SSAT Inhibits The Proliferation, Migration And Invasion Of Hepatocellular Cell Via AKT/GSK-3?/?-catenin Signaling Pathway

Background:Hepatocellular carcinoma(HCC) is one of the most common causes of cancer-related death worldwide.Early HCC is a symptomatic, and a majority of the patients are diagnosed when the disease is advanced and they are no longer candidates for surgical curative therapy. Therefore, there are extremely limited choices of advanced liver cancer treatment. Moreover, due to the high rate of recurrence and metastasis, five-year survival rate of liver cancer patients is still not satisfactory. Therefore, understanding the mechanism of HCC metastasis has a very important significance for the treatment of liver cancer. Polyamines, putrescine(PUT), spermidine(SPD) and spermine(SPM), a group of biogenic small molecules existing abundantly in mammalian cells, possess intriguing functions in cell growth, differentiation and other physiological processes. Owing to the established close association between upregulated polyamine levels and tumor growth, polyamine has been a very potential anti-tumor targets. Spermidine/spermine acetyl transferase(SSAT) is a polyamine catabolism rate-limiting enzyme in eukaryotic cell and maintains the concentration of polyamines. But now, the specific biological function of SSAT in HCC remains unclear.Objective:Three hepatoma cell lines(SMMC7721, Hep G2 and Bel7402) were used to determine the potential role of SSAT in HCC and the intrinsic molecular mechanisms about it.Methods:Western blotting assay was used to test the expression of SSAT in SMMC7721, Hep G2 and Bel7402of; Using the transfection method to build SSAT over-expression SMMC7721, Hep G2 cell lines and SSAT knockdown cell lines in Bel7402; High performance liquid chromatography was applyed to check the polyamine content in SSAT changes hepatoma cell; MTT and colony formation assay was used to detect liver cancer cell proliferation and colony formation capability; Wound healing experiments and Transwell with or wtihout Matrigel assay results showed the role of SSAT on migration and invasion ability of liver cancer cell; Western blotting assay the effect of SSAT on AKT / ?-catenin pathway; The added polyaminesand the suppression of GSK-3? help us to confirm the resultsResults:1. Western blotting results found that the expression of SSAT in SMMC7721, Hep G2 has low levels,while Bel7402 is higher. After the constructing of SSAT high expression SMMC7721, Hep G2 cell lines,and SSAT knockdown Bel7402 cell lines, HPLC experiments found that high expression of SSAT significantly reduced hepatocellular carcinoma polyamine content, however, polyamine content increased in SSAT knockdown cell;2. MTT and colony formation showed SSAT can significantly inhibit the proliferation and colony-forming ability of liver cancer cells;3. Wound healing experiments and transwell with or without Matrigel found SSAT can inhibit the migration and invasion of HCC cells;4. Western blotting experiments results showed that up-regulation of SSAT expression noticeably inhibit the the expression of AKT, p-AKT, p-GSK-3?, ?-catenin and N-cadherin in SMMC7721 and Hep G2 cells. In contrast, the blockage of SSAT dramatically promote the protein level of AKT, p-AKT,p-GSK-3?, ?-catenin and N-cadherin in BEL-7402 cells;5. The exogenous polyamines could increased the polyamine content and reverse the effect of SSAT on AKT/GSK-3?/?-catenin pathway in SSAT high expression SMMC7721 and Hep G2 cell lines;6. Upon treatment with GSK3? inhibitor LiCl, the inhibitory effect of SSAT on invasion was dramatically released. Furthermore, Western blotting experiments results showed that the expression of?-catenin increased following the increased expression of p- GSK3?.Conclusions:SSAT significantly inhibits the proliferation, migration and invasion of liver cancer cells via AKT/GSK-3?/?-catenin signal pathway by decreasing of intracellular polyamines.