Interaction of colon cancer cells with glycoconjugates triggers complex changes in gene expression, glucose transporters and cell invasion

  • Authors:
    • Romana Křivohlavá
    • Valika Grobárová
    • Eva Neuhöferová
    • Anna Fišerová
    • Veronika Benson
  • View Affiliations

  • Published online on: January 25, 2018
  • Pages: 5508-5517
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Glycan metabolism balance is critical for cell prosperity, and macromolecule glycosylation is essential for cell communication, signaling and survival. Thus, glycotherapy may be a potential cancer treatment. The aim of the present study was to determine whether combined synthetic glycoconjugates (GCs) induce changes in gene expression that alter the survival of colon cancer cells. The current study evaluated the effect of the GCs N‑acetyl‑D‑glucosamine modified polyamidoamine dendrimer and calix[4]arene scaffold on cancer cell proliferation, apoptosis, invasion and sensitivity to immune cell‑mediated killing. Using reverse transcription‑quantitative polymerase chain reaction, the expression of genes involved in the aforementioned processes was measured. It was determined that GCs reduce the expression of the glucosaminyltransferases Mgat3 and Mgat5 responsible for surface glycosylation and employed components of the Wnt signaling pathway Wnt2B and Wnt9B. In addition, the calix[4]arene‑based GC reduced cell colony formation; this was accompanied by the downregulation of the metalloproteinase Mmp3. By contrast, the dendrimer‑based GC affected the expression of the glucose transporter components Sglt1 and Egfr1. Therefore, to the best of our knowledge, the present study is the first to reveal that N‑acetyl‑D‑glucosamine‑dendrimer/calix[4]arene GCs alter mRNA expression in a comprehensive way, resulting in the reduced malignant phenotype of the colon cancer cell line HT‑29.


Cell surface glycans are molecules that regulate interactions among neighboring cells or contact between cells and the extracellular matrix during cell adhesion, recognition and communication (1). An aberrant glycosylation pattern promotes the development and progression of certain pathologies, including cancer (1). Glycan alterations may result in tumor development and progression, as well as tumor-cell dissociation and invasion, which subsequently promote metastasis and tumor-associated neoangiogenesis (2). Importantly, aberrant glycosylation of the tumor cell surface results in the impairment of its recognition by cells of the immune system, including natural killer (NK) cells (2,3).

Alterations in glycosylation primarily arise from changes in the expression of N-acetyl-D-glucosamine (GlcNAc) transferases in the Golgi system (4). The family of β-1,4-mannosyl-glycoprotein 4-β-N-acetyl glucosaminyltransferases consists of several 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 intercellular recognition (5). Mgat5 is responsible for adding β1-6 GlcNAc residues and forming branched structures, which are particularly abundant in cancer tissues with high metastatic potential (4). Mgat3 facilitates the addition of β1-4 GlcNAc residues and constructs a bisecting structure that inhibits further addition of GlcNAc by other glucosaminyltransferases, including Mgat5 (6). Mgat3 and Mgat5 enzymes tend to be overexpressed in tumor cells (2,7). Song et al (8) reported that bisecting GlcNAc on N-glycans inhibits the signaling of growth factors and attenuates the progression of mammary tumors. Therefore, the resulting effect of Mgat3/Mgat5 activity is likely to be tumor specific. The expression of Mgat5 is regulated via the NM23 regulator, which is encoded by Nme1 gene (9) and the expression of glucosaminyltransferases, including Mgat3 and Mgat5, is associated with their surface glycosylation activity (10).

In vitro, cell glycosylation may be studied using synthetic glycoconjugates (GCs) that modulate cell surface glycosylation. GCs are able to interfere with cancer cell processes and the cancer microenvironment; therefore they may be able to modulate the malignant phenotype of cancer cells (11).

The present study focused on GC-triggered alterations in the mRNA expression of surface glycosylation regulators, as well as key components of signaling pathways responsible for cell-extracellular matrix adhesion, neovascularization and invasion, each of which contribute to the metastatic potential of cancer cells. The GCs assessed included GlcNAc moieties linked with a calix[4]arene or with a polyamidoamine dendrimer (PAMAM) core. Calix[4]arenes possessing distinctive geometry act as carriers of anticancer drugs (12) or as direct anticancer agents via enzyme inhibition (13), angiogenesis inhibition (14) or innate immunity modulation (15). PAMAM dendrimers have previously been used to deliver genes and drugs (16).

Cells internalize exogenous glucosamine via the glucose transporter and process it, as well as cellular glucosamine, via lysosomal degradation into uridine diphosphate glucose (UDP)-GlcNAc (17,18). This UDP-GlcNAc is further used in the post-transcriptional modification of glycosylate substrates, such as proteins. Glycosylation of nuclear proteins, including histone H2B, contributes to transcriptional regulation (19). The genes encoding the sodium glucose cotransporter 1 (Sglt1) and epidermal growth factor receptor (Egfr1) are components of a glucose co-transporter. Sglt1 is expressed primarily in the human intestine and kidney (20). An example of a Egfr1 ligand is N-acetylglucosamine (21). Glycosylation of the epidermal growth factor receptor improves its ability to bind to epidermal growth factor (22). The coupling of Egfr1 to Sglt1 further stabilizes the whole complex and enables cancer cells to take up high levels of glucose (23).

The authors of the present study previously reported that a GC consisting of four GlcNAc residues on a N-acetyl-D-glucosamine-coated calix[4]arene core (GN4C) alters the glycosylation of human NK cells and promotes the cell-mediated cytotoxicity of human NK cells against K562 and HT-29 target cells via the phosphoinositide 3-kinase pathway (2). In addition, it has been demonstrated that a GC consisting of eight GlcNAc residues on a PAMAM core N-acetyl-D-glucosamine-coated polyamidoamine dendrimer (GN8P) mediates alterations in cytokine profile specific to mouse NK T-cells and macrophages (24). Therefore, the present study aimed to determine whether these GCs directly affect cancer cells by modulating the regulation of gene expression and causing changes in the phenotypes of malignant cells. The effect of synthetic GCs on the mRNA expression of the cell glucosaminyltransferases Mgat3 and Mgat5, members of the Wnt signaling family (Wnt2B and Wnt9B), regulators of glucose metabolism (Sglt1 and Egfr1) and regulators of cell adhesion and invasion matrix metalloproteinase 3 (Mmp3) or transforming growth factor-β1 (Tgfb1) were measured.

Materials and methods

Preparation and treatment of cells with GCs

The HT-29 adenocarcinoma cell line (ATCC number: HTB-38) was obtained from American Type Culture Collection (Manassas, VA, USA) and was authenticated following the guidelines of the International Cell Line Authentication Committee ( Cells were maintained in 37°C and in an RPMI-1640 medium with 10% fetal calf serum (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Peripheral blood mononuclear cells (PBMC) were obtained from peripheral blood of healthy donors (4 males, mean age 46.3±9.4; samples collected August 2012) using a Ficoll-Paque (GE Healthcare Life Sciences, Little Chalfont, UK) at density 1,077 g/ml and centrifugation for 40 min at room temperature and 400 × g. Blood samples were taken from material remaining following a routine donor check-up at the transfusion unit of the Thomayer Hospital (Prague, Czech Republic). All donors signed informed consent for the use of their blood for experimental purposes. The current study was approved by the Ethical Committee of Thomayer Hospital. The chemically defined GC calix[4]arene containing of four terminal N-acetyl-D-glucosamine moieties (GN4C) was synthesized and kindly provided by Vladimir Křen and Karel Křenek (Czech Academy of Sciences, Prague, Czech Republic). A GC consisting of PAMAM with eight terminal N-acetyl-D-glucosamine moieties (GN8P) was synthesized and kindly provided by Thisbe Lindhorst (Christiana Albertina University, Kiel, Germany). The synthesis, purity, nuclear magnetic resonance data and dose-dependent effect of the GCs on human PBMC cells have been previously described (2,2426). The optimal concentration of 10 nM GN4C or GN8P were used for the experiments. Fludara® (1 mM, FLU; Genzyme, Cambridge, MA, USA), which is a conventional anticancer drug, was used to compare the anticancer effect of the tested GCs with a known anticancer agent.

Glyco-gene profiling array

The HT-29 cell line was incubated with GN8P for 24 h and total RNA was isolated using an RNAeasy Mini kit involving DNAse I treatment following the manufacturer's protocol (DNAse I was a component of RNAeasy Mini kit; Qiagen GmbH, Hilden, Germany). A Glyco-gene Chip array (GLYCOv3 Gene Chip; Affymetrix; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing probe sets of 2,000 human transcripts was provided by the Consortium for Functional Glycomics ( Microarray experiments were conducted by The Microarray Gene Core of Consortium for Functional Glycomics, National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS), both Bethesda, MD, USA ( and were performed in triplicate. BRB ArrayTools version 4.3.0 beta 3 [Biometric Research Branch, NIH/National Cancer Institute (NCI)] were used to filter and analyze experimental data sets. Class comparison used a two-sample t-test with a random variance model. A P-value of log-ratio <0.05 was considered significant.

Gene Ontology analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) software version 6.7 available from NCI ( The Gene Card database was used for basic gene identification and characterization (; Weizmann Institute of Science, Rehovot, Israel).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

The HT-29 cell line was incubated with GCs and FLU for 24 h and total RNA was isolated using an RNAeasy Mini kit involving DNAse I treatment, as described by the manufacturer (Qiagen). A total of 5 µg RNA was transcribed into cDNA using a cDNA Archive kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed using PowerSybr Green Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) on an iCycler5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). PCR product specificity was analyzed by melt curve analysis. The cycling conditions recommended by the Master mix manufacturer were followed: initiation 95°C 10 min and 40 cycles of 95°C 15 sec and 60°C 1 min. The primers used for PCR were designed using Primer3 Input software version 0.4.0 (National Center for Biotechnology Information, Bethesda, MD, USA). The sequences of primers were as follows: Mgat5, forward, 5′-CTTCTTTCTTCCAGCACCTCAAC-3′ and reverse, 5′-AAACACACAGTGCTTATTCTTAGGG-3′; Nme1, forward, 5′-ACCTTCATTGCGATCAAACC-3′ and reverse, 5′-GGCCCTGAGTGCATGTATTT-3′; Siglec5, forward, 5′-CAAGTGCAGAAGTCGGTGAC-3′ and reverse, 5′-GGGTCTCTGGCTTCACTCTT-3′; Siglec8, forward, 5′-TGCAACCCTCAGCTTCCATA-3′ and reverse, 5′-ACTTCTTTGCTGGAGGGGTT-3′; Wnt9B, forward, 5′-TGGGCAGACTGTCATCACAT-3′ and reverse, 5′-AACAAGGTTGGGGATGCTTG-3′. Sequences of the primers used to amplify B2M, Egfr1 and Sglt1, Mki67, Mmp3, Tgfb1 and Wnt2B were described previously (2731): B2M, forward, 5′-GAGTATGCCTGCCGTGTG-3′ and reverse, 5′-AATCCAAATGCGGCATCT-3′; Egfr1, forward, 5′-TTTCGATACCCAGGACCAAGCCACAGCAGG-3′ and reverse, 5′-AATATTCTTGCTGGATGCGTTTCTGTA-3′; Sglt1, forward, 5′-TGGCAGGCCGAAGTATGGTGT-3′ and reverse, 5′-ATGAATATGGCCCCCGAGAAGA-3′; Mki67, forward, 5′-GGAGGCAATATTACATAATTTCA-3′ and reverse, 5′-CAGGGTCAGAAGAGAAGCTA-3′; Mmp3, forward, 5′-ATGCCCACTTTGATGATGATGAAC-3′ and reverse, 5′-CCACGCCTGAAGGAAGAGATG-3′; Tgfb1, forward, 5′-TGACAGCAGGGATAACACACT-3′ and reverse, 5′-GTAGGGGCAGGGCCCGAGGCA-3′; Wnt2B, forward, 5′-CACCTGCTGGCGTGCACTCTCAGA-3′ and reverse, 5′-GGGCTTTGCAAGTATGGACGTCCACAGTA-3′. Primers for Mgat3 were obtained from SA Biosciences; Qiagen (product ID: PPH01058A). The expression of the genes of interest were normalized to that of the control gene B2M and quantified using the 2-ΔΔCq method (32). Differences in the expression of genes between the untreated and GC-treated cells were evaluated using Bio-Rad iQ5 version 2.0 (Bio-Rad Laboratories, Inc.).

Cell proliferation and colony formation assays

To determine the effect of GCs on cell growth, 5×103 cells/well were seeded in triplicate on a 96-well plate and incubated with GCs for 126 h. FLU (1 mM) was used as a positive control of tumor cell growth inhibition. Negative controls represent cells with no additional treatment. Cell growth was monitored continuously using a Real-Time Cell Analyzer (RTCA; xCELLigence System; Acea Biosciences, San Diego, CA, USA; and evaluated using RTCA software version 1.2 (Acea Biosciences).

To study colony formation, a standard colony forming assay and an impedance-based assay using the RTCA instrument (Acea Biosciences) were performed. For colony formation assays, 1×103 were seeded in a Petri dish 100 mm in diameter. After 2 weeks, colonies were fixed for 30 min with 70% ethanol (Sigma-Aldrich; Merck KGaA) at room temperature and subsequently stained for 10 min at room temperature with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA). RTCA was also used to evaluate colony growth in order to obtain quantitative data. Here, 100 cells/well were seeded in triplicate on a 96-well plate and colony growth was monitored using RTCA for 2 weeks.

Apoptosis assay and cell-mediated cytotoxicity test

GC-mediated toxicity in the HT-29 cancer cell line was assessed using propidium iodide (PI) staining and subsequent flow cytometry with a BD LSRII (BD Biosciences, Franklin Lakes, NJ, USA). Apoptosis was measured using an FITC Annexin V Apoptosis Detection kit I (Annexin V-fluorescein isothiocyanate, BD Biosciences) following the manufacturer's protocol. The results were analyzed using FlowJo version 7.2.2 software (FlowJo LLC, Ashland, OR, USA).

Cell-mediated cytotoxicity was performed using a standard chromium release assay, as previously described (2). Briefly, HT-29 cells (2×104 cells/well) pretreated with or without GCs for 30 min were incubated with 51Cr for 2 h and served as target cells for PBMCs (total volume 0.1 ml/well). All samples were tested in triplicate. The effector:target ratio of 16:1 was optimal for the experimental conditions. Following 4 h co-incubation of target and PBMC cells, 51Cr release was measured in cell-free supernatants (obtained as supernatants following centrifugation of cells at 2,000 × g for 5 min at room temperature) using a Wallac Microbeta Trilux scintillation counter (PerkinElmer, Inc., Waltham, MA, USA). The percentage specific lysis was calculated using the following formula: [Experimental counts per minute (cpm) - spontaneous cpm]/(maximum cpm - spontaneous cpm) × 100, where maximum cpm was determined by addition of 10% Triton X-100.

Statistical analysis

Statistically significant differences in the parameters tested in HT-29 cells cultured in the presence or absence of GCs were assessed using a one-way analysis of variance followed by Dunnett's post hoc test and a confidence interval of 95%. Statistical analysis was conducted using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.


GC stimulation modulates the mRNA expression of glucosaminyltransferases, components of glucose metabolism and adhesion molecules

Previous research has noted alterations in the phenotype of cancer cells following treatment with GCs. Therefore, the present study investigated a wide range of candidate molecules and the results revealed that the interaction of cancer cells with GCs induced complex alterations in gene expression.

Candidate molecules were selected based on the novel results of a glyco-gene array investigating GN8P, as well as previously published results of glyco-gene profiling of GN4C in NK-92 cancer cells (2). Regarding the glyco-gene array performed on cell samples incubated for 24 h in the presence or absence of GN8P, the present study excluded all genes in which the percentage of absent data (all data where two out of three parallels were missing) >50% and where P>0.05. Subsequently, class comparison identified 22 genes that exhibited significantly different expression between control and GN8P-treated HT-29 cells (P<0.05). The responsive genes were functionally categorized according to Gene Ontology classification and the Gene Card database. A total of 64% of the genes listed were linked to cancer and 14% of the genes were linked to inflammation. Differentially expressed genes were primarily involved in signal transduction (28%), carbohydrate binding (23%), proliferation (14%) and immune processes (14%). A complete list of the differentially expressed genes, including their function and disease association, is presented in Table I. Along with other genes, GN8P mediated the downregulation of Wnt signaling molecules that serve an important role in cancer progression; thus inhibition of their expression is of particular interest. Upregulated genes included protein tyrosine phosphatase Ptprt and tyrosine kinase Flt3 (Table I). Candidate genes were validated by RT-qPCR (data not shown).

Table I.

List of GN8P-responsive genes in the HT-29 cell line.

Table I.

List of GN8P-responsive genes in the HT-29 cell line.

GeneEntrez gene IDBiological functionDisease association(Refs.)
Upregulated genes
  Mpl4352GFR, ST, CPCAMT, TR, PV, MD(3335)
  Flt32322Ig, TK, ST, CPC-L (AML, ALL)(36,37)
  Bmp7655GF, TGFRD, OS, PA(3840)
  Ptprt11122ST, PPCoCa, glioma(41,42)
  Ppbp5473GF, GTTA, CP, IIN, ET, TP(4345)
  Cxcl22920I, CPC, N, S(46,47)
  Gpc32719CPHepCa, WT(48,49)
Downregulated genes
  Siglec1289858CB, adheze(50)
  Nrg13084GF, TFC, SC(51)
  Ccl76354I, STIN, AS(52)
  Lgals1329124CB, GAL, LPLPre-eclampsia(53)
  Csf2ra1438ST, CPC, N(54)
  Clec4C170482CB, I(56)
  Mrc14360CB, L, RITBC, ALL(58,59)
  Cxccl94283ST, IN, IN(60)
  Sgsh6448hydrolase, GBMPS(61)
  Clec3B7123CB, L, PAPC(62)
  Gdf1110220TGF, GFALL(63)

[i] CAMT, congenital amegakaryocytic thrombocytopenia; TR, thrombocytosis; PV, polycythemia vera; MPS, mucopolysaccharidosis; C-L, leukemia; MD, myeloproliferative disorders; OS, osteosarcoma; PA, pseudarthrosis; RD, renal disease; CoCa, colon cancer; HepCa, hepatocellular carcinoma; WT, Wilm's tumor; KS, Kaposhi sarcoma; SC, schizophrenia; IN, inflammation; ET, essential hypertension; TP, thrombocytopenia; C, cancer; N, necrosis; S, sepsis; DA, dermatitis atopic; AS, asthma; GFR, growth factor receptor; TK, tyrosin kinase; CP, cell proliferation; ST, signal transduction; GTTA, glucose transmembrane transporter activity; I, immune processes; CB, carbohydrate binding; T, transferase; GB, glycan biosynthesis; SFT, sulfotransferase; Ig, immunoglobulin protein domain; L, lectin pr dom; RI, ricin B lectin pro dom; GAL, galectin pr dom; PAP, pancreatis associated protein; TGF, transforming growth factor β receptor binding; PP, protein tyrosine phosphatase activity; UDP, glucuronosyl/UDP glucosyl transferase; LPL, lysophospholipase activity.

For a detailed examination of the effect of GCs on the glycosylation mechanism in HT-29 cells, the expression of glucosaminyltransferases involved in terminal glycan elongation and genes involved in glucose uptake and cell adhesion, were measured. When compared with the NTC group, treatment with the GN4C and GN8P significantly downregulated expression levels of Mgat3 (P=0.0002 and P=0.0001, respectively) and Mgat5 (each, P=0.0001) glucosaminyltransferases. FLU also inhibited the expression of Mgat5 (P=0.0001) compared with the NTC group. The Nme1 gene encodes a transcription regulator controlling expression of the Mgat5 gene (9). Compared with the NTC group, the mRNA expression of this transcription factor was significantly downregulated by GN8P and FLU (P=0.0009 and P=0.002, respectively; Fig. 1A).

Sglt1 and Egfr1 are components of a glucose cotransporter in human cells. The mRNA expression of these genes was significantly reduced by GN8P (P=0.0054 for SGLT1). However, the reduction of EGFR1 expression following exposure to GN8P was of borderline significance (P=0.2) (Fig. 1B). Furthermore, the mRNA expression of the glycan binding adhesion molecules Siglec5 and Siglec8 were downregulated by GN4C (P=0.0078 and P=0.0001, respectively) as well as by GN8P (P=0.0009 and P=0.0081, respectively; all Fig. 1C). However, FLU only inhibited the mRNA expression of Siglec8 (P=0.0001; Fig. 1C).

GCs inhibit HT-29 cell proliferation, induce apoptosis and promote PBMC-mediated cytotoxicity

The present study questioned whether GCs alter the proliferation of cancer cells. To test this hypothesis, real-time monitoring of cell proliferation was performed for 5 days. A proliferation protocol was optimized using an RTCA system allowing the continuous monitoring of treated and untreated cells for a set period of time. The results indicated that the proliferation of HT-29 cells treated with GN4C, GN8P or FLU was significantly inhibited from ~5 h following induction. The greatest inhibition of cell growth was detected 60 h following treatment in the cells treated with FLU, GN8P and GN4C (P=0.0001 for all treatments vs. NTC group; Fig. 2A). A significant reduction in the expression of the proliferation antigen Mki67 48 h following treatment with GN4C (P=0.0017), GN8P (P=0.0005) and FLU (P=0.0003; Fig. 2B), was observed, compared with the NTC group.

To determine the toxicity of GCs in the HT-29 cell line, Annexin V positivity (specific marker of apoptosis) and the incorporation of PI into cells were measured. The percentage of early apoptotic, Annexin V-positive and PI-negative cells was significantly increased following treatment with GN4C, GN8P and FLU (P=0.0001, P=0.002 and P=0.0001, respectively), compared with the NTC group. Treatment with FLU induced a significant decrease in the population of cells in the late apoptotic and necrotic phases compared with the NTC group (all P=0.0001; Fig. 2C) However, treatment with GCs had no effect on the proportion of cells in the late apoptotic and necrotic stages of apoptosis (Fig. 2C). The proportion of untreated HT-29 cells that underwent spontaneous necrosis (PI positive, Annexin V-negative population) was 5.8%; comparable with the percentage of necrotic cells found in GN4C and GN8P treated cells (Fig. 2C). These results indicate that GCs preferably induce apoptosis over necrosis.

Surface glycosylation is a tool facilitating the recognition of cancer cells by immune cells. The current study questioned whether alterations in cancer cell glycosylation sensitizes them to PBMC-mediated toxicity. HT-29 cells, acting as target cells, which were naturally resistant to human PBMC-mediated cytotoxicity, were pretreated with GCs and subsequently exposed to PBMCs from healthy donors, acting as effector cells. Significantly higher cytolytic activity was observed in HT-29 cells pretreated with GN4C and GN8P (P=0.0002 and P=0.0001, respectively; Fig. 2D). The rate of spontaneous toxicity in untreated HT-29 cells against human PBMCs was ~1.2% (Fig. 2D).

Synthetic GCs reduce cancer cell colony formation

As cell invasion is associated with extracellular matrix remodeling and neoangiogenesis, levels of regulatory components, including Mmp3, Tgfb1, Wnt2B and Wnt9B, were measured. There was a significant decrease in the expression of Mmp3 mRNA following treatment with GN4C and FLU (P=0.0005 and P=0.0006, respectively; Fig. 3A). GN4C inhibited the expression of Tgfb1 (P=0.0016; Fig. 3A). GN4C and GN8P decreased the expression of Wnt2B (P=0.0002 and P=0.0066, respectively) and Wnt9B (P=0.0004 and P=0.0101, respectively; Fig. 3B). FLU significantly decreased the expression of Wnt9B (P=0.01; Fig. 3B) but not Wnt2B. Treatment of HT-29 cells with GN4C and GN8P led to decreases in cell colony formation by 39 and 18%, respectively (P=0.0001 vs. NTC). FLU treated (positive control) cells exhibited a decrease in cell colony formation of 96% compared with the NTC group (P=0.0001; Fig. 3C). These changes in colony formation were confirmed following standard crystal-violet staining. There was a reduction in colonies in samples incubated with GCs and FLU (Fig. 3D).


Previous studies have focused on the identification of genes involved in the response of NK cells isolated from PBMC and the permanent NK-92 cell line to the synthetic GC GN4C (2), or on the role of GN8P in the activation of immune cells (24).

Phenotypic alterations in cancer cells following exposure to GCs has been observed. Calix[6]arenes exhibit an anticancer effect by modulating AXL and Mer tyrosine kinase receptor gene expression (66); therefore, the present study questioned whether the phenotypic changes in cancer cells were modulated by changes in gene expression. The current study specifically focused on the expression of cancer development-related genes and how gene expression is affected by two GlcNAc-modified GCs that contain different cores: First generation PAMAM or calix[4]arene.

Previous studies have reported that exogenous glucosamine, the terminal moiety of the tested GCs, may be internalized via a glucose transporter (17,18). In HT-29 cells, clathrin-mediated endocytosis has been described as a mechanism of uptake of third generation PAMAM (67). Cancer cells, such as HT-29, exhibit a high glucose intake due to high-energy requirements (22). The sodium-glucose cotransporter (SGLT1) transports glucose into cells independent of its concentration (68). SGLT1 is stabilized by interaction with EGFR1, facilitating cancer cell survival (27). In colon cancer, the high expression of Sglt1 and Egfr1 is associated with poor patient prognosis (69). The results of the present study demonstrated that the PAMAM-based GC significantly decreased the expression of the two genes Sglt1 and Egfr1 that code for the glucose cotransporter complex. This, in turn, inhibited cell growth and reduced levels of the proliferation antigen Mki67.

It has been reported that higher generation dendrimers (G2, G4 and G6) promote cell growth at lower concentrations but induce cell death at higher concentrations. The critical concentration at which dendrimers induced cell death was 500 nM. Also, toxicity was enhanced by higher generation dendrimers (70). The current study observed that a first generation PAMAM dendrimer with eight GlcNAc moieties induced alterations in gene expression and altered the properties of cancer cells even at low concentrations (10 nM). Comparing the responses of cells to GCs and FLU identified the different underlying mechanisms of action of each compound. FLU is a potent inducer of apoptosis and affected cells undergo rapid disintegration, thus explaining the decrease in the number of cells in the late apoptotic and necrotic phases following treatment with FLU.

The growth of cancer cells was markedly affected by the GC GN8P and changes in cell adhesion and invasion were associated with the altered expression of specific mRNAs. The Siglec family contains proteins that serve an important role in cancer cell adhesion and invasion. Human cell-surface-receptors, including sialic acid-binding Ig-like lectin (SIGLEC) 5 and SIGLEC8, are members of the cluster of differentiation 33-related Siglec subfamily, expressed predominantly by immune cells (71) and they are overexpressed in acute myeloid leukemia, chronic eosinophilic and myelogenous leukemias (72,73). In solid tumors, Siglecs are overexpressed in tumor-associated immune cells, such as macrophages; however there is little evidence regarding their overexpression in actual cancer cells (74). The alteration in Siglec mRNA expression observed in the present study following the incubation of tumor cells with GC may be due to alterations in Mgat5 expression.

In colon cancer, glycosylation performed by MGAT5 may regulate colon cancer stem cells and tumor progression via Wnt signaling (75). As GCs downregulate the expression of Mgat5 and exhibit reduced colony formation, the present study measured the expression of the Wnt family members Wnt2B and Wnt9B, the metalloproteinase Mmp3, which is responsible for extracellular matrix remodeling (76) and Tgfb1, which is involved in tumor neoangiogenesis (77). These genes were selected based on the preliminary results of cDNA profiling in GN8P-treated cancer cells (unpublished results). GCs reduced the expression of Wnt2B and Wnt9B; however, only GN4C inhibited the expression of Mmp3 and Tgfb1. This may be due to the fact GN4C is more effective at inhibiting cell invasion; indeed, it has been demonstrated that calix[4]arene inhibits cancer angiogenesis (78).

The present study identified a specific pattern in gene expression, common to the two GCs, which included downregulation of i) the glucosaminyltransferases Mgat3 and Mgat5, ii) the adhesion molecules Siglec5 and Siglec8, iii) Wnt2B and Wnt9B and iv) the proliferation marker Mki67. The two GCs increased the proportion of cells in the early apoptotic phase and the sensitivity of cancer cells to PBMCs. The current study focused the effect of GCs in cancer cells and revealed associations that may allow for the investigation of individual components in a different perspective or focus on specific signaling pathways induced by the GCs. It has been demonstrated that glycosylation in NK cells involves the phosphoinositide 3-kinase signaling pathway (2). Based on information from a recently published report (79), alterations regarding the sensitivity to PBMCs may be due to the GlcNAc section of the GCs. In the case of the downregulation of the Sglt1/Egfr1, the PAMAM core may induce this change since GN8P significantly lowered expression of the Sglt1 component but GN4C demonstrated no such effect. In myotubes, Wnt signaling affects glucose transport via a glucose transporter (80). In the present study, the same members of the Wnt pathway (Wnt2B and Wnt9B) were downregulated by GN4C. No differences in Sglt1/Egfr1 expression following treatment with GN4C were detected. This suggests that these factors are not involved in colon cancer cell signaling, however, further analysis may reveal if there is a similar association between the SGLT1/EGFR1 cotransporter and other members of Wnt pathway.

It is likely that other members of the Wnt signaling pathway are involved and responsible for this distinctive response to particular cores. The present study demonstrated that Wnt signaling may be involved in the response of cancer cells to synthetic GCs, possibly via modulation of glucosaminyltransferases. Subsequent detailed studies focusing on the Wnt pathway may identify response-specific members of the Wnt family.

The evolutionarily conserved Wnt signaling mechanism is an important pathway and Wnt proteins undergo post-translational glycosylation. Therefore, anticancer therapies that target Wnt-signaling members, based on glycosylation modulation, may be developed as a novel therapeutic strategy.

In conclusion, the present study demonstrated that the interaction of colon cancer cells with specifically designed GCs results in a complex commitment of different cellular pathways and induces alterations in the phenotypes of cells. The results of the current study revealed that alterations in the expression of particular genes following treatment with GCs are associated with specific outcomes in cancer cells, including their higher sensitivity to immune cell-mediated killing. The GCs used in the current study exhibited multiple effects following their application to cancer cells. These results, together with those of previous studies determining the immunostimulatory effects of GCs, support the importance of glycosylation-targeted anticancer therapy and provides a basis for further studies.


The authors wish to thank The Consortium for Functional Glycomics/NIGMS/Gene Microarray Core (grant no. GM62116) for their resources and collaboration. The authors thank Cory Benson for editing the English language of the manuscript, Professors Vladimır Křen and Thisbe Lindhorst for providing GCs and Dr Lucie Vondráčková for providing the xCELLigence system. The present study was supported by the Ministry of Health of the Czech Republic (grant no. 15-33094A) and the Czech Science Foundation (grant no. 14-10100S).









N-acetyl-D-glucosamine-coated calix[4]arene


N-acetyl-D-glucosamine-coated polyamidoamine dendrimer




peripheral blood mononuclear cells


Real-Time Cell Analyzer



Kobata A and Amano J: Altered glycosylation of proteins produced by malignant cells and application for the diagnosis and immunotherapy of tumours. Immunol Cell Biol. 83:429–439. 2005. View Article : Google Scholar : PubMed/NCBI


Benson V, Grobarova V, Richter J and Fiserova A: Glycosylation regulates NK cell-mediated effector function through PI3K pathway. Int Immunol. 22:167–177. 2010. View Article : Google Scholar : PubMed/NCBI


Lau E, Feng Y, Claps G, Fukuda MN, Perlina A, Donn D, Jilaveanu L, Kluger H, Freeze HH and Ronai ZA: The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation. Sci Signal. 8:ra1242015. View Article : Google Scholar : PubMed/NCBI


Pinho SS and Reis CA: Glycosylation in cancer: Mechanisms and clinical implications. Nat Rev Cancer. 15:540–555. 2015. View Article : Google Scholar : PubMed/NCBI


Cipolla L, La Ferla B and Gregori M: Combinatorial approaches to iminosugars as glycosidase and glycosyltransferase inhibitors. Comb Chem High Throughput Screen. 9:571–582. 2006. View Article : Google Scholar : PubMed/NCBI


Abbott KL, Nairn AV, Hall EM, Horton MB, McDonald JF, Moremen KW, Dinulescu DM and Pierce M: Focused glycomic analysis of the N-linked glycan biosynthetic pathway in ovarian cancer. Proteomics. 8:3210–3220. 2008. View Article : Google Scholar : PubMed/NCBI


Kang X, Wang N, Pei C, Sun L, Sun R, Chen J and Liu Y: Glycan-related gene expression signatures in human metastatic hepatocellular carcinoma cells. Exp Ther Med. 3:415–422. 2012. View Article : Google Scholar : PubMed/NCBI


Song Y, Aglipay JA, Bernstein JD, Goswami S and Stanley P: The bisecting GlcNAc on N-glycans inhibits growth factor signaling and retards mammary tumor progression. Cancer Res. 70:3361–3371. 2010. View Article : Google Scholar : PubMed/NCBI


Chakraborty AK, Sousa Jde F, Chakraborty D, Funasaka Y, Bhattacharya M, Chatterjee A and Pawelek J: GnT-V expression and metastatic phenotypes in macrophage-melanoma fusion hybrids is down-regulated by 5-Aza-dC: Evidence for methylation sensitive, extragenic regulation of GnT-V transcription. Gene. 374:166–173. 2006. View Article : Google Scholar : PubMed/NCBI


Lau KS and Dennis JW: N-Glycans in cancer progression. Glycobiology. 18:750–760. 2008. View Article : Google Scholar : PubMed/NCBI


Yoshinaga A, Kajiya N, Oishi K, Kamada Y, Ikeda A, Chigwechokha PK, Kibe T, Kishida M, Kishida S and Komatsu M: NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation. Eur J Pharmacol. 782:21–29. 2016. View Article : Google Scholar : PubMed/NCBI


Galindo-Murillo R, Sandoval-Salinas ME and Barroso-Flores J: In Silico design of monomolecular drug carriers for the tyrosine kinase inhibitor drug imatinib based on calix- and thiacalix [n]arene host molecules: A DFT and molecular dynamics study. J Chem Theory Comput. 10:825–834. 2014. View Article : Google Scholar : PubMed/NCBI


Cherenok S, Vovk A, Muravyova I, Shivanyuk A, Kukhar V, Lipkowski J and Kalchenko V: Calix[4]arene alpha-aminophosphonic acids: Asymmetric synthesis and enantioselective inhibition of an alkaline phosphatase. Org Lett. 8:549–552. 2006. View Article : Google Scholar : PubMed/NCBI


Zhou H, Wang DA, Baldini L, Ennis E, Jain R, Carie A, Sebti SM and Hamilton AD: Structure-activity studies on a library of potent calix[4]arene-based PDGF antagonists that inhibit PDGF-stimulated PDGFR tyrosine phosphorylation. Org Biomol Chem. 4:2376–2386. 2006. View Article : Google Scholar : PubMed/NCBI


Geraci C, Consoli GM, Galante E, Bousquet E, Pappalardo M and Spadaro A: Calix[4]arene decorated with four Tn antigen glycomimetic units and P3CS immunoadjuvant: Synthesis, characterization and anticancer immunological evaluation. Bioconjug Chem. 19:751–758. 2008. View Article : Google Scholar : PubMed/NCBI


Esfand R and Tomalia DA: Poly(amidoamine) (PAMAM) dendrimers: From biomimicry to drug delivery and biomedical applications. Drug Discov Today. 6:427–436. 2001. View Article : Google Scholar : PubMed/NCBI


Schleicher ED and Weigert C: Role of the hexosamine biosynthetic pathway in diabetic nephropathy. Kidney Int Suppl. 77:S13–S18. 2000. View Article : Google Scholar : PubMed/NCBI


Hinderlich S, Berger M, Schwarzkopf M, Effertz K and Reutter W: Molecular cloning and characterization of murine and human N-acetylglucosamine kinase. Eur J Biochem. 267:3301–3308. 2000. View Article : Google Scholar : PubMed/NCBI


Phatnani HP and Greenleaf AL: Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 20:2922–2936. 2006. View Article : Google Scholar : PubMed/NCBI


Gorboulev V, Schürmann A, Vallon V, Kipp H, Jaschke A, Klessen D, Friedrich A, Scherneck S, Rieg T, Cunard R, et al: Na+-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion. Diabetes. 61:187–196. 2012. View Article : Google Scholar : PubMed/NCBI


Ferguson KM, Berger MB, Mendrola JM, Cho HS, Leahy DJ and Lemmon MA: EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization. Mol Cell. 11:507–517. 2003. View Article : Google Scholar : PubMed/NCBI


Wang XQ, Sun P, O'Gorman M, Tai T and Paller AS: Epidermal growth factor receptor glycosylation is required for ganglioside GM3 binding and GM3-mediated suppression (correction of suppresion) of activation. Glycobiology. 11:515–522. 2001. View Article : Google Scholar : PubMed/NCBI


Hanahan D and Weinberg RA: Hallmarks of cancer: The next generation. Cell. 144:646–674. 2011. View Article : Google Scholar : PubMed/NCBI


Grobárová V, Benson V, Rozbeský D, Novák P and Cerný J: Re-evaluation of the involvement of NK cells and C-type lectin-like NK receptors in modulation of immune responses by multivalent GlcNAc-terminated oligosaccharides. Immunol Lett. 156:110–117. 2013. View Article : Google Scholar : PubMed/NCBI


Krenek K, Kuldová M, Hulíková K, Stibor I, Lhoták P, Dudic M, Budka J, Pelantová H, Bezouska K, Fiserová A and Kren V: N-acetyl-D-glucosamine substituted calix[4]arenes as stimulators of NK cell-mediated antitumor immune response. Carbohydr Res. 342:1781–1792. 2007. View Article : Google Scholar : PubMed/NCBI


Vannucci L, Fiserová A, Sadalapure K, Lindhorst TK, Kuldová M, Rossmann P, Horváth O, Kren V, Krist P, Bezouska K, et al: Effects of N-acetyl-glucosamine-coated glycodendrimers as biological modulators in the B16F10 melanoma model in vivo. Int J Oncol. 23:285–296. 2003.PubMed/NCBI


Weihua Z, Tsan R, Huang WC, Wu Q, Chiu CH, Fidler IJ and Hung MC: Survival of cancer cells is maintained by EGFR independent of its kinase activity. Cancer Cell. 13:385–393. 2008. View Article : Google Scholar : PubMed/NCBI


Han Y, San-Marina S, Yang L, Khoury H and Minden MD: The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells. Breast Cancer Res. 9:R432007. View Article : Google Scholar : PubMed/NCBI


Ullmannová V, Stöckbauer P, Hradcová M, Soucek J and Haskovec C: Relationship between cyclin D1 and p21Waf1/Cip1 during differentiation of human myeloid leukemia cell lines. Leuk Res. 27:1115–1123. 2003. View Article : Google Scholar : PubMed/NCBI


Klatt AR, Paul-Klausch B, Klinger G, Kühn G, Renno JH, Banerjee M, Malchau G and Wielckens K: A critical role for collagen II in cartilage matrix degradation: Collagen II induces pro-inflammatory cytokines and MMPs in primary human chondrocytes. J Orthop Res. 27:65–70. 2009. View Article : Google Scholar : PubMed/NCBI


Van Den Berg DJ, Sharma AK, Bruno E and Hoffman R: Role of members of the Wnt gene family in human hematopoiesis. Blood. 92:3189–3202. 1998.PubMed/NCBI


Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI


Besancenot R, Roos-Weil D, Tonetti C, Abdelouahab H, Lacout C, Pasquier F, Willekens C, Rameau P, Lecluse Y, Micol JB, et al: JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation. Blood. 124:2104–2115. 2014. View Article : Google Scholar : PubMed/NCBI


Moliterno AR, Hankins WD and Spivak JL: Impaired expression of the thrombopoietin receptor by platelets from patients with polycythemia vera. N Engl J Med. 338:572–580. 1998. View Article : Google Scholar : PubMed/NCBI


Moliterno AR, Williams DM, Rogers O and Spivak JL: Molecular mimicry in the chronic myeloproliferative disorders: Reciprocity between quantitative JAK2 V617F and Mpl expression. Blood. 108:3913–3915. 2006. View Article : Google Scholar : PubMed/NCBI


Marhäll A, Kazi JU and Rönnstrand L: The Src family kinase LCK cooperates with oncogenic FLT3/ITD in cellular transformation. Sci Rep. 7:137342017. View Article : Google Scholar : PubMed/NCBI


Rousset D, Agnès F, Lachaume P, André C and Galibert F: Molecular evolution of the genes encoding receptor tyrosine kinase with immunoglobulinlike domains. J Mol Evol. 41:421–429. 1995. View Article : Google Scholar : PubMed/NCBI


Higgins DF, Ewart LM, Masterson E, Tennant S, Grebnev G, Prunotto M, Pomposiello S, Conde-Knape K, Martin FM and Godson C: BMP7-induced-Pten inhibits Akt and prevents renal fibrosis. Biochim Biophys Acta. 1863:3095–3104. 2017. View Article : Google Scholar : PubMed/NCBI


Anticevic D, Jelic M and Vukicevic S: Treatment of a congenital pseudarthrosis of the tibia by osteogenic protein-1 (bone morphogenetic protein-7): A case report. J Pediatr Orthop B. 15:220–221. 2006. View Article : Google Scholar : PubMed/NCBI


Honda Y, Knutsen R, Strong DD, Sampath TK, Baylink DJ and Mohan S: Osteogenic protein-1 stimulates mRNA levels of BMP-6 and decreases mRNA levels of BMP-2 and −4 in human osteosarcoma cells. Calcif Tissue Int. 60:297–301. 1997. View Article : Google Scholar : PubMed/NCBI


Zhao Y, Scott A, Zhang P, Hao Y, Feng X, Somasundaram S, Khalil AM, Willis J and Wang Z: Regulation of paxillin-p130-PI3K-AKT signaling axis by Src and PTPRT impacts colon tumorigenesis. Oncotarget. 8:48782–48793. 2017.PubMed/NCBI


Bourgonje AM, Verrijp K, Schepens JT, Navis AC, Piepers JA, Palmen CB, van den Eijnden M, Hooft van Huijsduijnen R, Wesseling P, Leenders WP and Hendriks WJ: Comprehensive protein tyrosine phosphatase mRNA profiling identifies new regulators in the progression of glioma. Acta Neuropathol Commun. 4:962016. View Article : Google Scholar : PubMed/NCBI


Yeo L, Adlard N, Biehl M, Juarez M, Smallie T, Snow M, Buckley CD, Raza K, Filer A and Scheel-Toellner D: Expression of chemokines CXCL4 and CXCL7 by synovial macrophages defines an early stage of rheumatoid arthritis. Ann Rheum Dis. 75:763–771. 2016. View Article : Google Scholar : PubMed/NCBI


Sullam PM, Frank U, Yeaman MR, Täuber MG, Bayer AS and Chambers HF: Effect of thrombocytopenia on the early course of streptococcal endocarditis. J Infect Dis. 168:910–914. 1993. View Article : Google Scholar : PubMed/NCBI


Aziz H, Zaas A and Ginsburg GS: Peripheral blood gene expression profiling for cardiovascular disease assessment. Genomic Med. 1:105–112. 2007. View Article : Google Scholar : PubMed/NCBI


De Plaen IG, Han XB, Liu X, Hsueh W, Ghosh S and May MJ: Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: Independence from endogenous TNF-alpha and platelet-activating factor. Immunology. 118:153–163. 2006. View Article : Google Scholar : PubMed/NCBI


Sarvaiya PJ, Guo D, Ulasov I, Gabikian P and Lesniak MS: Chemokines in tumor progression and metastasis. Oncotarget. 4:2171–2185. 2013. View Article : Google Scholar : PubMed/NCBI


Wang HL, Anatelli F, Zhai QJ, Adley B, Chuang ST and Yang XJ: Glypican-3 as a useful diagnostic marker that distinguishes hepatocellular carcinoma from benign hepatocellular mass lesions. Arch Pathol Lab Med. 132:1723–1728. 2008.PubMed/NCBI


Tretiakova M, Zynger DL, Luan C, Andeen NK, Finn LS, Kocherginsky M, Teh BT and Yang XJ: Glypican 3 overexpression in primary and metastatic Wilms tumors. Virchows Arch. 466:67–76. 2015. View Article : Google Scholar : PubMed/NCBI


Mitra N, Banda K, Altheide TK, Schaffer L, Johnson-Pais TL, Beuten J, Leach RJ, Angata T, Varki N and Varki A: SIGLEC12, a human-specific segregating (pseudo)gene, encodes a signaling molecule expressed in prostate carcinomas. J Biol Chem. 286:23003–23011. 2011. View Article : Google Scholar : PubMed/NCBI


Marballi K, Quinones MP, Jimenez F, Escamilla MA, Raventós H, Soto-Bernardini MC, Ahuja SS and Walss-Bass C: In vivo and in vitro genetic evidence of involvement of neuregulin 1 in immune system dysregulation. J Mol Med. 88:1133–1141. 2010. View Article : Google Scholar : PubMed/NCBI


Girkin J, Hatchwell L, Foster P, Johnston SL, Bartlett N, Collison A and Mattes J: CCL7 and IRF-7 mediate hallmark inflammatory and IFN responses following rhinovirus 1b infection. J Immunol. 194:4924–4930. 2015. View Article : Google Scholar : PubMed/NCBI


Than NG, Romero R, Xu Y, Erez O, Xu Z, Bhatti G, Leavitt R, Chung TH, El-Azzamy H, LaJeunesse C, et al: Evolutionary origins of the placental expression of chromosome 19 cluster galectins and their complex dysregulation in preeclampsia. Placenta. 35:855–865. 2014. View Article : Google Scholar : PubMed/NCBI


Waghray M, Yalamanchili M, Dziubinski M, Zeinali M, Erkkinen M, Yang H, Schradle KA, Urs S, Pasca Di Magliano M, Welling TH, et al: GM-CSF mediates mesenchymal-epithelial cross-talk in pancreatic cancer. Cancer Discov. 6:886–899. 2016. View Article : Google Scholar : PubMed/NCBI


Akpa MM, Iglesias DM, Chu LL, Cybulsky M, Bravi C and Goodyer PR: Wilms tumor suppressor, WT1, suppresses epigenetic silencing of the β-catenin gene. J Biol Chem. 290:2279–2288. 2015. View Article : Google Scholar : PubMed/NCBI


Riboldi E, Daniele R, Parola C, Inforzato A, Arnold PL, Bosisio D, Fremont DH, Bastone A, Colonna M and Sozzani S: Human C-type lectin domain family 4, member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides. J Biol Chem. 286:35329–35333. 2011. View Article : Google Scholar : PubMed/NCBI


Shi Q, Liu H, Han P, Li C, Wang Y, Wu W, Zhu D, Amos CI, Fang S, Lee JE, et al: Genetic variants in WNT2B and BTRC predict melanoma survival. J Invest Dermatol. 137:1749–1756. 2017. View Article : Google Scholar : PubMed/NCBI


Lin SY, Li K, Stewart GS and Elledge SJ: Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation. Proc Natl Acad Sci USA. 101:pp. 6484–6489. 2004; View Article : Google Scholar : PubMed/NCBI


Focarelli ML, Soza S, Mannini L, Paulis M, Montecucco A and Musio A: Claspin inhibition leads to fragile site expression. Genes Chromosomes Cancer. 48:1083–1090. 2009. View Article : Google Scholar : PubMed/NCBI


Ohta K, Shigeishi H, Taki M, Nishi H, Higashikawa K, Takechi M and Kamata N: Regulation of CXCL9/10/11 in oral keratinocytes and fibroblasts. J Dent Res. 87:1160–1165. 2008. View Article : Google Scholar : PubMed/NCBI


Sidhu NS, Schreiber K, Pröpper K, Becker S, Usón I, Sheldrick GM, Gärtner J, Krätzner R and Steinfeld R: Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA. Acta Crystallogr D Biol Crystallogr. 70:1321–1335. 2014. View Article : Google Scholar : PubMed/NCBI


Zhang X, Wan JX, Ke ZP, Wang F, Chai HX and Liu JQ: TMEM88, CCL14 and CLEC3B as prognostic biomarkers for prognosis and palindromia of human hepatocellular carcinoma. Tumour Biol. 39:10104283177089002017. View Article : Google Scholar : PubMed/NCBI


Yu H, Gaochao Z, Huaquan W, Limin X, Lijuan L, Huijuan J, Wei Z, Jinglian T and Zonghong S: GDF11 is increased in patients with myelodysplastic syndrome. Int J Clin Exp Pathol. 9:6031–6038. 2016.


Safaee M, Ivan ME, Oh MC, Oh T, Sayegh ET, Kaur G, Sun MZ, Bloch O and Parsa AT: The role of epidermal growth factor-like module containing mucin-like hormone receptor 2 in human cancers. Oncol Rev. 8:2422014. View Article : Google Scholar : PubMed/NCBI


Fernández-Vega I, García-Suárez O, García B, Crespo A, Astudillo A and Quirós LM: Heparan sulfate proteoglycans undergo differential expression alterations in right sided colorectal cancer, depending on their metastatic character. BMC Cancer. 15:7422015. View Article : Google Scholar : PubMed/NCBI


Pelizzaro-Rocha KJ, de Jesus MB, Ruela-de-Sousa RR, Nakamura CV, Reis FS, de Fátima A and Ferreira-Halder CV: Calix[6]arene bypasses human pancreatic cancer aggressiveness: Downregulation of receptor tyrosine kinases and induction of cell death by reticulum stress and autophagy. Biochim Biophys Acta. 1833:2856–2865. 2013. View Article : Google Scholar : PubMed/NCBI


Saovapakhiran A, D'Emanuele A, Attwood D and Penny J: Surface modification of PAMAM dendrimers modulates the mechanism of cellular internalization. Bioconjug Chem. 20:693–701. 2009. View Article : Google Scholar : PubMed/NCBI


Wright EM, Loo DD and Hirayama BA: Biology of human sodium glucose transporters. Physiol Rev. 91:733–794. 2011. View Article : Google Scholar : PubMed/NCBI


Guo GF, Cai YC, Zhang B, Xu RH, Qiu HJ, Xia LP, Jiang WQ, Hu PL, Chen XX, Zhou FF, et al: Overexpression of SGLT1 and EGFR in colorectal cancer showing a correlation with the prognosis. Med Oncol. 28 Suppl 1:S197–S203. 2011. View Article : Google Scholar : PubMed/NCBI


Parimi S, Barnes TJ, Callen DF and Prestidge CA: Mechanistic insight into cell growth, internalization and cytotoxicity of PAMAM dendrimers. Biomacromolecules. 11:382–389. 2010. View Article : Google Scholar : PubMed/NCBI


Crocker PR, McMillan SJ and Richards HE: CD33-related siglecs as potential modulators of inflammatory responses. Ann N Y Acad Sci. 1253:102–111. 2012. View Article : Google Scholar : PubMed/NCBI


Virgo P, Denning-Kendall PA, Erickson-Miller CL, Singha S, Evely R, Hows JM and Freeman SD: Identification of the CD33-related Siglec receptor, Siglec-5 (CD170), as a useful marker in both normal myelopoiesis and acute myeloid leukaemias. Br J Haematol. 123:420–430. 2003. View Article : Google Scholar : PubMed/NCBI


Hudson SA, Herrmann H, Du J, Cox P, Haddad el-B, Butler B, Crocker PR, Ackerman SJ, Valent P and Bochner BS: Developmental, malignancy-related and cross-species analysis of eosinophil, mast cell and basophil siglec-8 expression. J Clin Immunol. 31:1045–1053. 2011. View Article : Google Scholar : PubMed/NCBI


Potapenko IO, Haakensen VD, Lüders T, Helland A, Bukholm I, Sørlie T, Kristensen VN, Lingjaerde OC and Børresen-Dale AL: Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression. Mol Oncol. 4:98–118. 2010. View Article : Google Scholar : PubMed/NCBI


Guo H, Nagy T and Pierce M: Post-translational glycoprotein modifications regulate colon cancer stem cells and colon adenoma progression in Apcmin/+ mice through altered WNT receptor signaling. J Biol Chem. 289:31534–31549. 2014. View Article : Google Scholar : PubMed/NCBI


Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE, Leake D, Godden EL, Albertson DG, Nieto MA, et al: Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature. 436:123–127. 2005. View Article : Google Scholar : PubMed/NCBI


Ferrari G, Cook BD, Terushkin V, Pintucci G and Mignatti P: Transforming growth factor-beta 1 (TGF-beta1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis. J Cell Physiol. 219:449–458. 2009. View Article : Google Scholar : PubMed/NCBI


Dings RP, Miller MC, Nesmelova I, Astorgues-Xerri L, Kumar N, Serova M, Chen X, Raymond E, Hoye TR and Mayo KH: Antitumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding. J Med Chem. 55:5121–5129. 2012. View Article : Google Scholar : PubMed/NCBI


Liu WC, Lin YL, Spearman M, Cheng PY, Butler M and Wu SC: Influenza virus hemagglutinin glycoproteins with different N-glycan patterns activate dendritic cells in vitro. J Virol. 90:6085–6096. 2016. View Article : Google Scholar : PubMed/NCBI


Abiola M, Favier M, Christodoulou-Vafeiadou E, Pichard AL, Martelly I and Guillet-Deniau I: Activation of Wnt/beta-catenin signaling increases insulin sensitivity through a reciprocal regulation of Wnt10b and SREBP-1c in skeletal muscle cells. PLoS One. 4:e85092009. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

Volume 17 Issue 4

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
Spandidos Publications style
Křivohlavá R, Grobárová V, Neuhöferová E, Fišerová A and Benson V: Interaction of colon cancer cells with glycoconjugates triggers complex changes in gene expression, glucose transporters and cell invasion. Mol Med Rep 17: 5508-5517, 2018
Křivohlavá, R., Grobárová, V., Neuhöferová, E., Fišerová, A., & Benson, V. (2018). Interaction of colon cancer cells with glycoconjugates triggers complex changes in gene expression, glucose transporters and cell invasion. Molecular Medicine Reports, 17, 5508-5517.
Křivohlavá, R., Grobárová, V., Neuhöferová, E., Fišerová, A., Benson, V."Interaction of colon cancer cells with glycoconjugates triggers complex changes in gene expression, glucose transporters and cell invasion". Molecular Medicine Reports 17.4 (2018): 5508-5517.
Křivohlavá, R., Grobárová, V., Neuhöferová, E., Fišerová, A., Benson, V."Interaction of colon cancer cells with glycoconjugates triggers complex changes in gene expression, glucose transporters and cell invasion". Molecular Medicine Reports 17, no. 4 (2018): 5508-5517.