Cell culture
The human breast epithelial cell lines MCF-7 and MDA-MB-231 were used in this study. Both were grown in Dulbecco's Modified Eagle's Medium (Gibco BRL, MD). The medium was enhanced with heat-inactivated 10% fetal bovine serum (Gibco, South America), antibiotics (1% penicillin and streptomycin; Capricorn, Germany), and 1% L-glutamine (Thermo, ABD). Cells were then routinely propagated at 37°C and 5% CO2 atmosphere.
Preparation Of Hesperidin
HES powder (Sigma, MO, USA) was suspended in dimethyl sulfoxide (Sigma, MO, USA). The 1 mM stock solution was prepared.
Cell Viability Assay
Cell growth was assessed in 96-well plates at the 24, 48, and 72 h points using the methylthiazolyl tetrazolium (MTT) assay (Onder et al. 2022). 100 ml of complete medium containing 5x103 cells were seeded in each well. The following day (day 0), cells were treated with different concentrations of HES (20, 40, 60, 80 and 100 µM). Cell growth was assessed at the 24, 48, and 72 h points using the methylthiazolyl tetrazolium (MTT) assay (Onder et al. 2022). HES's half maximal inhibitory concentration (IC50) was determined. After the IC50 dose was determined, five groups were identified: control, cells that received the IC50 dose of HES were identified as HES1, cells that received half the IC50 dose of HES were identified as HES2, cells that received the ratio of DMSO used in the preparation of HES in the HES1 group were identified as D1, cells that received the ratio of DMSO used in the preparation of HES in the HES2 group were identified as D2.
Cell-cycle Analysis
Muse Cell Cycle Kit (Luminex, TX, USA) was used to analyze the cell cycle. HES-treated cells were trypsinized from the culture medium, centrifuged at 300 g for 5 min. Then phosphate-buffered saline (PBS) was used to wash the cells. 70% cold ethanol were used to fixed the cells in at -20°C. The cells were then centrifuged and exposed to cell cycle reagents for 30 minutes without light. A Muse cell analyzer and Muse analysis software (Merck, Darmstadt, Germany) were used to analyze the data.
Immunocytochemical Analysis For Detecting Apoptosis
To assess HES-mediated apoptotic cell death, the ApopTag Fluorescein In Situ Apoptosis Detection Kit (EMD Millipore, Darmstadt, Germany) was as previously described (Baran et al. 2020). An Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) was used to observe the apoptotic cells (magnification, x400). A total of 10 fields of view were randomly selected for analysis.
Immunocytochemical Analysis Of Bax And Bcl-2
To determine Bax and Bcl-2 expression profiles, an immunofluorescence staining was used as previously described (Onder et al. 2022). Cells were incubated on coverslips in a 24-well plate. After cells were treated with HES for 24 hours, they were fixed in 10% formaldehyde. Then, the procedure of immunofluorescence staining was applied. Cells were treated with Bax (1:100, primary antibody, Novus Biologicals, Littleton, USA) and then Bcl-2 (1:30, primary antibody, Thermo, Rockford, USA). For each group, 400x images were obtained from ten separate microscopic fields using an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan). The intensity of Bax and Bcl-2 primary antibodies' immunoreactivity was measured using Image J software (Bethesda, USA).
Statistical analysis
GraphPad Prism 9.4.1 (CA, USA) was used to statistically analyze the data. The Shapiro-Wilk and Kolmogorov-Smirnov tests were used to determine whether the data were in accordance with the normal distribution. The one-sample t test was used to compare viability percentage values between dosage groups. The Kruskal-Wallis test and one-way analysis of variance (ANOVA) were employed to evaluate the quantitative variables in more than two groups. The multiple comparisons were done using the Tukey and Bonferroni tests. P values that were less than 0.05 were regarded as statistically significant.