Chemicals and reagents
HES, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), and 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The primers of cyclinD1 and cyclinE for real-time polymerase chain reaction (PCR) were purchased from Genscript (Nanjing, Jiangsu, China). Antibodies against GADD153/CHOP, GRP78, cytochrome c, apoptosis-inducing factor (AIF), cleaved-caspase-3, cyclinD1, cyclinE1, cyclin-dependent kinase 2 (CDK2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA). Fluorescence-conjugated secondary antibodies and Dulbecco’s modified Eagle’s medium were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were obtained in the highest purity commercially available.
Human cervical cancer HeLa cells and human colon cancer HT-29 cells were obtained from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % (v/v) fetal calf serum, 100 μg/mL streptomycin, and 100U/mL penicillin. Cultures were maintained at 37°C in a humidified incubator in an atmosphere of 5 % CO2. Confluent cells were used for the following experiments.
MTT cell proliferation assay
Cell proliferation was determined by MTT assay. In brief, HeLa cells and HT-29 cells in logarithmic growth phase were seeded into 96-well plates at 1 × 104 cells/well followed by incubation at 37 °C for 24 h to allow attachment. Then the cells were treated with HES (0, 20, 40, 60, 80, and 100 μM) for 24, 48, or 72 h. Six wells were included in each group. MTT (20 μL of 5 mg/mL) was added to each well and incubated at 37 °C for 4 h. The supernatant was discarded and the formazan precipitates were dissolved in 150 μL of dimethyl sulfoxide (DMSO) by gentle shaking for 10 min. After dissolution, absorbance (A) was measured at 490 nm on a microplate reader (Tecan, Meilen, Zurich, Switzerland). Background absorbance of the medium without cells was subtracted from all experimental samples. Percent viability was calculated as [value of drug-treated group (A)/control group (A)] × 100 %. Each assay was carried out three times, and the results were expressed as the mean (± SEM).
Detection of apoptotic cells by Hoechst 33342 staining and DNA laddering fragmentation
The apoptosis of HeLa cells and HT-29 cells was detected using the Hoechst 33342 assay kit (Beyotime Institute of Biotechnology, China). HeLa cells (2 × 105 cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then the attached cells were washed with phosphate buffered saline (PBS) and fixed with freshly prepared 4 % paraformaldehyde for 30 min. After fixation, the cells were washed with PBS and incubated with Hoechst 33342 staining solution for 5 min. After staining, cells were washed with PBS and anti-fade mounting medium (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) was added, then the cells were viewed with a fluorescence microscope (Nikon Corporation, Tokyo, Japan). Apoptosis, as indicated by condensed and fragmented nuclei, was observed and recorded with the fluorescence microscope.
The HeLa cells (2 × 105 cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then the attached cells were washed with PBS and the DNA was isolated from HES-treated and control cells use DNA isolation kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China), separated by 1.0 % agarose gel electrophoresis, viewed and photographed by an ultraviolet light gel documentation system.
Detection of ROS, intracellular Ca2+ concentrations and mitochondrial membrane potential (ΔΨm) in HeLa cells by flow cytometry
HeLa cells (1 × 106cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then cells were harvested for detection of ROS, intracellular Ca2+ concentration, and ΔΨm.
The level of ROS in HeLa cells was examined by flow cytometry (Becton Dickinson Corporation, USA), using DCFH-DA (Sigma). The cells were harvested and washed twice with PBS. The cells were then re-suspended in 500 μL of DCFH-DA (10 μM), incubated at 37 °C for 30 min, and the level of ROS in the HeLa cells was examined by flow fluorescence activated cell sorting (FACS).
The level of intracellular Ca2+ in HeLa cells was determined by flow cytometry, using Indo 1/AM (Calbiochem; La Jolla, CA, USA). The cells were harvested and washed twice with PBS. The cells were re-suspended in Indo 1/AM (3 μg/mL), incubated at 37 °C for 30 min, and then analyzed to detect the changes of cytoplasmic Ca2+ levels using flow cytometry.
The ΔΨm in HeLa cells was determined by flow cytometry using 3, 3-dihexyloxacarbocyanine iodide (DiOC6) (4 μM). The cells were harvested, washed twice, re-suspended in 500 μL of DiOC6 (4 μM) and incubated at 37 °C for 30 min before being analyzed by flow cytometry to detect the changes in ΔΨm.
To detect ROS, intracellular Ca2+ concentration, and ΔΨm in Hela cells pre-treated with BAPTA or NAC, 1 × 106cells/well of HeLa cells were plated into a 6-well plate and pre-treated with BAPTA (a Ca2+ chelator, 10 μM) or NAC (a ROS inhibitor, 1 mM) before adding 80 μM of HES for 24 or 4 h incubation. Then levels of ROS, intracellular Ca2+concentrations, and ΔΨm were measured using flow cytometry as the same methods described above.
Cell cycle assay by flow cytometry
The distribution of HeLa cells in the phases of the cell cycle was quantified using flow cytometry. HeLa cells (1 × 106 cells/well) were seeded into a 6-well plate and treated with HES (0, 40, 80, and 160 μM) for 48 h. Then the cells were harvested by treatment with trypsin and centrifuged at 7500 rpm for 5 min. The cells were washed with PBS, and stained with 500 μL of propidium iodide in the dark at room temperature for 15 min according to the manufacturer’s protocol (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The cells were then analyzed by flow cytometry. The percentage of cells in the different cell cycle phases (G0/G1, S, and G2/M phase) was calculated using Coulter Epicx XL-MCL DNA analysis software.
Western blot analysis
Following treatment with HES (0, 40, 80, and 160 μM) for 48 h, HeLa cells were washed with ice-cold PBS and collected in lysis buffer (50 mM Tris, pH 7.4, 150 mM sodium chloride, 1 % NP-40, 0.25 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1 μg/mL leupeptin). The supernatant was obtained by centrifugation at 13,500 rpm for 20 min. Total protein was extracted and protein concentration was determined by the Bradford assay.
For Western blots, 120 μg of protein from each sample were subjected to electrophoresis on 12 % SDS-PAGE and separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5 % non-fat milk powder (w/v) at room temperature for 2 h, then incubated with the primary antibodies against GADD153/CHOP (1:500), GRP78(1:500), cytochrome c (1:500), AIF (1:500), cleaved-caspase-3 (1:500), cyclinD1 (1:500), cyclinE1 (1:500), CDK2 (1:500), GAPDH (1:1000), and β-actin (1:500) at 4 °C overnight. After washing, the membrane was incubated with fluorescence-conjugated secondary antibodies (anti-rabbit or anti-mouse, 1:10,000) at room temperature for 50 min. GAPDH or β-actin was used as an internal control to account for protein loading and transfer from the gel to the membranes. Bands on the Western blots were quantified using the Odyssey infrared imaging system (LI-COR, USA). All results represent of three independent experiments.
Data were reported as means ± SEM of at least three independent experiments. For statistical analysis, one-way ANOVA was used for comparison of one variance among groups and two-way ANOVA was used for comparison of two independent variances among groups followed by the Tukey’s post hoc test. P values less than 0.05 were considered significant.