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Involvement of Highly Sulfated Chondroitin Sulfate in the Metastasis of the Lewis Lung Carcinoma Cells

2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês

10.1074/jbc.m806015200

ISSN

1083-351X

Autores

Fuchuan Li, Gerdy B. ten Dam, Sengottuvelan Murugan, Shuhei Yamada, Taishi Hashiguchi, Shuji Mizumoto, Kayoko Oguri, Minoru Okayama, Toin H. Van Kuppevelt, Kazuyuki Sugahara,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

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 Δ4,5HexUA-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. 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 Δ4,5HexUA-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. The poor prognosis of cancer is mainly due to the metastasis of malignant cells from the primary neoplasm. Metastasis is a selective process involving invasion, embolization, survival in the circulation, arrest in distant capillary beds, and extravasation into and multiplication within the target organ parenchyma (1Eccles S.A. Welch D.R. Lancet. 2007; 369: 1742-1757Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar, 2Fidler I.J. Nat. Rev. Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3532) Google Scholar). In the process of metastasis, tumor cells are involved in a series of interactions with surrounding extracellular matrix (ECM) 5The abbreviations used are: ECM, extracellular matrix; PG, proteoglycan; DS, dermatan sulfate; CS, chondroitin sulfate; GAG, glycosaminoglycan; CSase, chondroitinase; 2AB, 2-aminobenzamide; DMEM, Dulbecco's modified Eagle's medium; HPLC, high performance liquid chromatography; GlcUA, d-glucuronic acid; PBS, phosphate-buffered saline; HexUA, hexuronic acid; Δ4,5HexUA, 4,5-unsaturated hexuronic acid; E-unit, GlcUA;1-3GalNAc(4S,6S); 2S, 2-O-sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate. 5The abbreviations used are: ECM, extracellular matrix; PG, proteoglycan; DS, dermatan sulfate; CS, chondroitin sulfate; GAG, glycosaminoglycan; CSase, chondroitinase; 2AB, 2-aminobenzamide; DMEM, Dulbecco's modified Eagle's medium; HPLC, high performance liquid chromatography; GlcUA, d-glucuronic acid; PBS, phosphate-buffered saline; HexUA, hexuronic acid; Δ4,5HexUA, 4,5-unsaturated hexuronic acid; E-unit, GlcUA;1-3GalNAc(4S,6S); 2S, 2-O-sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate. components and nontumor cells such as platelets and endothelial cells. Proteoglycans (PGs), which bear heparan sulfate (HS) or chondroitin sulfate (CS)/dermatan sulfate (DS) side chains and are widely expressed on the cell surface and in the ECM, are important in modulating these interactions (3Tímár J. Lapis K. Dudás J. Sebestyén A. Kopper L. Kovalszky I. Semin. Cancer Biol. 2002; 12: 173-186Crossref PubMed Scopus (95) Google Scholar, 4Iida J. Meijne A.M. Knutson J.R. Furcht L.T. McCarthy J.B. Semin. Cancer Biol. 1996; 7: 155-162Crossref PubMed Scopus (71) Google Scholar, 5Gallagher J.T. Curr. Opin. Cell Biol. 1989; 1: 1201-1218Crossref PubMed Scopus (212) Google Scholar). It has been well documented that PGs with HS side chains play important roles in metastasis (6Sanderson R.D. Semi. Cell Dev. Biol. 2001; 12: 89-98Crossref PubMed Scopus (191) Google Scholar, 7Munesue S. Yoshitomi Y. Kusano Y. Koyama Y. Nishiyama A. Nakanishi H. Miyazaki K. Ishimaru T. Miyaura S. Okayama M. Oguri K. J. Biol. Chem. 2007; 282: 28164-28174Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 8Nakanishi H. Oguri K. Yoshida K. Itano N. Takenaga K. Kazama T. Yoshida A. Okayama M. Biochem. J. 1992; 288: 215-224Crossref PubMed Scopus (36) Google Scholar). In addition to HS-PGs, CS/DS-PGs have also been implicated in normal biological processes such as neuronal development, morphogenesis, growth factor binding, and cell signaling (9Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (588) Google Scholar, 10Sugahara K. Mikami T. Curr. Opin. Struct. Biol. 2007; 17: 536-545Crossref PubMed Scopus (223) Google Scholar) but also play a crucial role in the metastatic process (4Iida J. Meijne A.M. Knutson J.R. Furcht L.T. McCarthy J.B. Semin. Cancer Biol. 1996; 7: 155-162Crossref PubMed Scopus (71) Google Scholar). Human melanoma CS/DS-PG and its homologue NG2 in rats are involved in matrix adhesion, migration, and invasion, a role that is abolished by treatment with antibodies against CS/DS-PG (11Lynch S.A. Bouchard B.N. Vijayasaradhi S. Yuasa H. Houghton A.N. Cancer Metastasis Rev. 1991; 10: 141-150Crossref PubMed Scopus (18) Google Scholar, 12Pluschke G. Vanek M. Evans A. Dittmar T. Schmid P. Itin P. Filardo E.J. Reisfeld R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9710-9715Crossref PubMed Scopus (136) Google Scholar, 13Iida J. Meijne A.M. Oegema T.R. Jr. Yednock T.A. Kovach N.L. Furcht L.T. McCarthy J.B. J. Biol. Chem. 1998; 273: 5955-5962Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Moreover, CD44-related CS/DS-PG on the cell surface is essential in the invasion/migration of normal endothelial and melanoma cells (14Henke C.A. Roongta U. Mickelson D.J. Knutson J.R. McCarthy J.B. J. Clin. Investig. 1996; 97: 2541-2552Crossref PubMed Scopus (127) Google Scholar, 15Faassen A.E. Schrager J.A. Klein D.J. Oegema T.R. Couchman J.R. McCarthy J.B. J. Cell Biol. 1992; 116: 521-531Crossref PubMed Scopus (236) Google Scholar). The interaction between CS/DS-PG and membrane type 3 matrix metalloproteinase is important for the invasion of melanoma cells (16Iida J. Pei D. Kang T. Simpson M.A. Herlyn M. Furcht L.T. McCarthy J.B. J. Biol. Chem. 2001; 276: 18786-18794Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In addition, treatment with chondroitinase (CSase) AC (specifically cleaving CS) or CSase B (specifically cleaving DS) significantly inhibited the proliferation and invasion of melanoma cells (17Denholm E.M. Lin Y.Q. Silver P.J. Eur. J. Pharmacol. 2001; 416: 213-221Crossref PubMed Scopus (93) Google Scholar). Most recently, cell surface CS/DS was shown to participate in basic fibroblast growth factor-induced proliferation of human metastatic melanoma cell lines (18Nikitovic D. Assouti M. Sifaki M. Katonis P. Krasagakis K. Karamanos N.K. Tzanakakis G.N. Int. J. Biochem. Cell Biol. 2008; 40: 72-83Crossref PubMed Scopus (52) Google Scholar), the membrane type 3 matrix metalloproteinase-mediated activation of promatrix metalloproteinase-2 (19Iida J. Wilhelmson K.L. Ng J. Lee P. Morrison C. Tam E. Overall C.M. McCarthy J.B. Biochem. J. 2007; 403: 553-563Crossref PubMed Scopus (102) Google Scholar), and P-selectin-mediated metastasis of breast cancer cell lines (20Monzavi-Karbassi B. Stanley J.S. Hennings L. Jousheghany F. Artaud C. Shaaf S. Kieber-Emmons T. Int. J. Cancer. 2007; 120: 1179-1191Crossref PubMed Scopus (71) Google Scholar). These studies point to the fact that CS/DS side chains play crucial roles through binding to various ligands, although the core protein also has ligand binding capabilities (21Svee K. White J. Vaillant P. Jessurun J. Roongta U. Krumwiede M. Johnson D. Henke C. J. Clin. Investig. 1996; 98: 1713-1727Crossref PubMed Scopus (101) Google Scholar). CS chains are composed of repeating disaccharide units of GlcUA-GalNAc, where GlcUA and GalNAc represent d-glucuronic acid and N-acetyl-d-galactosamine, respectively, whereas DS is a stereoisomer of CS chains formed from precursor CS chains through the action of glucuronyl C5 epimerase (22Silbert J.E. Sugumaran G. IUBMB Life. 2002; 54: 177-186Crossref PubMed Scopus (256) Google Scholar, 23Maccarana M. Olander B. Malmstróm J. Tiedemann K. Aebersold R. Lindahl U. Li J-P. Malmstróm A. J. Biol. Chem. 2006; 281: 11560-11568Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). CS and DS chains are often found as co-polymeric structures (CS/DS) that tend to exhibit a periodic distribution of GlcUA-containing disaccharide repeats and l-iduronic acid-containing disaccharide repeats in a cell/tissue-specific manner (9Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (588) Google Scholar, 24Cheng F. Heinegárd D. Malmstróm A. Schmidtchen A. Yoshida K. Fransson L.-Á. Glycobiology. 1994; 4: 685-696Crossref PubMed Scopus (89) Google Scholar). CS/DS chains are further modified by the differential sulfation pattern of specific sulfotransferases at C-2 of GlcUA/l-iduronic acid and/or C-4 and/or C-6 of GalNAc to yield enormous structural diversity (25Kusche-Gullberg M. Kjellen L. Curr. Opin. Struct. Biol. 2003; 13: 605-611Crossref PubMed Scopus (243) Google Scholar). Hence, functional structures of CS/DS chains may be generated in tumor cells because of the differential expression of the individual modification enzymes during tumor progression, which may have a direct/indirect link with metastatic potential. Thus, identification of these altered functional structures would be a significant step in understanding the mechanism of involvement of CS/DS-PGs in metastasis and enable us to address the diagnosis and treatment of malignant tumors. Highly sulfated disaccharide units like E-unit, GlcUA;1-3GalNAc(4S,6S) (26Sugahara K. Yamada S. Trends Glycosci. Glycotechnol. 2000; 12: 321-349Crossref Scopus (95) Google Scholar), where 4S and 6S stand for 4-O- and 6-O-sulfate, respectively, are rare, and E-unit-rich CS preparations show remarkable biological activities like the promotion of neurite outgrowth and high affinity binding to growth factors (9Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (588) Google Scholar, 10Sugahara K. Mikami T. Curr. Opin. Struct. Biol. 2007; 17: 536-545Crossref PubMed Scopus (223) Google Scholar, 27Deepa S.S. Umehara Y. Higashiyama S. Itoh N. Sugahara K. J. Biol. Chem. 2002; 277: 43707-43716Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Increasing evidence suggests that the expression pattern of cell surface CS/DS-PGs is related to metastatic potential (3Tímár J. Lapis K. Dudás J. Sebestyén A. Kopper L. Kovalszky I. Semin. Cancer Biol. 2002; 12: 173-186Crossref PubMed Scopus (95) Google Scholar, 12Pluschke G. Vanek M. Evans A. Dittmar T. Schmid P. Itin P. Filardo E.J. Reisfeld R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9710-9715Crossref PubMed Scopus (136) Google Scholar, 13Iida J. Meijne A.M. Oegema T.R. Jr. Yednock T.A. Kovach N.L. Furcht L.T. McCarthy J.B. J. Biol. Chem. 1998; 273: 5955-5962Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, the involvement of highly sulfated structures of CS/DS chains in the metastatic process remains obscure. In this study, E-unit-containing epitopes specifically recognized by the antibody GD3G7 (28ten Dam G.B. van de Westerlo E.M. Purushothaman A. Stan R.V. Bulten J. Sweep F.C. Massuger L.F. Sugahara K. van Kuppevelt T.H. Am. J. Pathol. 2007; 171: 1324-1333Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29Purushothaman A. Fukuda J. Mizumoto S. ten Dam G.B. van Kuppevelt T.H. Kitagawa H. Mikami T. Sugahara K. J. Biol. Chem. 2007; 282: 19442-19452Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) were identified as crucial for the highly metastatic potential of the Lewis lung carcinoma-derived LM66-H11 clone. 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 single chain antibody GD3G7 was selected for reactivity with rat embryo-derived GAGs by the phage display technique (28ten Dam G.B. van de Westerlo E.M. Purushothaman A. Stan R.V. Bulten J. Sweep F.C. Massuger L.F. Sugahara K. van Kuppevelt T.H. Am. J. Pathol. 2007; 171: 1324-1333Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The monoclonal anti-vesicular stomatitis virus tag antibody P5D4 and porcine intestinal mucosal heparin were from Sigma. Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) was obtained from Invitrogen. Actinase E was from Kaken Pharmaceutical Co. (Tokyo, Japan). The Diff-Quick solution was purchased from International Reagent Corp. (Kobe, Japan). 2-Aminobenzamide (2AB) was purchased from Nacalai Tesque (Kyoto, Japan). Sodium cyanoborohydride (NaBH3CN) was from Aldrich. All other chemicals and reagents were of the highest quality available. Size-defined even-numbered CS-E oligosaccharides were prepared by enzymatic fragmentation of a commercial squid cartilage CS-E with sheep testicular hyaluronidase (Sigma), followed by fractionation using gel filtration column chromatography on Bio-Gel P-10 as described previously (30Kinoshita A. Yamada S. Haslam S.M. Morris H.R. Dell A. Sugahara K. J. Biol. Chem. 1997; 272: 19656-19665Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). For RNA extraction, a QuickPrep™ total RNA extraction kit was purchased from GE Healthcare. RNA-qualified RNase-free DNase, RNasin® ribonuclease inhibitor, and Moloney murine leukemia virus reverse transcriptase were obtained from Promega (Madison, WI). A Platinum® SYBR® Green qPCR Supermix-UDG kit was purchased from Invitrogen. Animals and Cell Lines—Six-week-old male C57BL/6 mice and 9-week-old female C3H/HeN mice were obtained from Japan SLC (Hamamatsu, Japan) and kept in standard housing. All of the experiments were performed under the experimental protocol approved by the local animal care committee of Hokkaido University. The low metastatic P29 and high metastatic LM66-H11 cells cloned from a murine Lewis lung carcinoma 3LL were prepared as reported (31Itano N. Oguri K. Nakanishi H. Okayama M. J. Biochem. (Tokyo). 1993; 114: 862-873Crossref PubMed Scopus (14) Google Scholar) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Thermo Trace, Melbourne, Australia), streptomycin (100 ;g/ml), and penicillin (100 units/ml) at 37 °C in a humidified 5% CO2 atmosphere. The cells were harvested after incubation with 2 mm EDTA in phosphate-buffered saline (EDTA/PBS) for 10 min at 37 °C by gentle flushing with a pipette and subcultured twice a week. Extraction of GAGs from P29 and LM66-H11 Cells—The cells were dehydrated and delipidated by extraction with acetone, air-dried, and used for extraction of GAGs essentially as described previously (32Li F. Shetty A.K. Sugahara K. J. Biol. Chem. 2007; 282: 2956-2966Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) with some modifications. Briefly, 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. Following incubation, each sample was treated with 5% trichloroacetic acid, and the resultant precipitate was removed by centrifugation. The supernatant was extracted with ether to remove trichloroacetic acid. After neutralization with 1.0 m sodium carbonate, the aqueous phase was adjusted to contain 80% ethanol and 1% sodium acetate and kept at 4 °C overnight. The precipitated crude GAGs were recovered by centrifugation, desalted on a PD-10 column (GE Healthcare) using 50 mm pyridine acetate, pH 5.0, as an eluent, and evaporated dry. 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 (32Li F. Shetty A.K. Sugahara K. J. Biol. Chem. 2007; 282: 2956-2966Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, the sample was dissolved in water, and an aliquot was digested with CSase ABC (33Saito H. Yamagata T. Suzuki S. J. Biol. Chem. 1968; 243: 1536-1542Abstract Full Text PDF PubMed Google Scholar), the digest was labeled with 2AB (34Kinoshita A. Sugahara K. Anal. Biochem. 1999; 269: 367-378Crossref PubMed Scopus (190) Google Scholar), and excess 2AB was removed by extraction with chloroform (35Kawashima H. Atarashi K. Hirose M. Hirose J. Yamada S. Sugahara K. Miyasaka M. J. Biol. Chem. 2002; 277: 12921-12930Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The 2AB-labeled digest was analyzed by anion exchange HPLC on a PA-03 silica column (YMC-Pack PA, Kyoto, Japan). Identification and quantification of the resulting disaccharides were achieved by comparison with the elution positions of CS-derived authentic unsaturated disaccharides (Ref. 34Kinoshita A. Sugahara K. Anal. Biochem. 1999; 269: 367-378Crossref PubMed Scopus (190) Google Scholar and Table 1).TABLE 1Disaccharide composition of CS/DS chains in Lewis lung carcinoma (3LL)-derived low metastatic P29 and high-metastatic LM66-H11 cellsUnsaturated disaccharideP29LM66-H11pmol (mol %)pmol (mol %)ΔO: ΔHexUA-GalNAc43.3 (3.7)31.5 (27.4)ΔC: ΔHexUA-GalNAc(6S)7.0 (0.6)0.8 (0.7)ΔA: ΔHexUA-GalNAc(4S)1,104.7 (94.5)75.4 (65.6)ΔD: ΔHexUA(2S)-GalNAc(6S)NDcND, not detected.NDΔB: ΔHexUA(2S)-GalNAc(4S)12.9 (1.1)1.3 (1.1)ΔE: ΔHexUA-GalNAc(4S,6S)1.1 (0.1)6.0 (5.2)ΔT: ΔHexUA(2S)-GalNAc(4S,6S)NDNDTotalaThe amount in 1 mg of dry cells delipidated with acetone.1,169 (100)115 (100)S/unitbS/unit, a molar ratio of sulfate to disaccharide.0.980.79Molar ratio of disaccharide10:110:1a The amount in 1 mg of dry cells delipidated with acetone.b S/unit, a molar ratio of sulfate to disaccharide.c ND, not detected. Open table in a new tab Immunocytochemistry—E-unit-containing epitopes on the cell surface of P29 and LM66-H11 cells were stained using a single chain phage display antibody, GD3G7 (28ten Dam G.B. van de Westerlo E.M. Purushothaman A. Stan R.V. Bulten J. Sweep F.C. Massuger L.F. Sugahara K. van Kuppevelt T.H. Am. J. Pathol. 2007; 171: 1324-1333Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29Purushothaman A. Fukuda J. Mizumoto S. ten Dam G.B. van Kuppevelt T.H. Kitagawa H. Mikami T. Sugahara K. J. Biol. Chem. 2007; 282: 19442-19452Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly, the carcinoma cells (5 × 104 cells/well) were plated on 8-well Lab-Tech chamber slides (Nalge Nunc International), cultured in DMEM supplemented with 10% fetal bovine serum for 24 h, and fixed with the Diff-Quick reagent A. After being blocked with PBS containing 3% bovine serum albumin for 1 h at room temperature, the fixed cells were incubated with 100 ;l of the primary antibody GD3G7 (diluted 1:100 (10 ;g/ml) in 0.1% bovine serum albumin/PBS) for 1 h at room temperature and washed with PBS containing 0.5% Tween 20 three times. In a control experiment, GD3G7 was preincubated with CS-E (5 or 10 ;g) at 4 °C for 30 min to demonstrate the specificity of GD3G7 toward CS-E. After being incubated with anti-vesicular stomatitis virus antibody (diluted 1:5,000 in 0.1% bovine serum albumin/PBS) for 1 h at room temperature, the cells were washed with PBS containing 0.5% Tween 20. To detect the anti-vesicular stomatitis virus antibodies, the cells were stained with the Alexa Fluor 488-conjugated third antibody (diluted 1:500 in 0.1% bovine serum albumin/PBS) and visualized with a laser-scanning confocal microscope, FLUOVIEW (Olympus, Tokyo, Japan). To evaluate the specificity of the antibody, the cells were treated with CSase ABC in the presence of a protease inhibitor mixture (Nacalai Tesque, Inc., Kyoto, Japan) and then processed for immunostaining as described above. Assays for Lung Metastasis—To prepare LM66-H11 cells for injection, the cells were harvested by brief exposure to EDTA/PBS, and the cell viability in single-cell suspensions was determined by trypan blue exclusion. A total of 4 × 105 cells (>90% viability) suspended in 200 ;l of DMEM were injected into a lateral tail vein of C57BL/6 or C3H/HeN mice. Although the cell lines LM66-H11 and P29 were established from the C57BL/6 mouse, the C3H/HeN mouse strain was also used for metastasis experiments because of the better handling advantage, and the results obtained were similar to those obtained with a syngeneic C57BL/6 mouse. After 3 weeks, the animals were sacrificed, and the number of visible tumor cell parietal nodules in the lung was counted by two observers in a blinded fashion. The inoculation procedure described above was used for the subsequent experiments. To investigate the involvement of cell surface CS/DS in metastasis, LM66-H11 cells were pretreated with either DMEM (control) or DMEM containing protease-free CSase ABC (20 mIU/ml) for 30 min at 37 °C before being injected into C3H/HeN mice. In a separate series of experiments, C57BL/6 or C3H/HeN mice received the indicated amount (100 ;g/mouse) of commercial CS/DS preparations (CS-A, CS-B, CS-C, CS-D, and CS-E) or heparin 30 min before the tumor cell injection. CS-E digested with CSase ABC was also used as a control. For dose-dependent inhibition experiments and for inhibition experiments using size-defined CS-E oligosaccharides, various doses of CS-E (10, 50, 100, 200, and 300 ;g/mouse), and hexa-, octa-, deca-, or dodecasaccharides (25 ;g/mouse) were administered into C3H/HeN mice 30 min before the tumor cell injection. In other instances, to investigate the involvement of the antibody GD3G7 epitope in metastasis, LM66-H11 cells were preincubated with 200 ;l of serially diluted GD3G7 (0.5, 0.25, and 0.125 ;g/ml) or the irrelevant antibody MPB49V (0.5 ;g/ml) for 30 min at 37 °C and used for metastasis experiments in both C57BL/6 and C3H/HeN mice. Aliquots of the cell suspension were assessed for cell viability before the injection. Dynamic Monitoring of LM66-H11 Cell Proliferation Using the RT-CES™ System—The cell proliferation assay was done using the RT-CES™ system (ACEA Biosciences, San Diego, CA). Briefly, ACEA 96X microtiter plates (e-plate™) were coated with laminin (25 ;g/ml) at 37 °C for 30 min, and LM66-H11 cells (2 × 104/well) were seeded in 100 ;l of medium. In the first experiment, cell attachment was monitored up to 20 min, at which point various inhibitors, CS-A (100 ;g/ml), CS-E (100 ;g/ml), or GD3G7 (2 ;g/ml) in 150 ;l of DMEM were individually added. The spreading of the cells was continuously monitored up to 60 min using the RT-CES™ system. In other instances, ∼24 h after seeding, when the cells were in a log growth phase, serially diluted GD3G7 (2, 0.5, and 0.2 ;g/ml) in 150 ;l of DMEM was added into the corresponding wells. The proliferation was monitored for a period of 64 h and expressed as a cell index as per the manufacturer's instructions. The cell index is a quantitative measure of the spreading and/or proliferative status of the cells in an electrode-containing well. Cell Migration and Invasion Assays in Vitro—The ability of LM66-H11 and P29 cells to migrate and invade was assessed using the BD BioCoat™ chamber with or without Matrigel (BD Biosciences) in vitro. In some instances, single cell suspensions of LM66-H11 (5 × 104 cells/ml) were prepared by detaching and resuspending in serum-free DMEM. Before being added to the upper chamber (8 ;m PET pores), LM66-H11cells were preincubated with CS-A (100 ;g/ml), CS-E (100 ;g/ml), or GD3G7 (2 ;g/ml) in 500 ;l DMEM for 30 min at 37 °C in a CO2 incubator. The lower chambers were filled with DMEM containing 10% fetal bovine serum. After incubation for 24 h, the cells that had migrated and invaded through the membrane alone or the Matrigel-coated membrane remained bound to the underside of the membranes. These cells were stained with the Diff-Quik staining kit and counted in five random microscopic fields/filter. Biodistribution of Intravenously Injected Radiolabeled CS-E—3H-Labeled CS-E (4 × 105 cpm) was prepared as described previously (27Deepa S.S. Umehara Y. Higashiyama S. Itoh N. Sugahara K. J. Biol. Chem. 2002; 277: 43707-43716Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) and injected into C3H/HeN mice through a lateral tail vein. At 1, 1.5, and 2.5 h post-injection, the blood samples were collected from anesthetized animals via cardiac puncture. The animals were then sacrificed, and the organs including liver, lungs, kidneys, spleen, and brain were dissected out, quickly rinsed, and weighed. The blood and each tissue from all the animals were pooled and homogenized with 1% Triton X-100/PBS in a Polytron homogenizer while being cooled on ice. Aliquots of the tissue homogenates and the plasma were freeze-thawed (five times) using liquid nitrogen bath and treated with 5% trichloroacetic acid. The resultant supernatants were recovered by centrifugation at 15,000 rpm for 10 min, and the radioactivity was measured using a multi-purpose liquid scintillation counter (Beckman coulter LS6500). The counts were converted to disintegrations on the basis of a standard quench correction curve, and the distribution of 3H-CS-E in the plasma and tissues was expressed as dpm/ml and dpm/g, respectively. Relative Quantification of Gene Expression of Sulfotransferases and Epimerase—Total RNA was extracted from P29 and LM66-H11 cells in 100-mm culture plates using a QuickPrep total RNA extraction kit according to the manufacturer's instructions, and then each extract was treated with RNase-free DNase for 30 min at 37 °C. The cDNA was synthesized from ∼1 ;g of the total RNA using Moloney murine leukemia virus reverse transcriptase and an oligo(dT)16 primer (Hokkaido System Science, Sapporo, Japan) and then purified with a MinElute® PCR purification kit (Qiagen). Quantitative real time reverse transcription-PCR was performed using a Platinum® SYBR® Green qPCR Supermix-UDG kit in the iCyclear iQ™ (Bio-Rad). Briefly, the reaction mixture (25 ;l) contained a SYBR Green Supermix, each primer set at 0.2 ;m (as shown in Table 2), and each template cDNA. PCR was carried out for 40 cycles at 95 °C for 15 s and 60 °C for 1 min, and the amplified products were measured by the iCycler iQ real time PCR analyzing system (Bio-Rad). The expression level of each sulfotransferase and epimerase mRNA was normalized to that of the glyceraldehyde-3-phosphate dehydrogenase transcript.TABLE 2Primers utilized for quantitative reverse transcription-PCRGeneSizeSequencebpGalNAc4S-6ST153Forward5′-TATGACAACAGCACAGACGG-3′Reverse5′-TGCAGATTTATTGGAACTTGCGAA-3′UST151Forward5′-TGACCATGGACCACCTCCTA-3′Reverse5′-GTGAATGTCTGATGTGACCAAA-3′C4ST-1141Forward5′-ACCTCGTGGGCAAGTATGAG-3′Reverse5′-TCTGGAAGAACTCCGTGGTC-3′C4ST-2102Forward5′-GCACAAGGCTGAAGTGAAGG-3′Reverse5′-CATGAGAGCCGACCCTAGTA-3′D4ST-1175Forward5′-GCGTCCTGAACAACGTG-3′Reverse5′-TCTCCAAACTTGTTACGGTAAGC-3′C5E139Forward5′-AGCGCTGGTGCACGCTACAC-3′Reverse5′-GCAGGTAGTGCACTTCCATAAG-3′G3PDH205Forward5′-CATCTGAGGGCCCACTG-3′Reverse5′-GAGGCCATGTAGGCCATGA-3′ Open table in a new tab Comparison of Amount and Composition of CS/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.

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