Cell lines
The human GC cell line NCI-N87 was maintained in our lab while Hs-746T was purchased from American Type Culture Collection, and human umbilical vein endothelial (HUVEC) cells were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured at 37 °C in 5% CO2 and saturation humidity in RPMI-1640 medium with 10% fetal bovine serum containing penicillin and streptomycin.
Endothelial tube formation
HUVEC cells were plated in 96-well plate coated with 50 μl matrigel (BD Bioscience, CA, USA) at the density of 3 × 104 cells/well and treated with or without different concentrations of crocetin. Tubes were photographed by microscopy and evaluated by Image Pro Plus software.
Cell proliferation assay
Cell proliferation was monitored by Cell Counting Kit-8 (CCK-8). HUVEC cells or GC cells plated in 96-well plate at the concentration 2000 cells/well were treated with or without crocetin. Cell proliferation was measured every 24 h for 5 days after adding CCK-8 2 h at the absorbance 450 nm using Epoch Microplate Spectrophotometer (Bio Tek).
Migration and invasion assays
A number of 1 × 105 HUVEC cells or GC cells were suspended in serum-free RPMI-1640 medium containing crocetin or not and plated in transwell upper chamber (8 μm for 24-well plate; Corning Costar, NY, USA) coated with or without Matrigel (BD Bioscience, CA, USA) for migration or invasion assay respectively. Cells were fixed by 10% formalin and stained with 0.5% crystal violet after 24 h incubation in 24-well plates. Finally, cells in the lower chamber were photographed and counted by inverted microscopy.
Filamentous actin (F-actin) staining
HUVEC cells and GC cells were plated into 8-well glass (Merck Millipore) and treated with or without crocetin for 24 h, and then fixed using 4% freshly made paraformaldehyde for 10 minutes. After permeabilization with 0.2% Triton X-100, the cells were blocked using 3% bovine serum albumin for 30 minutes. Next, the cells were stained with rhodamine phalloidin (1:20; CST) to visualize the cytoskeleton while the nuclei were stained with DAPI. Slides were imaged and analyzed on a fluorescence microscope.
Vasculogenic mimicry (VM) formation assay
Serum-free RPMI-1640 medium and Matrigel were mixed according to 2:1. The mixture was then seeded onto 16 mm glass cover slides in 6-well plate and was allowed to polymerize for 2 h at 37 °C. Then 1 × 105 GC cells were seeded onto each slide and cultured for 3 days at 37 °C with 5% CO2. Cells then were fixed by 4% paraformaldehyde and stained by PAS stain according to the manufacturer's protocols.
After counterstained with hematoxylin, slides were dehydrated and covered.
Three-dimensional (3D) culture and mosaic vessel assays
A 24-well plate was coated with 100 μl Matrigel/well and then was allowed to polymerize for 2 h at 37 °C. Hs-746T GC cells labeled with EGFP and HUVEC cells without EGFP label were mixed according to 1:1. Next, mixed cells were plated into 24-well at the density of 1 × 105 cells/well and cultured in complete growth medium with or without crocetin. Tubes were photographed by an AMG fluorescence microscope after 24 h incubation at 37 °C with 5% CO2 and were evaluated by Image Pro Plus software.
Quantitative Real-time PCR (RT-qPCR)
RNA was extracted from GC cells treated with or without crocetin, and then RNA was reversed to cDNA. RT-qPCR was performed using SYBR-green according to manufacturer’s instructions. Primers for HIF-1α, forward 5’-GAACGTCGAAAAGAAAAGTCTCG-3’, reverse 5’- CCTTATCAAGATGCGAACTCACA-3’; Notch-1, forward 5’-GCTTGTGGTAGCAAGGAAGC-3’, reverse 5’-CCACATTCAAGTGGCTGATG-3′; Shh, forward 5’-CTGCTCGGTGAAAGCAGAGA-3’, reverse 5’- CGCGTCTCGATCACGTAGAA-3’; VEGF, forward 5’- AGGGCAGAATCATCACGAAGT-3’, reverse 5’-AGGGTCTCGATTGGATGGCA-3’; GAPDH, forward 5′-GGACCTGACCTGCCGTCTAG-3′, reverse 5′-GTAGCCCAGGATGCCCTTGA-3′.
Western blotting
The method was consistent with the previous[32]. In brief, proteins of cells were separated by SDS-PAGE and then transferred into PVDF membranes. Primary antibody (1:1000 dilutions) Shh, PTCH2, Sufu, GLi1, E-cadherin, β-catenin, vimentin were purchased from cell signaling technology (CST, USA). GAPDH (1:10000 dilutions) was purchased from Proteintech. After incubation with primary antibody, secondary antibody followed. Finally, the results were visualized by Tanon system.
Enzyme linked immunosorbent assay (ELISA)
Concentrations of Shh secreted from HUVEC cells and Hs-746T GC cells induced by crocetin were assayed by ELISA according to the manufacturer’s instructions. The absorbance at OD 450 nm was read and the level of Shh expression were calculated according to the standard curve.
Nude mice tumorigenesis and immunohistochemistry
Hs-746T GC cells (1 × 106 cells) were subcutaneously injected into 4-week-old male BALB/c nude mice (Institute of Zoology, China Academy of Sciences) to evaluate the role of crocetin in tumor growth in vivo. Tumor nodules were measured every week and were calculated using the formula: tumor volume = (Width2 × Length) / 2. Mice were killed 4 weeks after injection. Tumors were weighed and fixed for immunohistochemistry staining (IHC).
For IHC, sections staining was performed according to the DAKO protocol using primary antibody (1:200 dilutions) E-cadherin, vimentin and Ki-67.
Statistical analysis
Data are shown as mean ± SD. Differences between experimental groups were assessed by the Student's t test or one-way ANOVA. A two-tailed value of P < 0.05 was considered statistically significant. Statistical analysis was performed using IBM SPSS 19.0 software (SPSS Inc).