Investigating The Apoptosis And Proteomics Mechanism Of Dioscin Against Human Colon Cancer HCT-116Cells

Objective: To study the cytotoxic effect of dioscin on human colon cancerHCT-116cells and then to investigate the possible mechanism based on proteomics.Methods: In the present study, HCT-116, LoVo, Caco-2, SW620and LS cells weretreated by different concentrations of dioscin (1.25,2.5,5.0and10.0μg/ml) underdifferent treatment times (24,48and72h), and then the cell viabilities were detected byMTT assay. At the same time, the cell viability of human normal colon organizationCCD-18Co cells was also evaluated. In the test of HCT-116cells, AO/EB and DAPIfluorescence stainings were used to observe cell apoptosis and nucleus change. ROSgeneration and Ca2+level were evaluated by flow cytometry. The levels of iNOS andNO were tested. Meanwhile, TEM was used to observe cell ultrastructure and flowcytometry was used to measure cell cycle. In addition, single cell gel electrophoresisand TUNEL assays were applied to detect DNA damage and apoptosis induced bydioscin. In mechanism investigation, iTRAQ-based proteomics was employed to findthe differently expressed proteins caused by dioscin. Bioinformatics including GO andKEGG analysis were also applied to study the function of differently expressed proteins,signal pathways and protein-protein interactions. Some differentially expressed proteinswere further validated by western blotting and quantitative real-time PCR assays. Results: The cell vitalities of five kinds of colon cancer cells were significantlyreduced by dioscin with dose-and time-depedent manners, and the inhibition effect ofthe compound on CCD-18Co colon cells was slighter than cancer cells. In dioscin-treated HCT-116cells, AO/EB and DAPI stainings showed that dioscin induced cellapoptosis, invigorated the emergence of nucleosome, significantly increased intracell-ular ROS and Ca2+concentrations, the levels of NO and iNOS, destroyed cell ultrastruc-ture, arrested the cell cycle at G2/M phase, and caused DNA damaged and apoptosis.Total of288differentially expressed proteins (p <0.05) in HCT-116cells caused bydioscin were found. Bioinformatics analysis found that the differentially expressedproteins were involved in biological process, cell process, metabolism pocess and alsoparticipated in oxidative phosphorylation, Wnt, p53and Ca2+signaling pathways. Tenproteins involved in oxidative phosphorylation, Wnt, p53and Ca2+signaling pathwayswere validated, in which the expressions of ATP6V0C, Ppp2r5e, Gna11and COX6C,Cyc1, ND1and Camk2g were up-regulated, while the expressions of Rhoa, Ccnb1andGnaq were down-regulated by dioscin.Conclusions: Our study found that dioscin showed significantly cytotoxicity onhuman colon cancer HCT-116cells, and the action may be through affectingintracellular oxidative stress, DNA damage, cell cycle regulation and apoptosis and alsorelated to oxidative phosphorylation, Wnt, p53and Ca2+signaling pathways. Thepresent work elucidates that dioscin may be developed to be a new drug and theidentified targets may be useful for treatment of colorectal cancer in future.