| Euodia rutaecarpa(Juss.)Benth.(Rutaceae)is a traditional Chinese medicine,which has a strong pharmacological effect on heart system,gastrointestinal tract,and tumor cells.Evodiamine is a kind of tryptamine indole alkaloids extracted from Evodia rutaecarpa,which has the functions of immunoregulation,inhibition of adipocyte differentiation,anti-inflammation and anti-angiogenesis.Studies have reported that Evodiamine inhibits a variety of cancers,such as human cervical cancer,prostate cancer,and colon cancer.However,the mehanisms of Evodiamine-induce anti-tumor effect are unlear.Anti-angiogenesis drugs usually inhibit invasion of endothelial cell and the levels of vascular endothelial growth factor by suppressing the activation of extracellular regulated protein kinases.Wnt/?-catenin signaling pathway is closely related to vascular development and reconstruction.Data showed that ?-catenin could promote angiog,enesis by maintaining the stability of angiogenic factors,fibr-oblast growth factor and E4 local adenovirus.The anti-angiogenic effect of endostatin is also achieved by induction of ?-catenin degradation.Therefore,we investigated the association of anti-angiogenesis with ?-catenin signaling pathway in HCC about Evodiamine.The anti-angiogenesis effect of Evodiamine was tested by in vivo and in vitro experiments.Western Blot,Real-time PCR and EMSA were used to detect the effect of Evodiamine on hepatocellular carcinoma cell lines in vitro.The effect of Evodiamine on hepatocellular carcinoma cell lines was studied by different methods.We will investigate the relationship between Wnt/?-catenin and angiogenesis and their roles of Evodiamine-induce anti-tumor effect in order to understand the key molecular mechanisms of Evodiamine-induce anti-tumor effect.Our resluts will provide scientific basis for further development and application of Fvodiamine and looking for effective early biological markers and antitumor drugs for liver cancer.Part I Effects of Evodiamine on hepatocellular carcinomaObjectiveTo investigate the effects of Evodiamine on the occurrence and development of hepatocellular carcinoma in nude mice,the cell proliferation,cell cycle,apoptosis,and death of invasion and metastasis of hepatocellular carcinoma I-HepG2 and SMMC-7721 cells.MethodsH22 mice xenograft model and a nude mice xenograft model were used to observe the effect of Evodiamine on tumor weight,tumor volume,AFP,TSGF and histopathological changes.In addition,the effects of Evodiamine on HCCs were detected by MTT assay,flow cytometry,cell scratch assay and transwell assay.ResultsThe inhibitory rate of Evodiamine on the transplanted tumor of H22 was more than 40%.The growth curve showed that the Evodiamine group showed slower growth than the model group until the 14th day.Evodiamine can significantly reduce the concentration of tumor marker AFP and TSGF.The pathological examination showed that the tumor necrosis of the transplanted tumor in Evodiamine group was significantly lower than that in the control group.The degree of coagulation necrosis was higher than that of model group and positive group.In nude mice transplanted with SMMC-7721 cell line.the volume of tumor growth and the weight of the tumor in nude mice were less than those in the control group but there was no statistically significant difference.Histopathological examination revealed that tumor necrosis in Evodiamine group was significantly higher than the model group suggesting that drugs have significant anti-tumor activities.MTT assay showed that Evodiamine inhibited the growth of HepG2 and SMMC-7721 cells in a dose-dependent manner,and the inhibitory rate increased with the increase of concentration.The effect of Evodiamine on the cell cycle of HepG2 and SMMC-7721 cells was detected by flow cytometry.The results showed that the cells in G2/M phase increased after treated with Evodiamine in HepG2 and SMMC-7721 cells.Scratch assay showed that Evodiamine could inhibit the migration of HepG2 and SMMC-7721 cells.In transwell assay,the number of HepG2 cells was observed under microscope,and the number of cells in Evodiamine group was significantly decreased.ConclusionEvodiamine can inhibit the growth of hepatocellular carcinoma hepatransplanted tumors,and inhibit the growth,invasion,and metastasis of ICC cells by inducing cell cycle arrest and apoptosis.Part ? Effects of evodiamine on angiogenesisObjectiveTo investigate systematically the effects of Evodiamine on angiongenesis in three levels of cell,tissue,and animal.MethodsWe have observed the effects of Evodiamine on the blood vessel fornation of human umbilical vein endothelial cells(HUVEC),the formation of the aortic rings,and the classical matrix.ResultsEvodiamine could inhibit the angiogenesis of HUVECs,and completely inhibited the tube formation of HUVECs.At the same time,Evodiamine can significantly inhibit the formation of microvascular-like structure in aortic rings.Evodiamine could significantly inhibit the angiogenesis in the thrombus in matrigel models.The results of HE staining and immunolistochemical staining of matrigel indicated that the drug could significantly reduce VEGF-induced angiogenesis in vivo.The number of blood vessels and vascular density in administration group was significantly reduced.ConclusionEvodiamine can inhibit angiogenesis in three levels of cell,tissue,and animal.Part? Evodiamine inhibits ?-catenin-mediated angiogenesisObjectiveTo investigate the effect of Evodiamine on hepatocellular carcinoma cell(3-catenin and VEGFa and their roles of anti-angiogenesis and anti-liver cancer,which find some molecular mechanisms of Evodiamine on liver cancer.MethodsUsing the methods of Immuno histochemistry.Western Blot,Real-time PCR,and other methods,we have investigated the effects of Evodiamine on the ?-catenin signal pathway and angiogenesis related factors in H22 or SMMC-7721 nude mice xenograft models and HepG2 cells.ResultsEvodiamine inhibited the expression of angiogenesis-related proteins and the vascular endothelial markers,such as CD31 and CD34VEGFa,VEGFR1,and MMP9,as well as the protein levels and mRNA levels(3-catenin in tumor tissues of xenograft models and HepG2 cells.In hepatocellular carcinoma cells,VEGFa also increased with the expression of(3-catenin agonist LiCl and high level frizzled 7,and evodiamine could inhibit the expression of ?-catenin and VEGFa.Additionally,the effect of Evodiamine-treated HepG2 cells conditioned medium on the formation of HUVEC cells was investigated.In addition.EMSA was used to evaluate the binding activity between ?-catenin and EGFa promoter DNA.Results showed that evodiamine can significantly reduce the ?-catenin DNA bindinig activity.suggesting that evodiamine can inhibit ?-catenin activation and the transcriptional activity of VEGFa.ConcolusionEvodiamine can inhibit VEGFa transcription activity by inhibiting the expression of ?-catenin,hoicking angiongenesis,which is involved in anti-liver cancer of Evodiamine.In summary,the novel findings of this are as following:1.Evodiamine can inhibit the growth of hepatocellular carcinoma hepatransplanted tumors,and inhibit the growth,invasion,and metastasis of HCC cells by inducing cell cycle arrest and apoptosis.2.Evodiamine can inhibit angiogenesis in three levels of cell,tissue,and animal.3.Evodiamine can inhibit VEGFa transcription activity by inhibiting the expression of ?-catenin,bolcking angiongenesis,which is involved in anti-liver cancer of Evodiamine. |
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