Neogambogic acid suppresses characteristics and growth of colorectal cancer stem cells by inhibition of DLK1 and Wnt/β-catenin pathway

Abstract

Purpose: Targeting cancer stem cells (CSCs) may be an efficacious strategy against cancer. We were devoted to exploring the role of neogambogic acid in characteristics and growth of colorectal CSCs.

Methods: SW480 and HCT116 cells were treated with neogambogic acid at different concentrations and transfected with siDLK1 and pcDNA3.1-DLK1 plasmids. The effect of neogambogic acid on the viability of SW480 and HCT116 cells was assessed by MTT assay. Spheroid formation assay was adopted to enrich colorectal CSCs from SW480 and HCT116 cells. The effect of neogambogic acid on colony number, aldehyde dehydrogenase (ALDH) level, apoptosis and cell cycle of SW480 and HCT116 CSCs was detected by colony formation and flow cytometry assays. The expressions of CSC markers, proliferation marker (proliferation nuclear antigen (PCNA)), apoptosis markers (cleaved caspase-3, cleaved caspase-9), Wnt/β-catenin pathway markers (P-GSK3β, GSK3β, β-catenin and Wnt) and DLK1 were determined by qRT-PCR or Western blot.

Results: Neogambogic acid suppressed viability, the spheroid formation ability and the levels of CSC markers in colorectal cancer (CRC) cells, accompanied with inhibition of colony-formation and ALDH level, apoptosis induction and G0/G1 phase arrest. Furthermore, neogambogic acid inhibited expressions of PCNA, P-GSK3β, P-GSK3β/GSK3β, β-catenin and Wnt, but promoted those of cleaved caspase-3, cleaved caspase-9 and GSK3β in colorectal CSCs. DLK1 silencing caused opposite results. DLK1 overexpression abrogated the effects of neogambogic acid on colorectal CSCs.

Conclusion: Neogambogic acid could be an efficacious natural compound targeting colorectal CSCs via inhibition of DLK1 and Wnt/β-catenin pathway. Thus, neogambogic acid may be an attractive agent against CRC.

Keywords: Apoptosis; Colorectal cancer stem cells; DLK1; Neogambogic acid; Proliferation; Wnt/β-catenin pathway.