2.1 Cell Lines
Human colon HCT116 and breast MCF7 cancer cell lines were purchased from ATCC (Manassas, VA, USA). Each cell line was grown in a recommended medium, McCoy’s 5A medium for HCT116 and low glucose Dulbecco's Modified Eagle Medium for MCF7, supplemented with 10% fetal bovine serum and penicillin/streptomycin solution.
2.2 Bromodeoxyuridine Incorporation
HCT116 or MCF7 cells were cultured in 12-well plates (3 × 104 per well) and incubated for 24 h at 37°C in a 5% CO2 incubator. After 24 h, cells were treated with bromodeoxyuridine (BrdU, Sigma-Aldrich, MO, USA). The extent of BrdU incorporation in cells was examined using a fluorescence microscope (Nikon, Melville, NY, U.S.A) after mounting with DAPI . BrdU positive cells were counted with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.3 Senescence-Associated β-Galactosidase Assay
The amounts of SA-β-gal in Sen-HCT116 and Sen-MCF7 cells for 5 days were determined according to the method of Dimri et al . Cells were examined using the fluorescence microscope after mounting with DAPI . SA-β-gal positive cells were counted with ImageJ software.
2.4 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] Assay
The IC50 of TQ and COS were determined by seeding of HCT116 or MCF7 cells in 12-well plates (3 × 104 per well) and incubated for 24 h at 37°C in a 5% CO2 incubator. At day 5 of 100 nM Dox treatment, cells were treated with TQ (0, 5, 10, 25, 50, 60, 75, or 100 µM dissolved in DMSO) or COS (0, 10, 25, 50, 75, 100, or 200 µM dissolved in DMSO), incubated for 24 h, and then treated with MTT reagent (1.25 mg/ml) and incubated for 2 h. The resulting formazan crystals were dissolved in 1 ml DMSO and the optical density was determined using a microplate reader at 570 nm .
2.5 Flow Cytometric Assessment
HCT116 or MCF7 cell lines were cultured in T25 flasks (30 × 104 cells per flask) for 24 h and then treated with 10 ml of TQ (50 µM) or COS (30 µM) in each flask for proliferative cells. For Sen-cells, the same counts of cells were cultured and treated with 100 nM of Dox (10 ml) for 5 days after which 10 ml of TQ (50 µM) or COS (30 µM) were added. Treated cells with their corresponding control flasks were trypsinized, washed with PBS and suspended in 70% ethanol until flow cytometry assessment of senescence (p53 and p21), apoptotic (Bax), and anti-apoptotic (Bcl2) protein markers using a BD Accuri C6 Plus flow cytometer (Becton Dickinson, San Jose, CA, USA). Cells were treated with FITC-conjugated and mouse-raised anti-p53 (Cat # 554298), anti-p21 (Cat # 612236), and anti-Bcl2 (Cat # 340650) produced by Becton Dickinson and anti-Bax (Cat # sc-7480) produced by Santa Cruz Biotechnology (Dallas, TX, USA) . Data were analyzed using Accuri C6 software.
2.6 Reverse Transcription Polymerase Chain Reaction (RT-PCR)
HCT116 or MCF7 cell lines either proliferative or senescent were cultured in T25 flasks and treated the same way as in the flow cytometry assessment. Total RNA in pelleted cells was extracted using a Total RNA Extraction Kit (iNtRON Biotechnology, Inc., Gyeonggi-do, South Korea). cDNA reverse transcription of the extracted 1 μg of RNA was performed with a TIANScript RT Kit (Tiangen, Shanghai, China). RT-PCR was performed with iCycler-iQ Optical System Software version 3.0 A (Bio-Rad, CA, USA). The following primers were used for the quantitative RT-PCR analysis: for caspase 3 (forward): CATGGAAGCGAATCAATGGACT and reverse: CTGTACCAGACCGAGATGTCA, and the housekeeping GAPDH forward primer sequence is GAAGGTGAAGGTCGGAGTCA and the reverse sequence is TTGAGGTCAATGAAGGGGTC.
PCR amplification was done with RealMOD™ Green FAST qPCR Master Mix (S) (Tiangen) using the following conditions: 1 cycle at 95°C for 30 s, 40 cycles at 95°C for 5 s, 1 cycle at 95°C for 5 s. The relative mRNA expression levels of caspase 3 gene were calculated using the comparative (Ct) method .
2.7 Statistical Analysis
Statistical analyses were done with GraphPad Prism 5 (San Diego, CA, USA) using One-way ANOVA (Tukey's Multiple Comparison Test). The IC50 values were determined with nonlinear regression using log (inhibitor) vs. response-variable slope equation. Data are presented as a mean ± SD. A value of P < 0.05 was considered statistically significant. All data show the mean results from at least three independent experiments.