| Hepatocellular carcinoma(HCC)is one of the most common primary malignant tumors,It is characterized by low early diagnosis,high malignancy,rapid progression,high morbidity and mortality.According to statistics,HCC patients in China account for half of the global HCC patients and clinical data show that the 5-year survival rate of HCC patients is only 4%.With the improvement of medical research and the progress of surgical treatment,liver transplantation and surgical resection are currently the first choice for early HCC,but cancer cells may still remain in the patients,so the 5-years recurrence rate after surgical resection is more than 70%.Most of HCC patients are already in the mid-or late-stage at the time of clinical diagnosis,and they basically miss the opportunity of surgical resection.However,the curative effect of traditional chemotherapeutic drugs on HCC is not ideal,and the side effects are large,which easily lead to liver dysfunction and poor tolerance of chemotherapy.Eventually,patients have to give up treatment because they cannot bear the pain and suffering.The best conservative treatment for advanced HCC is the use of targeted drugs to fight tumors,but there are only two kinds of targeted drugs approved in China for the treatment of HCC Sorafenib and Lavatinib,which are expensive and difficult to obtain.Therefore,it is urgent to find cheap,low toxic and efficient drugs for treatment of HCC.Evodiamine(Evo),a biologically active alkaloid extracted from the fruit of the traditional Chinese medicine evodia rutaecarpa(Juss.)Benth,which is a naturally occurring indole alkaloid.It has the characteristics of lowering blood pressure,losing weight,analgesic,anti-inflammatory and anti-tumor,etc.Previous experiments have confirmed that evodiamine can effectively control the development of tumors,play an anti-tumor role by inhibiting the proliferation and promoting apoptosis of tumor cells.In recent years,a large number of studies have suggested that Evo exerts anti-HCC effects through inhibiting ?-catenin-mediated angiogenesis and WWOX dependent signaling pathway.However,the precise mechanisms remain unclear.The Hippo-YAP signaling pathway,a critical regulator of tissue regeneration and organ size,is also involved in preventing tumor formation.The upstream core molecules of this pathway are serine/threonine kinases,mammalian sterile 20-like kinase 1/2(Mst1/2),and large tumor suppressor 1/2(Lats1/2).The transcriptional co-activator,Yes Activated Protein(YAP),is a downstream target of the Hippo pathway.A number of studies have shown that overexpression of YAP in cancer cells was shown to induce a significant promote cells proliferation and inhibit its apotosis.Accordingly,agents affecting the Hippo-YAP signaling pathway may have the ability to alleviate and treat HCC.We proposed the hypothesis that Evo might regulate the proliferation and apotosis of HCC through Hippo-YAP signaling pathway.Objective To investigate the possibility pathophysiological mechanism of Evodiamine promoting the apopotosis and of liver cancer cell,two human hepatocellular carcinoma cell(HCC)lines,Hep G2 and Bel-7402,and nude mice with xenograft tumors were used in this study.The effect of Evodiamine on the proliferation and apoptosis of HCC were observed.And we further investigated whether Evo exerts anti-HCC activity through Hippo-YAP signaling pathway in vitro.Our researches might provide a new direction for the future research and diagnosis of liver cancer.Method First part: The effect of evodiamine on the proliferation and apoptosis of two HCC cell lines and nude mice with xenograft tumors.CCK-8 kit was used to observe the effect of evodiamine on the cell proliferation and explore the IC50 of evodiamine on two HCC cell lines;Ed U staining and cloning formation assay were used to observe the effect of evodiamine on HCC proliferation;The effect of evodiamine on the apoptosis and cell cycle of HCC were detected by flow cytometry(FCM);TUNEL staining was employed to observe the effects of evodiamine on apoptosis of HCC;The expression of apoptosis-related proteins(Bax,Bcl-2,p53)in HCC cells after treated with evodiamine were measured by Western blotting;The nude mice with xenograft tumors was established,and observed the effect of evodiamine on mice body weight,tumor size and weight;HE staining was used to observe the effect of evodiamine on tumor cell morphology in vivo;TUNEL assay was used to observe the effect of evodiamine on tumor cell apoptosis in vivo.Second part: The effect of evodiamine on the Hippo-YAP signaling pathway in two HCC cell lines.Bioinformatics IPA(Ingenuity pathway analysis)database was used to explore the potential correlation among Evo,Hippo-YAP signaling pathway,and HCC.The effect of evodiamine on the expression levels of YAP and Mst1 in vitro were observed by Imunofluorescence assay;The effect of evodiamine on the expressions of key molecules of Hippo-YAP signaling pathway in vitro were investigated by RT-q PCR chip;Western blotting experiment was used to measure the expression levels of YAP in cytoplasmic and nucleus,and other key molecules of Hippo-YAP signaling pathway in vitro.Third part: whether evodiamine affects HCC development via Hippo-YAP signaling pathways.Pre-treat two HCC cell lines with the Hippo-YAP signaling pathway inhibitor XMU-MP-1,then FCM,cell clone formation assay,Ed U assay,TUNEL experiment,Western blotting and Immunofluorescence experiment were used to measure the effect of evodiamine combined with XMU-MP-1 on proliferation,apoptosis and Hippo-YAP signaling pathway of HCC cell lines.Results First part: 1.CCK-8 analysis indicated that evodiamine inhibited the growth of HCC cells in a time-and dose-dependent manner(P <0.05),and the IC50 values of Hep G2 and Bel-7402 cells at 48 h were 14.7 ?M and 16 ?M,respectively.The same concentration of evodiamine had no obvious cytotoxicity to normal hepatocytes(P <0.05);2.FCM test revealed that the HCC cell apoptosis ratio significantly increased in a concentration-dependent manner,and the cell cycle was significantly arrested in the G2/M phase(P <0.05)after treated with evodiamine;3.Ed U staining showed that compared with the control group,the cell proliferation was significantly reduced in evodiamine treated group(P <0.01);4.Cloning formation experiment found that with the increased concentration of evodiamine,the numbers of clonal formation gradually decreased in a concentration-dependent manner compared with control group(P <0.01);5.TUNEL staining revealed that the cells in the evodiamine group showed obvious green fluorescence,while there was no significant green fluorescence and the nuclei were intact in the control group.6.Western blotting confirmed that evodiamine could effectively inhibit the expression of Bcl-2(P <0.05),and promote the expression of apoptosis protein,including Bax and p53(P <0.05),in a concentration-dependent.7.In nude mice with xenograft tumors,no significantly difference of mice body weight were observed between groups(P> 0.05),while the tumor volume and weight in evodiamine treated group were significantly lower than the control group(P <0.05);HE staining indicated that the tumor tissue in the evodiamine group had large areas of necrosis,obvious nucleus contraction and fragmentation,and rare intact cells;Meanwhile,the result of TUNEL staining showed that there was obvious apoptosis in the evodiamine group.Second part: 1.IPA database speculates that the physiological function of evodiamine on HCC may be through the YAP protein;2.Immunofluorescence staining confirmed that evodiamine significantly reduced the expression level of YAP,especially in nucleus,while significantly increased the expression level of Mst1 in the cytoplasm,a key molecule upstream of the Hippo-YAP signaling pathway;3.RT-q PCR array further confirmed that evodiamine increased the gene level of Mst1 while decreased the YAP gene level.4.Western blotting found that the YAP protein was significantly down-regulated in evodiamine group when compared with the control group(P <0.05),and the YAP expression level in nucleus was reduced(P <0.01).In addition,compared with the control group,the protein levels of Hippo-YAP signal pathway,including Mst1/2,Lats1,p-Lats1 and p-YAP all were significantly increased with the concentration of evodiamine increased(P <0.05),while the protein expression levels of YAP,CTGF and Survivin were significantly decreased(P <0.05).Third part: 1.Cell clone formation experiment and the Ed U assay indicated that after pretreatment with the Hippo-YAP signaling pathway inhibitor XMU-MP-1,the numbers of colony forming and proliferating cells were significantly increased when compared with the evodiamine group(P <0.05);2.FCM analysis showed that compared with the evodiamine group,the G2/M cell phase and the ratios of cells apoptosis were significantly reduced in XMU-MP-1 combined with evodiamine group(P <0.05);3.TUNEL staining showed that XMU-MP-1 combined with evodiamine significantly reduced the numbers of apoptotic cell when compared with the evodiamine group;4.Western blotting confirmed that compared with the evodiamine group,XMU-MP-1 combined with evodiamine significantly increased the Bcl-2 protein expression level(P <0.05),while significantly reduced the Bax and p53 protein expression level(P <0.05);In addition,compared with the control group,the expression level of Mst1/2,Lats1,p-Lats1,and p-YAP protein were significantly increased in evodiamine group(P <0.05),while YAP and its downstream transcription factors CTGF and Survivin protein expression all were significantly reduced(P <0.05),XMU-MP-1 effectively reversed these effects(P <0.05);5.Immunofluorescence staining analysis indicated that compared with the evodiamine group,XMU-MP-1 reduced the Mst1 fluorescence expression level while increased the YAP fluorescence expression level,especially in cell nucleus.Conclusion 1.Evodiamine inhibits the HCC cells proliferation and promotes its apoptosis;2.YAP is highly expressed in HCC cells.Evodiamine treatment promotes the phosphorylation of YAP,while reduces its nuclear translocation;3.Evodiamine can inhibit tumor formation and promote tumor cell apoptosis in vivo;4.Evodiamine regulates the proliferation and apoptosis of HCC via Hippo-YAP signaling pathway. |
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