The Significance Of Notch1Protein Expression In Hepatocellular Carcinoma

Part I The putative role of Notch1receptor in human hepatocarcinoma carcinogenesis and progression.OBJECTIVE:Notchl signaling is associated with tumorigenesis. This study was carried out to investigate the putative role of Notchl receptor in the Livercancer carcinogenesis and prog ression. Since HBV is the prima ry cause in the development of Liver cance r. the interaction between Notchl and HBV infection was examined. In addition. We also discussed the potentials of using Notchl as an ea rly diagnostic marker and possible the rapeutic target for Liver Cancer.Methods:The expression of Notchl p rotein was analyzed by Western-blotting approach in80liver cance r and80normal liver tissues, some of which were examined using RT-PCR for determing mRNA levels. Celluar localization of Notch1Protein in the pa raffin-embedded Liver tissues was also analyzed by immunohistochemistry using the antibody to Notch1. All the samples were examined for the existence of HBX by PCR. Through the analysis of HBX infection rate, the relationship between Notch1receptor and HBX in carcInogenesis of liver cancer was cha racterizedResults:Notchl protein was detected in72of80normal liver tissues. In contrast, only8samPles of80Liver cance r tissues had the Notchl expression. The level of Notchl Protein expression was significantly lower in liver cance r tissues than that in normal tissue samples. In agreement with these observations, levels of Notch1mRNA we re found to be substantially down-regulated in liver cancer tissues. In immunohistochemistry staining assay. Notchl protein was showed to localize p redomjnantly at the cytoplasm and nucleus in the normal liver epithelium of the liver. but no staining was observed in livercancer cells. Of80tumor samples there were65cases found having HBX (positive rate77.5%). while no HBX DNA was detected in the normal one. Our resuIts indicate that the Notch1protein differential expression level is significantly higher in the carcinomas without HBX DNA than thse with HBV infection (the expression positive rate is46.67%and1.54%respectively, P=0.001). Interestingly, down-modulation of Notchl mRNA level in ca rcinoma was demonstrated to associate with HBX transcription. Moreover, The correlation between cllnicopat-hological parameters of liver cancer and Notchl expression was analyzed. Notch1expression was observed to correlate to the clinical stages of the disease, but no correlations to age, tumor size. Differe-ntiation, lymph node metastasis and treatment effect(P>0.05).Conclusion:Notchl plays a role as a tumor suppressor in liver tumori-genesis. Notch1may exe rt p rotective effects against HBV-induced transformation. Down-modulation of Notch1expression is likely to play an impo rtant role in the late stages of HBV-induced ca rcinogen esis. Determination of Notch1expression may be helpful for preoperative diagnosis and accu racy of staging, Whether Notch1can be used as a tumor marker and therapeutic target in liver cance r requires further investigation. PART Ⅱ The Effect and Possible Mechanism of Notch1Gene on Proliferation and Cell Cycle in Human hepatocarcinoma HepG2Cellobjective:1、To investigate the effect of Notchl gene on the change of proliferation and cell cycle in human hepatocarcinoma HepG2Cell;2、To research the possible mechanisms of Notchl gene involved in proliferation and cell cycle in human hepatocarcinoma HepG2Cell.Methods:1、Eukaryocyte expression vector, which expressed Notchl shRNA or Notch1, were transfcctcd into U251cell respectively. RT-PCR and Western Blotting were applied to monitor the validity of down-regulation of Notchl expression and over-expression.2、MTT assay Was performed to examine the cell proliferation. Flow cytometric analysis Was used to detect the cell cycle.3、RT-PCR and Western Blotting were applied to monitor the expression of NF-κβ. EMS A Was performed to assay the NF-κβ DNA-binding activity.Results:1、Eukaryocyte expression vector, which expressed Notchl shRNA or Notchl wereefficient in down-regulation of Notchl expression and over-expression respectively.2、Up-regulation of Notchl gene by pNotchl-IRES2-EGFP inhibited cell proliferation remarkably(P<0.01), arrested cell cycle at G1phase(P<0.01)and decreased the cellnumber of S phase(P<0.01). Down-expression of Notch1enhanced the cell proliferation significantly(P<0.01), promoted cell cycle at G1phase(P<0.05)and increased the cell number of S phase(P<0.01).3、Up-regulation of Notchl gene by pNotchl-IRES2-EGFP inhibited NF-κβ expression(P<0.05) and decreased NF-κβ DNA-bindingactivity(P<0.01). Down-expression of Notchl enhanced NF-κβ(P<0.01) and increased NF-κβ DNA-binding activity(P<0.01) in HepG2cell.Conclusion:1、Notchl gene leads to the change of proliferation and cell cycle in human hepatocarcinoma HepG2Cell2、Notch1gene mybe inhibitor the cell proliferation via increasing NF-κβ DNA-binding activity in HepG2cell. PART III The effect of saffron on human liver cancer HepG2cellObjective:To study the effect and molecular mechanism of saffron on human liver cancer HepG2cells lines.Methods:The growth inhibition rates of HepG2cell were measured by MTT assay. The cell cycle were determined by flow cytometry; The proteins expressions of notch1、c-myc、p21、cdk2and cyclin E were assayed by western blotting.Results:Saffron significantly inhibited the proliferation of human liver cancer cell line (HepG2)in a dose-dependent and time-dependent manner; Cell cycle analysis revealed that HepG2cells treated by saffron were blocked in G0/G1phase; western blotting indicated that the proteins expressions of p21and Notch1was increased, while the levels of cyclinE、cdk2and c-myc proteins expressions were decreased in saffron-treated HepG2cells.Conclusion:Saffron can inhibit proliferation of liver cancer HepG2cells and induce HepG2cells G0/G1arrest, apoptosis and necrosis. The mechanism of cell cycle arrest may be associated with activation of notch1signaling pathway.