Investigation Of Apoptotic Effect Induced By Casticin In Colon Cancer Stem Like Cells

ObjectiveThe objective of this study was to investigate whether casticin, apolymethoxyflavone, induces apoptotic cell death of the sphere forming cells derivedfrom human colon cancer cell line HCT-116cultured in vitro, namely colon cancerstem cell like cells(CCSLCs), and to explore whether this effect of casticin is involvedin a decrease of the level of Akt protein phosphorylation in CCSLCs.MethodsHuman colon cancer HCT-116cell line was cultured in vitro. The stem cellconditioned culture system was used to isolate and enrich CCSLCs that was used asthe tested object. The self-renewal was assessed using tumorsphere formation assayfollowing exposure of CCSLCs to various concentrations of casticin. Apoptotic rate ofCCSLCs indyced by casticin was analyzed by flow cytometry (FCM) usingpropidium iodide (PI) staining. The apoptotic effect of casticin in CCSLCs wascertified by DNA agarose gel electrophoresis. the expression levels of CD133as astem cell related protein in the HCT-116sphere-forming cells was determined usingFCM analysis with FITC-antibody against CD133. Finally, the expression of Aktprotein phosphorylation in CCSLCs was examined by western blot analysis.ResultsThe cells cultured and amplified from human colon cancer line HCT-116in vitro were used isolated the sphere-forming cells namely CCSLCs in the stem cellconditioned culture system for10days. In this condition, cells grew as nonadherent,three-dimensional sphere clusters, called spheres. The results from tumorsphereformation assay indicated that treatment with casticin (5.0～10.0μM) significantlydecreased the amount of sphere forming cells, in a concentration-dependent manner(P＜0.05). FCM analysis using PI staining showed that casticin increased the apoptoticrate of CCSLCs, in a concentration-dependent manner(P＜0.05). Treatment withcasticin at5.0μM or10μM for24h resulted in the formation of a DNA ladder. Thedata from FCM analysis with FITC-antibody against CD133indicated that casticindownregulated the level of CD133protein expression in CCSLCs(P＜0.05). Inaddition, casticin inhibited the expression of Akt protein phosphorylation inCCSLCs(P＜0.05).Conclusion1. Casticin possesses the inhibitory effect on the self-renewal of CCSLCs and theapoptotic effect in CCSLCs.2. Inhibition of the cancer stem cell properties of CCSLCs by casticin is associatedwith decrease the level of Akt protein phosphorylation...