The Inhibitory Effects Of Axl Deglycosylation On The Lymphatic Metastasis In Murine Hepatocarcinoma Cell Lines

Metastasis is the important factor of malignant tumor causing death. The study oftumor metastasis mechanism is important significance. The modes of tumor metastasishave lymphatic metastasis, implantation metastasis and hematogenous metastasis.Lymphatic metastasis is the most common, but the mechanism of metastasis has not yetbeen thoroughly explored. Several previous studies have demonstrated that tumormetastasis is not only related with organism microenvironment, but also associated withtumor cells property. The glycosylation of cell surface glycoprotein play significantroles in regulating tumor metastasis.Axl is a highly glycosylated the receptor tyrosine kinases superfamily (RTKs)transmembrane protein, which is characterized by a combination of twoimmunoglobin-like domains and dual fibronectin type III repeats in the extracellularregion, and a cytoplasmic kinase domain. Some had reported that Axl has thetransforming potential when over-expressed. The over-expression of Axl is clearlyassociated with invasiveness and metastasis in several cancer cell types, includingmyeloid leukemia, lung cancer, colon cancer, prostatic carcinoma, breast cancer andgastric cancer. These findings suggest that Axl may play an important role on tumorprogression, but its mechanisms of action have not been understood so far.Protein glycosylation is one of the major types of posttranslational modifications withprofound biological implications. Specific changes in the glycosylation pattern of cellsurface glycoproteins have been shown to correlate with the enhancement of the metastaticefficiency of tumour cells. In particular, protein N-glycosylation is increasingly beingrecognized as one of the most prominent biochemical alterations involved in tumorigenesisand metastatic spread of tumor cells. Therefore, it is necessary to research the effects of Axldeglycosylation on the lymphatic metastasis in murine hepatocarcinoma cell lines The mouse hepatocarcinoma cell lines Hca-F with a highly lymphatic metastasisrate over80%and Hca-P with a low lymphatic metastasis rate less than30%wereseparated from615type murine hepatocarcinoma cell line H22. Hca-F and Hca-P cellsmetastasize only to the lymph nodes, and not to other organs. Therefore, they are theideal model to study the mechanism of lymphatic metastasis.Objective: To research the possible effect of Axl deglycosylation on regulation oftumor cells proliferation, invasion and the lymphatic metastasis in murinehepatocarcinoma.Methods: Western blot analysis was used to evaluate the expression profile of Axlglycoprotein in the mouse hepatocarcinoma cell lines Hca-F which were treatmentedwith tunicamycin and PNGase F. MTT assay, ECM invasion assay in vitro and tumormetastasis assay in vivo were utilized to evaluate the effect of Axl deglycosylation onthe Hca-F cells proliferation, invasion and the lymphatic metastasis.Results: By treatment with tunicamycin and PNGase F, the expression level ofAxl glycoprotein was markedly changed. The new bands that appeared were consistentwith the size of the core protein, which were the result of tunicamycin and PNGase Finhibiting Axl glycosylation. The Hca-F cells proliferation, invasion and the lymphaticmetastasis capability decreased, comparing with control (*P<0.05).Conclusions: the N-glycosylation process in Hca-F cells was highly sensitive toinhibition by tunicamycin and PNGase F. Axl deglycosylation inhibits tumor cellsproliferation, invasion and the lymphatic metastasis in murine hepatocarcinoma Hca-F.