The Inhibition Effect And Mechanism Study Of Cardiac Glycoside In Human Colon Cancer Cells In Vitro

In a previous high-throughput screening study,we found that a number of cardiac glycosides could kill colon cancer cells specifically.Among them,oleandrin is a cardiac glycoside derived from Nerium oleander.PurposeEvidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer.However,the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown.The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells.MethodsThe cytotoxicity and IC50 of five small molecule compounds(oleandrin,neriifolin,strophanthidin,gitoxigenin,and convallatoxin)in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays,respectively.Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide,followed by flow cytometry.Intracellular Ca2+ was determined using Fluo-3,AM,glutathione(GSH)levels were measured using a GSH detection kit,and the activity of caspase-3,-9 was measured using a peptide substrate.BAX,pro-caspase-3,-9,cytochrome C and BCL-2 expression were determined by Western blotting.ResultsOleandrin significantly decreased cell viabilities in SW480,HCT116 and RKO cells.The IC50 for SW480 cells was 0.02μM,whereas for NCM460 cells was 0.56μM.More interestingly,the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells.Oleandrin downregulated protein expression of pro-caspase-3,-9,but it enhanced caspase-3,-9 activities.These effects were accompanied by upregulation of protein expression of cytochrome C and BAX,and downregulation of BCL-2 protein expression in a concentration-dependent manner.Furthermore,oleandrin increased intracellular Ca2+concentration,but it decreased GSH concentration in the cells.ConclusionsThe present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway.Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.