2.1 Chemicals and Cell Culture Materials
MDA-MB-231 cells, a triple negative breast cancer cell line, were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and stored in our laboratory and resuscitated once every half year. Human umbilical vein endothelial cells (HUVECs)were kindly provided by Shanghai Whelab Bioscience Limited. Both cells were cultured in DMEM medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with 2 mmol/l L-glutamine, 100 U/ml penicillin, 0.1 µg/ml streptomycin and 10% fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.) at 37˚C in a humidified atmosphere containing 5% CO2. The culture medium was replaced once every two days. DHA was purchased from Dalian Meilun Biotechnology Co., Ltd. (Liaoning, China).
Cells were seeded in a 96-well plate at a concentration of 4×103 cells/well. While incubated at 37˚C and humidified atmosphere containing 5% CO2 for 12h, culture medium with different concentrations of DHA (0, 6.25, 12.5, 25, 50, 100, 200, 400μM) was replaced in each well. After 24 or 48 h of incubation, 200μL PBS with 10% CCK-8 solution (Beyotime, Shanghai, China) was replaced in each well, followed by incubating at 37℃ for 2 hours. The cell viability was measured at an absorbance of 490 nm using a microplate reader (Thermo Fisher Scientific, US). Cell viability=OD 490 nm of the treatment/OD 490 nm of the control.
2.3 Chick chorioallantois membrane (CAM) assay
To assess the effect of DHA on the tumor cells induced vasculature, we performed a CAM assay according to the previously described method . Briefly, embryonic eggs were incubated in a humidified (65-70%) incubator at 37.6℃. The normally developing eggs were randomly divided into four groups of five. Group without tumor cells was named as control group, and group with tumor cells pretreated by DHA (50and 100μM) or not were name as DHA (50μM) group, DHA (100μM) group and MDA-MB-231group separately as follows. After 7-days incubation, a small window was cut on the top of each egg to expose the CAM on which was put an absorbable gelatin sponge loaded with basal medium or indicated MDA-MB-231 cells as above. Then the window was sealed with laboratory film and incubated for an additional 2 days. CAM photos were taken under a stereoscopic microscope equipped with a digital camera.Excellent as the CAM model for assessing tumor angiogenesis, the scientific statistics and assessment of vascular patterning in it are challenging. In view of this, vessels around the seed point are divided into three grades according to their branches, with the last capillary being grade III. Total vascular area and the number of vessels of each class in visual field are measured using ImageJ Software.
It is interesting to note that there are blood vessels drilling into the gelatin sponge in some group. To find it out, the sponge was fixed with paraformaldehyde and carefully isolate. 5μm thick paraffin-embedded sections were used to stain with hematoxylin and eosin (H&E).Sections after staining are observed and photographed under a microscope，the number of vessels in 5 random field were counted.
2.4 Aortic ring angiogenesis assay of angiogenesis
MDA-MB-231 cells (5×106 cells/culture flask) were exposed to different concentrations of DHA (0, 50, and 100μM) for 24 h then the medium was replaced with fresh medium containing 1% FBS after being rinsed with PBS (pH 7.4) to remove unreacted drug. Pretreated MDA-MB-231 cells were cultured for another 24 h, and the supernatant was collected and referred toMDA-MB-231-derived conditioned medium (MDA-MB-231 CM)
Aortic rings are prepared from the thoracic aorta of 2-month-old rats. The aorta is cleaned of blood and fibroadipose tissue under a dissecting microscope and then cross-sectioned into 1 mm long rings using a scalpel blade. Aortic ring cultures are set in a 96 -well culture plate with clotting media of Matrigel with serum-free medium mixed at 1:1. Then the growth medium (DMEM with 20% FBS) was added to the wells. After 3 days of culture in a humidified CO2 incubator at 37 °C, rings were randomly divided into four groups of five, and the growth media was replaced with indicated MDA-MB-231 CM or serum-free medium in each group . After another 7–days in incubator, the images were taken under a microscope and the average microvessel sprouting number was counted from each group discharging the highest and lowest sample.
HUEVC cells were cultured in low-serum media supplemented with 1% FBS for 24 h, and then were suspended in the upper chamber of an 8μm aperture transwell in a density of 2×105 cell/well with MDA-MB-231 CM or basal medium 200μL. The lower chamber was filled with 10% FBS medium (700μl) and incubated at 37°C for 12 hours. Subsequently, the migrated cells on the lower surface were stained with crystal violet. The number of migratory cells of each well was counted at five random fields. Results were from triplicate experiments.
2.6 Tube formation assay
Growth factor-reduced Matrigel (Corning, New York, US) was coated on a pre-cooled 96-well culture plate, and incubated for 30 min at 37°C. HUVEC cells were suspended in MDA-MB-231 CM pretreated with DHA or not and then seeded into each well at a density of1×105 cells/well. After 4 h in the incubator, the tubular structure was visualized at random fields and photographs were taken using Nikon’s Eclipse TS 100 microscope. Tubulogenic was assessed by counting the number of closed intercellular compartments (closed rings or pro-angiogenic structures) according to reported methods using ImageJ Software. Results were from triplicate experiments, and representative views were shown.
2.7 Gelatin zymography for the detection of MMPs
Gelatin zymography was used to assess the enzymatic activities of MMPs as described previously. Briefly, MDA-MB-231 CM was collected as mentioned above, and 30 μL CM in each group was resuspended in nonreducing laemmli sample buffer and resolved by 8% SDS-PAGE containing 1 mg/mL of gelatin (Yuanyebio, Shanghai, China). Following electrophoresis, gels were washed with 2.5% Triton X-100 to remove SDS and incubated in substrate buffer (50 mM, pH 8 Tris buffer containing 5 mM CaCl2) for 18 h at 37°C. Gels were then stained with 0.5% Coomassie brilliant blue R-250 (Service, Wuhan, China), followed by de-staining for 2 h. The gelatinolytic activity was visualized as negative staining bands using a digital camera.
2.8 Cellular immunofluorescence assay
MDA-MB-231 cells were set in a 6-well plat with a coverslip in each well. After being treated with different concentrations of DHA for 24 h, the cells were fixed by using 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton-X-100 for 20 min, and blocked with standard goat serum albumin for 1 h at room temperature. The cells were then incubated with primary anti-NF-κB monoclonal anti- body at 4°C overnight. the secondary anti- rabbit Cyanine 3 (Cy3) anti- body (Cell Signaling Technology, USA) was incubated on another day at room temperature for 1 h. subsequently the 4’,6-diamidino-2-phenylindole (DAPI) was stained for 5min. The coverslips were taken out and set on the slide, captured, and analyzed by a confocal laser scanning microscopy system using a 400 × magnification (LSM710, Carl Zeiss, Germany).
2.9 Western blot analysis
Cells were seeded in a 6-well plate and treated with DHA at indicated concentration for 24 h. Whole cell lysates were prepared using RIPA lysis buffer (INVITROGEN, LLC) following the manufacturer’s instructions. While proteins were boiling property about 40μg of total protein was resolved in 8-12% SDS-PAGE. After transferring resolved protein onto PVDF membrane (Millipore), 10% BSA were used to block the membrane. The membranes were then incubated with primary antibodies in 4℃ overnight, washed, incubated with appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Finally, the membranes were visualized using the chemiluminescence detection substrate (Beyotime, Wuhan China) on Image Lab (Bio Chem. USA). The protein bands were quantified using ImageJ software and normalized with respective β-actin.
2.10 Statistical Analysis
GraphPad Prism (Version 4.00, 1992-2003 GraphPad Software Inc.; San Diego, CA, USA) was used for statistical analysis using oneway ANOVA plus Tukey post hoc test to determine the significant changes. P< 0.05 or P< 0.01 were considered significant.