N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma

Abstract

Background: Correlations of disease phenotypes with glycosylation changes have been analysed intensively in the tumor biology field. Glycoforms potentially associated with carcinogenesis, tumor progression and cancer metastasis have been identified. In cancer therapy, drug resistance is a severe problem, reducing therapeutic effect of drugs and adding to patient suffering. Although multiple mechanisms likely underlie resistance of cancer cells to anticancer drugs, including overexpression of transporters, the relationship of glycans to drug resistance is not well understood.

Results: We established epirubicin (EPI)--and mitoxantrone (MIT)--resistant cell lines (HLE-EPI and HLE-MIT) from the human hepatocellular carcinoma cell line (HLE). HLE-EPI and HLE-MIT overexpressed transporters MDR1/ABCB1 and BCRP/ABCG2, respectively. Here we compared the glycomics of HLE-EPI and HLE-MIT cells with the parental HLE line. Core fucosylated triantennary oligosaccharides were increased in the two resistant lines. We investigated mRNA levels of glycosyltransferases synthesizing this oligosaccharide, namely, N-acetylglucosaminyltransferase (GnT)-IVa, GnT-IVb and alpha1,6-fucosyltransferase (alpha1,6-FucT), and found that alpha1,6-FucT was particularly overexpressed in HLE-MIT cells. In HLE-EPI cells, GnT-IVa expression was decreased, while GnT-IVb was increased. Both GnT-IVs were downregulated in HLE-MIT cells. HLE-MIT cells also showed decreases in fucosylated tetraantennary oligosaccharide, the product of GnT-V. GnT-V expression was decreased in both lines, but particularly so in HLE-MIT cells. Thus both N-glycan and glycosyltransferase expression was altered as cells acquired tolerance, suggesting novel mechanisms of drug resistance.

Conclusion: N-glycan and glycosyltransferase expression in HLE-EPI and HLE-MIT were analysed and presented that glycans altered according with acquired tolerance. These results suggested novel mechanisms of drug resistance.

Figure 1

Chromatograms of PA-N-glycans identified on ODS columns. Letters and asterisks over peaks correspond with Table 1.

Figure 2

Alterations of complex type oligosaccharide ratios in cell lines. Ratios were calculated based on amide column isolation following separation using ODS columns.

Figure 3

Biosynthesis pathway of complex type N-glycans. The reactions of α1,6-FucT, GnT-IV and GnT-V and complex type N-glycan structures in this study are highlighted. GnT-IV and GnT-V increase number of antenna, and some of them are modified by α1,6-FucT. Then, they are galactosylated by β1,4- galactosyltransferase as shown as black arrows. The products of GnT-V were modified by GnT-IV in this study, so we have indicated that GnT-V works after GnT-IV in the biosynthetic pathway. However, it is possible that GnT-V may act at an earlier step than GnT-IV.

Figure 4

Glycosyltransferase activities estimated by ratios of N-glycans. Alpha1,6-FucT, (210.4+310.8+410.16)/(200.4+300.8+400.16); GnT-IV, (300.8+310.8+400.16+410.16)/(200.4+210.4); and GnT-V, (400.16+410.16)/(300.8+310.8).

Figure 5

RT-PCR analysis of glycosyltransferase mRNA expression in each cell line. Detailed experimental procedures are in Materials and Methods. Human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) is reported for internal controls.