Proanthocyanidins Inhibited Colorectal Cancer Stem Cell Characteristics Through Wnt/β-catenin Signaling

Research background and purpose:Colon cancer stem cells(CSCs)are the key factors for tumor cell growth,drug resistance,recurrence and metastasis.Procyanidine(PC)is a polyphenolic compound that widely exists in plants and has strong antioxidant and anti-aging effects.Recent studies have found that PC has an inhibitory effect on tumor growth.However,the role and mechanism of PC in colorectal cancer stem cells are still unclear.The aim of this study is to clarify the regulatory effect of PC on CRC progression and stem cells,and to explore the possible mechanism of PC inhibiting CRC stem cell characteristics.Materials and Methods:(1)Colorectal cancer cell lines HT29 and HCT116 were treated with different concentrations of PC(0μM,5μM,10μM,20μM,40μM and 80μM),and the effects of PC on cell proliferation and sensitivity to oxaliplatin were detected by CCK-8 assay.Suspension sphere formation assay was used to detect the effect of PC on the ability of cell suspension sphere formation.Plate cloning assay was used to detect the effect of PC on cell colony formation ability.Western blotting was used to detect the effect of PC on the expression of cancer stem cell-related proteins c-Myc,OCT-4,Nanog,CD44 and CD133.(2)HT29 cells were used to establish a transplanted tumor model in nude mice.The nude mice were divided into two groups and treated with PC(100 mg/kg)or drug solvent by gavage for 21 days,respectively.(3)To explore the possible mechanism of action of PC,Colorectal cancer cell lines HT29 and HCT116 were treated with different concentrations of PC(0μM,5μM,10μM,20μM,40μM and 80μM),Western The protein expression levels of β-catenin,p-GSK3β(ser9),GSK3β,DVL1,DVL2 and DVL3 were detected by blotting.(4)HT29 and HCT116 cells were treated with Wnt/ β-catenin agonist Li Cl(10 m M)and PC(10μM),and the expression changes of Wnt/β-catenin signal transduction factors were analyzed by Western blotting.CCK-8 assay was used to analyze the changes in cell proliferation and oxaliplatin sensitivity.Suspension sphere formation,plate cloning and Western blotting were used to detect the changes in the characteristics of cancer stem cells.Result:(1)PC can inhibit the proliferation of HT29 and HCT116 cells,improve the chemosensitivity of cells to oxaliplatin,and reduce the tumorigenicity of colorectal cancer cells in nude mice.(2)PC down-regulated the expression of stem cell characteristics related proteins c-Myc,OCT-4,Nanog,CD44 and CD133 in HT29 and HCT116 cells,and inhibited the ability of suspension sphere formation and colony formation of cells.(3)PC decreased the protein expression of p-GSK3β,β-catenin and DVL1-3.Treatment of the cells with Wnt/β-catenin agonist Li Cl significantly reduced the inhibition of Wnt/β-catenin by PC.(4)HT29 and HCT116 cells were treated with Wnt/ β-catenin agonist Li Cl and PC(10 μ M),and the expression changes of Wnt/ β-catenin signal transduction factors were analyzed by Western blotting.CCK-8 assay was used to analyze the changes in cell proliferation and oxaliplatin sensitivity.Suspension sphere formation,plate cloning and Western blotting were used to detect the changes in the characteristics of cancer stem cells.Conclusions:This study demonstrated that PC could inhibit CRC stem cell properties and increase the sensitivity to chemotherapeutic drugs through Wnt/ β-catenin signaling pathway,which may be a potential novel natural agent for the treatment of CRC.