Source and preparation of snail mucus
Snail mucus was collected from Achatina fulica and supplied by Dr. Yao-Tern Li (Hsin-Ling Tang Chinese Medicine Clinic, Chia-Yi, Taiwan). 3% NaCl was used to stimulated mucus production, and mucus was collected from the surface of live snails. The stock of snail mucus is defined as 100% (6 mg/mL). The stock solution was diluted to 30–600 µg/mL as the working solution and was filtered out the impurities with 0.45mm pore size membrane disc filters.
Cell line and cell culture
The human breast cancer MDA-MB-231cell line was purchased from American Type Culture Collection (ATCC). All of the cell lines were cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum, 100 unit/mL penicillin, and 100 µg/ mL streptomycin. Cells were incubated at 37°C, 95% air and 5% CO2 (18).
Breast cancer cells were seeded in a 96-well plate at 0.5x104 per well. Cells were treated with various concentrations of the snail mucus for 2 days followed by incubation with 20% of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, MO, USA) for 3 hours, and then dissolved with DMSO for 30 minutes (18). The absorbance was measured at the wavelength of 570 nm by using an ELISA reader.
IncuCyte Live-cell analysis
Breast cancer cells were seeded in a 96-well plate at 0.5x104 per well. Cells were incubated in IncuCyte after treatment with snail mucus and/or doxorubicin. The growth rate was measured with IncuCyte Live-Cell Analysis System.
Cell cycle analysis
Breast cancer cells were seeded in a 6-well dish at the density of 0.1x106/well. After treatment with snail mucus for 48 hours, the cells were washed with PBS and collected by trypsinization. Then cells were fixed in 75% ethanol and stored at -20°C for 2 hours. Cells were centrifuged at 1000 rpm for 5 minutes to remove the supernatant and stained with 0.1mg/ mL RNase (Cat no.12091-021), 1% 100X Triton, 20 µg/mL propidium iodide in 1X PBS at 37oC for 30 minutes. The sample was detected for ten thousand cells and analyzed by using Flow Cytometry.
Breast cancer cells were seeded in a 6-well dish at the density of 0.1x106/well. Following treatment with snail mucus for 24 hours, cells were collected by trypsinization and washed with PBS. Cells were stained with an Annexin V-FITC Apoptosis Detection Kit (Bio Vision). The sample was detected for ten thousand cells and analyzed by using Flow Cytometry (BD Biosciences).
DNA laddering assay
After the treatment for 24 and 48 hours, cells were collected and centrifugated at 1600g for 5 minutes. The cell lysate was prepared with lysis buffer (1% NP-40 in 20mM EDTA, 50mM Tris-HCl at PH 7.5) and was centrifuged at 10000g for 10 min. The supernatant was added with 1% SDS and was incubated for 2 hours at 37oC. Then, the supernatant was added with 5 µg/µL proteinase K followed by incubation at 37oC for 2 hours and was further added with 3 M sodium acetate to precipitate genomic DNA. DNA was mixed thoroughly with 100% ethanol then placed at -80oC for 1 hour. The sample was centrifuged at 12000 rpm for 20 minutes to obtain the pellet. The pellet was washed with 75% cold ethanol and air-dried at room temperature. The dried pellet was dissolved with double-distilled H2O. DNA laddering was observed in agarose gel electrophoresis.
Western blot analysis
Cells were washed with ice-cold PBS, and lysates were collected in RIPA buffer (50 mM Tris, 150mM NaCl, 10 mM EDTA, 1% Triton X-100, 0.1% SDS) containing protease inhibitors. Cell lysates were centrifuged at 15000 rpm for 15 minutes after sonication, and the supernatant was collected. The concentration of protein was measured by using Bradford protein assay (Bio-Rad). An equal amount of protein was separated by SDS-PAGE. After transfer to PVDF membrane (0.45 µM, GE Healthcare), the membrane was blotted with 5% milk in TBST buffer for 1 hour followed by hybridization with indicated primary antibodies and HRP-labeled secondary antibodies. The expression levels of target proteins were determined by detecting the chemoluminescence signal, which was catalyzed with ECL (18) (GE Healthcare or Millipore).
General of AFC components analysis
1H-NMR spectra were recorded using a Avance Neo 400 NMR spectrometer (Bruker Instruments, Karlsruhe, Germany) with CD3OD or D2O as the solvent. MPLC was performed using a reverse-phase column (iLOK™ (12g, I.D. 21.4×H76 mm, C18, 460 mm × 36 mm i.d.) on a PuriFlash XS 420 series apparatus (Interchim Inc., Montlucon, France). The UV detector was set at 210, 254, and 280 nm, and the UV absorption spectra were recorded in the range of 210–400 nm.
Isolation of the active fractions
The Achatina fulica mucus (AFM, 45.3 mg) was separated with MPLC [C18 gel (460 mm × 36 mm i.d.), column (21.4×H76 mm i.d.)] using water (A) and acetonitrile (B) under gradient conditions (0–2 min, 2 % B; 2–3 min, linear gradient 2–5% B; 3–17: min, linear gradient 5–15% B; 17–32 min, linear gradient 15–100% B; 32–50 min, 100% B) as the mobile phase at a flow rate of 20.0 mL/min. The sample was dry-load onto the column and fractionated into seven fractions (AFM-1 ~ 7).
Data were presented as mean ± S.E.M. The difference between the experimental and control groups was assessed by Student’s t-test and reached significance if the p-value is < 0.05.