Abstract
The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.
Highlights
The poor prognosis of cancer is mainly due to the metastasis of malignant cells from the primary neoplasm
Comparison of Amount and Composition of chondroitin sulfate (CS)/dermatan sulfate (DS) between P29 and LM66-H11 Cells—To investigate the possible alteration in the expression of CS/DS in the carcinoma clones with different metastatic potentials, GAGs were extracted from P29 and LM66-H11 cells, as described under “Experimental Procedures,” and the amount and composition of CS/DS in both GAG preparations were determined by digestion with CSase ABC followed by anion exchange HPLC
The results showed that the deca- and dodecasaccharides were substantial inhibitors of the metastasis of LM66-H11 cells compared with the hexa- and octasaccharides (Fig. 6), suggesting that E-unit-containing decasaccharides recognized by the antibody GD3G7 act as minimal sized functional domains of CS/DS chains in the metastatic process
Summary
Materials—CSase ABC (EC 4.2.2.4), highly purified CSase ABC (protease-free CSase ABC), standard unsaturated disaccharides, CS-A from whale cartilage, CS-B from porcine skin, CS-C and CS-D from shark cartilage, and CS-E from squid cartilage were purchased from Seikagaku Corp. (Tokyo, Japan). The dried acetone powder was digested with heat-activated (60 °C, 30 min) actinase E in 200 l of 0.1 M sodium borate, pH 8.0, containing 10 mM calcium acetate at 60 °C for 48 h. Disaccharide Composition Analysis of CS Chains—The disaccharide composition of the GAG preparations from both Lewis lung carcinoma cell lines was determined as previously described [32]. The 2AB-labeled digest was analyzed by anion exchange HPLC on a PA-03 silica column (YMC-Pack PA, Kyoto, Japan). Disaccharide composition of CS/DS chains in Lewis lung carcinoma (3LL)-derived low metastatic P29 and high-metastatic LM66-H11 cells. The GAG preparation from each carcinoma cell line was digested with chondroitinase ABC and analyzed by anion exchange HPLC after labeling with a fluorophore 2AB as detailed under “Experimental Procedures.”
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