The Effect And Mechanism Of Evodiamine On Hepatocellular Cancer Via NOD1 Pathway

Hepatocellular cancer（HCC）is the most common primary malignancy of the liver and is the third leading cause of cancer-related death worldwide.It is characterized by high morbidity and mortality,high risk of intrahepatic recurrence,and poor prognosis.At present,the main way of the treatment is surgical resection and chemotherapy.But there are some problems,with surgical resection such as easy metastasis and recurrence;with leading conventional chemotherapeutic agents such as poor targeting efficiency,strong adverse effects and poor tolerance.Thus,it is urgent for us to search an agent with better treating efficiency and poor adverse effects.Evodiamine（Evo）,an active ingredient isolated from the fruit of Evodia rutaecarpa Bentham,has anti-obesity,anti-inflammation and anti-tumor effects.Recent years,there are many researchers have found that Evo could inhibit various of tumor cells.Meanwhile,it has been reported that the antitumor activity through the suppression of nuclear factor-κB（NF-κB）and mitogen-activated protein kinase（MAPK）activation.However,it is necessary for us to study further the exact mechanism of the effect of Evo on the proliferation and apoptosis of HCC cells.NOD1（Nucleotide-binding oligomerization domain 1）,one of the PRRs（pattern-recognition-receptors）which attend the process of the innate immune.In the NOD1 pathway,NOD1 could initiate NF-κB-dependent and MAPK-dependent activation.Furthermore,in the GEO database,NOD1 is expressed in most tissues,including cancer cells and the expression differed significantly between tumor and non-tumor tissue.Therefore,we hypothesize that Evo exerts anti-hepatocellular carcinoma activity maybe via NOD1 pathway.Objective:The study set HepG2,SMMC-7721 and xenograft tumor model as the research objects to detect the effect of Evo on the proliferation and apoptosis of HCC in vitro and vivo.Furthermore,the levels of NOD1 were measured in two HCC lines and normal liver cell line HL-7702,and investigated the exact mechanism of the effect of Evo on the proliferation and apoptosis of HCC cells,which provided theoretical and experimental basis for the clinical application of Evo in the treatment of HCC.Methods:1.HCC lines（HepG2 and SMMC-7721）was cultured for 24h invitro,then cocultrued with Evo at the concentration of 0.25,0.5,1,2,4?mol·L^-11 for 12,24 and 48h,respectively.Theproliferation of cells was detected by CCK-8.The cells weretreated with Evo at the concentration of 0.5,1?mol·L^-1 for 24h,then morphological changes of apoptosis were observed byfluorescence microscope after Hoechst staining.The effect ofEvo on the proliferation of HCC cells was detected by colonyformation and the levels of apoptosis related proteins P53,Baxand Bcl-2 were examined by Western Blot.2.We injected HepG2 cells into nude mice to establish the tumormodel,and detected the effect of Evo on HCC by recordingtumor volume and weight,HE and TUNEL staining.3.In vitro and vivo,the levels of NOD1 in the HCC cells andnormal liver cell line HL-7702 were detected by RT-PCR andWestern Blot.Western Blot and the immunofluorescence wereused to detect the effect of Evo on the expression of NOD1pathway-associated proteins in HCC cells in vitro and in vivo.4.We added the group which has the pre-treatment of the NOD1specific agonist,IE-DAP,before being treated with Evo,changes of the proliferation in HCC cells were checked by Edu,the cell cycle was detected by flow cytometry and theexpression of apoptosis,cell-cycle and NOD1 pathway relatedproteins were measured by Western Blot.Results:1.HepG2 and SMMC-7721 were treated with differentconcentration of Evo,viability of cells was significantly reducedin a time and dose-dependent manner.Changes in nuclearmorphology of Evo-exposed cells were observed under afluorescence microscope and featured a marked increase in thequantity of apoptotic chromatin condensation and nuclearfragmentation.The effect of Evo on colony formation indicateda significant and dose-dependent inhibition of colony formationwith HepG2 and SMMC-7721 cells relative to untreated controls.Compared to controls,Evo treatment showed significantincreases in Bax and P53 levels and decrease in Bcl-2 level.2.The qualified measurement results showed that small tumorvolumes and reduced tumor weights in Evo-treated mice.Thehematoxylin-eosin staining（HE）staining and TdT-mediateddUTP Nick-End Labeling（TUNEL）results showed that therewas a lot of cell necrosis and damage in experimental micecompared with control mice.3.The RT-PCR and Western Blot results indicated that in contrastto that in HL-7702 cells,NOD1 levels were significantlyincreased in HepG2 and SMMC-7721 cells.In vitro and vivo,Evo decreased the expression of NOD1,and suppressed theactivation of NF-κB and MAPK.4.Pre-treatment with IE-DAP rescued HepG2 and SMMC-7721cells from Evo-induced suppression of proliferation andarresting of cell cycle measured by 5-ethynyl-20-deoxyuridine（EdU）and FCM.The Evo-induced changes in levels of proteinsrelated to apoptosis,cell cycle and NOD1 pathway were rescuedby IE-DAP with different degree.Conclusion:1.1.Evo could inhibit the proliferation and induced the apoptosisin HepG2 and SMMC-7721 cells,it may be related to thedownregulation of Bcl-2,upregulation of Bax and activation ofP53.2.NOD1 is up-regulated in HCC cell lines,Evo could decrease theexpression of NOD1,resulting in the suppression of NF-κB andMAPK pathways.Evo exerted anti-proliferation and induced-apoptosis activity in HCC cells maybe via this way.3.Evo exerted anti-hepatocellular carcinoma activity maybe via the NOD1 pathway in vivo.