Mechanism Of Dictamnine Inhibiting Cell Epithelial-mesenchymal Transition By Down-Regulating HIF-1α And Slug Signaling Pathway

Objective:Dictamnine(DTM)is a natural alkaloid isolated from the root of Dictamnus dasycarpus Turcz and has been shown to exhibit multiple biological functions,including anti-inflammatory,antifungal,anti-angiogenic and anticancer activity.However,the mechanisms by which dictamnine inhibits tumor growth are not fully understood.In this study,we investigated the effectiveness of dictamnine as a treatment for cancer and to identify the underlying mechanisms of its anticancer activity.Methods:Molecular docking experiments were used to simulate the interactions between dictamnine and HIF-1α or Slug;We demonstrated the effect of dictamnine on HIF-1α and Slug protein expression which was detected by western blot;Immunofluorescence assay was used to detect HIF-la and Slug nuclear expression;It was confirmed that dictamnine reduced the expression of HIF-1α by inhibiting HIF-la protein synthesis by western blot;We next investigated the effects of dictamnine on the mTOR/p70S6K/4E-BP1/eIF4E,MAPK and GSK-3β/Slug signaling pathways by western blot;Western blot and RT-PCR assays were used to detect the effects of EMT-related proteins on protein and mRNA levels in dictamnine and siRNA-treated HCT116 cells targeting HIF-1α and Slug;The effect of dictamnine on cell migration was detected by a scratch test;The effect of dictamnine on cell migration was detected by cell scratch test;Matrigel invasion assay was used to detect the effect of dictamnine on cell invasion ability;Flow cytometry was used to detect the effect of dictamnine on cell apoptosis;EdU assay and Colony formation assay were used to detect the effect of dictamnine on cell proliferation;Moreover,the effect of dictamnine on transplanted tumors in vivo was examined by zenograft model experiments in nude mice.Result:Dictamnine markedly decreased the hypoxia-induced accumulation of HIF-la and Slug protein in a dose-dependent manner.Further analysis revealed that Dictamnine inhibited HIF-la protein synthesis,without affecting its degradation.Our results demonstrated that dictamnine reduced HIF-1α protein synthesis by downregulating the mTOR/p70S6K/eIF4E and MAPK pathways,and reduced the expression of Slug by inhibiting the GSK-3β/Slug signaling pathway.Moreover,epithelial-mesenchymal transition(EMT)was inhibited in dictamnine-treated tumors by downregulation of HIF-la and Slug,as reflected by the upregulation of E-cadherin and Occludin,and the downregulation of N-cadherin and Vimentin.Phenomenological experiments showed that dictamnine reduced migration and invasion,inhibited HCT116 cell proliferation and promoted HCT116 cell apoptosis by downregulating HIF-1α and Slug.In vivo studies further confrmed that dictamnine treatment caused signifcant inhibition of tumor growth in a xenograft tumor model.Conclusion:Dictamnine promoted apoptosis and inhibited EMT,migration,invasion and proliferation by downregulating the HIF-la and Slug pathways.These findings suggest that dictamnine is a potent cancer inhibitor,providing a rationale for anticancer pathway-targeted therapy.