HeLa (CCL-2), HCT116 (CCL-247), A549 (CCL-185), A427 (HTB-53), WI-38 (CCL-75) and CCL-211 cell lines were purchased from American Type Culture Collection (Manassas, VA). Cells were grown in complete growth medium (Dulbecco's modified Eagle's medium for HeLa, A549 and CCL-211; Eagle's Minimum Essential Medium for WI-38; Roswell Park Memorial Institute's medium for HCT116 and A427) supplemented with 10% fetal bovine serum, 10 units/ml penicillin and 10 μg/ml streptomycin at 37°C and 5% CO2.
A natural product library NPL 400 (Timtect Inc., Newark, DE) was used to screen possible CK2 inhibitors. This library is composed of 400 highly pure, rationally selected drug-like small-molecule compounds with molecular weights ranging from 183 to 832 Da. All compounds were 0.5 mg powder form in individual wells of 96-well plates. After dissolved with 100 μL of dimethyl sulfoxide (DMSO), compounds were stored at -20°C with final concentrations of 5 mg/ml. TBB was purchased from Sigma-Aldrich Co (St. Louis, MI).
Cell proliferation and viability assay
The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI) was used to evaluate growth of normal and cancer cells after treatment by different compounds. Exponentially growing cells were plated in 96-well microtiter plates at 5 × 103 cells/well with indicated concentrations of compounds. After incubation with indicated amount of compounds for 48 hours, 20 μl of the CellTiter 96® AQueous One Solution Reagent was added directly to culture wells. Absorbance at 490 nm was recorded with a 96-well plate reader after 2 hours incubation.
CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) was used to evaluate the cytotoxicity of hematein. After incubation with indicated amount of compounds for 48 hours, 100 μl of the CellTiter-Glo reagent was added directly to culture wells. Following procedures recommended by the manufacture, the luminescence produced by the luciferase-catalyzed reaction of luciferin and ATP was measured using a laminator.
The CK2 Kinase Assay/Inhibitor Screening Kit (CycLex Co. Ltd., Japan) was used for further screening of compounds for their CK2 inhibition effects in vitro
according to manufacture's manual[16
For determination of the dose dependent inhibition response of hematein, Casein Kinase 2 Assay Kit (Millipore, Bedford, MA) was used according to manufacture's protocol. Kinase assay was carried out in the presence of increasing amount of hematein in a final volume of 50 μl containing 20 mM MOPS (pH 7.2), 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 15 mM Mgcl2, 200 μM for CK2 substrate peptide: RRRDDDSDDD (Millipore, Bedford, MA), 0.05 μg purified active CK2 (Millipore, Bedford, MA) and [γ-33P]-ATP. After incubation in 30°C for 20 minutes, assay was stopped by adding of 20 μl 4% trichloroacetic acid and transferred 25 μl to P81 phosphocellulose squares. After washing with 0.75% phosphoric acid for 6 times and with acetone for 1 time, phosphocellulose squares were dried and transferred to scintillation vials for counting.
Western blot analysis
After treated with indicated concentration of hematein for 48 hours, whole cell protein were extracted from A549 cells with M-PER Mammalian Protein Extraction Reagent (Pierce, Rockfold, IL) added with Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA) and Complete Protease Inhibitor Cocktails (Roche, Switzerland) according to manufactures' protocols. The proteins were used for further CK2 kinase activity assay or western blot analysis. For western blot analysis, the proteins were separated on 4–15% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Following primary antibodies: Akt, PARP (Cell Signaling Technology, Danvers, MA), phospho-Akt S129 (Abcam Inc., Cambridge, MA) and β-actin (Sigma, St. Louis, MO) were used. After binding to indicated secondary antibodies, an enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare Life Sciences, Piscataway, NJ) was used for antigen-antibody detection.
The occurrence of apoptosis was determined by ApoTarget™ annexin V-FITC kit (BioSource International, Inc., Camarillo, CA). Briefly, cells were treated with indicated amounts of DMSO or hematein for 48 hours. The cells were washed with cold PBS and then re-suspended in binding buffer. The cells were aliquot to 100 μl in a concentration of 1 × 106/ml. After adding 5 μl of annexin V-FITC and 20 μl of PI and incubated in dark for 15 minutes, the cells were resuspended in 400 μl of binding buffer. Accuri's C6 Flow Cytometer™ System (Accuri Cytometers, Ann Arbor, MI) was used for analysis.
The data shown represent mean values ± standard error of deviation (SD). Student's t-test was used for comparing of cell viability in different treatments. Statistical analysis was carried out using SPSS (version 10.0, Chicago, IL). Significance was defined as p < 0.05 with two sided analysis. The half maximal inhibitory concentration (IC50) values was determined using GraphPad Prism® log (inhibitor) vs. response (variable slope) software (version 5, La Jolla, CA).