| Objective:Tenacissoside H(TDH)is a Chinese medicine monomer extracted from Marsdenia tenacissima extract(MTE)which has been confirmed to have anti-tumor effects,but its mechanism of action is still unclear.The aim of this study was to investigate the role and mechanism of TDH against human colon cancer LoVo cell proliferation and migration,and to explore the correlation of TDH with expression of GOLPH3 gene and cell signaling pathway in LoVo cells.Methods:1.LoVo cells were treated with TDH at 0.1,1,10 and 100 μg/mL for 24,48 and 72 h.The proliferation rate of LoVo cells was detected by MTT assay.2.The cells were divided into the following groups:(1)Control: LoVo cells;(2)TDH:TDH-treated LoVo cells;(3)Experimental Group 1: LoVo cells treated with TDH and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/m TOR)signaling pathway agonist insulin-like growth factor-1;(4)Experimental Group 2: LoVo cells treated with TDH and Wnt/β-catenin signaling pathway agonist Wnt agonist 1;(5)p-CMV-2: LoVo cells transfected with p-CMV-2empty plasmid;(6)GOLPH3: LoVo cells transfected with p-CMV-2-GOLPH3 recombinant plasmid;(7)Experimental Group 3: TDH-treated LoVo cells transfected with empty plasmid;and(8)Experimental Group 4: TDH-treated LoVo cells overexpressing GOLPH3 recombinant plasmid.3.Recombinant plasmid p-CMV-2-GOLPH3 was constructed,and p-CMV-2-GOLPH3 and p-CMV-2 empty plasmids were transfected into LoVo cells by lipofection.Western blotting was used to detect the effect of transfection.4.The annexin V–fluoresecein isothiocyanate/propidium iodide method was used to detect apoptosis.5.Transwell assay was used to detect cell migration ability.6.western blotting was used to detect expression of p-p70S6K/p70S6 K,β-catenin and Golgi phosphorylated protein(GOLPH)3.Results:1.TDH increased inhibition of LoVo cells in a concentration-dependent manner,but no obvious time-dependent effect within 72 h.The IC50 of TDH in LoVo cells at 24,48 and72 h was: 40.24,13.00 and 5.73 μg/mL,respectively.2.The apoptosis rate of the TDH Group was significantly higher than that of the Control Group.The apoptosis rate of Experimental Group 1 and Experimental Group 2was significantly lower than that of the TDH Group(P<0.05).3.The cell migration number of the TDH group was significantly lower than the control group while that of Experimental Group 1 and Experimental Group 2 was significantly higher than that of the TDH Group(P<0.05).4.Compared with the Control Group,the expression of p-p70S6K/p70S6 K,β-catenin,and GOLPH3 protein was significantly decreased in the TDH Group(P<0.05).Expression of β-catenin and p-p70S6K/p70S6 K was significantly higher in Experimental Group 1 and Experimental Group 2 than in the TDH Group TDH(P<0.05).5.Expression of GOLPH3 protein increased significantly in the GOLPH3 Group(P<0.05).Compared with Experimental Group 3,expression of p-p70S6K/p70S6 K,β-catenin and GOLPH3 protein in Experimental Group 4 was increased significantly,and the difference was significant(P<0.05).6.The apoptosis rate of Experimental Group 3 was significantly higher than that of the p-CMV-2 Group,while the apoptosis rate of Experimental Group 4 was significantly lower than that of Experimental Group 3(P<0.01).7.Cell migration number of Experimental Group 3 was significantly lower than that of the p-CMV-2 Group,and cell migration of Experimental Group 4 was significantly higher than that of Experimental Group 3(P<0.01).Conclusion:1.TDH significantly inhibited growth of human colon cancer LoVo cells in a concentration-dependent manner.2.TDH significantly inhibited migration of human colon cancer LoVo cells and induced apoptosis.3.Overexpression of GOLPH3 gene in LoVo cells can antagonize the effects of TDH on apoptosis and migration in LoVo cells.4.The antitumor effect of TDH on LoVo cells is to inhibit the activity of PI3K/AKT/m TOR and Wnt/β-catenin signaling pathways by down-regulating of GOLPH3 protein expression. |
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