br Tube formation assay br Matrigel
2.6. Tube formation assay
Matrigel (50 μL, growth factor reduced and phenol red-free, BD Bio-science) was added into 96-well plates in the ice bath and subsequently allowed to polymerize for 30 min at 37 °C in the incubator. Cell suspen-sion of HUVECs at density of 2 × 105 cells/well was added onto Matrigel and then incubated with cell medium containing free NCTD (40 μg/mL)
and different concentrations of CNC (12.5, 50, 200 μg/mL) in 5% CO2 at 37 °C. After incubation for 8 h, cell morphology and organization were observed through an inverted microscope. The closed networks of vessel-like tubes were quantified by counting and averaging tubular length from five random fields (100×).
2.7. In vitro apoptosis assay
The cellular apoptosis assay was detected using confocal laser scan-ning microscopy (CLSM) and flow cytometry. The nuclear morphology of Iberiotoxin incubated with CNC was investigated by staining the cells with Hoechst 33258. Briefly, SGC-7901 cells were seeded in 6-well plates and incubated for 24 h at 37 °C. After the cells reached 90% confluence, the culture medium was replaced with fresh medium containing differ-ent concentrations of CNC and free NCTD. After incubation for 24 h, cells
Elemental analysis results of CMCS and CNC.
were washed twice with cold PBS and fixed with methanol for 20 min, followed by staining with Hoechst 33258 (10 mg/mL) for 5 min in the dark. The stained cells were then washed with cold PBS and photographed under CLSM (Nikon, Japan).
Cell apoptosis was determined with an Annexin V-FITC/PI apoptosis-detection kit. The SGC-7901 cells were seeded in 6-well plates at a den-sity of 2 × 105 cells/well and preincubated for 24 h. Then, the medium was replaced with 2 mL fresh medium containing different
B Merge Fluorescence Brightfield
Fig. 2. In vitro cytotoxicity, cellular uptake, anti-metastasis and anti-angiogenesis effects of CNC. (A) Cell viability of SGC-7901 cells. (B) Cellular uptake of the FITC-CNC in SGC-7901 cells at different time points (original magnification, 100×). (C) Photographs of HUVECs migrated through the membranes of transwell chambers (original magnification, 100×). (D) Inhibition rate on the migration of HUVECs. (E) Photographs of HUVECs on Matrigel after incubation for 8 h. (F) Relative tubular formation rate of HUVECs. (a) cells treated with complete medium; (b) cells treated with CNC at the concentration of 12.5 μg/mL; (c) cells treated with CNC at the concentration of 50 μg/mL; (d) cells treated with CNC at the concentration of 200 μg/mL; (e) cells treated with NCTD at the concentration of 40 μg/mL. Data represents mean ± SD (n = 6), *P b 0.05, **P b 0.01 significant difference compared with control group; #P b 0.05, ##P b 0.01 significant difference compared with NCTD group.
concentrations of CNC and free NCTD. The cells were incubated for an-other 24 h and then harvested with trypsinization, washed with PBS and resuspended in 1 mL binding buffer. Finally, the cell suspension was incubated with 5 μL Annexin V-FITC and 10 μL propidium iodide (PI) for 10 min, and the fluorescence intensity was analyzed using a flow cytometer (FACS Calibur, BD, USA).
2.8. In vivo antitumor efficacy
Female BALB/c nude mice were subcutaneously injected with SGC-7901 cells (1 × 107) in the right armpit, aiming to establish human gas-tric cancer xenografts. After inoculation, the mice were randomly assigned into five groups, including control group (physiological saline), DDP (5 mg/kg body weight) treatment group, free NCTD (6.524 mg/kg body weight) treatment group, CNC high dose (32.62 mg/kg body weight) treatment group and CNC low dose (16.31 mg/kg body weight) treatment group. Mice in each group were injected with different for-mulations via tail vein every other day for 24 days in total. Meanwhile, body weight changes were measured and recorded every other day. After the treatments, mice in all groups were sacrificed after collection of the blood. Tumor tissues were weighted, fixed in 4%
paraformaldehyde and processed with routine hematoxylin and eosin (H&E) staining using standard methods.
2.9. Immunohistochemical assay
To investigate the metastasis and apoptosis of tumor cells, immuno-histochemical staining was conducted to access the expressions of CD34, Bcl-2, Bax and Caspase-3 protein with the avidin-biotinylated peroxidase complex method. The paraffin-embedded tumor sections were deparaffinized, rehydrated and incubated in 3% hydrogen perox-ide (H2O2) for 10 min to eliminate the intrinsic peroxidase activity. After washing with phosphate buffered saline, the sections were blocked in bovine serum albumin (BSA) solution and then incubated with primary antibody against CD34 (1:150), Bcl-2 (1:200), Bax (1:200) and Caspase-3 (1:200) at 4 °C overnight. Subsequently, bio-tinylated goat anti-rabbit antibodies were added and incubated at 37 °C for 30 min, followed by incubation with an ABC reagent for 60 min at 37 °C according to the instructions. The sections were then stained with the chromogen 3, 3′-diaminobenzidine (DAB) for 5–10 min, and fi-nally counterstained with hematoxylin. The positive stained areas were photographed using the E200 microscope (Nikon Co. Japan) and the