Effects Of SFRP2 On Biological Behaviors Of Human Hepatoma Cell Line HepG2

Development and progess of tumor is a complex biologic process that is involved in various factors and numerous genes. Tumor cells are characterized by deregulated proliferation, attributed to the abnormality of signalling pathways. Wnt/β-catenin pathway is one of these signalling pathways. In this pathway,β-catenin, axin, adenomatous polyposis coli (APC), glycogen synthase kinase -3β(GSK-3β), and dishevelled (DVL) are the major components which regulate the degradation ofβ-catenin. Secreted Wnt ligands act on the cell surface receptor Frizzled and activate DVL. Activated DVL inhibits the phosphorylation ofβ-catenin by GSK-3β, and then blocksβ-catenin degradation so thatβ-catenin accumulates and translocates to the nucleus, where it interacts with the T cell factor 4 (TCF4) transcription factor and induces the transcription of target genes such as c-jun, c-myc, and cyclin D1. Defects in different components of the Wnt/β-catenin signalling pathway, upregulateβ-catenin/TCF4 activity, therefore promote tumorigenesis and tumour progression. Upregulatedβ-catenin/TCF4 activity is a pivotal event in most human tumors.Secreted frizzled-related protein (sFRP) is a group of proteins which regulate the Wnt-frizzled signaling pathways. SFRP has abundant cysteine-rich domains and hydrophilic carboxyl terminal. sFRP has no transmembrane domains. Thus, it binds Wnt, and interfers the Wnt signaling pathway.Hepatoma is one of the most common malignant tumors, expecially in China. The human hepatoma cell line HepG2 was chosen as the cell model, and the HepG2 cells were treated with the recombinant adenovirus which carries the sFRP2 gene. The effects of the sFRP2 protein on HepG2 cells were evaluated by various methods.The adenovirus which carries the sFRP2 cells was used to infect the HepG2 cells in order to express sFRP2 proteins in the cells. The Western Blotting method was used to test for the expression ofβ-catenin proteins; and the MTT method was used to assess the antiproliferative effect of the sFRP2 protein on HepG2 cells. The Flow cytometry (FCM) method was used to measure the inhibition rates of HepG2 cells for assessing the effect of sFRP2 proteins on these cells. An immunohistochemistry method was used to assess the expression of CD44, E-cadherin, CD82 and EMMPRIN proteins which are important for judging the effect of the sFRP2 protein on HepG2 cells. The Transwell method was used to observe the effect of the sFRP2 protein on metastasis.RESULTS:Western Blotting results demonstrated that theβ-catenin protein was expressed in HepG2 cells. Results from the MTT method showed that the sFRP2 protein caused the proliferation speed of the HepG2 cells to drop obviously. The FCM method showed that there was an obvious increase in the rate of G0/G1 period cells. This observation supported the results of the MTT method above. Immunohistochemistry results: The CD44, E-cadherin and CD82 proteins which can inhibit the cells capability for invasion and metastasis was enhanced obviously. However, the EMMPRIN protein's expression which can promote the cells'capability for invasion and metastasis was weakened obviously by sFRP2 protein. This gives a good explanation at the molecular level of how the sFRP2 protein inhibits the process of invasion and metastasis. The results of the Transwell test also show that the cells capablity for invasion and metastasis was markedly inhibited by the sFRP2 protein.CONCLUSIONS:The HepG2 cells were successfully infected by the adenovirus which carries the sFRP2 gene. The infected cells capability for invasion and metastasis was reduced markedly by the sFRP2 protein.