Identification of Sialylated Glycoproteins in Doxorubicin-Treated Hepatoma Cells with Glycoproteomic Analyses

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.