title: Identification of sialylated glycoproteins in Doxorubicin-treated hepatoma cells with glycoproteomic analyses.
abstract: Sialylation is one of the most important types of glycosylation involved in carcinogenesis and establishment of cancer stemness. We previously showed that increased sialylation is a characteristic glycan change in cancer stem cells (CSCs) from hepatocellular carcinoma. However, the identities of glycoproteins targeted for sialylation remain unknown. In the present study, we identified glycoproteins targeted for sialylation in doxorubicin (DXR)-treated hepatocarcinoma cell line, Huh7, using glycoproteomic analyses. Since CSCs constitute a small subset of cells within carcinoma cell lines, it is difficult to identify sialylated proteins using general glycoproteomic strategies. It is known that treatment with anticancer drug can condense CSCs, we used DXR to concentrate CSCs. In DXR-treated Huh7 cells, isobaric tag for relative and absolute quantitation (iTRAQ) analysis identified 17 sialylated glycoproteins. Most of the identified glycoproteins were cancer-associated proteins. Furthermore, two proteins of approximately 70 kDa were detected using Sambucus sieboldoana agglutinin (SSA) blot analysis and identified as beta-galactosidase and alpha-2-HS-glycoprotein (fetuin-A) by SSA precipitation followed by liquid chromatography-tandem mass spectrometry analyses. Sialylation levels of fetuin-A were increased in DXR-treated Huh7 cell lysates. These changes in sialylation of glycoproteins might be involved in the establishment of cancer stemness.
authors.affiliation: Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine , Osaka 565-0871, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Azuma
authors.forename: Satoshi
authors.initials: S
authors.lastname: Serada
authors.forename: Shinji
authors.initials: S
authors.lastname: Takamatsu
authors.forename: Naoko
authors.initials: N
authors.lastname: Terao
authors.forename: Shunsaku
authors.initials: S
authors.lastname: Takeishi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kamada
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Naka
authors.forename: Eiji
authors.initials: E
authors.lastname: Miyoshi
mesh_terms: D000373:Agglutinins; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004317:Doxorubicin / Q000494:pharmacology; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D037102:Lectins; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012794:Sialic Acids / Q000378:metabolism; D018709:Statistics, Nonparametric; D053719:Tandem Mass Spectrometry
chemical_list: D000373:Agglutinins; D006023:Glycoproteins; D037102:Lectins; D012794:Sialic Acids; D004317:Doxorubicin
keywords: cancer stem cells; doxorubicin; glycoproteomics; hepatoma; sialylation