Human Patient Samples
Dr. Susanne Fuqua (Baylor College of Medicine) provided human breast cancer tissue RNA samples. Dr. George C. Prendergast (Lankenau Institute for Medical Research) provided the plasma samples along with the corresponding tissue slides. All the human samples used were in accordance with the IRB procedures at the respective institutions.
Cell Lines and Cell Culture
The breast cancer cell lines MCF-7, ZR-75, MDA-MB-231, MDA-MB-468 and SK-BR-3 and colon cancer cell line Caco-2 were obtained from ATCC. All cells were maintained at 37°C and 5% CO2 in DMEM/F12-50/50 (Mediatech, Inc.) supplemented with 10% FBS (Atlanta Biologics) and 1% antibiotics (Gibco®, Invitrogen). During serum-starvation, cells were incubated with serum-free DMEM supplemented with 1% antibiotics.
The triple-negative breast cancer (TNBC) cell line MDA-MB-231 was chosen for generating stable clones. The cells were transfected with the following plasmids: pcDNA 3.1 (Invitrogen) (control), ERα and HER-2. The stable clone cell line (pcDNA, ER and HER-2) were maintained in DMEM/F12-50/50, supplemented with 10% FBS, 1% antibiotics and 0.5μg/ml of G418 (Sigma Aldrich).
For lactoferrin treatment, cells were maintained in serum-free DMEM for 48 hrs and then treated with 20μg/ml of lactoferrin (Sigma Aldrich). Protein samples were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a nitrocellulose membrane (Bio-Rad). The following primary antibodies were used: anti-ERα (Bethyl Laboratories), anti-HER-2 (Bethyl Laboratories), anti-PR (Dako), anti-IGF-IRβ (Santa Cruz Biotechnology, Inc.), anti-RXRα (Santa Cruz Biotechnology, Inc.), anti-cyclinD1 (Labvision/Thermo Fisher Scientific), anti-tubulin (Sigma-Aldrich) and anti-vinculin (Sigma-Aldrich).
Immunohistochemistry and Confocal Microscopy
Deparaffinized sections were treated with 0.3% hydrogen peroxide in methanol and subjected to antigen retrieval by boiling the sections in antigen unmasking solution (Vector Laboratories). The sections were then blocked with 5% skimmed milk in PBS (Mediatech Inc.) and incubated with lactoferrin antibody (1: 500) or endothelin antibody (1: 100) at 4°C overnight, followed by incubation with EnVision (Dako) for 1 hr at room temperature and visualization with liquid DAB+ substrate chromogen system (Dako). Immunostained sections were lightly counterstained with hematoxylin, dehydrated in graded ethanol, cleared in xylene, and mounted with the use of the permount mounting medium.
For confocal imaging, ZR-75 and MCF-7 cells were plated onto cover slips in six-well plates, starved for 48 hrs and treated with 20μg/ml of lactoferrin. After a 12-hour treatment, the cells were fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked in 10% normal goat serum in PBS. The cells were incubated with primary antibodies, washed three times in PBS, and then incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated with Alexa-488 or Alexa-555 (Molecular Probes). 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) was used for nuclear staining. The slides were then examined using a Zeiss LSM 710 confocal microscope and images were acquired with the help of Zen 2009 software. Images were transferred to TIFF format using Image J software.
For Northern Blotting, MCF-7 as well as MDA-MB-231 and MDA-MB-468 cells were treated with 100μg/ml of lactoferrin for 6 hrs. A RNA sample amount of 20μg was loaded onto a formaldehyde denaturing gel (1.5% agarose, 2.2M formaldehyde). Subsequently, RNA was transferred to a Hybond™-N membrane (GE Healthcare UK Limited) and then immobilized through covalent linkage to the membrane by UV cross linker. A complimentary RNA ET-1 probe was prepared using Ambion’s In Vitro Transcription Kit (Ambion, Inc.) with radio-labeled UTP. Finally, the membrane was exposed to a phosphor imager and the autoradiogram was developed using the Storm™ 865 Imager (GE Healthcare UK Limited). For another experiment, MDA-MB-231 cells were plated and then serum-starved for 48 hrs. The cells were either treated with 20μg/ml of Cycloheximide (CHX) or 100μg/ml Lactoferrin (Lf) or both. CHX was administered an hour before Lf treatment. After 6 hrs of treatment with Lf, RNA was isolated from the cells using TRIzol® method and RNA levels were detected using the Northern blotting protocol as described above.
To analyze the effect of lactoferrin with or without tamoxifen on cell migration, MCF-7 cells were plated with 10% fetal calf serum-DMEM. Before treatment, each plate received multiple “wounds” with a 200μl pipette tip. Treatments were done with low-serum medium supplemented with 100μg/ml lactoferrin and 10−7 tamoxifen (Sigma Aldrich). In another set, the cells were treated with and without lactoferrin in addition to 10nM of Herceptin (Genentech, Inc.) and in combination. After 24 hrs, each plate was examined for the amount of wound closure by measuring the physical separation remaining between the original wound widths using the Olympus DP2-BSW digital camera software (Olympus). Ten separate measurements were made per plate, and each experiment was performed in triplicates.
ZR-75 cells were serum-starved for 48 hrs and then resuspended in the presence of 0.1% BSA. Subsequently, 1 × 105/well were loaded onto the upper well of an uncoated Boyden chamber. ET-1 and BQ-123 were diluted in plain medium before plating. The lower chamber was supplemented with conditioned medium of NIH-3T3 fibroblasts grown in DMEM/F-12 medium with 0.1% BSA. After fixation with methanol and staining with 0.05% crystal violet, the number of cells that successfully migrated through the filter was counted. Data were analyzed using the Olympus DP2-BSW digital camera software.
Cells were serum-starved for 48 hours and then seeded at a density of 1 × 105 cells/well in the upper well of the Matrigel™ Invasion Chambers (BD Biosciences). At the time of the cell seeding, each well was treated with 100 μg/ml of lactoferrin. For E2 treatment, cells were serum starved in phenol red-free medium for 48 hours. At 6 hours before trypsinizing for collection, the cells were treated with 100 μg/ml of lactoferrin. After 24 to 48 hrs incubation, the number of the invaded cells was counted and the results were expressed as percentages of control. For MDA-MB-231 and MDA-MB-468, the invaded cells at the bottom were fixed with 4% paraformaldehyde and stained for DAPI after 48 hrs.
Microarray Analysis of Lactoferrin-Regulated Genes
MDA-MB-231 and MDA-MB-468 cells were plated in triplicates, serum-starved for 48 hrs and then treated with 100μg/ml of lactoferrin for 6 hrs. RNA was isolated using TRIzol® following the manufacturer’s protocol. cDNA samples were generated and hybridized onto an Affymetrix Human Exon 1.0 ST Array chip. GeneSpring GX was used to process the data and statistical analysis was carried out by means of an unpaired t test. A fold change of ≥1.5 was employed to identify differentially regulated genes and those with a p-value of ≤ 0.05 were considered statistically significant. Heat map analysis of the identified genes for individual arrays was performed using MultiExperiment viewer version 4.4 (Dana-Farber Cancer Institute).
MCF-7 as well as MDA-MB-231 and MDA-MB-468 cells were grown to 50% confluency. ET-1 promoter, β-galactosidase and control plasmids were transfected into cells using FuGENE® 6 according to the manufacturer’s protocol (Roche). After lactoferrin treatment, cell lysates were prepared using 200μl of Tropix® Lysis solution (Applied Biosystems). For luciferase promoter activity, 10μl of lysate was incubated with 100μl Luciferase Assay Substrate solution (Promega) and measurements were taken using LUMAT LB 9507 luminometer (Berthold Technologies). β-galactosidase was used as a transfection control.
Electrophoretic Mobility Shift Assay
PCR amplified DNA fragments or oligos containing lactoferrin consensus sites were 5′-end labeled using γ32p and PNK enzyme. End labeled fragments were purified using sephadex G-50 spin columns (GE Healthcare UK Limited). Each fragment was incubated with purified holo- or apo-lactoferrin (GenWay Biotech) or lactoferrin-treated and untreated nuclear extracts for 15 min at room temperature. The reaction samples were resolved on a 5% native PAGE for 2 hrs. Gels were removed and dried and exposed to phosphor imager. The final images were developed using the Storm™ 865 Imager (GE Healthcare UK Limited).
Plasma Protein Detection using ELISA
For detection of lactoferrin, the AssayMax Human Lactoferrin ELISA Kit (AssayPro) was used with a 1:100 dilution of serum samples. ET-1 levels were measured using the QuantiGlo Human Endothelin-1 Kit (R&D Systems) following the manufacturer’s manual. For ET-1 levels, the 1450 Microbeta Jet Microplate Scintillation and Luminescence Counter (Perkin Elmer Life and Analytical Sciences) were used to measure the luminescence. A dilution of 1:5 for the serum was used for the ET-1 ELISA assay.
RNA Isolation and Real-time Quantitative PCR
Total RNA from human breast tumor tissue samples as well as human breast cancer cell lines was isolated using TRIzol® (Invitrogen) according to the manufacturer’s protocol. A 2μg RNA sample was used to synthesize cDNA using Invitrogen’s SuperScript® III First-Strand Synthesis SuperMix following the manufacturer’s manual. The following primers (synthesized by Sigma Aldrich) were used for target genes: Lf Forward: 5′-ACCGCAGACATGAAACTTGT-3′ and Lf Reverse: 5′-GGGGGAGTCTCTCTTTATGC-3′; ET-1 Forward: GCCAAGGAGCTCCAGAAACAGCAG ET-1 Reverse: AGCAGGAGCAGCGCTTGGAC; Actin Forward: 5′ –TCCCTGGAGAAGAGCTACGA 3′ and Actin Reverse: 5′-GTACTTGCGCTCAGGAGGAG-3′. Normalized expression of target genes were calculated relative to endogenous expression of actin (Relative expression= 2(−ΔC(t))).
Paired Student’s t test was used for statistical significance of differences in numerical data. All the statistical tests were two sided. P-value of less than 0.05 was considered to be statistically significant.