ORIGINAL ARTICLE Inhibition of N-linked glycosylation by tunicamycin induces E-cadherin-mediated cell–cell adhesion and inhibits cell proliferation in undifferentiated human colon cancer cells Julio Cesar Madureira de Freitas Junior • Ba ´rbara Du Rocher D’Aguiar Silva • Waldemir Fernandes de Souza • Wallace Martins de Arau ´jo • Eliana Saul Furquim Werneck Abdelhay • Jose ´ Andre ´s Morgado-Dı ´az Received: 6 July 2010 / Accepted: 21 September 2010 / Published online: 7 October 2010 Ó Springer-Verlag 2010 Abstract Purpose Aberrant protein glycosylation and disassembly of E-cadherin-mediated cell–cell adhesion are characteris- tics 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. Com- plex-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 a- mannosidase II, and tunicamycin, a drug that inhibits the biosynthesis of N-glycans, via western blot, immunofluo- rescence, 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 adhe- sion in undifferentiated HCT-116 cells, as shown by the increased association of E-cadherin with the actin cyto- skeleton. 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. Keywords Tunicamycin Á Swainsonine Á Colorectal cancer Á Adherens junctions Á E-cadherin Á N-glycosylation Introduction Colorectal cancer (CRC) is among the most common human neoplasms. It is the third most frequent cancer worldwide, with an estimated one million new cases and half a million deaths each year [1]. In Brazil, it is also the third most common cancer, according to recent data from the Instituto Nacional de Caˆncer [2]. The progression of CRC is char- acterized by a series of clinical and histopathological stages [3], which range from single-crypt lesions, through small benign tumors (adenomatous polyps), to malignant cancers (carcinomas); in this final stage, the disruption of adherens junctions (AJs) is frequently observed [4]. AJs and tight junctions (TJs) constitute the apical junctional complex, which is responsible for maintaining the epithelial archi- tecture [5]. AJs are dynamic multiprotein complexes in which E-cadherin, a transmembrane glycoprotein and the main mediator of cell–cell adhesion in the epithelium, is J. C. M. de Freitas Junior Á W. F. de Souza Á W. M. de Arau ´jo Á J. A. Morgado-Dı ´az (&) Divisa ˜o de Biologia Celular, Coordenac ¸a ˜o de Pesquisa, Instituto Nacional de Ca ˆncer, 37 Andre ´ Cavalcanti street, 5th floor, Rio de Janeiro, RJ CEP 20230-051, Brazil e-mail: jmorgado@inca.gov.br B. D. R. D’AguiarSilva Á E. S. F. W. Abdelhay Centro de Transplante de Medula O ´ ssea, Instituto Nacional de Ca ˆncer, 23 Cruz Vermelha square, 6th floor, Rio de Janeiro, RJ CEP 20230-130, Brazil 123 Cancer Chemother Pharmacol (2011) 68:227–238 DOI 10.1007/s00280-010-1477-8