摘要
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 hepa-tocellular carcinoma cell line Hca-F treated with tunicamycin and PNGase F 3-(4,5)-dimethylthiazol(-zyl)-3,5- diphenyltetrazolium bromide (MTT) 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 μg/mL: 68 ± 8 vs 80 ± 9, P=0.0222; 10 μg/mL: 50 ± 6vs 80 ± 9,P=0.0003; 20 μg/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 μg/mL: 0.84 ± 0.21 g vs 0.72 ± 0.19 g, P=0.3237; 10 μg/mL: 0.84 ± 0.21 g vs 0.54 ± 0.11 g, P=0.0113; 20 μg/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 μg/mL: 815 ± 61 mm 3 vs 680 ± 59 mm 3 , P=0.0613; 10 μg/mL: 815 ± 61 mm 3 vs 580 ± 29 mm 3 , P=0.0001; 20 μg/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 at-tenuate neoplastic lymphatic metastasis. Axl N-glycans may be a universal target for chemotherapy.
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 hepa-tocellular carcinoma cell line Hca-F treated with tunicamycin and PNGase F 3-（4,5）-dimethylthiazol（-zyl）-3,5- diphenyltetrazolium bromide （MTT） 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 μg/mL： 68 ± 8 vs 80 ± 9, P=0.0222; 10 μg/mL： 50 ± 6vs 80 ± 9,P=0.0003; 20 μg/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 μg/mL： 0.84 ± 0.21 g vs 0.72 ± 0.19 g, P=0.3237; 10 μg/mL： 0.84 ± 0.21 g vs 0.54 ± 0.11 g, P=0.0113; 20 μg/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 μg/mL： 815 ± 61 mm 3 vs 680 ± 59 mm 3 , P=0.0613; 10 μg/mL： 815 ± 61 mm 3 vs 580 ± 29 mm 3 , P=0.0001; 20 μg/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 at-tenuate neoplastic lymphatic metastasis. Axl N-glycans may be a universal target for chemotherapy.
基金
Supported by Creating Team Item of Liaoning Province, No.2008T033
the Technological Natural Fund Item of Liaoning Province, China, No. 20092164
