Caco 2

The human colon carcinoma cell line Caco-2 was obtained from Jiangsu KeyGen BioTech (Nanjing, China). Caco-2 cells were grown in DMEM containing 10% FBS and 1% penicillin–streptomycin. The incubator was kept at 37°C with 5% CO₂ and the medium was replaced every 2 days until confluence reached 80%.
The trial was set up including the following three parts. In the first part, cells were treated with BaP (1, 2, 5, 10, 20, 50 and 100 μM) for different times (3, 6, 12, 24 and 48 h) and 6 types of polyphenols (1, 2, 5, 10 and 20 μM) for 24 h to evaluate the cytotoxicity of BaP and polyphenols. In the second part, cells were Pre-treated or co-treated with eriodictyol (5, 10 and 20 μM), naringenin (20 μM) and BaP (50 μM) for 24 h to assess the protective effects of the polyphenols on BaP-induced cytotoxicity. In the third part, cells were co-treated with eriodictyol (20 μM) and BaP (50 μM) to elucidate the possible mechanism by which eriodictyol may counteract the detrimental effects of BaP using proteomics. The doses of BaP used to treat the cells were selected according to previous in vitro studies and do not reflect the amounts attainable by oral intake.

Minimal Essential Media (MEM), Penicillin-Streptomycin (10,000 U/ml) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc., U.S.A. Oxycodone hydrochloride tablet (Sankyo Pharmaceutical Co. Ltd., Japan) was obtained from the Department of Pharmacy, Wuhan Central Hospital. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-fmk), α-tocopherol and N-acetyl cysteine (NAC) were purchased from Sigma, U.S.A. All antibodies were purchased from Santa Cruz Biotechnology Inc., U.S.A. MDA-468, SKBR and Caco2 (KeyGen Biotech Co., Ltd. China) were grown in 75 cm² culture flask using MEM supplemented with final concentration of 10% FBS and 100 U/ml Penicillin–Streptomycin under 37°C, 100% air and humid atmosphere.