Cell culture and cell lines
Colon adenocarcinoma cell line HT-29 was purchased from American Type Culture Collection. The cells were grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) with 10% fetal bovine serum (Sigma-Aldrich), 1% antibiotic mixtures of penicillin and streptomycin at 37°C in a 5% CO2 humidified incubator.
Cell viability test
Using XTT test assay (Roche Diagnostic, MA, USA), the effect of lycopene on the viability of HT-29 cell line was investigated. These cells were cultured at a concentration of 1×104 cells per well and incubated overnight before the addition of lycopene. After that lycopene (L9879, Sigma-Aldrich) dissolved in tetrahydrofuran (THF) (final concentration of 2.5, 5, 10 and 20 μM) was applied to cells for 24 h. Untreated cells were used as a control. After incubation, 50 μL of XTT mixture was added to each well. After 4-hour incubation, the cells were shaken and the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Altrincham, United Kingdom). Cell viability was assessed as a percentage of live cells versus control cells after each experiment was performed three times (10,11).
The measurement of BCL-2, cleaved caspase 3, BAX, cleaved PARP and 8-oxo-dG levels
The human ELISA kits of BCL-2 (BT Lab, catalog #E1832HU), cleaved caspase 3 (BT Lab, catalog # E6970HU), Bax (BT Lab, catalog #E1825HU), cleaved PARP (BT Lab, catalog #E6971HU) and 8-hydroxy-desoxyguanosine (8-oxo-dG) (BT Lab, catalog #E1436HU) were used to measure the levels of BCL-2, cleaved caspase 3, BAX, cleaved PARP and 8-oxo-dG in lycopene-treated and untreated control HT-29 cells. HT-29 cells were cultivated into a 6-well plate and treated with 4.382 μM lycopene for 24 hours. HT-29 cells that had been treated with lycopene and those that had not were gathered and diluted in Phosphate-buffered saline (PBS). Then they were frozen and thawed three times. Following that, the quantities of BCL-2, cleaved caspase 3, BAX, cleaved PARP and 8-oxo-dG in cell lysates were assessed following the manufacturer’s instructions. Bradford protein assay kit (Merck Millipore, Darmstadt, Germany) was used to calculate the total protein quantities in both experimental and control HT-29 cells (12).
Immunofluorescence staining
Cells were fixed using methanol for 5 minutes at -20°C and washed with PBS. They were then incubated with PBS containing 0.1% Triton X-100 for 15 minutes at room temperature. After washing, they were incubated with PBS containing 2% BSA for 60 minutes at room temperature. After rewashing, they were incubated overnight with monoclonal anti-gamma H2AX (Abcam, Catalog no. ab26350) and monoclonal anti-Cytochrome C (Abcam, Catalog no. ab110325) primary antibodies at a dilution ratio of 1/300 at +4ºC. Cells were washed with PBS then incubated with goat anti-mouse FITC secondary antibody at a dilution ratio of 1/50 for 45 minutes at room temperature in the dark. Finally, 4′,6-diamidino-2-phenylindole (DAPI) was applied on the washed cells and examined under fluorescence microscope. During the evaluation, positivity in the cells in the whole field was evaluated semi-quantitatively as follows; severe (+++), moderate (++), mild (+), absent (-).
Statistical analysis
The results were stated as a mean ± standard error of the mean (SEM). Statistical evaluation of the data was done with SPSS Version 23.0 for Windows using One Way ANOVA and a postdoc Tukey test. The results obtained from BCL-2, cleaved caspase 3, BAX, cleaved PARP and 8-oxo-dG levels tests were examined using Independent Samples t Test. For anti-gamma H2AX and anti-Cytochrome C staining statistical evaluation of the data was performed by One Way ANOVA. Differences were evaluated statistically significant at *p < 0.05, and **p < 0.01. GraphPad Prism 8.0 software (USA) was used for data analysis and graphical presentations.