River and were maintained under “specific pathogen-free” conditions based on the guidelines established by the University of Minnesota Institutional Animal Care and Use Committee. Mice were divided into five groups: 1) untreated vehicle group (n = 15); 2) 20 mg 3CAI/kg of body weight (n = 15), 3) 30 mg 3CAI/kg body weight (n = 15); 4) 100 mg I3C/kg of body weight (n = 15); 5) no cells and 30 mg 3CAI/kg of body weight (n = 15). HCT116 cells (3×106 cells/100 μl) were suspended in serum free McCoy's 5A medium and inoculated subcutaneously into the right flank of each mouse. 3CAI, I3C or vehicle was administered orally 5 times per week for 21 days. Tumor volume was calculated from measurements of 2 diameters of the individual tumor base using the following formula: tumor volume (mm3) = (length × width × height × 0.52). Mice were monitored until tumors reached 1 cm3 total volume, at which time mice were euthanized and tumors were extracted.
All quantitative results are expressed as mean values ± S.D. Statistically significant differences were obtained using the Student's t test or by one-way ANOVA. A value of p < 0.05 was considered to be statistically significant.