Kinase experiment:

The kinase assay is performed. Briefly, the reaction is carried out in the presence of 10 μCi of [γ-32P]ATP with each compound (e.g., 3CAI, 0.5, 1, 2 and 4 μM) 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 is stopped by adding 10 μL protein loading buffer and the mixture is separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The relative amounts of incorporated radioactivity are assessed by autoradiography[1].

Cell experiment:

HCT116 or HCT29 colon cancer cells are plated into 60-mm culture dishes (1×105 cells/dish) and incubated for 1 day in medium containing 10% FBS. The culture medium is then replaced with a 1% serum medium and cultured for 4 days with 3CAI (4 μM), I3C or a commercial AKT inhibitor. The cells are collected by trypsinization and washed with phosphate buffered saline (PBS). The cells are resuspended in 200 μL of binding buffer. Annexin V staining is accomplished. The cells are observed under a fluorescence microscope using a dual filter set for FITC and propidium iodide and then analyzed by flow cytometry[1].

Animal experiment:

Mice[1]Athymic mice [Cr:NIH(S), NIH Swiss nude, 6-9 wk old] are 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) are suspended in serum free McCoy's 5A medium and inoculated subcutaneously into the right flank of each mouse. 3CAI, I3C or vehicle is administered orally 5 times per week for 21 days. Tumor volume is calculated. Mice are monitored until tumors reach 1 cm3 total volume, at which time mice are euthanized and tumors are extracted.

Target: Akt

IC50: 1 μM

3CAI is a potent allosteric and specific inhibitor of AKT, which directly binds with AKT1 or AKT2 in an ATP noncompetitive manner and shows 60% inhibition of AKT1 kinase activity at 1 μM [1]. The serine/threonine kinase AKT includes three members, AKT1, AKT2, and AKT3. AKT plays a critical role in promoting transformation and chemoresistance through inducing proliferation and inhibiting apoptosis. Therefore, AKT is regarded as a potential target for cancer therapy.

In vitro: 3CAI (1 and 4 μM) significantly inhibited AKT1 kinase activity and decreased expression of AKT direct downstream targets including mTOR and GSK3β, respectively. 3CAI (4 μM) efficiently induced growth inhibition and apoptosis in HCT116 or HCT29 colon cancer cells [1].

In vivo: 3CAI (30 mg/kg, oral administration) significantly suppressed colon cancer growth in an in vivo xenograft mouse model and inhibited the expression of AKT-target protein such as mTOR and GSK3βin HCT116 colon tumor tissues [1].

Reference:
1. Kim DJ, Reddy K, Kim MO, Li Y, Nadas J, Cho YY, et al. (3-Chloroacetyl)-indole, a novel allosteric AKT inhibitor, suppresses colon cancer growth in vitro and in vivo. Cancer Prev Res (Phila). 2011;4(11):1842-51.