Susceptibility to proteases of anti-Tn-antigen MLS128 binding glycoproteins expressed in human colon cancer cells

Oura, Fumie; Yajima, Yukiko; Nakata, Munehiro; Taniue, Kenzui; Akiyama, Tetsu; Yamamoto, Kazuo; Fujita‐Yamaguchi, Yoko

doi:10.5582/bst.2014.01127

Cited by 3 publications

(6 citation statements)

References 14 publications

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“…Western blotting of LS180 and HT29 colon cancer cells with MLS128 antibody clearly showed the detection of a 110 kDa protein (12,14). Purification of the 110 kDa GP from these cells, however, has been unsuccessful although our laboratory has extensive experience in affinity chromatography (17).…”

Section: Discussionmentioning

confidence: 98%

“…C e l l l i n e s u s e d i n t h i s s t u d y, h u m a n c o l o n adenocarcinoma LS180 cells, human breast carcinoma MCF-7 cells, and human normal mammary epithelial MCF-10A cells, were obtained from American Tissue Type Culture Collection. LS180 colon cancer cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 mg/mL D-glucose, and 110 μg/mL pyruvic acid as described (12). MCF-7 breast cancer and MCF-10A cells were cultured in DMEM containing 10% FBS.…”

Section: Cell Lines and Culturementioning

confidence: 99%

“…LS180 cells were cultured in 75 cm flasks as previously described (12). Whole cell extracts were prepared by scraping followed by centrifugation at 200 × g for 5 min and solubilized on ice for 15 min in solubilization buffer (50 mM Tris-HCl buffer, pH 7.4, containing 1% NP40, 2 mM EDTA, 100 mM NaCl, 10 mM sodium orthovanadate, 1 mM PMSF, and protease inhibitors).…”

Section: Preparation Of Whole Cell Lysates and Membrane And Cytosolicmentioning

confidence: 99%

“…Previous studies in our laboratory revealed that MLS128 binds a membrane glycoprotein (GP) with a molecular weight of 110 kDa. This 110 kDa GP contains N-glycans in addition to O-glycans consisting of 2 or 3 consecutive Tn-antigens which are recognized by MLS128 (12). Tn-antigens are found in over 67% of colon cancer cell lines tested, but not in MCF-7 breast cancer cell lines (12,13).…”

mentioning

confidence: 99%

“…This 110 kDa GP contains N-glycans in addition to O-glycans consisting of 2 or 3 consecutive Tn-antigens which are recognized by MLS128 (12). Tn-antigens are found in over 67% of colon cancer cell lines tested, but not in MCF-7 breast cancer cell lines (12,13). MLS128 antibody treatment inhibited growth of LS180 and HT29 colon cancer cells (13,14).…”

mentioning

confidence: 99%

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MLS128 antibody-induced suppression of colon cancer cell growth is mediated by a desmocollin and a 110 kDa glycoprotein

Shuck¹,

Hong

Kalkum

et al. 2019

BST

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Section: Discussionmentioning

confidence: 98%

Section: Cell Lines and Culturementioning

confidence: 99%

Section: Preparation Of Whole Cell Lysates and Membrane And Cytosolicmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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MLS128 antibody-induced suppression of colon cancer cell growth is mediated by a desmocollin and a 110 kDa glycoprotein

Shuck¹,

Hong

Kalkum

et al. 2019

BST

Self Cite

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Production of a mouse monoclonal IgM antibody that targets the carbohydrate Thomsen-nouveau cancer antigen resulting in in vivo and in vitro tumor killing

Trabbic

Kleski

Shi

et al. 2018

Cancer Immunol Immunother

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The construction of a tumor-associated carbohydrate antigen-zwitterionic polysaccharide conjugate, Thomsen-nouveau-polysaccharide A1 (Tn-PS A1, where Tn = D-GalpNAc), has led to the development of a carbohydrate binding monoclonal antibody named Kt-IgM-8. Kt-IgM-8 was produced via hybridoma from Tn-PS A1 hyperimmunized Jackson Laboratory C57BL/6 mice, splenocytes and the murine myeloma cell line Sp2/0Ag14 with subsequent cloning on methyl cellulose semi-solid media. This in-house generated monoclonal antibody negates binding influenced from peptides, proteins, and lipids and preferentially binds monovalent Tn antigen as noted by ELISA, FACS, and glycan array technologies. Kt-IgM-8 demonstrated in vitro and in vivo tumor killing against the Michigan Cancer Foundation breast cell line 7 (MCF-7). In vitro tumor killing was observed using an LDH assay that measured antibody-induced complement-dependent cytotoxicity and these results were validated in an in vivo passive immunotherapy approach using an MCF-7 cell line-derived xenograft model. Kt-IgM-8 is effective in killing tumor cells at 30% cytotoxicity, and furthermore, it demonstrated approximately 40% reduction in tumor growth in the MCF-7 model.

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Glycoprotein screening in colorectal cancer based on differentially expressed Tn antigen

et al. 2016

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Colorectal cancer (CRC) is one of the most common cancers worldwide, and the identification of new biomarkers for CRC is valuable for its diagnosis and treatment. We aimed to screen differentially expressed glycoproteins (especially O-glycoproteins) and to identify diagnostic or therapeutic candidates for colorectal cancer (CRC) based on different Tn antigen expression levels. Fresh cancer tissues and adjacent healthy tissues were obtained from CRC patients and classified into three groups based on their Tn antigen expression: CRC with negative Tn expression (CRC Tn‑), CRC with positive Tn expression (CRC Tn+) and normal control without Tn expression (NC). Protein extractions were separated and identified by iTRAQ technology. Glycoproteins and O-glycoproteins were selected using UniProt and DAVID. Deep bioinformatic analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KO), was used to annotate this O-glycoprotein interaction network. Subsequently, two O‑glycoproteins were verified by western blotting and immunohistochemistry in either LS174T cells or CRC tissues. We found that 330 differentially expressed proteins were identified by iTRAQ between CRC Tn‑ and NC tissues, 317 between CRC Tn+ and NC tissues, and 316 between CRC Tn‑ and Tn+ tissues. Of the 316 proteins, 55 glycoproteins and 19 O‑glycoproteins were identified and analyzed via deep informatics. Namely, different Tn antigen expression levels in CRC led to differential protein expression patterns, especially for glycoproteins and O‑glycoproteins. Decorin and SORBS1, two representative functional O-glycoproteins, were significantly downregulated in the CRC Tn+ tissues compared with the level in the CRC Tn‑ or NC tissues. Based on this deep bioinformatic analysis, Decorin and SORBS1 are hypothesized to be involved in the TGF‑β and PPAR‑γ signaling pathways, respectively.

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Susceptibility to proteases of anti-Tn-antigen MLS128 binding glycoproteins expressed in human colon cancer cells

Cited by 3 publications

References 14 publications

MLS128 antibody-induced suppression of colon cancer cell growth is mediated by a desmocollin and a 110 kDa glycoprotein

MLS128 antibody-induced suppression of colon cancer cell growth is mediated by a desmocollin and a 110 kDa glycoprotein

Production of a mouse monoclonal IgM antibody that targets the carbohydrate Thomsen-nouveau cancer antigen resulting in in vivo and in vitro tumor killing

Glycoprotein screening in colorectal cancer based on differentially expressed Tn antigen

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