Axl glycosylation mediates tumor cell proliferation, invasion and lymphatic metastasis in murine hepatocellular carcinoma

AIM: To investigate the effects of Axl deglycosylation on tumor lymphatic metastases in mouse hepatocellular carcinoma cell lines. METHODS: Western blotting was used to analyze the expression profile of Axl glycoprotein in mouse hepatocellular carcinoma cell line Hca-F treated with tunicamycin and PNGase F 3-(4,5)-dimethylthiazol(-zyl)-3,5-diphenyltetrazolium bromide (MU) assay, extracellular matrix (ECM) invasion assay (in vitro) and tumor metastasis assay (in vivo) were utilized to evaluate the effect of Axl deglycosylation on the Hca-F cell proliferation, invasion and lymphatic metastasis. RESULTS: Tunicamycin and PNGase F treatment markedly inhibited Axl glycoprotein synthesis and expression, proliferation, invasion, and lymphatic metastasis both in vitro and in vivo. In the MTT assay, proliferation was apparent in untreated Hca-F cells compared with treated Hca-F cells. In the ECM invasion assay (in vitro), treated cells passed through the ECMatrix gel in significantly smaller numbers than untreated cells (tunicamycin 5 mu g/mL: 68 +/- 8 vs 80 +/- 9, P = 0.0222; 10 mu g/mL: 50 +/- 6 vs 80 +/- 9, P = 0.0003; 20 mu g/mL: 41 +/- 4 vs 80 +/- 9, P = 0.0001); (PNGase F 8 h: 66 +/- 7 vs 82 +/- 8, P = 0.0098; 16 h: 49 +/- 4 vs 82 +/- 8, P = 0.0001; 24 h: 34 +/- 3 vs 82 +/- 8, P = 0.0001). In the tumor metastasis assay (in vivo), average lymph node weights of the untreated Hca-F group compared with treated Hca-F groups (tunicamycin 5 mu g/mL: 0.84 +/- 0.21 g vs 0.72 +/- 0.19 g, P = 0.3237; 10 mu g/mL: 0.84 +/- 0.21 g vs 0.54 +/- 0.11 g, P = 0.0113; 20 mu g/mL: 0.84 +/- 0.21 g vs 0.42 +/- 0.06 g, P = 0.0008); (PNGase F 8 h: 0.79 +/- 0.15 g vs 0.63 +/- 0.13 g, P = 0.0766; 16 h: 0.79 +/- 0.15 g vs 0.49 +/- 0.10 g, P = 0.0022; 24 h: 0.79 +/- 0.15 g vs 0.39 +/- 0.05 g, P = 0.0001). Also, average lymph node volumes of the untreated Hca-F group compared with treated Hca-F groups (tunicamycin 5 mu g/mL: 815 +/- 61 mm(3) vs 680 +/- 59 mm(3), P = 0.0613; 10 mu g/mL: 815 +/- 61 mm(3) vs 580 +/- 29 mm(3), P = 0.0001; 20 mu g/mL: 815 +/- 61 mm(3) vs 395 +/- 12 mm(3), P = 0.0001); (PNGase F 8 h: 670 +/- 56 mm(3) vs 581 +/- 48 mm(3), P = 0.0532; 16 h: 670 +/- 56 mm(3) vs 412 +/- 22 mm(3), P = 0.0001; 24 h: 670 +/- 56 mm(3) vs 323 +/- 11 mm(3), P = 0.0001). CONCLUSION: Alteration of Axl glycosylation can attenuate neoplastic lymphatic metastasis. Axl N-glycans may be a universal target for chemotherapy.