Effect Of Lectin PSA On Apoptosis Of Human Hepatocellular Carcinoma Cell And Research On Related Mechanism

Hepatocellular carcinoma(HCC)is a malignant tumor with high morbidity worldwide,and it is the fifth most common type of tumor.HCC jeopardizes human health and safety seriously,and The fatality of HCC ranks third in all cancers,second in male patients,and sixth in female patients.Currently,the treatment of HCC contains surgery,ablation therapy,chemoembolization and chemotherapeutic therapy.Besides,the application of computed tomography(CT)and magnetic resonance imaging(MRI)for the early diagnosis of HCC has greatly improved the prognosis of patients.However,there is still no effective treatment because of the easy recurrence and metastasis of HCC.Therefore,it is of great significance to find new potential drugs or new treatments to treat HCC.PART1:Screening of differentially expressed glycopatterns of human normal hepatocytes HL7702 and HCC cell Hep3 B,MHCC97L,MHCC97 H,HCCLM3,Hep G2,SMMC7721 cells by lectin microarray,we found that the glycopatterns recognized by lectin PSA,LTL,MPL,and SNA were up-regulated in HCC cells,while the glycopatterns recognized by lectin STL were down-regulated in HCC cells.The results of the immunofluorescence are consistent with the lectin microarray,among which the expression of glycopatterns recognized by lectin PSA was most significantly up-regulated.PART2:In this part we regarded HCCLM3 and SMMC7721 as the object to investigate the effects of lectin PSA on the biological behavior of HCC cells.It was found by MTT assay that lectin PSA had significant inhibition effect on HCC cell survival,but it had no obvious effect on human normal hepatocytes.When the concentration of PSA was 40 μg/ml,the cell survival rate was about 50%.Transwell assay experiments showed that 40 μg/ml and 80 μg/ml PSA could significantly inhibit the migration and invasion of HCC cells,and the more the PSA concentration is,the stronger the inhibitory ability will be.And Hoechst stain experiment demonstrated that lectin PSA will induces the apoptosis of HCC cells.PART3:In this part we regarded HCCLM3 as object to investigate the molecular mechanism of PSA in promoting the apoptosis of HCC cells.The results of westen blotting showed that AKT/GSK-3β/β-catenin pathway molecules p-AKT(Ser473),p-GSK-3β(Ser9)and β-catenin were down-regulated,and the expression of AKT and GSK3βhave no significant changes.The down-regulated expression of p-AKT,p-GSK3β,and β-catenin correlated positively with the concentration of PSA.To further confirm that PSA promotes apoptosis through the AKT/GSK-3β/β-catenin pathway,we added GSK-3β inhibitors Li Cl and CHIR-99021 to do a confirmatory experiments.The results showed that the addition of 0.2 μM CHIR-99021 and 4 m M Li Cl both attenuate the apoptosis of HCC cell induced by PSA,and attenuate the inhibition of AKT/GSK-3β/β-catenin pathway by PSA.The expression of AKT,GSK-3β,and β-catenin were also significantly increased.PART4:The PSA-binding glycoprotein were enriched by PSA-magnetic particle complex and identified by mass spectrometry.The result indicated that there are 52 glycoproteins in HCCLM3 cells can specifically bind to PSA,55 glycoproteins in SMMC7721 cells.We found that 38 glycoproteins in HCCLM3 and SMMC7721 cells can both bind to PSA,14 glycoproteins specific expressed in HCCLM3 cells,and 17 glycoproteins specific expressed in SMMC7721.Furthermore,there are 4 glycoproteins that are related to the cell apoptosis.