Due to the hepatocarcinogenic property of both AFB1 and ST, the human hepatoma HepG2 cell was used as the cell model to investigate their co-proapoptotic activity and related mechanisms, and it is expected that the basic toxicity property
and mechanism obtained from a model system might facilitate developing interventive measures to reduce their vivo toxicity to the body. Human hepatoma HepG2 cells lines were obtained Tanespimycin from American Type Culture Collection (ATCC, Beijing, China). AFB1(purity ≥98%), ST (purity ≥98%), DMSO, Sulforhodamine B (SRB), TCA, H33258, DCFH-DA, DCF, rhodamine 123, JC-1 dye, and calf thymus DNA were purchased from Sigma Aldrich (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, HBSS, and phosphate-buffered saline (PBS) were purchased from Gibco Life Technologies (Shanghai, China). ATP assay kit, Annexin V-FITC cell apoptosis assay kit, and mitochondria membrane potential assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). DAB was purchased from Genetech Inc (Shanghai, China). All the antibodies for Caspase-3, p53, Bax and Bcl-2 were from Germany AbioB, LTM.(Shanghai, China). HepG2 cells were cultured in DMEM medium containing FBS (10%), penicillin (100 units/ml) and streptomycin (100 μg/mL) under a Bioactive Compound Library humidified incubator
with CO2 (5%) and air (95%) at 37 °C. Every 5–7 days, the adherent cells were suspended after treatment with 1 mL of 0.25% trypsin-EDTA solution for 2-3 min at 37 °C, and then were subcultured at a 1:3 split ratio. The culture medium was changed every 2 days. Stocks of cells were routinely frozen and stored in liquid nitrogen. Cells with 15-20 passages were used for experiments to ensure cell line stability. The cell viability was measured by a sulforhodamine B colorimetric assay (SRB) . Briefly, log phase HepG2 cells (200 μL) were seeded at a density of 3 × 104 cells/mL in a 96-well plate. After incubation for 24 h, culture medium
containing AFB1 or ST (dissolved in DMSO) was used to treat the cells for 24 or 48 h. Then, the cells were fixed by adding 100 μL cold (4 °C) trichloroacetic acid (TCA) solution (10% w/v) and incubated Carbohydrate at 4 °C for 1 h, and then gently washed with deionized water 4-5 times. After the plate was air-dried, 100 μL SRB reagent (4 g/L) was added and incubated at room temperature for 30 min, then the plate was washed with 1% acetic acid 4-5 times and air-dried. The OD reading at 490 nm was carried out by adding 200 μL 10 mM Tris-HCl buffer (pH 7.4) to each well. Cell growth inhibition rate in percentage was calculated by comparing with the control sample (without AFB1 and ST treatment). HepG2 cells were seeded at a density of 5 × 104 cells/mL in a 96-well plate.