Inhibition of N-linked glycosylation by tunicamycin induces E-cadherin-mediated cell-cell adhesion and inhibits cell proliferation in undifferentiated human colon cancer cells.

Purpose: Aberrant protein glycosylation and disassembly of E-cadherin-mediated cell-cell adhesion are characteristics of epithelial cancer. However, the relationship between these two events in colorectal cancer remains to be defined. In this study, we analyzed whether N-glycan expression is crucial for the loss of E-cadherin-mediated cell-cell adhesion in human colorectal cancer cells.

Methods: Differentiated Caco-2 and undifferentiated HCT-116 colon cancer cells were used as models of stable and unstable adherens junctions (AJs), respectively. Complex-type N-glycans were detected using the lectins E-PHA (Phaseolus vulgaris E.) and L-PHA (Phaseolus vulgaris L.). To study E-cadherin-mediated AJ assembly, we examined the effects of swainsonine, an inhibitor of α-mannosidase II, and tunicamycin, a drug that inhibits the biosynthesis of N-glycans, via western blot, immunofluorescence, differential extraction in Triton X-100, and electron microscopy. Cell proliferation and apoptosis were examined by crystal violet staining and flow cytometry, respectively.

Results: We observed positive labeling for E-PHA and L-PHA lectins in both cell lines; however, HCT-116 cells had increased E-cadherin-linked complex-type N-glycans. Interestingly, tunicamycin, but not swainsonine, was able to induce functional E-cadherin-mediated cell-cell adhesion in undifferentiated HCT-116 cells, as shown by the increased association of E-cadherin with the actin cytoskeleton. Moreover, in HCT-116 cells, tunicamycin also induced the formation of tight cell-cell contacts, and it inhibited cell proliferation without triggering apoptosis.

Conclusions: Collectively, our results demonstrate for the first time that altered N-glycan expression plays an important role in the loss of AJ stability in undifferentiated colorectal cancer cells and that this loss may be associated with the progression of colorectal cancer.