Migration was quantified and analyzed with Image-pro plus version 6.0 software (Media Cybernetics, Silver Spring, MD, USA). results showed that petasin decreased the phosphorylation of Akt (1.01??0.16 0.32??0.11, 0.85??0.14, 0.74??0.12, 1.98??0.22, 1.51??0.36, 0.82??0.11, 0.94??0.15, 577.67??75.12 mm3 at day 28, 36.0??4.9%, inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy. reported that petasin inhibits testosterone production and release of corticosterone from rat zona fasciculata-reticularis cells, and Bretazenil obstructs proliferation of human T24 bladder carcinoma cells.[11,12] These authors also found that petasin induces apoptosis in prostate cancer Bretazenil cells, suggesting that S-petasin and iso-S-petasin could be useful as anticancer agents. However, the activity of petasin against colon cancer cells remains unknown. This study investigated the antiproliferative properties of petasin using a human colon carcinoma cell line. Target endpoints included cytotoxicity, apoptosis, cell migration, and cell invasion. The effects of petasin on the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway involved in colon carcinogenesis were also investigated. Finally, in this study, the anti-proliferation activity of petasin was studied using Balb/c nude mice bearing tumors of a pre-established subcutaneous SW-620 cell line. Methods Ethical approval All animal protocols were approved by the Institutional Animal Care and Use Committee of Lanzhou University Second Hospital and the research protocol complied with institutional guidelines of the Animal Care and Use Committee at Lanzhou University Second Hospital. Cell lines and cell culture Human colon carcinoma cell line Caco-2 was purchased from CoBioer biotechnology Co., Ltd. (Nanjing, China). The LoVo cell line was purchased from SunBio Biotechnology Co., Ltd. (Shanghai, China). SW-620 cell line was obtained from the School of Basic Medical Sciences of Lanzhou University (Lanzhou, China). The HT-29 cell line was obtained from Cell Resource Center in the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences (Beijing, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (100 IU/mL penicillin and 100 IU/mL streptomycin). All cell lines were grown in a humidified atmosphere with 5% CO2 at 37C. Cell viability assay The 3-(4,5-dimethylthiazol-2-yl)C2,5-diphenyltetrazolium bromide (MTT, Beyotime Biotechnology, Suzhou, China) assay was implied to detect the proliferation of human colon carcinoma cells. Each cell line was cultured in 96-well plates at a density of 5.0??104 per well. After 24 h of incubation for attachment, the cells were treated for 24, 48, or 72 h with different concentrations of petasin (1, 5, and 25 mol/L) or with the same volume of phosphate-buffered saline (PBS). Petasin was purchased from Tianrui Biotech Co., Ltd. (Xian, China); the purity of petasin was 98% as determined by high-performance liquid chromatography. Cell proliferation was assessed at each time point. Spent medium was replaced with fresh medium containing 10 L MTT. After incubation at 37C for another 4 h, the medium was removed and 100 L of DMSO was added to each well, and plates agitated for 10 min. Absorbance was measured at 570 nm. Experiments were performed using triplicate wells and repeated at least three times. Results are presented as a percentage inhibition compared to untreated control. Cell apoptosis assay Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining (Nan Jing KeyGen Biotech Co., Ltd., Bretazenil Nanjing, China) was used to assess Bretazenil apoptosis. Briefly, SW-620 cells were seeded in six-well plates at a density of 1 1.0??105 cells per well and incubated for 24 h. Subsequently, cells were treated with 25 mol/L petasin or PBS for another 48 h. Cells were collected and centrifuged at 2000?for 5 min, then washed in cold PBS, resuspended in 500 L binding buffer, and incubated with 5 L Annexin V-FITC and 5 L PI. After 10 min of incubation in the dark at room temperature, cell counts were obtained using a flow cytometer. Morphological changes to cell nuclei were visualized using Hoechst 33258 (Beyotime Biotechnology) staining. Cells were treated as above. After 48 h treatment with 25 mol/L petasin or PBS, cells were IL6R incubated with 1 mL of Hoechst 33258 dye at 37C for 20 to 30 min, then washed twice with PBS. Cells were examined using fluorescence microscopy. All experiments were repeated three times. Wound-healing migration assay Cell migration.