The Effect Of Evodiamine On Migration And Invasion Of Colorectal Cancer Cells And Its Mechanism

Evodiamine（EVO）has various biological effects,such as vasodilatory,anti-inflammatory and antitumor^[1-3].EVO can suppress the pain caused by Taxol treatment through repressing inflammatory reaction,so it is a potential drug for treating chemotherapy-induced neuropathic pain.Yang et al.find that EVO can significantly suppress the growth of hepatoma carcinoma cells by inhibiting phosphorylation of STAT3 and restraining the activation of STAT3 induced by IL-6^[4].Although some references have shown that Evodiamine induces apoptosis,inhibits drug resistance and proliferation as well,the in vitro and in vivo mechanisms of Evodiamine on migration and invasion are still indistinct^[5-7].As a NAD-dependent histone deacetylase,Sir2（silent information regulator 2）can silence chromatin,extend life span and strengthen genetic stability in yeast.In mammal,Sirtuins has the similar function as the orthologues of Sir2,and it has seven isoforms,Sirt1 to Sirt7.In addition,Sirtl has the highest homology with Sir2,and it belongs to the family of classⅢhistone deacetylases^[8].Because of cytoplasmic and nuclear localizations,Sirt1 can deacetylate various histones and non-histones,and these non-histones include p53,p300,c-MYC and Nuclear factor-κB（NF-κB）etc.^[9-11].NF-κB is a nuclear transcription factor which exists in cytoplasm generally,and it stays in a silence state by combining with IκBα,an inhibitory protein of NF-κB.In recent years,studies reveal that NF-κB is related to the occurrence,proliferation,differentiation,apoptosis,migration and invasion of tumors^[12-14].Acetylated NF-κB can’t combine with IκBα,as a result it will expose nuclear localization signal and be transported into nucleus.Acetylated NF-κB combines with promoter region of specific gene,and activates expression of specific gene which is related to proliferation,invasion,metastasis of cancer cells,such as IL-6,TNF-α,VEGF,IL-1β,TF and MMPs^[15,16].Sirt1 can deacetylate NF-κB p65 subunit under the assist of NAD+,and repress nuclear localization signal and transcription activity of NF-κB p65.Our study focused on the interrelation between Sirt1 and the transcription activity of NF-κB p65 to explore the underlying mechanism of Evodiamine on migration and invasion of CRC cells in vitro and in vivo.Objective:The first part of this study aims to investigate the effect of Evodiamine on migration and invasion of colorectal cancer cells in vitro and in vivo;the second part of the study is to explore the mechanism of Evodimaine on metastasis of colorectal cancer.Methods:The first part:human colorectal cancer cells（HT-29 and HCT-116）were cultured in vitro,and the proliferation effect of Evodiamine was detected by CCK-8 assay;the migration ability was analyzed by Wound Healing assay;the invasion ability was detected by Transwell assay;HCT-116 cells were injected into the tail vein of BALB/c nude mice to establish colorectal cancer metastasis model,and EVO was administered by filling the stomach.The weight of mice was recorded.After the treatment,mice were sacrificed,and the nodules in the lung and liver were counted.The second part:the mRNA of MMP-9 was detected by RT-PCR;the NAD+/NADH ratio was detected by fluorescence assay kit;Western blot was used to analyze the expression of Sirt1,NF-κB p65,acetyl-NF-κB p65and MMP-9;Immunofluorescence assay was used to investigate the expression and cellular location of Sirt1;After HT-29 and HCT-116 cells were pretreated with NAM,an inhibitor of Sirt1,Western blot was used to analyze the expression of Sirt1,NF-κB p65,acetyl-NF-κB p65 and MMP-9in EVO group or EVO combined with NAM group.Results:The first part1.CCK-8 result revealed Evodiamine could inhibit the proliferation of HT-29 and HCT-116 cells in a time and concentration dependent manner;2.Wound Healing assay showed that Evodiamine inhibited the migration ability compared with the control group;3.Transwell result further showed that the invasion ability of HT-29 and HCT-116 cells were significant decreased by Evodiamine compared with the control group.4.The recording of mice weight revealed that the mice weight was recovered by Evodiamine;the numbers of lung and liver nodules were significantly decreased by EVO.The second part1.RT-PCR result revealed that EVO could markedly repress the mRNA expression of MMP-9 in HT-29 and HCT-116 cells in a concentration dependent manner.2.Fluorescence assay result indicated that Evodiamine could increase the NAD+/NADH ratio.3.Western Blot result revealed that in Evodiamine treated cells,the expression of NF-κB p65 was not change while the expression of Sirt1 was increased significantly,acetyl-NF-κB p65 and MMP-9 were decreased dramatically.4.Immunofluorescence showed that Evodiamine could increase the expression of Sirt1 and transport Sirt1 from cytoplasmic to nucleus.5.Western Blot result displayed that in the Evodiamine combined with NAM group,NAM could inhibit the expression of Sirt1,and the expression of acetyl-NF-κB p65 and MMP-9 was increased markedly compared with Evodiamine group.Conclusion1.Evodiamine significantly inhibited the proliferation of colorectal cancer cells in a time and concentration dependent manner.Migration and invasion abilities were markedly decreased by Evodiamine.Evodiamine could dramatically restrain the metastasis of colorectal cancer cells in vivo.2.Evodiamine could dramatically restrain the metastasis of colorectal cancer cells through promoting the expression of Sirt1 and increasing the ratio of NAD⁺/NADH to activate Sirt1which can deacetylate NF-κB p65 and suppress the transcription activity of NF-κB p65.This effect can be abolished by NAM,an inhibitor of Sirt1.