gamma-Glu-Cys br Cell apoptosis analysis br Cell
4.5. Cell-apoptosis analysis
Cell apoptosis was assessed by flow cytometry (FCM) analysis. The gamma-Glu-Cys were treated with concentrations of BA (0, 5, 10, 20 μg/mL) for 48 h in a humidified incubator with 5% CO2 at 37 °C. Then, cells were harvested and washed with ice-cold phosphate-buffered saline (PBS), then stained with apoptosis detection kits (BD Biosciences, San Diego, CA). The stained cells were measured using a flow cytometer (BD Biosciences).22 The apoptotic rate represented as sum of annexin V+/ PI+ and annexin V+/PI− Data are representative of three independent experiments.
Detection of mitochondrial membrane potential (Ψm) and reactive oxygen species (ROS).
A mitochondrial membrane potential assay was performed, and examined by FCM using Rh123 staining. Cells were treated with de-signed different concentrations of BA (0, 5, 10, 20 μg/mL) for 48 h and incubated with 10 μM RH123 at 37 °C in the dark place for 20–30 min.
Soon after, the stained cells were harvested and washed twice with cold PBS. RH123 fluorescence was detected by FCM analysis.22,23 For the intracellular,22,23 cells were harvested and incubated with 10 μM
DCFH‐DA dye for 20–30 min at 37 °C after treated with different con-centrations of BA (0, 5, 10, and 20 μg/mL) for 48 h. The stained cells were then washed twice with cold PBS and analyzed by FCM. Data are representative of three independent experiments.
4.6. Transwell migration and invasion assays
were fixed with 95% ethanol and stained with 0.5% crystal violet, and counted under an inverted microscope. For cell migration assays, the upper chamber was coated with Matrigel. Subsequent operations were similar to cell migration assays. Data are expressed as migrative and invasive rate compared with the untreated group.
4.7. Western blot analysis
To determine the effect of BA on the signaling pathway involved, some proteins in HCT116 cells were evaluated by western blot. The western blot analysis was performed as described previously,21–23 with minor modification. Briefly, HCT116 cells were treated with BA in different concentrations (0, 5, 10, 20 μg/mL) for 48 h; then cells were washed twice with cold PBS. Proteins were extracted using a kit (SAB, Nanjing). Then, we also use the kit (TIANGEN BIOTECH, Beijing) to make concentration determination. Then equal amounts of protein from each sample were subjected to sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) gels, following transfer onto poly-vinylidene difluoride (PVDF) membranes (Amersham Bioscience, Pis-cataway, N.J.). Then, the membranes were blocked with 5% skimmed milk at 37 °C for 2 h and incubated with peculiar primary antibodies overnight at 4 °C. After incubation with the relevant secondary anti-bodies, the reactive bands were identified using an enhanced chemi-luminescence kit (Amersham Bioscience, Piscataway, N.J.).
4.8. Xenograft tumor model in nude mice
The animal study protocol was approved by the Institutional Animal Care and Use Committee of China Medical University. Female BALB/c nude mice (6–8 week) were purchased from Beijing HFK Bioscience Co, Ltd (Beijing, China). After the mice had adapted to the environment for a week, they were randomized into three groups. Then we inoculated the mice with HCT116 cells (1 × 107 cells per mouse). Mice were in-traperitoneal administered various concentrations of BA (control, 10, and 20 mg/kg) once daily for 21 days 1 day post implantation. Mice of the vehicle group were treated with corn oil. Tumor volume and body weight were measured every three days. The tumor volume was cal-culated according to the following formula: tumor volume (mm3) = length (mm) × width (mm)2/2. All mice were euthanized at
day 21 post treatment, and their tumor nodules were excised and weighed.21–23 4.9. Immunohistochemistry
IHC staining of tumor sections were described previously.21–23 The sections were excised, fixed, embedded in paraffin, and cut to 4 mm sections into glass slide as paraffin sections. One part of paraffin tumor sections was stained with Ki-67 (ab197234), CC-3 (Asp175) and MMP-2 (ab37150) antibodies. Images were taken with Leica microscope (Leica, DM4000B).
4.10. Histological examination
Visceral organs (heart, liver, spleen, lung, and kidney) were col-lected from mice killed at day 21 post treatment, fixed in 10% (v/v) neutral‐buffered formalin for 24 h and then dehydrated in a graded ethanol series. Sequential paraffin‐embedded tissue sections (5 μm) were prepared and stained with routine hematoxylin and eosin for histological examination. Then make an attached digital camera and NIS‐Elements Advanced Research system (Eclipse 80i; Nikon, Tokyo, Japan).