2.7. Apoptosis and Cell Cycle Analysis by Flow Cytometry

Early changes in cell surfaces, associated with apoptosis and cell cycle arrest of cancer cells, treated with willow organic-soluble fractions were investigated by use of flow cytometry with the FITC-Annexin V/Propidium Iodide (PI) Apoptosis Detection Kit I (BD Biosciences Company, NJ, USA) by use of fluorescence-activated cell sorter analysis (FACS) Canto II flow cytometry system as previously described by us [19]. MCF-7 cells were treated with IC₅₀ concentrations of F2 (128.1 µg/mL) and F3 (111.72 µg/mL) for 72 h. F2 and F3 were chosen as they exhibited the most potent anti-proliferation potential out of all the seven prepared S. salix fractions and extracts. In order to set up the compensation and quadrant parameters, an unstained reference population of cells was employed, whereas the untreated control cell population was utilized to define the basal level of apoptotic and dead cells. BD Diva software version 6.0 was used for flow cytometric analysis. To determine the role of oxidative stress and intracellular reactive oxygen species (ROS) generation in apoptosis of cells, caused by exposure to fractions of willow leaves, the intracellular peroxide-dependent oxidation of 2′,7′-dichlorodihydrofluoresceindiacetate (DCFH-DA) into a fluorescent compound, 2′,7′-dichlorofluorescein (DCF) was carried out as previously described [17]. The willow EtOAc-soluble fraction (F3) was specifically chosen as it exhibited the most potent anti-proliferative activity against MCF-7 cells (Table 1). In brief, MCF-7 cells were treated by the 100, 400 and 800 µg/mL concentrations of F3 for 72 h at 37 °C in 5% CO₂ atmosphere. Then, cells were washed and stained appropriately by 5 μM of DCFH-DA at 37 °C for 10 min. Cell detection at multiple wavelengths (485 and 530 nm) was conducted using a fluorescence microscope (Nikon, Eclipse E600). WISH cells were utilized for the study of cell cycle progression and cyto-protective effects of F3 against hydrogen peroxide (H₂O₂) exposure according to our previously published protocol [20]. The design employed WISH cells treated with 0.1% DMSO as a solvent control, 1 mM H₂O₂ as positive control and co-treated with F3 (100, 400 and 800 µg/mL) plus 1 mM H₂O₂ for 24 h. Cells were harvested and centrifuged at 3600× g for 5 min. Pellets were resuspended in 500 µL of PBS. Cells were fixed with equal volume of chilled 70% ice-cold ethanol and incubated at 4 °C for 1 h. After two successive washes with PBS at 3600× g for 5 min, cell pellets were resuspended in PBS and stained with 50 µg PI/mL containing 0.1% Triton X-100 and 0.5 mg/mL RNAase A for 1 h at 30 °C in the dark. Fluorescence of the PI was measured by FACS by use of a Beckman Coulter flow cytometer (Coulter Epics XL/Xl-MCL, Miami, USA) through a FL-4 filter (675 nm) and 10,000 events were acquired as previously reported by us [21]. Data were analyzed by Coulter Epics XL/XL-MCL, System II Software, Version 3.0. Cell debris was characterized by a low FSC/SSC and was excluded from the analysis.

IC₅₀ values for willow leaves fractions/extracts against cancer cell lines.

IC₅₀ is the concentration (µg/mL) of a given fraction/extract that results in 50% inhibition of cellular growth. * NE indicates that no anti-proliferative effect was observed for the corresponding fraction/extract against the tested cancerous cell line. IC₅₀ values were calculated using trendline equation [17]. F1, F2 and F3 are petroleum ether, CHCl₃ and EtOAc sequential fractions, respectively. E1, E2, E3 and E4 are EtOH extract, 70% hydro-acetone extract, aqueous infusion and aqueous decoction, respectively.