N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.     

N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients

Glycosylated proteins play important roles in a broad spectrum of biochemical and biological processes, and prior reports have suggested that changes in protein glycosylation occur during cancer initiation and progression. Ovarian cancer (OC) is a fatal malignancy, most commonly diagnosed after the development of metastases. Therefore, early detection of OC is key to improving survival. To this end, specific changes of the serum glycome have been proposed as possible biomarkers for different types of cancers. In this study, we extend this concept to OC. To characterize differences in total N-glycan levels, serum samples provided by 20 healthy control women were compared to those acquired from patients diagnosed with late-stage recurrent OC who were enrolled in an experimental treatment trial prior to receiving therapy (N=19) and one month later, prior to the second treatment cycle (N=11). Additionally, analyses of the N-glycans associated with IgG and characterization of the relative abundance levels of core vs outer-arm fucosylation were also performed. The N-linked glycomic profiles revealed increased abundances of tri- and tetra-branched structures with varying degrees of sialylation and fucosylation and an apparent decrease in the levels of "bisecting" glycans in OC samples compared to controls. Increased levels of a-galactosylation structures were observed on N-linked glycans derived from IgG, which were independent of the presence of fucose residues. Elevated levels of outer-arm fucosylation were also identified in the OC samples. These results allowed the control samples to be distinguished from the baseline ovarian cancer patients prior to receiving the experimental treatment. In some cases, the pre-treatment samples could be distinguished from the post-experimental treatment samples, as many of those patients showed a further progression of the disease.     

Comparison of sialylated N‐glycopeptide levels in serum of pancreatic cancer patients,acute pancreatitis patients,and healthy controls

Serum protein glycosylation is known to be affected by pathological conditions, including cancer and inflammatory diseases. Pancreatic cancer patients would benefit from early diagnosis, as the disease is often detected in an advanced stage and has poor prognosis. Searching for changes in serum protein site‐specific glycosylation could reveal novel glycoprotein biomarkers. We used Sambucus nigra lectin affinity chromatography to enrich α‐2,6 sialylated tryptic N‐glycopeptides from albumin‐depleted sera of pancreatic cancer patients, acute pancreatitis patients, and healthy individuals, and compared their relative abundance using ultra performance LC‐MS. Relative quantitation was done using the spectrum processing software MZmine. Identification was performed on the web‐based tool GlycopeptideID, developed for in silico analysis of intact N‐glycopeptides. Seventeen high‐abundance serum proteins, mainly acute‐phase proteins, and immunoglobulins, with total 27 N‐glycosylation sites, and 62 glycoforms, were identified. Pancreatitis patient sera contained 38, and pancreatic cancer patients sera contained 13 glycoform changes with statistical significance (p < 0.05). In pancreatitis, up to tenfold changes were found in some glycoforms, and in pancreatic cancer, threefold. Analysis showed that the changes often concerned one or two, but not all, N‐glycosylation sites in a specific glycoprotein. In conclusion, the analysis shows that pancreatic cancer, and acute pancreatitis are associated with changes in concentrations of intact sialylated N‐glycopeptides derived from acute‐phase proteins, and immunoglobulins, and that changes are site specific.     

Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis: application to pancreatic cancer serum

A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of alpha(2,3) and alpha(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to elucidate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using microLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using microLC-ESI-TOF-MS where the N83 glycosylation of alpha1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer.     

Novel glycan biomarkers for the detection of lung cancer

Lung cancer has a poor prognosis and a 5-year survival rate of 15%. Therefore, early detection is vital. Diagnostic testing of serum for cancer-associated biomarkers is a noninvasive detection method. Glycosylation is the most frequent post-translational modification of proteins and it has been shown to be altered in cancer. In this paper, high-throughput HILIC technology was applied to serum samples from 100 lung cancer patients, alongside 84 age-matched controls and significant alterations in N-linked glycosylation were identified. Increases were detected in glycans containing Sialyl Lewis X, monoantennary glycans, highly sialylated glycans and decreases were observed in core-fucosylated biantennary glycans, with some being detectable as early as in Stage I. The N-linked glycan profile of haptoglobin demonstrated similar alterations to those elucidated in the total serum glycome. The most significantly altered HILIC peak in lung cancer samples includes predominantly disialylated and tri- and tetra-antennary glycans. This potential disease marker is significantly increased across all disease groups compared to controls and a strong disease effect is visible even after the effect of smoking is accounted for. The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease.     

Selective enrichment of sialic acid-containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry

The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO(2)) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO(2) toward SA-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO(2) resin. By combining TiO(2) enrichment of sialylated glycopeptides with HILIC separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO(2)/HILIC protocol requires 2 d and the entire procedure from protein isolation can be performed in <5 d, depending on the time taken to analyze data.     

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides

Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α₁-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.     

Glycoproteomic analyses of ovarian cancer cell lines and sera from ovarian cancer patients show distinct glycosylation changes in individual proteins

Ovarian cancer is difficult to diagnose in women because symptoms of the disease are often not noticed until the disease has progressed to an advanced untreatable stage. Although a serum test, CA125, is currently available to assist with monitoring treatment of ovarian cancer, this test lacks the necessary specificity and sensitivity for early detection. Therefore, better biomarkers of ovarian cancer are needed. A glycoprotein analysis approach was undertaken using high resolution Fourier transform ion cyclotron resonance mass spectrometry to analyze glycosylated proteins present in the conditioned media of ovarian cancer cell lines and in sera obtained from ovarian cancer patients and normal controls. In this study, glycosylated proteins were separated by gel electrophoresis, and individual glycoproteins were selected for glycosylation analysis and protein identification. The attached glycans from each protein were released and profiled by mass spectrometry. Glycosylation of a mucin protein and a large glycosylated protein isolated from the ES2 ovarian cancer cell line was determined to consist of mostly O-linked glycans. Four prominent glycoproteins of approximate 517, 370, 250, 163 kDa from serum samples were identified as two forms of apolipoprotein B-100, fibronectin, and immunoglobulin A1, respectively. Mass spectrometric analysis of glycans isolated from apolipoprotein B-100 (517 kD) showed the presence of small, specific O-linked oligosaccharides. In contrast, analysis of fibronectin (250 kD) and immunoglobulin A1 (163 kD) produced N-linked glycan fragments in forms that were sufficiently different from the glycans obtained from the corresponding protein band present in the normal serum samples. This study shows that not only a single protein but several are aberrantly glycosylated, and those abnormal glycosylation changes can be detected and may ultimately serve as glycan biomarkers for ovarian cancer.     

Increase in Sialylation and Branching in the Mouse Serum N-glycome Correlates with Inflammation and Ovarian Tumour Progression

Ovarian cancer is the most lethal gynaecological cancer and is often diagnosed in late stage, often as the result of the unavailability of sufficiently sensitive biomarkers for early detection, tumour progression and tumour-associated inflammation. Glycosylation is the most common posttranslational modification of proteins; it is altered in cancer and therefore is a potential source of biomarkers. We investigated the quantitative and qualitative effects of anti-inflammatory (acetylsalicylic acid) and pro-inflammatory (thioglycolate and chlorite-oxidized oxyamylose) drugs on glycosylation in mouse cancer serum. A significant increase in sialylation and branching of glycans in mice treated with an inflammation-inducing compound was observed. Moreover, the increases in sialylation correlated with increased tumour sizes. Increases in sialylation and branching were consistent with increased expression of sialyltransferases and the branching enzyme MGAT5. Because the sialyltransferases are highly conserved among species, the described changes in the ovarian cancer mouse model are relevant to humans and serum N-glycome analysis for monitoring disease treatment and progression might be a useful biomarker.     

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.     

Changes in protein glycosylation during chick embryo development

To investigate the molecular changes in cell-surface glycoproteins during chick embryo development, fibroblasts from 8- and 16-day embryos were extensively digested by pronase after (i) metabolic labeling with radioactive precursors and (ii) external labeling. Two main classes of glycopeptide pronase digestion product were distinguished by Sephadex G-50 column chromatography. The large material excluded was mostly composed of glycosaminoglycans. The small retarded glycopeptides underwent age-related modifications. Those in the 8-day cells were mainly N-linked, whereas 16-day cells contained both O- and N-linked glycopeptides. The evolution of high-mannose chains in younger cells to complex-type chains in the older cells is suggested by (i) the decrease in the mannose-to-galactose and mannose-to-N-acetylglucosamine ratio with embryo development, and (ii) the fact that endo-β-N-acetylglucosaminidase H treatment released more oligomannosyls from younger than from older embryo cell glycopeptides. Small glycopeptides were also more highly sialylated in 16-day cells than in 8-day cells. The present results provide the first biochemical evidence that both quantitative and qualitative modifications occur in cell-surface glycoconjugates during the late stages of chick embryo development.     

Dynamic Sialylation in Transforming Growth Factor-β (TGF-β)-induced Epithelial to Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development and organ formation. Aberrant regulation of EMT often leads to tumor progression. Changes in cell surface sialylation have recently been implicated in mediating EMT. Herein we report the visualization of dynamic changes of sialylation and glycoproteomic analysis of newly synthesized sialylated proteins in EMT by metabolic labeling of sialylated glycans with azides, followed by click labeling with fluorophores or affinity tags. We discovered that sialylation was down-regulated during EMT but then reverted and up-regulated in the mesenchymal state after EMT, accompanied by mRNA expression level changes of genes involved in the sialic acid biosynthesis. Quantitative proteomic analysis identified a list of sialylated proteins whose biosynthesis was dynamically regulated during EMT. Sialylation of cell surface adherent receptor integrin β₄ was found to be down-regulated, which may regulate integrin functions during EMT. Furthermore, a global sialylation inhibitor was used to probe the functional role of sialylation during EMT. We found that inhibition of sialylation promoted EMT. Taken together, our findings suggest the important role of sialylation in regulating EMT and imply its possible function in related pathophysiological events, such as cancer metastasis.     

Consequences of the expression of sialylated antigens in breast cancer

Changes in cell surface glycosylation are common modifications that occur during oncogenesis, leading to the over-expression of tumour-associated carbohydrate antigens (TACA). Most of these antigens are sialylated and the increase of sialylation is a well-known feature of transformed cells. In breast cancer, expression of TACA such as sialyl-Lewis^x or sialyl-Tn is usually associated with a poor prognosis and a decreased overall survival of patients. However, the specific role of these sialylated antigens in breast tumour development and aggressiveness is not clearly understood. These glycosylation changes result from the modification of the expression of genes encoding specific glycosyltransferases involved in glycan biosynthesis and the level of expression of sialyltransferase genes has been proposed to be a prognostic marker for the follow-up of breast cancer patients. Several human cellular models have been developed in order to explain the mechanisms by which carbohydrate antigens can reinforce breast cancer progression and aggressiveness. TACA expression is associated with changes in cell adhesion, migration, proliferation and tumour growth. In addition, recent data on glycolipid biosynthesis indicate an important role of G_D3 synthase expression in breast cancer progression. The aim of this review is to summarize our current knowledge of sialylation changes that occur in breast cancer and to describe the cellular models developed to analyze the consequences of these changes on disease progression and aggressiveness.     

Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry

Pools of O-glycopeptides prepared from trypsin-digested reduced and alkylated human serum IgA1 have been analyzed using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-ToF-MS) in the positive-ion mode, using 2,4,6-trihydroxy acetophenone-ammonium citrate matrix. Dozens of such pools prepared from normal serum IgA1 and from serum of patients with a number of different medical conditions have been routinely analyzed in this manner. The glycopeptides present in these pools possess identical amino acid sequences but are substituted with a variety of neutral and sialylated glycans and the spectra obtained were such that individual compositional glycoforms were baseline resolved. In addition, the spectra were reproducible, exhibiting a relative peak intensity and area variation of around 11-16%, enabling the technique to be used for the relative quantitation of the different compositional glycoforms present. This could be achieved manually or by applying a Java program especially developed for this purpose. The MS analysis described here is a major improvement over present MALDI methods used for profiling the O-glycosylation of IgA1. The MS methodology together with the Java data analysis are expected to be generally applicable for profiling O-linked glycopeptides derived from other glycoproteins and probably for N-linked glycopeptide pools.     

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.     

Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented.     

Tissue and serum α2-3- and α2-6-linkage specific sialylation changes in oral carcinogenesis

Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects α2-6- and α2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and α2,3-ST activity compared to the adjacent normal tissues. α2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, α2-6-sialoproteins and α2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of α2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.     

Serum sialylation changes in cancer

Cancer is a major cause of death in both developing and developed countries. Early detection and efficient therapy can greatly enhance survival. Aberrant glycosylation has been recognized to be one of the hallmarks of cancer as glycans participate in many cancer-associated events. Cancer-associated glycosylation changes often involve sialic acids which play important roles in cell-cell interaction, recognition and immunological response. This review aims at giving a comprehensive overview of the literature on changes of sialylation in serum of cancer patients. Furthermore, the methods available to measure serum and plasma sialic acids as well as possible underlying biochemical mechanisms involved in the serum sialylation changes are surveyed. In general, total serum sialylation levels appear to be increased with various malignancies and show a potential for clinical applications, especially for disease monitoring and prognosis. In addition to overall sialic acid levels and the amount of sialic acid per total protein, glycoprofiling of specific cancer-associated glycoproteins, acute phase proteins and immunoglobulins in serum as well as the measurements of sialylation-related enzymes such as sialidases and sialyltransferases have been reported for early detection of cancer, assessing cancer progression and improving prognosis of cancer patients. Moreover, sialic-acid containing glycan antigens such as CA19–9, sialyl Lewis X and sialyl Tn on serum proteins have also displayed their value in cancer diagnosis and management whereby increased levels of these factors positively correlated with metastasis or poor prognosis.