br expression of PIM in
expression of PIM3 in normal organs is low [7–9]. PIM3 was found to be an oncogene given thatPIM3 overexpression correlated with enhanced tumor growth and cell survival whereas PIM3 siRNA or inhibitor de-layed tumorigenesis . Abnormal Pim-3 expression has also been found in the invasion and metastasis of gastric cancer . These findings imply that PIM3 might function as a potential therapeutic target for GC.
Cinobufacini (Huachansu), which is extracted from the skin and parotid venom gland of the toad Bufo bufo gargarizans Cantor, has been used as a folk medicine in China for decades to treat swelling, pain, and heart failure . In the past decade, cinobufacini injection has also been tested in clinical trials in combination with chemotherapeutic agents to treat lung cancer, hepatocellular carcinoma, prostate cancer and gallbladder cancer. The results of such studies have evidenced that Cinobufacini possess anti-neoplastic activity [13–15]. Bufadienolides are bioactive compounds of Cinobufacini responsible for its anti-cancer activities [16,17]. Bufothionine is a potent bufadienolide which has been found to exert anti-neoplastic activities in hepatocellular
Corresponding author at: NO.1 Jianshe Road, Zhengzhou 450052, Henan, China. E-mail address: [email protected] (G. Wang).
Fig. 1. Bufothionine reduces cell viability and disrupts cell membrane integrity in GC cells. A. The chemical structure of bufothionine. B. Treatment with bu-fothionine at 20, 50 and 100 μg/ml for 24 or 48 h followed by cell viability assessment by CCK-8 assay. C. GES-1 丝裂霉素 C were treated with bufothionine at 20, 50 and 100 μg/ml for 24 or 48 h. Cell viability was assessed by CCK-8 assay. D. MKN28 and AGS cells were treated with bufothionine at 20 and 50 μg/ml for 48 h. The eﬀect of bufothionine on cell membrane integrity was assessed by LDH assay. *P < 0.05, **P < 0.01.
carcinoma cell lines [12,18]. However, the eﬀect of bufothionine on other human malignancies has not been reported. In the present study, the eﬀect of bufothionine on GC was explored and the underlying molecular mechanisms were investigated. The results showed that bu-fothionine suppresses GC growth in vitro and in vivo. Moreover, PIM3 was identified as the primary molecular target that mediates the anti-cancer activities of bufothionine against GC.
2. Materials and methods
The human GC cell line MKN28was purchased from GeneChem (Shanghai, China), AGS was purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and GES-1 (the normal gastric epithelial cells) was purchased from the American Type Culture Collection (Rockville, MD, USA). All cells were cultured in RPMI 1640, supplemented with 10% foetal bovine serum (Hyclone, Logan, UT, USA) in a humidified CO2 (5%) incubator at 37 °C.
2.2. Viability assay
Briefly, 100 μL cells were seeded in 96-well plate at the density of 1.2 × 105 cells/ml. Following treatment with bufothionine, the cell viability was detected using the nonradioactive cell proliferation assay kit (Promega Corporation, Madison, WI) according to the manufac-turer's protocol. The absorbance at 570 nm was examined by a micro-plate reader.
LDH activity assay kit (Jiancheng Biotech, Nanjing, Jiangsu, China) was utilized to evaluate LDH level. Briefly, GC cells were seeded in 6-well plates at a density of 2 × 105cells/well. Following treatment with bufothionine, NADH and pyruvate were added to the culture medium
samples and incubated for 15 min at 37 °C, 0.4 M NaOH was then added to stop enzymatic reaction. A microplate reader was used to detect absorbance at 450 nm.
After treatment, GC cells were rinsed twice with cold 1× PBS and incubated with Hoechst/PI staining buﬀer for 15 min at 25 °C in the dark. Then the level of apoptotic cell death was visualized.
2.5. Flow cytometry analysis
Cell apoptosis was detected using an Annexin V-FITC/PI apoptosis kit (BD pharmingen, NJ, USA). Briefly, after treatment, the cells were seeded at a density of 2 × 105, and then incubated with Annexin V FITC and PI staining solution for 15 min. Apoptotic cell population was then quantified using a flow cytometer.
2.6. Western blotting
Briefly, the proteins in cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membrane. Subsequently, the membranes were incubated with the primary antibodies overnight at 4 °C. Goat anti-rabbit IgG-HRP (Beyotime, Shanghai, China) served as the second an-tibodies and GAPDH served as an internal control to normalize protein levels. The signals were then visualized using chemiluminescent sub-strate (KPL, Guildford, UK).