| BackgroundPrimary liver cancer is a common tumor with high mortality and the third malignant tumor of death in our country. The disease is usually diagnosed at an advanced stage, recurrence rate is very high approximately30to40with5-year in well selected patients. Patients with advanced HCC have a median survival of about6-8months, there is no effective treatment for these patients. For this reason, novel therapeutic strategies are essential to improve clinical management of patients with HCC. Experimental studies show that bufalin, the effective component of Chan su which is used in clinical in China, has the effects of proliferation inhibition, inducing apoptosis, reversing multidrug resistance, anti-invasion in several tumor cell lines. Besides, it is reported that bufalin can inhibit the phosphorylation of Akt. The activated of Akt by phosphorylation modifies at least ten major regulatory proteins which play a central role in a variety of oncogenic processes including cell growth, proliferation, apoptotic cell death, motility, epithelial mesenchymal transition (EMT), angiogenesis and metastasis. However, the inhibition of bufalin on the invasion and migration of human hepatoma cell lines have not been reported. Our pre-experiment showed that bufalin can inhibit the migration of HCCLM3cell line, but the mechanism is unclear. Whether inhibition the phosphorylation of Akt which modifies regulatory proteins is related to the inhibition of cell motility is unknown. We will explore the effect and the mechanism of bufalin on the proliferation, migration, invasion, adhesion of human hepatoma cell lines in vitro experiment. So, we hope to provide experimental bases for the antitumor effect of Chan su especially in anti-metastasis.PurposeThe effects of bufalin on the proliferation, migration, invasion, adhesion of HCCLM3and HepG2were to be observed. Then the effect of bufalin on the expression of Akt was to be observed, and the expression of downstream signal molecules pGSK-3β (Ser-9), GSK-3β,β-catenin(Ser33/37),β-catenin, E-cadherin and the nuclear translocation of β-catenin were to be observed too. We explored the effect of bufalin on the Akt—GSK-3β—β-catenin—E-cadherin signaling pathway and the mechanism of inhibition of proliferation, migration, invasion, and adhesion. MethodsWe evaluated the effects of bufalin on the proliferation, migration, invasion and adhesion of HCCLM3and HepG2in vitro. We evaluated the inhibition of bufalin on the proliferation human hepatoma cell lines using CCK8, the inhibition of migration by would healing test and TranswellTM-migration, the inhibition of invasion by TranswellTM-invasion and the inhibition of adhesion by cell adhesion assay. The expression of pGSK-3β (Ser-9), GSK-3β,β-catenin (Ser33/37),β-catenin and E-cadherin were detected by Western bolt. The nuclear translocation of β-catenin and the expression of E-cadherin were observed by immunofluorescence analysis.Results1. The inhibition of bufalin on the proliferation of HCCLM3and HepG2cell linesAfter treatment of Onmol/L,1nmol/L, lOnmol/L,100nmol/L,1000nmol/L,10000nmol/L,100000nmol/L of bufalin, the proliferation inhibition rate of HCCLM3was0.071±0.076,0.241±0.024,0.485±0.009,0.777±0.005,0.834±0.175,0.871±0.007respectively. And the proliferation inhibition of HepG2was0.208±0.029,0.350±0.050,0.486±0.019,0.793±0.011,0.835±0.013,0.886±0.016. The IC50of HCCLM3(48h) was66.90nmol/L calculated by GraphPad Prism5.0, and the IC50of HepG2(48h) was97.43nmol/L.2. The inhibition of bufalin on the migration of HCCLM3and HepG2cell linesAfter24h treatment of Onmol/L, lnmol/L,10nmol/L,100nmol/L of bufalin, the mobility rate of HCCLM3was0.453±0.028,0.251±0.036,0.175±0.022,0.117±0.027. And after48h, the mobility rate of HCCLM3was0.650±0.048,0.468±0.052,0.419±0.014,0.236±0.046respectively. All the experimental groups had obvious difference compared with the control group (P<0.01). The24h mobility rate of HepG2was0.684±0.026,0.653±0.035,0.604±0.022,0.325±0.010, the experimental groups over10nmol/L had difference with the control group(P<0.05,P<0.01), the48h mobility rate of HepG2was0.840±0.050,0.761±0.026,0.661±0.029,0.416±0.014, the experimental groups over10nmol/L had difference with the control group (P<0.01).The cell number of HCCLM3after treatment of Onmol/L,1nmol/L,10nmol/L,100nmol/L of bufalin using TranswellTM-migration (48h) was105±12,92.7±12.2,41±5.6,29.7±4.2(200X,5fields), the experimental groups over10nmol/L had difference with the control group (P<0.01). And the cell number of HepG2was408±20.7,307±15.7,220±16,205.3±10(200X,5fields). All the experimental groups had obvious difference compared with the control group (P<0.01).3. The inhibition of bufalin on the invasion of HCCLM3and HepG2cell linesThe cell number of HCCLM3after treatment of Onmol/L, lnmol/L,10nmol/L,100nmol/L of bufalin using TranswellTM-invasion (48h) was69.7±8.5,35.3±4.7,23.7±4.2,14.7±2.5(200×,5fields), the experimental groups had obvious difference compared with the control group (P<0.01). And the cell number of HepG2was91±11.5,65.3±8.5,47±6.3,37±2.6, all the experimental groups had obvious difference compared with the control group (P<0.05, P<0.01, P<0.01).4. The inhibition of bufalin on the adhesion of HCCLM3and HepG2cell linesAfter48h treatment of Onmol/L, lnmol/L,10nmol/L,100nmol/L of bufalin, the adhesion number of HCCLM3was497±20,398±16,125±14,102±5(200×, per field). The adhesion number of HepG2was514±17,417±22,217±7,145±13(200×, per field). All the experimental groups over10nmol/L had obvious difference compared with the control group (P<0.01).5. Detecting the expression of Akt, pAkt(Ser473), pGSK-3β (Ser-9), GSK-3β,β-catenin(Ser33/37),β-catenin and E-cadherin by Western boltAfter the treatment of bufalin and LY294002in HCCLM3and HepG2, the pAkt (Ser473) and pGSK-3β (Ser-9) were reduced and the GSK-3β were increased.There were no change in total Akt and total β-catenin in the two cell lines.6. Detecting the nuclear translocation of β-catenin and the expression of E-cadherinWe found that bufalin and LY294002can inhibit the nuclear translocation of β-catenin in HCCLM3and HepG2by immunofluorescence analysis, and the expression of E-cadherin were increased after treatment.Conclusion1.Bufalin can inhibit the proliferation,migration, invasion and adhesion of HCCLM3and HepG2.2.Bufalin may inhibit the proliferation, migration, invasion and adhesion of HCCLM3and HepG2through Akt—GSK-3β—β-catenin signaling pathway.3. The bufalin’s inhibition of proliferation may relate to inhibiting the phosphorylation of Akt, GSK-3β and the nuclear translocation of β-catenin. The bufalin’s inhibition of migration and invasion may relate to inhibiting nuclear translocation of β-catenin, reversing the tumor cell’s phenotype of mesenchymal cell and increasing the expression of E-cadherin. And the bufalin’s inhibition of adhesion may relate to inhibiting nuclear translocation of β-catenin which induce the adhesion ability between the cells and the matrix. |
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