Abstract:
Objective: To probe the role of neogambogic acid in the features of colorectal cancer stem cells(CRC-CSCs) through the Wnt/β-catenin signaling pathway. Methods: The SW480 and HCT166 colorectal cells were divided into neogambogic acid groups(1.5, 3, 6 and 12 μmol/L) and control groups;the viability of CRC-CSCs was detected by methy thiazolyl tetrazolium(MTT);spheroid formation assay and clone formation assay were selected to assess the capacity of spheroid formation and self-renewal ability of the cells;the mRNA expression levels of relative markers(CD133, CD44, ALDH1, Oct4 and Nanog)of CRC-CSCs were shown on Real-time quantitative PCR; the impact of neogambogic acid on apoptosis and cell cycle of CRC-CSCs were analyzed by using flow cytometry assays;Western blot was chosen to analyse the protein expression levels of the self-renewal marker(PCNA), apoptosis markers(cleaved caspase-3 and cleaved caspase-9)and Wnt/β-catenin signaling pathway markers(P-GSK3β, GSK3β, β-catenin and Wnt). Results: Compared with control group, after neogambogic acid treatment, the viability of SW480 and HCT116 cells decreased(P<0.05). Neogambogic acid inhibited the spheroid forming ability and the clone numbers of CRC-CSCs(P<0.001, P<0.01), but increased the cell apoptosis rate(P<0.01), accompanied with G0/G1 phase arrest.Moreover, neogambogic acid down-regulated the mRNA and protein expression of relative markers of CRC-CSCs(CD133, CD44, ALDH1, Oct4 and Nanog), PCNA, P-GSK3β, β-catenin and Wnt(P<0.05), while up-regulated the expression of caspase-3, cleaved caspase-9 and GSK3β(P<0.01). Conclusion: Neogambogic can inhibit stem cell properties of colorectal cells via inhibition of DLK1 and Wnt/β-catenin pathway.As a result, neogambogic acid may be an attractive agent against colorectal cancer.
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