Cell Culture and Reagents
The breast cancer cell lines MCF7 and MDA-MB231 were obtained from American Type Culture Collection (ATCC, VA). MDA-MB231 LM2-4175 (231 LM) was a generous gift from Dr. J. Massagué [25
]. 231 LM is the variant of MDA-MB231 which preferentially metastasizes to the lung. MCF10A, which is a spontaneously immortalized, non-tumorigenic epithelial cell line, was also purchased from ATCC. Normal human mammary epithelial cell (HMEC) was purchased from Lonza Inc., MD.
Stable clone of MDA-MB231 cells was established in our lab by the lentivirus infection which constitutively expresses Luciferase gene and designated as MDA-MB231 Luc. MCF7 was cultured in DMEM (ATCC) containing 10% FBS. MDA-MB231, MDA-MB231Luc and 231 LM were cultured in RPMI (Invitrogen, NY) containing 10% FBS. MCF10A and HMEC cells were cultured in MEBM media with Bullet kit supplements (Lonza Inc., MD). Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO. Resveratrol, TOFA and Fumonisin B1 were purchased from Sigma-Aldrich Co, MO.
Lentivirus production and Infection
Lentivector expression system was purchased from System Biosciences (Mountainview, CA). Packaging and production of GFP (control) and FAS-expressing lentiviruses were performed according to the manufacturer’s protocol. The virus culture supernatants were collected, concentrated, and used to infect the MDA-MB231 cells.
Cell proliferation assay (MTS assay)
10000 cells/well were seeded in 96-well plates and cultured for 48 hours. The cells were then treated with or without resveratrol for further 48 hours. Cell viability was measured using CellTiter 96 Aqueous one solution Cell Proliferation Assay kit (Promega corp., WI), composed of the novel tetrazolium compound, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphonyl)-2H-tetrazolium, inner salt (MTS) and an electron coupling reagent, phenazine ethosulfate (PES), following the manufacturer’s instructions. Absorbance was read at 490 nm using an ELISA reader which was directly proportional to the number of living cells in culture. Cell viability was expressed as a percentage relative to untreated cells.
Measurement of lipid content
Intracellular lipid content was measured by using the AdipoRed assay reagent kit (Lonza Inc., MD). AdipoRed reagent is a solution of the hydrophilic stain Nile Red which enables the quantification of intracellular lipid droplets. In brief, 10000 cells/well were cultured in 96 well plates, and the cells were treated with or without resveratrol for 48 hours. The control cells were treated with vehicle only. After 48 hours, cells were assayed for the quantification of intracellular lipid content following the manufacturer’s instructions.
In situ apoptosis assay
Cells were cultured on 96 well plates and were treated with different concentrations of resveratrol and further incubated for 48 hours. The terminal deoxynucleotidyl transferase, dUTP nick-end labeling (TUNEL) assay was performed using In situ Cell Death detection kit/TMR Red (Roche Inc., IN) according to the manufacturer’s instructions. The number of apoptotic cells in each well was counted under a fluorescence microscope and represented in bar diagrams.
Cells after treatment with or without resveratrol were collected and proteins were extracted by homogenizing the pellet in the protein lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) under reducing conditions. The cell lysate was boiled for 10 min and the proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and then incubated with antibodies to FAS (0.2 μg/ml, Immuno-biological Laboratories Co., Japan), β-tubulin (1:10000, Upstate Biotechnology, NY) and Actin (1:250, Santa Cruz Biotechnology, CA) at 4°C. The membranes were then incubated with corresponding HRP-conjugated secondary antibodies followed by visualizing bands using ECL Plus Western blotting detection system (Amersham Life Sciences, UK).
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from the cells using RNeasy mini kit (Qiagen, CA) and they were reverse transcribed with Reverse Transcriptase kit (Fermentas Life Sciences, MD). qRT PCR was carried out using Maxima SYBR Green kit (Fermentas Life Sciences, MD) in DNA engine opticon-2 system (MJ Research, MA) and β-actin was used as an internal control. All reactions were performed at least in triplicate. The following pairs of primers were used for the gene amplification; for FASN: 5′-CATCCAGATAGGCCTCATAGAC-3′ and 5′-CTCCATGAAGTAGGAGTGGAAG-3′, for p21: 5′-CCGTGGACAGTGAGCAGTT-3′ and 5′ CCAATCTGCGCTTGGAGTGA-3′, for BNIP3: 5′ CCTGGGTAGAACTGCACTTCAGCAAT-3′ and 5′ TTCATGACGCTCGTGTTCCTCATGCT-3′, for DAPK2: 5′ CTTTGATCTCAAGCCAGAAAAC-3′ and 5′-CTCGTAGTTCACAATTTCTGGAG 3′, for β-actin: 5′-TGAGACCTTCAACACCCCAGCCATG-3′ and 5′-CGTAGATGGGCACAGTGTGGGTG-3′). The thermal cycling conditions comprised of an initial denaturation step at 95°C for 2 min followed by 40 cycles of PCR using the following profile: 94°C: 30s, 57°C: 30s, 72°C: 30s.
We designed four individual siRNAs (small interfering RNAs) against the FAS gene (sense strand sequences: GAGCGUAUCUGUGAGAAAC, GACGAGAGCACCUUUGAUG, UGACAUCGUCCAUUCGUUU, CCAUGGAGCGUAUCUGUGA) and custom synthesized by Dharmacon Inc. (Lafayette, CO). One siRNA duplex targeting the GFP (green fluorescent protein) gene was also synthesized to use as a negative control in all the experiments. The siRNA was transfected into the breast cancer cells using the Trans-TKO transfection reagent (Mirus Corp., WI) according to the manufacturer’s protocol. The cells were then harvested for real time PCR experiments to determine the mRNA level.
Isolation of cancer stem-like cells from breast cancer cells and Mammosphere assay
Breast cancer stem-like cells from MDA-MB231Luc or 231 LM were isolated using the cell surface phenotype CD24−
by MACS separation columns (Miltenyi Biotec, Germany) [26
]. Cells were trypsinized and washed with PBS. Biotin-conjugated CD24 (StemCell Technologies, Canada) and allophycocyanin (APC)-conjugated CD44 (BioLegend, CA) antibodies were added to the sample and incubated for 15 min on ice. After washing the sample with PBS, CD24 negative cells were isolated by anti-biotin microbeads (Anti-Biotin MultiSort Kit; Miltenyi Biotec). The cells were then mixed with anti-APC microbeads (Anti-Biotin MultiSort Kit; Miltenyi Biotec) and biotin-conjugated ESA antibody (GeneTex Inc., TX) for 15 min on ice. After washing the cells, CD44 and ESA positive fractions were collected through MACS separation columns. Trypan Blue Solution (Sigma Co, MO) was used for measurement of cell viability. The single cell suspension was cultured in mammosphere forming medium (DMEM/F12 containing B27 supplement; Invitrogen) with or without resveratrol for the following assays.
Immunohistochemistry (IHC) analysis
Formalin-fixed paraffin-embedded (FFPE) breast cancer tissue specimens were obtained from the pathology archives of Iwate Medical School (Iwate, Japan). IHC staining was performed using antibodies to FAS (0.2 μg/ml, Immuno-biological Laboratories Co., Japan) and BNIP (Biocarta, CA). Briefly, tumor sections were deparaffinized, rehydrated and immersed in sodium citrate buffer (pH 6.0) at 90°C water bath for 30 min. After cooling down the sections to room temperature, the slides were dipped in 3% H2O2 for 15 min at room temperature to block endogenous peroxidase activity. After washing with the 0.1% Tween 20/PBS (PBST) solution, the sections were incubated with 2% BSA/PBST for 20 min. The sections were incubated with each primary antibody for 12 hours at 4°C. The sections were then processed using EnVision+ System (DAKO, CA) according to the manufacturer’s protocol. Tissue sections were incubated for 1hr with secondary anti-rabbit HRP-conjugated polymer antibody. After three washings, sections were dipped in distilled water for 5 min, subsequently counterstained with hematoxylin and dehydrated with ethanol.
Anti-tumorigenic effect of resveratrol in in vivo
In order to determine the anti-tumor effects of resveratrol in in vivo, cancer stem-like cells isolated from MDA-MB231Luc (1×104 cells) was mixed with Matrigel (BD biosciences, MA) and the mixture was injected into the mammary fat pad of 4-week old female nude mice. Resveratrol suspended in PBS (22.4 kg/body weight) was administered to these mice via oral gavage or intraperitoneal (IP) route every two days for a period of 4 weeks. The control group received the vehicle only. In vivo imaging was performed to monitor the tumor growth using the IVIS Imaging System (Xenogen, MA). Mice were sacrificed at the endpoint and tumors were harvested, and the paraffin block sections of tumors were later used for IHC analysis of FAS, DAPK2 and BNIP3.
Values were expressed as the mean ± SE of at least three independent experiments. Statistical significance was determined by Student’s t test or one way ANOVA. P values < 0.05 were considered significant. Statistical procedures were carried out using Graphpad prism software.