An experimental in vitro study was designed on breast cancer cell lines. MCF-7 breast cancer cell line and breast cancer stem cell line (CelProgen, CA, USA, Cat No: 36102-29-T25), which is positive for such stem cell markers as CD133, CD44, SSEA3/4, and aldehyde dehydrogenase, were used in the current study.
Cell culture and reagent
PD-0332991-isethionate was purchased from Sigma-Aldrich (Sigma-Aldrich, USA). MCF-7 breast cancer cell line was purchased from ATCC (ATCC, HTB-22, Wesel, Germany). RPMI 1640 medium, containing 10% FBS, was used for MCF-7 cells. Human breast cancer stem-like cell line was purchased from CelProgen (San Pedro, CA, USA). Human breast cancer stem cell complete growth media, including all components to maintain cell culture, was used. Cells were maintained at 37oC under a humidified atmosphere of 5% CO2 in air. The passage numbers of BCSC and MCF-7 cell lines were five and seven, respectively.
WST-1 cell proliferation assay
WST-1 test (Roche Diagnostic GmbH, Mannheim, Germany) was performed to show cytotoxicity of PD-0332991 on MCF-7 and BCSC cell lines. Before the cytotoxicity assay was performed, the cell numbers per well were optimized via WST-1 test in order to eliminate a deceptive apoptotic effect due to doubling time differences between cell lines. The cells, starting with 1x106 in a well and diluted in 1/2 ratio, were seeded on 96 well-plate. After 72h incubation with the media, without agent, proliferation characteristic was determined with WST-1 test for BCSCs and MCF-7 cells. Thus, in assay, MCF-7 cell line was seeded as 1x104 cells per well in 96 well-plates. For BCSC cell line, 7x103 cells per well were seeded in 96 well-plates. PD-0332991 treatments for both cell lines were depending on time and concentrations. Cells were treated with the logarithmic dilution of PD-0332991 ranging from 100μM to 0.0001μM for 24h, 48h, and 72h. WST-1 reagent, 10μl, was added to 100μl medium containing PD-0332991. Absorbance values at 450nm were taken with the microplate reader (Thermoscientific, Multiskan FC, Finland) after incubation periods. Absorbance value at 620nm was used as the reference wavelength. The viability of the cells was calculated according to absorbance value made up of WST-1 reduction (21, 22). The absorbance of control cells was assumed as showing 100% viability for normalization.
Cell cycle assay by flow cytometry
IC50 values of PD-0332991 at 48th h were used in cell cycle experiment for both cell lines. Non-treated cells were used as control. BD Cycletest Plus DNA Reagent Kit (BD Pharmigen, CA, USA) was used for cell cycle assay. Protocol was performed according to manufacturers instructions. For the determination of cell cycle pattern by flow cytometry, BCSCs and MCF-7 cells were seeded at a density of 5x105 cells per 25cm2 cell culture flasks. For defined concentrations, depending on IC50 values at 48th h, 8 ml media with agent was prepared and the cells were treated. Media, 8ml without agent, was used for the control group which has the same cell number as the treatment group. After 48h incubation, cells were removed from surface of cell culture flask and cells were taken to falcon tubes. The cell suspensions were centrifuged at 300xg for 5 min. After removing the supernatant, the pellet was resuspended with 1ml washing buffer. Washing step was repeated twice. Washing buffer was removed without disturbing the pellet. 250μl of solution A (trypsin buffer) was added to the pellet and mixed gently. Tubes were incubated at room temperature for 10 min. After incubation, 200μl of solution B was added to tubes without removing solution A. Incubation step was repeated. 200μl of solution C (propidium iodide stain solution with RNase enzyme) kept in 4ºC was added to tubes. Samples were read at the flow cytometry (BD Accuri C6 flow cytometry, BectonDickinson, USA). The argon laser light source, emitting at 488nm wavelength, was used for excitation. A fluorescence detector equipped with the 585/40 band-pass filter was used to show the PI-DNA content (FL2-A) for the analysis.
Apoptosis assay by flow cytometry
Both cell lines were treated with IC50 values of PD-0332991 at 48th h. Non-treated cells were used as control. BCSCs and MCF-7 cells were seeded at a density of 5x105 cells per 25cm2 cell culture flasks. The control group had the same cell number as treatment group. PE-Annexin V Apoptosis Detection Kit I (BD Pharmigen, San Diego, CA, USA) was used to detect apoptosis after PD-0332991 treatment in both cell lines. Protocol was performed according to manufacturers instructions. Cells were washed twice with 1X PBS. After washing step, 1X binding buffer was added to tubes. 5μl of PE-Annexin V and 7-AAD antibodies were added to tubes and samples were incubated for 15 min at room temperature in the dark. After incubation, 400μl of binding buffer was added to samples and the prepared samples were read at the flow cytometry (BD Accuri C6 Flow Cytometer, BectonDickinson, USA) within 1h. The argon laser light source, emitting at 488nm wavelength, was used for excitation. The 670nm band-pass filter for 7-AAD fluorescence (FL3-A) and a fluorescence detector equipped with the 585/40 band-pass filter for FL2A were used for analysis. A total of 20.000 events were acquired for analysis with using Cell Quest Software.
Gene expression analysis
In order to detect mRNA expression of cell cycle regulatory genes, total RNA were isolated from PD-0332991 treated and non-treated cell lines. Total RNA purification Kit (Jena Bioscience, Jena, Germany) was used to isolate total RNA from cell lines according to manufacturers instructions. Total RNA (1-5μg) was converted to cDNA with using SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany). For the detection of gene expression, 1.0 μL cDNA were mixed with 10 μl qPCR GreenMaster with UNG Mix (Jena Bioscience, Jena, Germany) and sterile bidistilled water was added to PCR mix. The expression studies of CDKN1A, CHEK1, CDKN2A, CDC25A, and CCND1 genes were performed with LightCyler (Roche 480 LightCycler, Mannheim, Germany) according to the GreenMaster protocol (Jena Bioscience, Jena, Germany). Relative quantification method described by Livak et al. (23) was used to analyze data. ACTB, HPRT1, 18SRNA, and GAPDH genes were chosen as housekeeping genes for normalization. Ct values were obtained for both the target genes and the housekeeping genes in all cell lines. The expression data was normalized to Ct values obtained from non-treated cell lines to calculate 2-ΔΔCt. Primers for gene expression were designed with Probe Finder Software (Roche Applied Science). Primers were given in Table-1.
GraphPad Prism 6 (GraphPad Software, Inc., CA, USA) was used for statistical analysis. The two-way ANOVA test was performed with Dunnett's multiple comparisons test to compare the mean differences between treatment concentrations and time in the cytotoxicity assay. The same test was also used to compare the mean differences for two independent variables, cell cycle phase and treatment, in the data analysis of cell cycle assay. A p value under 0.05 was accepted as significant.