2.1. Cell lines and cell culture
The Human BCSCs MCF-7/SC were established from MCF-7 cells by sorting CD44+/CD24−
population in the Stem cell Institute, Vietnam. MCF-10A cells were obtained from the
American Type Culture Collection (ATCC, Rockville, MD, USA). 7, MC-7/SC,
MCF-10A cells were maintained in DMEM, RPMI-1640, FBMTM Fibroblast Growth Basal Medium
(Lonza Group Ltd, Switzerland), respectively. All media were mixed with fetal bovine
serum (10% FBS) and antibiotics containing 100 U/mL penicillin and 100 µ g/mL
streptomycin (1%).
2.2. Cell viability assay
In this investigation, 96-well plates were used for culturing cells. Initially, 200 µ L of MCF-7/SC (104 cells/mL,) were cultured for 24 h following by treating with different concentrations of C15:0, C17:0, C18:1 or C18:2. All the fatty acids were obtained from the Sigma, St. Louis, MO, USA and filtered-EtOH was used as solvent. MTT assay was then conducted as previously described [17]. The GraphPad Software (GraphPad Prism 7.00) was used to determine the half maximum inhibitory concentration (IC50) of pentadecanoic acid in each breast cancer cell line.
2.3. Cell migration assay
Cells (1.5 × 105 cells/well) were cultured in 6‑well plates until the cells reach confluency (90%). Then, using a
sterilized 200 μL pipette tip
, scratches in monolayer (width, ~1 mm)were made. Cells were then washed with PBS until detached cells were removed. In the next step, MCF-7/SC were exposed to pentadecanoic acids for 48h. After incubation, the width of the wound areas were observed and measured through an inverted phase-contrast microscope at 4× magnification.
2.4. Cell invassion assay
The invasive capacity of cells was evaluated using 24-well plates. This is a Transwell system purchased from Corning, Cambridge, MA, USA. The upper chambers were precoated with Matrigel diluted in cold serum-free media before seeding. MCF-7/SC were suspended in serum-free medium at a density of 6 × 105 cells/mL). 200 µ L of cells were added to each Transwell comprising with or without 50 µ M pentadecanoic acid. Then, the lower chamber was filled with 750 µ L of RPMI-1640 (10% FBS). After 48 h of treatment, cells were washed with PBS twice. 4% paraformaldehyde were used for fixing MCF-7/SC, and following by methanol. In the last step, invading cells were stained with crystal violet (2%) and captured by using a phase-contrast microscope.
2.5. Flow Cytometric Analysis of the CD44+/CD24− Population
To investigate the effect of pentadecanoic acid on the cell surface expression of CD24 and CD44, the flow cytometry (BD Biosciences, San Diego, CA, USA) was used. MCF-7/SC were seeded into 60 mm dishes at a density of 2 × 104 cells/mL for 24 h. Different concentrations of pentadecanoic acid then was added to PRMI 1640 (10% FBS, 1%
antibiotics). After 48 h of treatment, cells were harvested and washed twice with PBS. Then, MCF-7/SC cells were re-suspended in immunofluorescence staining buffer (100 μL) and incubated at 4 °C (10 - 15 minutes). This buffer contained PE-conjugated anti-human CD24
antibody and FITC-conjugated anti-human CD44 antibody. Both the antibodies were obtained from BD Pharmingen, San Diego, CA, USA. After harvesting, the stained cells were washed with 0.5 mL PBS and performed the analysis.
2.6. Aldefluor Assay
ALDEFLUOR Kit was obtained from Stemcell Technologies, Vancouver, BC, Canada.
ALDH enzyme activity was analyzed using ALDEFLUOR Kit following the manufacturer’s instruction. Diethylaminobenzaldehyde (DEAB) was used as the negative control. MCF-7/SC were seeded at density 2 × 104 cells/dishes (60 mm dish). MCF-7/SC cells were treated with non-lethal concentration of pentadecanoic acid for 48 h. Then, the analysis was carried out using the FACSCalibur flow cytometer.
2.7. Mammosphere Formation Assay
The ultralow-attachment dishes were used to cultured MCF-7/SC (2 × 104 cells/mL).
MammoCult Human Medium (Stemcell Technologies) was used as the media for mammosphere formation assay. After 10 days, mammospheres were observed under a phasse-contrast microscope. Mammospheres that have a diameter larger than 60 μm were counted.
2.8. Cell cycle assay
MCF-7/SC cells were cultured at density 3 × 104 cells/dish (60 mm dish) and then treated with or without pentadecanoic acid. Then, MCF-7/SC cells were harvested and washed with PBS twice. Following centrifugation, 70% EtOH was used to fix MCF-7/SC. Before performing the analysis, fixed cells were stained with 500 µ l solution consist of 25 ng/mL RNase A, 30 μg/mL PI, and 2 mM EDTA-PBS (RNase A: PI: EDTA-PBS = 10 µ l: 1 µ l: 3 mL)
and incubated at 37 °C at least 10 minutes in the dark. The stained cells were analyzed the cell cycle by using the FACSCalibur flow cytometer.
2.9. Annexin V/PI staining assay
The annexin V-FITC Apoptosis Detection Kit I was used to conduct the annexin V-FITC assay according to manufacturer’s protocol. Initially, MCF-7/SC cells were cultured at density 3 × 104 cells/dish (60 mm dish). Then, different concentrations of pentadecanoic acid was supplemented in RPMI 1640 (10% FBS, 1% antibiotics) for 48h. Then, cells were trypsinized, and washed with PBS twice. Propidium iodide (PI) and annexin V-FITC were diluted 50 times and 20 times in 1x binding, respectively. PI and annexin – FITC conjugate then were added in the cells, mixed gently. Stained cells were incubated at room temperature in the dark (15 minutes). The analysis was conducted using the FACSCalibur flow cytometer.
2.10. Western blot analysis
After 48 h treatment with pentadecanoic acid, MCF-7/SC were lysed with RIPA lysis buffer containing protease inhibitors and PMSF (Beyotime, China) to extract total protein.
BCA assay was used to quantify protein.
Western blot experiments were performed as previously mentioned in our recent publication [17].
The primary antibodies were obtained from Cell Signaling Technology, Inc, United States. The secondary antibodies, the anti-rabbit and the anti-mouse immunoglobulin G (IgG) were purchased from Vector Laboratories, Burlingame, CA, USA). The BS ECL Plus Kit was purchased from Biosesang, Seongnam, South Korea to detect bands.2.11. ROS Generation Analysis
ROS generation assay was conducted using the FACSCalibur flow cytometer. Briefly, cells were seeded at a density of 3 × 104 in 60 mm dishes. Following 48 h of incubation, harvesting and staining, the cells were treated with 2′,7′-dichlorofluorescein diacetate (H2DCFDA) for 15 minutes. After, PBS was used to wash stained cells, and then performed the ROS generation analysis.
2.12. Analysis
Group comparisons were performed using the Student's t-test and one-way analysis of
variance with GraphPad Prism 7.0 (La Jolla, CA, USA) software. P-values < 0.05 were
considered statistically significant.