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  • br Methodology br Compound and cell lines br The

    2020-08-12


    2. Methodology
    2.1. Compound and cell lines
    The compound 8-prenylnaringenin (Fig. 1A) was purchased from Santa Cruz Biotechnology, USA with molecular weight of 340.37 g/mol and purity > 98% (lot # A3014). Colon cancer (HCT-116) and normal colon (CCD-841) cell lines were purchased from ATCC, USA, whereas Dulbecco's modified Eagle's medium (DMEM/high glucose) was ob-tained from HyClone, USA. Fetal Bovine Serum (FBS) and penicillin-streptomycin were purchased from Biowest, France.
    2.2. Cell viability assay
    Cell viability of HCT-116 (colon cancer cells) and CCD-841 (normal colon cells) were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) assay [25]. Cells with density of 5000 cells/well were seeded in 96 transparent plates and incubated overnight at 37 °C. Cells were treated with different concentrations of 8-PN with the highest concentration of 100 μg/ml and serial dilution factor of 2 for 9 concentrations. The anticancer drug 5-fluorouracil (5-FU) was used as a positive control. Three wells in each plate were treated with the solvent (0.02% DMSO) and served as negative control. Cells were incubated for 24, 48 and 72 h at 37 °C in 5% CO2. Subse-quently, 20 μl of MTT was added to each well and further incubated for 4 h. Following the incubation process, the solution was aspirated and 100 μl of DMSO was added into each well. The cytotoxicity effect of 8-PN against HCT-116 and CCD-841 208538-73-2 was measured using microplate reader (Tecan, Infinite M1000) at 570 nm. Data were calculated as  Life Sciences 232 (2019) 116633
    percentage of cell inhibition using the following formula:
    Cell viability assay was performed in triplicates in three in-dependent experiments. The concentration of 8-PN with 50% cell growth inhibition was defined as the IC50 value, which was determined through use of a linear plot.
    The viability state of HCT-116 cells was further assessed by using acridine orange (AO) and propidium iodide (PI) fluorescent dye [26,27]. Briefly, 106 cells were seeded in 25 cm2 tissue culture flask and treated with the IC50 of 8-PN and incubated at 37 °C for 24, 48 and 72 h. Untreated flask was kept as a control. After that, cells were detached and washed with cold phosphate buffered saline (PBS) for three times. After centrifugation, the cell pellets were suspended in 100 μl of PBS, 10 μg/ml of AO and 10 μg/ml of PI. Fluorescence inverted microscope (Nikon, Japan) was used to detect the morphological changes of the cells in which green color stained cells represent normal or cells at early stage of apoptosis, while necrotic cells are stained with red color.
    Flow cytometry was used to determine the distribution of HCT-116 cells at different phases of the cell cycle in the presence of 8-PN. Content of DNA in the cells was detected with propidium iodide (PI) staining of HCT-116 cells using flow cytometry measurements [28]. Briefly, cells were seeded in a well plate at a density of 106 cells in a complete medium and were treated with 8-PN for different periods of time (24, 48 and 72 h). Negative control was treated with 0.02% DMSO. The cells were collected and fixed with 70% ethanol at −20 °C over-night. Cells were washed and stained with 500 μl of PI/RNase staining buffer (Becton Dickinson, USA) and then incubated for 30 mins at room temperature. Each experiment was conducted in triplicates. Cell cycle phase distribution was determined using BD FACSCanto II flow cyt-ometer instrument and BD FACS Diva software (Becton Dickinson, USA). Totally 15,000 events per sample were recorded for analysis. ModFit LT version 3.0 software was used for analyzing the data.
    Annexin V assay was used to study the cellular apoptosis of the treated and untreated cells. Conjunction of propidium iodide (PI) with annexin V helps to determine if the cells are dead, apoptotic or viable due to their cell membrane permeability and integrity [29,30]. In this assay 106 HCT-116 cells were seeded in a 6-well plate with complete medium at 37 °C overnight. Cells were treated with 8-PN at different time points (24, 48 and 72 h). Then, cells were washed with cold PBS and suspended in 1× binding buffer at the concentration of 1 × 106 cells/ml. A 100 μl volume of this suspension was transferred to 6 ml polystyrene round-bottom FACS tubes and incubated with 5 μl of PI and 5 μl of FITC-Annexin V for 20 min at room temperature. Subsequently, 400 μl of 1× binding buffer was added to this suspension and in-troduced to the FACS Caliber instrument (Becton Dickinson, USA) to investigate the apoptosis.
    2.6. Caspase activity assay
    Caspase-Glo 3/7, Caspase-Glo 8 and Caspase-Glo 9 (Promega, USA) are luminescence based assays which were used to evaluate the effect of 8-PN on caspase cascades in treated HCT-116 cells [22]. Briefly, white plate (SPL, Korea) containing 1 × 104 cells/well was incubated at 37 °C overnight. The IC50 of 8-PN (23.83 μg/ml) was used to treat the cells for 24, 48 and 72 h. This assay was performed in triplicates for each period