2 − 1. Cell Culture
Human umbilical vein endothelial cells (HUVECs) were provided from Pasteur Institute (Tehran, Iran). HUVECs were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all reagents were purchased from Bio-Idea Tehran, Iran). Cells were incubated at 37 ºC with 95% humidity and 5% CO2.
2.2. Preparation of treatments
5-FU at 50 mg/ml was obtained from Ebewe Pharmacy (Unterach, Austria). By adding the right amount of drug as a supplement to the medium, we obtained the desired concentration. Que was purchased from Sigma–Aldrich (St. Louis, Missouri, United States) and dissolved in dimethylsulfoxide (DMSO, Bio-Idea), and the stock solution was prepared at 200 µM, which diluted with medium to prepare different concentrations. The concentration of DMSO in the cell culture medium was < 0.1%. Thus, it has not affected any cell functions.
2–3. Cell viability assay (MTT)
The MTT assay was used to measure the cell viability in culture; for this purpose, 6× 103 (HUVECs) cells per well were seeded in 96-well plates and incubated for 24 h. Then the cells were treated with different concentrations of 5-FU (2.5, 5, 10, 20, 40, 80, and 160µM), Que (10; 40; 70; 100; 130 and 160µM), and various combination state with 2.5 and 5µM of 5-FU plus 70, 100, and 130µM of Que for 24h and 48h. After that, the medium was replaced by MTT solution at a final concentration of 0.5mg/ml and the cells were incubated for 4h in a dark place. Then MTT solution was removed and 150µl DMSO (Merck, Darmstadt, Germany) was added and shacked for 15 minutes. The plates were read using a microplate reader (BioTek ELx800 Winooski, Vermont, United States) at a wavelength of 570 nm. IC50 values were calculated using computer software Graphpad Prism 8 (La Jolla, CA). The assay was performed in triplicate with four replicates per sample.
2–4. Wound-healing Migration Assay
Cell migration was evaluated by wound healing assay. To this end, 2 × 105 Cells / well were seeded in 6-well plates and incubated at 37 ºC to reach 90% confluency. Then cells were scratched using the sterile yellow tip and washed with PBS to remove debris and subsequently treated with 5µM 5-FU, 130µM Que, and their combination. The wells were photographed at 0 h, 24 h, and 48 h, in random microscopic zones, and the width of the scratch was measured by NIH Image J software (National Institutes of Health, Bethesda, USA). Subsequently, the percentage of wound closure was calculated according to the following formula: %wound closure = [(T0 – T48)/T0] × 100.
2–5. Real-time RT-PCR
Total RNA was extracted with Hybrid-R RNA isolation kit from harvested cells according to manufacturer’s instructions (GeneAll, Songpa-gu, Seoul, South Korea). RNA purity and integrity were confirmed with A260/A280 ratio (~ 1.8-2.0) and agarose gel electrophoresis, respectively. cDNA was synthesized from the extracted RNAs using the cDNA synthesis kit based on the manufacturer’s instructions (Yehta Tajhiz Azma, Iran). In brief, 2µl of cDNA was amplified in each 20µl PCR reaction mix containing 10µl of 2x SYBR Green Master Mix (Yehta Tajhiz Azma, Iran), 1µl of each 10µM forward and reverse primers and 6µl DEPC water. HPRT was preferred as the internal reference. Results were analyzed using an Applied Biosystem with software version 2.3 (StepOne™, USA). The reaction conditions were as follows: 94˚C for 3 min; 94˚C for 40 sec, 59˚C for 30 sec, 72˚C for 30 sec for 40 cycles; 72˚C for 5 min. The primer sequences were shown in Table 1.
2–6. CAM assay
To evaluate in vivo angiogenesis CAM assay was performed. Fertilized chicken eggs (purchased from the Department of Poultry, School of Veterinary Medicine, Shiraz University) were incubated at 37 ºC and 60% humidity and randomly divided into 4 groups: control, 5µM 5-FU, 130µM Que, and 5-FU plus Que (n = 4 per group). Briefly, on the second day of incubation 1 cm2 square window was made at the top of the live eggs and 2ml of albumin was aspirated at the opposite side. On the 5th day, the chorioallantoic membrane (CAM) is developed, so sterile methylcellulose discs, which were loaded with the drug, were applied to the CAMs. The drug-treated eggs were incubated at 37 ºC and 60% humidity for 48 hours. Then the CAM tissues under the filter discs were removed, washed in phosphate-buffered saline (PBS), and fixed by Formaldehyde 4%. Lastly, images (150X) was taken using a stereomicroscope (Leica Zoom 2000). The images were analyzed using online Wimasis Image Analysis Software. The number of total branch points and total vessel network lengths were used as indicators of angiogenesis.
2–7. Statistical analysis
Values are presented as the mean ± S.E.M and results were analyzed using SPSS software version 22. Data were compared by one-way analysis of variance (ANOVA) and LSD analysis. P < 0.05 was considered to indicate a statistically significant difference.