Cells and reagents
MCF-7 (ER+/PR+) human breast ductal adenocarcinoma and MCF-10A (ER-/PR-) non-tumorigenic epithelial (as the control) cell lines were acquired from ATCC (American Type Culture Collection, Rockville, MD, USA). PBS was purchased from Gibco by Invitrogen of Thermo Scientific (Rockford, USA.). Cell culture media RPMI 1640 was purchased from Biochrome (Bremen, Germany), which was later supplemented with 10% FCS (fetal calf serum) as well as 1% penicillin (100 U/mL) and streptomycin (100 µg/mL) before use. 0.25% Trypsin-EDTA was purchased from Biochrome (Bremen, Germany). HBSS buffer was purchased from ThermoFisher Scientific (Gloucester, UK), which was supplemented with 2% FBS as needed. Fluorochrome FITC-conjugated CD44 and fluorescent PE-conjugated CD24 clones of monoclonal antibodies were purchased from ThermoFisher Scientific, (Gloucester, UK). FITC Annexin V Apoptosis Detection Kit II was purchased from BD Biosciences (Heidelberg, Germany). 0.04% trypan blue dye (2X) was purchased from ThermoFisher Scientific (Gloucester, UK). DMEM/F-12 medium was purchased from ThermoFisher Scientific (Gloucester, UK). Culture plates were purchased from ThermoFisher Scientific (Gloucester, UK). MTT kit was purchased from Promega (Madison, WI, USA). TRIZOL was purchased from ThermoFisher Scientific (Gloucester, UK). Cell culture dishes were purchased from ThermoFisher Scientific (Gloucester, UK). RevertAid First Strand cDNA Synthesis kit was purchased from ThermoFischer Scientific (Gloucester, UK). Binding buffer was purchased from BD Biosciences (San Jose, CA, USA).
Cell Culture And Cell Stock
Two cell lines, MCF-7 (ER+/PR+) human breast ductal adenocarcinoma and MCF-10A non-tumorigenic epithelial (as the control) cell lines, were used to study cell inhibition, cell death and gene expression. Cells were cultured in 75 cm2 cell/tissue culture flaks, containing 9 mL RPMI 1640 culture medium (Biochrome, Bremen, Germany), at 37oC in a humidified 5% CO2 + 95% air incubator. Cells grown up to 80% of the culture flasks were washed once with 1x PBS solution (pH 7.4). Cells were then harvested by addition of 2 mL of 0.25% trypsin-EDTA enzyme solution. The trypsin enzyme activity in the harvested cell culture was inhibited by addition of equal volume of RPMI 1640 culture medium. The harvested cells were then used for IC50 and flow cytometry studies. Harvested cells not in use were added freezing medium, containing 10% DMSO and 90% FCS, and archived at -80oC for later use.
Cultured Cell Count
100 µL of a suspension of cultured cells was transferred to a 1.5 mL Eppendorf tube to which 100 µL 0.4% trypan blue dye (2x) was added and gently pipetted in and out. After keeping the dyed cell suspension at room temperature for 5 minutes, 20 µL of the suspension was pipetted onto a hematocytometric lamina. Cells were then counted in 5 allocated areas under an inverted-microscope at 100x magnification. Cell counts in 5 allocated areas were then averaged out. Total viable and dead cell counts were computed as follows:
Cell count/mL = averaged cell count x 20 (dilution factor) x 104
Isolation Of Stem Cells
DOX [IC50 at optimal incubation time] treated and untreated MCF-10A and MCF-7 cells were harvested, washed with PBS buffer (pH 7.4), re-suspended in HBSS buffer (containing 2% FBS) followed by addition of fluorochrome FITC-conjugated CD44 (10 µL per 106 cells) and fluorochrome PE-conjugated CD24 clones of monoclonal antibody (10 µL per 106 cells) solutions (in PBS buffer with sucrose) and incubation in dark for 40 minutes. Unbound antibodies were then washed out with PBS buffer (pH 7.4). The remaining antibody-bound cells were centrifuged and re-suspended in HBSS buffer (containing 2% FBS) for use in flow cytometry studies.
Fluorochrome (FITC-conjugated CD44 and/or fluorescent PE-conjugated CD24 clone of monoclonal) antibody tagged MCF-7 (CD44+/CD24¯/low) and MCF-10A (CD44+/CD24¯/low) stem cells were counted and separated from other cells by a Fluorescence Activated Cell Sorting (FACS/flow cytometry) instrument (BD FACSAriaTM III cell sorter, San Jose, CA, USA).
Colony Formation Of Stem Cells
Colony formation of CD44+/CD24¯/low stem cells was determined by Soft Agar Assay. To prepare base agar plates, 1% agar pre-heated in a microwave was cooled down in a 40oC water-bath. DMEM/F12 (2x) medium pre-heated in a 40oC water-bath was mixed with the 1% agar in the water-bath at 40oC (1:1 v/v), from which 1.5 mL aliquots were transferred to 35 mL petri-dishes. To culture cells, a top agar was prepared by heating 0.7% agar in a microwave and then cooling it down in a 40oC water-bath. DMEM/F12 (2x) medium was pre-warmed in the same 40oC water-bath. Five-thousand (CD44+/CD24¯/low) stem cells/1 mL DMEM/F12 (2x) pre-warmed in a culture medium, five-thousand harvested cells/1 mL DMEM/F12 (2x) pre-warmed in a culture medium (as the positive control), and 1 mL pre-warmed DMEM/F12(2x) culture medium (as the negative control) were separately mixed gently with 0.7% agar, from which 1.5 mL aliquots were poured onto the base agar in dishes. Inoculated plates were incubated in a humidified incubator at 37oC for 10–14 days. Incubated plates were then stained by 0.005% Crystal Violet 0.5 mL per well) for more than 1 hour to count colonies under a dissecting microscope.
Determination Of Ic Concentration
Inhibition of MCF-7 human breast adenocarcinoma cells by doxorubicin was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] Cell Proliferation Assay kit as described by company protocol (Promega, Madison, WI, USA). 5x103 cells were pipetted into the wells of a group of 92-well plates (5x103 cells/well), followed by incubation at 37°C for 24 h, 48 h, 72 h and 96 h. After incubation, 10 µL of MTT solution was added to each well on the 92-well plates, followed by incubation for 2.5 hours under CO2 atmosphere at 37oC. Then, 200 µL of solutions from each wells were transferred to the wells of fresh 96-well plates, whose absorbances were colorimetrically measured at 490 nm by an Elisa Microplate Reader. Plates with the least apoptosis values under optimal condition were selected to be used for MTT inhibition studies. Increasing concentrations of doxorubicin were added sequentially into the wells of selected plates, followed by incubation for 72 h at 37oC. Wells on the plates were then added 10 µL of MTT solution, whose absorbances were colorimetrically measured at 490 nm by an Elisa Microplate Reader. Percent inhibition values (EQ.1), averaged out of three determinations, were then plotted versus corresponding concentrations of doxorubicin followed by generation of the best-fit trend line to determine the IC50 concentration at 50% cell inhibition.
Inhibition % = [1-(averaged OD490 [DOX+MCF-7] / averaged OD490 [MCF-7])]*100 EQ.1
Flow Cytometry Studies
FITC Annexin V Apoptosis Kit II (BD Pharmgen, USA) was used to determine percent apoptosis/necrosis on the MCF-7 and MCF-10A and their CD44+/CD24¯/low stem cell lines, all treated with DOX [IC50], by a Fluorescence Activated Cell Sorting (FACS/flow cytometry) instrument (BD FACSAriaTM III cell sorter).
DOX [IC50] treated cells were firstly harvested and washed with PBS buffer (pH 7.4), which were then re-suspended in HBSS (including 2% FBS) culture medium and incubated in dark for 40 minutes in fluorochrome FITC-conjugated CD44 (10 µL per 106 cells) and fluorochrome PE-conjugated CD24 monoclonal antibody (10 µL per 106 cells) solutions. Unbound antibodies were then washed out with PBS buffer (pH 7.4). The remaining antibody-bound cells were centrifuged, re-suspended in HBSS (including 2% FBS) culture medium and transferred to FACS tubes for flow cytometry studies.
In order to determine percent cell ratios undergoing cell death, apoptosis or necrosis, DOX [IC50] treated cells were harvested and transferred into FACS tubes, which were then washed with phosphate buffered saline (PBS, pH 7.4) solution and centrifuged at 1500g for 5 minutes. Cell pellets were then dissolved in 5 µL Annexin-V and 5 µl propidium iodide (PI) solutions followed by incubation for 20 minutes at room temperature. In order to facilitate antibody binding, incubated cell solutions were diluted with 1 mL binding buffer [BD Biosciences (San Jose, CA)] and 9 mL PBS buffer (pH 7.4), from which 400 µL aliquots were transferred to FACS tubes for flow cytometry analysis. 10,000 events were implemented for each Flow Cytometry experiment. Events for the Annexin-V-/PI+ cells were gated in Q1 quadrant for necrotic cells, events for the Annexin-V+/PI+ cells were gated in Q2 quadrant for late apoptotic (dead) cells, events for the Annexin-V-/PI- cells were gated in Q3 quadrant for viable cells, events for the Annexin-V+/PI- cells were gated in Q4 quadrant for early apoptotic cells.
Gene Expression Studies
Total RNA acquisition from cultured cells
Into the wells, containing about 5–10 x 106 cells, of cell culture dishes were added 1 mL TRIZOL (Invitrogen) and mixed by pipetting in and out. Cell lysates were then transferred to 1.5 mL Eppendorf tubes and kept at room temperature for 5–10 minutes. This followed addition of 0.2 mL chloroform and vigorous shaking of the Eppendorf tubes for 15 seconds. Eppendorf tubes were then let stand for 2–3 minutes and centrifuged at 12000xg at + 4oC for 15 minutes. The upper transparent phase (supernatant) in the tubes were transferred to 1.5 mL Eppendorf tubes, to which 0.5 mL isopropanol were then added and flipped over several times. The mixtures were kept at room temperature for 10 minutes and centrifuged at 12000xg at + 4 oC for 4–5 minutes. After the supernatant was removed from the Eppendorf tube, the remaining precipitate was added 75% ethanol followed by centrifugation at 7500xg at + 4 oC for 5 minutes. After the supernatant was removed, the remaining RNA was dried at room temperature for 15–20 minutes. Dry RNA was then dissolved in 50 µL RNase free ddH2O by pipetting in and out. Concentration of the total RNA solution, which also includes DNA, were determined using a NanoDrop 2000/2000c spectrophotometer (USA). The total RNA solution (contaminated with DNA) was then stored at -80oC until use.
cDNA synthesis
cDNAs were synthesized from purified total RNAs by using the RevertAid First Strand cDNA Synthesis kit (Thermoscientific, USA). In order for the DNA decontamination of total RNA solution, to 1 µg of total RNA (contaminated with DNA) was added 1 µL of 10X reaction buffer solution including MgCl2, 1 µL of DNase I solution, and appropriate amount of RNase free ddH2O to adjust the final volume to 10 µL. After incubation at 37oC for 30 minutes, DNase in the mixture was inhibited by addition of 1 µL of 50 mM EDTA and incubation at 65oC for 10 minutes. Decontaminated and non-degraded RNA was confirmed by 1% agarose gel electrophoresis as well as its OD260/280 measurement ratio (found to be between 1.8 and 2.2). To each DNA-free and intact RNA solution (~ 50 ng/µL) were sequentially added 20 µL cDNA synthesis solution with ingredients given in Table 1. The mixture was then consecutively incubated at 65oC for 5 minutes, at 42oC for 60 minutes, and at 25oC for 5 minutes. cDNAs synthesized were stored at – 20oC until use.
Quantitative determination of gene expression
Gene expressions were determined by quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) using a LightCyclerR 480 II (Roche, Germany) instrument, which conformed to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [27]. Optimized RealTime Ready UPL (Universal Probe Library) primer pairs (forward and reverse) for Bax, Bcl-2, p53, Oct-4, Sox-2, Nanog, GAPDH (the reference) gene specific probes were purchased from Roche (Germany), see Table 2 for primer sequences. LightCycler® 480 Probes Master kit, which includes Taq DNA Polymerase as well as dNTPs and probes, was purchased from Roche (Germany). A Universal Probe Library (UPL) RT-qPCR procedure provided by Roche® (Germany), was adapted for normalization of quantification cycle (Cq) as well as amplification of the genes of interest. Efficiencies of the primer pairs as well as the reference gene were confirmed by running RT-qPCR for five different logarithmic dilutions of each primer pairs and the reference gene in presence of equal amount of cDNA, where Cq increments were found to be consistent with dilution ratios. RT-qPCR experiments were implemented, in presence of a primer pair of the target gene and the LightCycler® 480 Probes Master solution, in two steps as follows: 1) a denaturation step at 95oC for 10 minutes followed by 2) a 40 cycles of amplification step, each cycle running consecutively at 95oC for 15 seconds (for denaturation), then at 63oC for 15 seconds (for annealing) and lastly at 72oC for 15 seconds (for extension).
Table 1.
ΔCq values for the target genes (in DOX [IC50] treated stem cells) and the control genes (in DOX [IC50] untreated stem cells) were determined by EQ.2, where CqTARGET/CONTROL is the Cq value for the target or the control gene, and CqREFERENCE is the Cq value for GAPDH, the reference gene. Cq values for each target and control genes were averaged out of six RT-qPCR determinations. -ΔΔCq values were determined by EQ.3. This followed the computation of fold regulation values as in EQ.4.
ΔCqTARGET/CONTROL = (CqTARGET/CONTROL – CqREFERENCE) EQ.2
-ΔΔCq = - [(ΔCqTARGET – ΔCqCONTROL)] EQ.3
Fold-regulation = 2(–ΔΔCt) EQ.4
Statistical analysis
Data were analysed by GraphPad PrismR v.5 software (http://www.graphpad.com) to determine P values using Fisher's exact test. P values lower than 0.05 were interpreted as statistically significant. Data were averaged out of at least three determinations and presented as the mean ± S.D.