5508 MOLECULAR MEDICINE REPORTS 17: 5508-5517, 2018 Interaction of colon cancer cells with glycoconjugates triggers complex changes in gene expression, glucose transporters and cell invasion ROMANA KŘIVOHLAVÁ, VALIKA GROBÁROVÁ, EVA NEUHÖFEROVÁ, ANNA FIŠEROVÁ and VERONIKA BENSON Laboratory of Molecular Biology and Immunology, Department of Immunology, Institute of Microbiology, Czech Academy of Sciences, 14220 Prague 4, Czech Republic Received September 21, 2017; Accepted December 15, 2017 DOI: 10.3892/mmr.2018.8490 Abstract. Glycan metabolism balance is critical for cell pros- mRNA expression in a comprehensive way, resulting in the perity, and macromolecule glycosylation is essential for cell reduced malignant phenotype of the colon cancer cell line communication, signaling and survival. Thus, glycotherapy HT-29. may be a potential cancer treatment. The aim of the present study was to determine whether combined synthetic glycocon- Introduction jugates (GCs) induce changes in gene expression that alter the survival of colon cancer cells. The current study evaluated the Cell surface glycans are molecules that regulate interac- effect of the GCs N‑acetyl‑D‑glucosamine modified polyami- tions among neighboring cells or contact between cells and doamine dendrimer and calix[4]arene scaffold on cancer cell the extracellular matrix during cell adhesion, recognition proliferation, apoptosis, invasion and sensitivity to immune and communication (1). An aberrant glycosylation pattern cell-mediated killing. Using reverse transcription-quantitative promotes the development and progression of certain patholo- polymerase chain reaction, the expression of genes involved in gies, including cancer (1). Glycan alterations may result in the aforementioned processes was measured. It was determined tumor development and progression, as well as tumor-cell that GCs reduce the expression of the glucosaminyltransfer- dissociation and invasion, which subsequently promote metas- ases Mgat3 and Mgat5 responsible for surface glycosylation tasis and tumor-associated neoangiogenesis (2). Importantly, and employed components of the Wnt signaling pathway aberrant glycosylation of the tumor cell surface results in the Wnt2B and Wnt9B. In addition, the calix[4]arene-based GC impairment of its recognition by cells of the immune system, reduced cell colony formation; this was accompanied by the including natural killer (NK) cells (2,3). downregulation of the metalloproteinase Mmp3. By contrast, Alterations in glycosylation primarily arise from changes in the the dendrimer-based GC affected the expression of the glucose expression of N-acetyl-D-glucosamine (GlcNAc) transferases in transporter components Sglt1 and Egfr1. Therefore, to the best the Golgi system (4). The family of β-1,4-mannosyl-glycoprotein of our knowledge, the present study is the first to reveal that 4-β-N-acetyl glucosaminyltransferases consists of several N-acetyl-D-glucosamine-dendrimer/calix[4]arene GCs alter members, including Mgat3 and Mgat5, which are involved in linking terminal residues to glycans on the cell surface. The competition between Mgat3 and Mgat5 results in the branching or bisecting of surface glycans and this final pattern influences Correspondence to: Dr Veronika Benson, Laboratory of intercellular recognition (5). Mgat5 is responsible for adding Molecular Biology and Immunology, Department of Immunology, β1-6 GlcNAc residues and forming branched structures, which Institute of Microbiology, Czech Academy of Sciences, are particularly abundant in cancer tissues with high metastatic Vídeňská 1083, 14220 Prague 4, Czech Republic potential (4). Mgat3 facilitates the addition of β1-4 GlcNAc E-mail: [email protected] residues and constructs a bisecting structure that inhibits further addition of GlcNAc by other glucosaminyltransferases, including Abbreviations: GC, glycoconjugate; GlcNAc, N-acetylglucosamine; Mgat5 (6). Mgat3 and Mgat5 enzymes tend to be overexpressed GN4C, N-acetyl-D-glucosamine-coated calix[4]arene; GN8P, in tumor cells (2,7). Song et al (8) reported that bisecting N-acetyl-D-glucosamine-coated polyamidoamine dendrimer; PAMAM, polyamidoamine; PBMC, peripheral blood mononuclear GlcNAc on N-glycans inhibits the signaling of growth factors cells; RTCA, Real-Time Cell Analyzer and attenuates the progression of mammary tumors. Therefore, the resulting effect of Mgat3/Mgat5 activity is likely to be tumor Key words: calix[4]arene, polyamidoamine dendrimer, glycosylation, specific. The expression of Mgat5 is regulated via the NM23 N-acetyl-glucosaminyl-transferase, WNT, colon cancer cell regulator, which is encoded by Nme1 gene (9) and the expres- sion of glucosaminyltransferases, including Mgat3 and Mgat5, is associated with their surface glycosylation activity (10). KŘIVOHLAVÁ et al: GLYCOCONJUGATES TRIGGER COMPLEX CHANGES IN COLON CANCER CELLS 5509 In vitro, cell glycosylation may be studied using synthetic VA, USA) and was authenticated following the guidelines glycoconjugates (GCs) that modulate cell surface glycosyl- of the International Cell Line Authentication Committee ation. GCs are able to interfere with cancer cell processes and (https://www.lgcstandards-atcc.org). Cells were maintained the cancer microenvironment; therefore they may be able to in 37˚C and in an RPMI‑1640 medium with 10% fetal calf modulate the malignant phenotype of cancer cells (11). serum (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The present study focused on GC-triggered alterations Peripheral blood mononuclear cells (PBMC) were obtained in the mRNA expression of surface glycosylation regulators, from peripheral blood of healthy donors (4 males, mean as well as key components of signaling pathways responsible age 46.3±9.4; samples collected August 2012) using a for cell-extracellular matrix adhesion, neovascularization Ficoll‑Paque (GE Healthcare Life Sciences, Little Chalfont, and invasion, each of which contribute to the metastatic UK) at density 1,077 g/ml and centrifugation for 40 min at potential of cancer cells. The GCs assessed included GlcNAc room temperature and 400 x g. Blood samples were taken moieties linked with a calix[4]arene or with a polyamido- from material remaining following a routine donor check-up amine dendrimer (PAMAM) core. Calix[4]arenes possessing at the transfusion unit of the Thomayer Hospital (Prague, distinctive geometry act as carriers of anticancer drugs (12) or Czech Republic). All donors signed informed consent for the as direct anticancer agents via enzyme inhibition (13), angio- use of their blood for experimental purposes. The current genesis inhibition (14) or innate immunity modulation (15). study was approved by the Ethical Committee of Thomayer PAMAM dendrimers have previously been used to deliver Hospital. The chemically defined GC calix[4]arene containing genes and drugs (16). of four terminal N-acetyl-D-glucosamine moieties (GN4C) Cells internalize exogenous glucosamine via the glucose was synthesized and kindly provided by Vladimir Křen and transporter and process it, as well as cellular glucosamine, Karel Křenek (Czech Academy of Sciences, Prague, Czech via lysosomal degradation into uridine diphosphate glucose Republic). A GC consisting of PAMAM with eight terminal (UDP)-GlcNAc (17,18). This UDP-GlcNAc is further used in N-acetyl-D-glucosamine moieties (GN8P) was synthe- the post‑transcriptional modification of glycosylate substrates, sized and kindly provided by Thisbe Lindhorst (Christiana such as proteins. Glycosylation of nuclear proteins, including Albertina University, Kiel, Germany). The synthesis, purity, histone H2B, contributes to transcriptional regulation (19). nuclear magnetic resonance data and dose-dependent effect The genes encoding the sodium glucose cotransporter 1 of the GCs on human PBMC cells have been previously (Sglt1) and epidermal growth factor receptor (Egfr1) are described (2,24-26). The optimal concentration of 10 nM components of a glucose co-transporter. Sglt1 is expressed GN4C or GN8P were used for the experiments. Fludara® primarily in the human intestine and kidney (20). An example (1 mM, FLU; Genzyme, Cambridge, MA, USA), which is a of a Egfr1 ligand is N-acetylglucosamine (21). Glycosylation conventional anticancer drug, was used to compare the anti- of the epidermal growth factor receptor improves its ability to cancer effect of the tested GCs with a known anticancer agent. bind to epidermal growth factor (22). The coupling of Egfr1 to Sglt1 further stabilizes the whole complex and enables cancer Glyco‑gene profiling array. The HT-29 cell line was cells to take up high levels of glucose (23). incubated with GN8P for 24 h and total RNA was isolated The authors of the present study previously reported using an RNAeasy Mini kit involving DNAse I treatment that a GC consisting of four GlcNAc residues on a following the manufacturer's protocol (DNAse I was a N-acetyl-D-glucosamine-coated calix[4]arene core (GN4C) component of RNAeasy Mini kit; Qiagen GmbH, Hilden, alters the glycosylation of human NK cells and promotes the Germany). A Glyco-gene Chip array (GLYCOv3 Gene cell-mediated cytotoxicity of human NK cells against K562 Chip; Affymetrix; Thermo Fisher Scientific, Inc., altham,W and HT-29 target cells via the phosphoinositide 3-kinase MA, USA) containing probe sets of 2,000 human tran- pathway (2). In addition, it has been demonstrated that a scripts was provided by the Consortium for Functional GC consisting of eight GlcNAc residues on a PAMAM core Glycomics (http://www.functionalglycomics.org/static/consor-