br Measurement of cell viability br
Measurement of cell viability
naringenin and hesperetin and then incubated for a further 48 or 72 h at 37 °C. WST-1 solution (100 µl) was added to each well, and absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Rich-mond, CA, USA) after a 5-min incubation at 37 °C.
Cell migration assays were performed using 8.0 µm pore size normal Transwell permeable supports (Corning Costar, Lowell, MA, USA) as described previously (Lee et al., 2015a). Briefly, Miapaca-2, Panc-1, and SNU-213 cells (1 × 105/well) were seeded in six-well plates (Nunc, Roskilde, Denmark) and then incubated for 36 h. After 24 h starvation with serum-free medium, a migration assay was performed for 6 h at 37 °C under diﬀerent ratios of naringenin and hesperetin treatment. The absorbance of the eluted dye at 560 nm was measured using an enzyme-linked immunosorbent assay reader (Bio-Rad, Richmond, CA, USA).
Western blot analysis
To evaluate the phosphorylation of focal adhesion kinase (FAK) and p38, Panc-1 cells (1 × 105/well) were seeded in six-well plates (Nunc, Roskilde, Denmark). After 48 h, the cells were treated with diﬀerent ratios of naringenin and hesperetin and then incubated for a further 24 h at 37 °C. To produce whole-cell lysates, cells were lysed in M-PER lysis buﬀer (Thermo Scientific, Bonn, Germany) with protease and phosphatase inhibitors. Total protein quantity was determined by the BCA quantification method (Bio-Rad). Cell lysates (10 µg) were sepa-rated by means of sodium dodecyl sulphate-polyacrylamide gel elec-trophoresis and transferred to a nitrocellulose membrane (Amersham Bioscience, Little Chalfont, Buckinghamshire, UK). The membranes were blocked with 5% bovine serum albumin. Antibodies specific for phospho-FAK (Tyr397), FAK, phospho-p38 (Ser473), p38, and GAPDH were diluted in blocking buﬀer at 1:1000 and incubated overnight at 4 °C. Secondary Galactose 1-phosphate included horseradish peroxidase-conjugated donkey anti-rabbit (1:4000) or donkey anti-mouse antibodies (1:4000) (Santa Cruz Biotechnology) and were used in conjunction with a normal Western blot detection reagent (iNtRON, Seoul, Korea) to visualize bands using X-ray film.
High performance liquid chromatography (HPLC) analysis
A HPLC system (Shimadzu Scientific Instruments, Columbia, MD, USA) equipped with a photo diode array (PDA) detector and a Luna C18(2) column (5 µm particle size, 4.6 mm X 250 nm; Phenomenex, Torrance, CA, USA) was used for HPLC analysis as previously reported (Lee et al., 2016c). The analysis was performed in a step gradient mode: 20% acetonitrile at 0 min, 40% acetonitrile at 10 min, 70% acetonitrile at 20 min, 70% acetonitrile at 25 min, 20% acetonitrile at 27 min, and 20% acetonitrile at 30 min at a column flow rate of 1 ml/min. Detection of peaks was performed at 270 nm. Quantification of extracted narir-utin, hesperidin, naringenin, and hesperetin in Citrus unshiu peels was performed by using limit of detection (LOD) and limit of quantification (LOQ) values from HPLC analysis as previously described (Lee et al.,
A Naringenin B Hesperetin
HO O HO OH
Fig. 1. Structure of narigenin and its hesperetin.
2018). The purity of the extracted compounds was compared with re-ference hesperidin, narirutin, hesperetin, and naringenin, respectively, using HPLC analysis.
Target protein analysis of naringenin or hesperetin
Target proteins of naringenin or hesperetin were identified from ChEMBL version 20 (Lee et al., 2016d).
Xenograft tumor model
BALB/c nude mice, 6–8 weeks of age, were obtained from Orient (Seongnam, Korea). Panc-1 cells (1 × 107) were injected sub-cutaneously into the flank as previously reported (Lee et al., 2017b). Once the tumors reached a size of approximately 50 mm3, mice were randomized to five experimental groups to receive phosphate buﬀered saline (PBS), 30 mg/kg naringenin, 30 mg/kg hesperetin, 10 mg/kg naringenin/hesperetin mixture, or 30 mg/kg naringenin/hesperetin mixture. Tumor volume (V) was calculated as 0.523 LW2 (L = length, W = width). Body weights were recorded regularly. Animal care and experiments were carried out in accordance with guidelines approved by the animal bioethics committee of Jeju National University (2016–0049).