Axl glycosylation mediates tumor cell proliferation, invasion and lymphatic metastasis in murine hepatocellular carcinoma.
OBJECTIVE
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 (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 ± 6 vs 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 mm³ vs 680 ± 59 mm³, P = 0.0613; 10 μg/mL: 815 ± 61 mm³ vs 580 ± 29 mm³, P = 0.0001; 20 μg/mL: 815 ± 61 mm³ vs 395 ± 12 mm³, P = 0.0001); (PNGase F 8 h: 670 ± 56 mm³ vs 581 ± 48 mm³, P = 0.0532; 16 h: 670 ± 56 mm³ vs 412 ± 22 mm³, P = 0.0001; 24 h: 670 ± 56 mm³ vs 323 ± 11 mm³, P = 0.0001).


CONCLUSIONS
Alteration of Axl glycosylation can attenuate neoplastic lymphatic metastasis. Axl N-glycans may be a universal target for chemotherapy.
Department of General Surgery, the Second Affiliated Hospital of Dalian Medical University, Dalian 116027, Liaoning Province, China.
Ji
J
Li
Li
L
Jia
Zhen-Hai
ZH
Ma
Qiu-Hong
QH
Ma
Xiao-Hong
XH
Yang
Yong-Fu
YF
Zhao
D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006031:Glycosylation; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D008207:Lymphatic Metastasis; D051379:Mice; D009361:Neoplasm Invasiveness; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D000094963:Axl Receptor Tyrosine Kinase
D014408:Biomarkers, Tumor; D011518:Proto-Oncogene Proteins; D020794:Receptor Protein-Tyrosine Kinases; D000094963:Axl Receptor Tyrosine Kinase; C000722290:AXL receptor tyrosine kinase, mouse