Reagents and Antibodies. Lysine was obtained from Beijing Solarbio Science and Technology Co. (Beijing, China). Gambogic acid (98%) was obtained from Nanjing Jingzhu Biotechnology Ltd. Gambogic acid lysinate (GAL) was synthesized according to established methods in our department. The molecular weight is 755.4 Da . 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was purchased from Sigma Aldrich. A Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific Inc. Primary antibodies against Bcl-2, Bax, CDK-2, P53 and P21 were purchased from Cell Signaling Technology. An anti-GAPDH antibody was purchased from Santa Cruz Technology. Secondary antibodies were also purchased from Cell Signaling Technology. Immobilon Western Chemiluminescent HRP substrate was bought from EMD Millipore.
Cell culture and a cytotoxicity Assay. The human cervical cancer SiHa and HeLa cell lines were obtained from the National Biomedical Medical Cell Resource Bank. Completed culture medium was composed of Dulbecco’s modified Eagle’s medium (DMEM), 100 units/ml penicillin/streptomycin and 10% fetal bovine serum (FBS; HyClone). Both the SiHa and HeLa cell lines were cultured in complete medium and incubated in a 95% air/5% CO2 humidified atmosphere at 37°C. The inhibitory of GAL on proliferation was detected by an MTT assay. Briefly, HeLa cells and SiHa cells were seeded in 96-well plates at 4 × 103 cells per well. After culturing overnight, different concentrations (0, 0.5, 1, 1.5 and 2 µmol/l) of GAL were added to each well. After incubating for 24 h, 48 h or 72 h, 20 µl of MTT solution (5 mg/ml) was added to each well. After 4 h of incubation, the medium was removed, and 150 µl of DMSO was added to each well. A Spectra Max 190 Absorbance Microplate Reader (Sunnyvale) was used to measure the optical density at 570 nm, and the half-maximal inhibitory concentration (IC50) was calculated with GraphPad Prism 5.0 (GraphPad).
Cell growth curve. Five hundred SiHa or HeLa cells were seeded in each well of a 6-well plate in 10% FBS DMEM. Saline or GAL (0.5 or 1.0 µmol/L) was added to each well after an overnight incubation. Triplicate wells are harvested by trypsinization every day for 6 days and counted with a cytometer. Growth curves were generated using Microsoft Excel 2010.
Cell cycle and cell apoptosis assays. SiHa cells were plated in a 25 cm2 flask and exposed to various concentrations of GAL (0, 1, and 2 𝜇mol/L) for 24 h or 48 h. For the apoptosis assay, cells were digested, washed and centrifuged. Then, 300 µl of binding buffer was added to the cells, which were a final density of 1 × 106 cells/ml. Annexin V-FITC and propidium iodide (PI) (Thermo Fisher Scientific, Inc.) were applied to stain the cells for 15 min in the dark. Then, flow cytometry was performed with a FACSCalibur and Cell Quest software (version 5.1; BD Biosciences) to detect apoptosis. For the cell cycle assay, cells were digested, washed, and fixed in 70% ethanol, and RNase (5 mg/ml) was added to treat the cells for 30 min. Following the addition of 50 mmol/l PI, flow cytometry was performed with a FACSCalibur and Cell Quest software to evaluate the cell cycle.
Western Blot Analysis. Different concentrations of GAL (0, 0.5, 1, and 2 µmol/l) was added to SiHa cells for 24 h or 48 h. The cells were rinsed twice with cold PBS, and RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail (Roche, Beijing, China) was applied to lyse the cells for 30 min on ice. The cell lysates were centrifuged, and the supernatant was collected. A BCA assay kit was used to determine the protein concentration. Equal amounts and volumes of protein (30 µg) were resolved by SDS–PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Then, the membranes were blocked with 5% nonfat skim milk and sequentially incubated with primary and secondary antibodies. Protein bands were developed using Immobilon Western Chemiluminescent HRP substrate with an enhanced ChemiImager 5500 chemiluminescence system (ProteinSimple). ImageJ software (version 1.0; National Institutes of Health) was applied to calculate the optical density of the bands, which were standardized to the band for GAPDH.
Caspase 3 Activity Assay. Caspase-3 activity was evaluated with a colorimetric caspase-3 assay kit (Beyotime Institute of Biotechnology). SiHa cells (5x105) were inoculated into 75 cm2 culture flasks and treated with GAL (0, 0.5, 1.0 and 2.0 µmol/l). After 24 or 48 h of treatment, the SiHa cells were collected and resuspended in 50 µl of lysis buffer. Follow-up experiments were performed according to the study by Chunlai Shi et al .
In vivo xenograft mouse model. Four-week-old female BALB/c nude mice (20-24 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing China) and maintained at the Experimental Animal Center of Beijing Bozhiyuan Biotechnology Co., Ltd. Nude mice were kept in a specific pathogen-free environment in which the temperature was 22°C and the humidity was 40–50%. Studies involving mice were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals of Beijing Bozhiyuan Biotechnology Co., Ltd. SiHa cells (1 × 107cells per mouse) were inoculated into the left armpit of nude mice by subcutaneous injection. Nine days after the inoculation, the mice were treated with either saline (control group) or 2.5 mg/kg GAL, every other day (GAL treated group). The body weight and tumor volume of the mice were measured every 2 days. After 10 days of treatment, the mice were euthanized, and their tumors were weighed recorded and photographed.
Statistical analysis. All data are represented as the mean ± standard deviation. SPSS 17.0 statistical software was used to perform statistical analysis. Efficacy was compared by one-way ANOVA or a two-sided unpaired Student’s t-test. When the p-value was less than 0.05, the difference was considered to be significant.