3-(4, 5-Dimethylthiazol–2-yl)–2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), 4’,6-diamidino–2-phenylindole (DAPI), and 2’,7’-dichlorofluorescein diacetate (DCFH-DA) were from Sigma-Aldrich. Dulbecco’s Modified Eagle Medium (DMEM, High Glucose) and Minimum Essential Medium were purchased from Thermo Fisher Scientific. MRC–5, HeLa and MCF–7 were purchased from cell culture center of Chinese Academy of Medical Sciences, the Institute of Basic Medical Sciences (Beijing, China). H1299 and UC2 were the gifts from Shi Cui-juan (Tianjin Medical University). Apoptosis detection kit was purchased from BD Biosciences. Antibodies against p53, p21, cyclin A1, cyclin A2, Bax, Bcl–2 and GAPDH were purchased from ABclonal.
2.2.1 Synthesis of the compounds
The bis(tri-tert-butylphosphine)palladium(0) (30.6 mg, 0.06 mmol), cesium fluoride (334 mg, 2.2 mmol) and 4- bromophenol (2.5 mmol) containing different substituents were added into a round-bottom flask at room temperature, and the tributylvinyltin (0.3 mL, 1 mmol) and toluene (2.0 mL) were then added by syringe. The solution was heated and refluxed for 6 h under argon. The reaction liquid was cooled to room temperature, KF (500 mg) and ethyl acetate (5 mL) were added and stirred for 30 min, then extracted with ethyl acetate for three times. The organic phases were washed with a solution of saturated KF and water, dried over Na2SO4. The solvent was evaporated and the residue was purified by column chromatography (silica gel; petroleum/ethyl acetate 5/1) to provide compound 1–4.
1H NMR and 13C NMR of compound 1–4
Compound 1 (E)-–4,4’-(ethene–1,2-diyl)bis(3,5-dimethylphenol): m.p.: 224–226℃; 1H NMR (400 MHz, acetone-d6): δ = 8.02 (s, 2H), 6.59 (s, 4H), 6.45 (s, 2H), 2.34 (s, 12H); 13C NMR (100 MHz, acetone-d6), δ = 156.6, 138.0, 132.1, 130.0, 115.6, 21.8.
Compound 2 (E)-–4,4’-(ethene–1,2-diyl)bis(2,6-dimethylphenol): m.p.: 249–251℃; 1H NMR (400 MHz, acetone-d6): δ = 7.29 (s, 2H), 7.14 (s, 4H), 6.88 (s, 2H), 2.24 (s, 12H); 13C NMR (100 MHz, acetone -d6), δ = 153.6, 130.5, 127.2, 126.5, 124.8, 16.6.
Compound 3 (E)-–4,4’-(ethene–1,2-diyl)bis(2,5-dimethylphenol): m.p.: 299–300℃; 1H NMR (400 MHz, acetone-d6): δ = 8.09 (s, 2H), 7.38 (s, 2H), 7.04 (s, 2H), 6.65 (s, 2H), 2.30 (s, 6H), 2.19 (s, 6H); 13C NMR (100 MHz, acetone -d6), δ = 153.6, 130.5, 127.2, 126.5, 124.8, 16.6.
Compound 4 (E)-–4,4’-(ethene–1,2-diyl)bis(2,3-dimethylphenol): m.p.: 301–303℃; 1H NMR (400 MHz, acetone-d6): δ = 8.15 (s, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.05 (s, 2H), 6.73 (d, J = 8.4 Hz, 2H), 2.28 (s, 6H), 2.17 (s, 6H); 13C NMR (100 MHz, acetone -d6), δ = 155.2, 136.1, 130.2, 127.7, 124.7, 123.3, 113.3, 15.8, 12.1
2.2.2 Cell culture
The cell lines were cultured in DMEM (MCF–7, HeLa, H1299, and UC2 involved) and MEM/NEAA (MRC–5) respectively, with 10% (v/v) fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 units/mL), and kept at 37 °C in a 5 % CO2 atmosphere. The cell lines were regularly subcultured.
DMSO, as the effective solvent, was used to dissolve the compounds at first. The concentration of DMSO in cell suspension was less than 0.1 % (v/v).
2.2.3 Cell viability assay
The cell lines (MCF–7, HeLa, H1299, MRC–5 and UC2 involved) viability was evaluated by the MTT assay [18–19]. The initial densities were 2.5×104 cells/mL. After cell culture for 24 h, the culture medium was replaced, and the cells were incubated with target compounds at gradient concentrations (1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, four wells were used for each concentration, and repeat 3 times) for 72 h in 96-well flat microtiter plates. Subsequently, 100 μL culture medium containing 10% MTT was added to each well. Incubation for another 4 h at 37 ℃ in the dark, remove the culture medium, and add 100 μL DMSO in each cell. The absorbance was determined at 570 nm by a microplate reader (Thermo Fisher 1510).
2.2.4 Cell apoptosis analysis
Cell apoptosis assay was carried out by flow cytometry to detect labelled annexinV- fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI). The initial densities of MCF–7 cells were 2×105/mL in six-well plates for 24 h (1.5×105/mL for 48 h). Then the culture medium was replaced by indicated concentrations of compound 1 (control, 5 μM, 10 μM and 20 μM) for another 24 h / 48 h. After incubation, the cells were collected, washed twice with pre-cooling PBS, and stained with annexin V-FITC/PI for 15 min in the dark. The cells were evaluated by flow cytometry (BD FACS Calibur).
2.2.5 Nuclear Staining with DAPI
The morphological changes of nucleus were observed by fluorescence microscopy with DAPI staining. Firstly, 8×104/mL MCF–7 cells were planked in a 6-well plate with slide in each cell for 24 h. Later, the cells were treated with gradient concentration (control, 5μM, 10μM, 20μM) of compound 1 for another 24 h. Then the treated cells were fixed with 4 % paraformaldehyde overnight. DAPI staining for 10 min and the cells were determinated by fluorescence microscopy (Olympus BX41).
2.2.6 Cell Cycle analysis
MCF–7 cells (2×105 /mL density) were seeded in six-well plates for 24 h. Discarding the culture medium, the MCF–7 cells were treated by different concentrations of compound 1 for another 24 h (control, 5μM, 10μM, 20μM) or 48 h (control, 5μM, 10μM, 20μM) respectively. Then the cells were collected and fixed with 70% ethanol at 4 °C overnight. Washed the cells with pre-cooling PBS later, and incubated with PI staining buffer for 30 min in the dark. Then the cell cycle distribution was analyzed by flow cytometry (BD FACSCalibur).
2.2.7 Measurement of ROS
DCFH-DA produces fluorescent signal after oxidation by ROS, which is used to reflect the intracellular level of ROS. MCF–7 cells were seeded in a 6-well plate for 24 h, and were treated with gradient concentration (control, 5μM, 10μM, 20μM, 30μM and 40μM) of compound 1 for another 6 h or 9 h. Discarded the culture medium, collected and washed the MCF–7 cells, which were resuspended in PBS (3μM DCFH-DA containing) for 0.5 h at 37 °C in the dark. Then washed the cells by PBS and detected the fluorescence intensity by flow cytometry (BD FACSCalibur) at once.
2.2.8 Western blot analysis
The lysates of treated MCF–7 cells with different concentration of compound 1 were extracted by RIPA Lysis Buffer containing PMSF (P0020, Solarbio, China) following the instructions. After centrifugation, the protein supernatants were collected, and the protein concentration was measured by BCA protein assay kit (R0020, Beyotime Institute of Biotechnology, China). Then the SDS-PAGE loading buffer (1:1 v/v) was added into the proteins sample, and boiled for 5min. The extracted proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane by a wet transfer method. After blocking with 5% nonfat milk in TBST for 1.5 h at room temperature, the PVDF membrane was incubated with primary antibodies (1:2000 against p53, p21, Bax, Bcl–2, Cyclin A1, Cyclin A2 and 1:10000 against GAPDH) overnight at 4 °C, followed by the incubation with secondary antibody at room temperature for another 1.5 h. The immunoblots were visualized with an Ultra ECL kit (LK-U1421, MultiSciences Biotech Co., Ltd, China).
2.2.9 Statistical Analysis
All experiments were repeated at least three times. The results were shown as mean ± SD and the differences were analyzed by ANOVA using SPSS 22.0.