Cell lines and drugs
Human cancer cell lines of colorectal (HCT-8 and HT-29), head and neck (FaDu and A253) and prostate (PC3 and C42) were purchased from American type cell culture (ATCC, Manassas, VA) and maintained in RPMI 1640 with 10% fetal bovine serum (FBS). The cell lines were tested regularly using Stratogene's mycoplasma plus PCR Primer set (La Jolla, CA) and they were free from Mycoplasma. SN-38, docetaxel, 5-FU, paclitaxel, oxaliplatin, lithium chloride (LiCl) and cycloheximide (CHX) were purchased from Sigma Aldrich (St. Louis, MO). MSeA (CH3SeO2H) was purchased from PharmaSe Inc. (Lubbock, TX). Toptecan was obtained from GlaxoSmithKline (Durham, NC). Puromycin dihydrochloride, plasmid transfection medium and transfection reagents were purchased from Santa Cruz biotechnology Inc. (Santa Cruz, CA).
Schedules and drug doses
Cells were treated with various doses of MSeA (0.05, 0.1, 0.5, 1, 5 and 10 μM) for various times (2, 4, 6, 8, 16 and 24 h). MSeA and SN-38 or docetaxel were given alone or in sequential combination. In sequential combination, 2 h treatments with SN-38 (0.7, 1.3, 0.07 and 0.3 μM against HCT-8, HT-29, FaDu and A253 respectively) or docetaxel (2 nM against PC3 and C42) started 22 h after treatment with MSeA (1 or 5 μM). HCT-8 parental and β-catenin knockout cells were treated with SN-38 (2 h), docetaxel (2 h), paclitaxel (24 h), oxaliplatin (24 h), 5-FU (24 h) and topotecan (2 h) using various doses. The doses were for SN-38 (0.5, 1 and 5 μM), for docetaxel (0.05, 0.1 and 0.5 μM), for paclitaxel (0.5, 1 and 5 μM), for oxaliplatin (1 and 5 μM), for 5-FU (50, 100 and 500 μM) and for topotecan (0.5 and 1 μM). LiCl was applied for 24 h in multiple doses (0.5, 1, 5, 10, 20 and 25 mM) alone or in combination with MSeA (5 μM).
Cycloheximide (CHX) was applied for 5, 10, 20, 30 minutes and 24 hours at a nontoxic concentration of 100 μM alone or in combination with MSeA (5 μM). Puromycin dihydrochloride was used at concentration of 20 μM for clones' selection.
Preparation of cytoplasm and nuclear extract
Cytoplasm and nuclear extracts were prepared as previously described . Briefly, to obtain cytoplasm extract, untreated and treated HCT-8 cells were harvested and suspended in lysis buffer (0.08 M KCl, 35 mM HEPES, pH 7.4, 5 mM potassium phosphate, pH 7.4, 5 mM MgCl2, 25 mM CaCl2, 0.15 M sucrose, 2 mM PMSF, 8 mM dithiothreitol). After overnight storage at -80°C, cells were passed through a 28-gauge needle, centrifuged and the supernatant collected to represent the cytoplasm extract. The remaining pellet was re-suspended in lysis buffer, sonicated, centrifuged and the supernatant collected to represent the nuclear extract.
Silencing the expression of β-catenin
HCT-8 cells were utilized to generate a stable transfection using small hairpin β-catenin RNA (β-catenin ShRNA) purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Transfection was carried out following the manufacture's instructions. Briefly, HCT-8 cells were plated 5 × 105 cells/well (6-well plate) one day before the transfection. A well with 70-80% cells confluence was transfected with control shRNA Plasmid-A (a negative control) that encodes a scrambled shRNA sequence that does not inhibit β-catenin to generate HCT-8 scrambled control (HCT-8SC). Another well with the same cells confluence, was transfected with β-catenin shRNA plasmid DNA, a β-catenin-specific lentiviral vector plasmid to knock down expression and generate HCT-8 recombinant clones (HCT-8R). Clones of stable transfectants were selected using 20 μM of puromycin dihydrochloride. After selection, 10 individual clones were evaluated using western blots and the 2 clones (HCT-8RH7 and HCT-8RF4) that demonstrated the most effect of survivin suppression were selected for further studies.
Western blots analyses
Western blots were performed as described previously  to determine the effects on the intracellular protein levels. Briefly, untreated and treated cells were collected and digested using RIPA buffer (1 M Tris, 1 M NaCl, Triton X-100 and distilled water) with fresh protease inhibitor cocktail. Protein level was measured using Bio-Rad DC protein assay and a synergy HT spectrophotometer (BioTek Instruments, Winooski, VT). Equal amount of protein (50 μg) was loaded on 4-20% SDS-PAGE. After transfer, nitrocellulose membrane was rinsed with PBS-T, blocked with 5% milk and hybridized with the selected antibody. The following primary anti-bodies were used: anti-β-catenin, anti-GSK-3β (BD Biosciences, San Jose, CA) and anti-p-GSK-3β (cell signaling technology, Danvers, MA). The following secondary antibodies were used: goat anti-mouse IgG and goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with the primary and secondary antibodies, membrane was rinsed and incubated with chemilluminescence or enhanced chemilluminescence and developed using Kodak X-OMAT 2000A (First Source Inc., Rochester, NY). Anti-β-actin (Sigma Aldrich, St. Louis, MO) was used as loading controls.
Cell growth assay
Cell growth was evaluated using sulforhodlamine B (SRB) assay as previously described and performed . Briefly, after drug treatment, HCT-8WT, HCT-8SC and HCT-8RH7 and HCT-8RF4 cells were incubated in a drug-free medium for 5 days, fixed, washed and stained with SRB dye. The optical density of bound dye was measured at 570 nm using synergy HT multi-mode microplate reader (BioTek Instruments, Winooski, VT).
Each experiment has been repeated at least 3 times. Values were presented as the mean plus or minus standard deviation. Statistical analyses were performed comparing all treatments groups using unpaired t-student test. Significant difference between groups was noted when the p value was less than 0.05.