Shin Yazawa

α1‐Acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis

Serum α1‐acid glycoprotein (AGP), an acute‐phase protein secreted by the liver, carries α(1,3)‐fucosylated structures on its 5 highly branched, N‐linked sugar chains.

α1,2Fucosylation Is a Superior Predictor of Postoperative Prognosis for Colorectal Cancer Compared with Blood Group A, B, or Sialyl Lewis X Antigen Generated within Colorectal Tumor Tissues

BackgroundWe have previously demonstrated tumor-specific α1,2fucosylation, which is associated with resistance of tumor cells to anticancer treatment in human colorectal tumor tissues. By using the YB-2 monoclonal antibody, the resulting products have been identified as Y, Leb, and H type 2 antigens in colorectal tumor tissues.MethodsImmunohistochemical analyses of colorectal cancer tissues (74 specimens) were performed with a newly established mouse monoclonal antibody, YB-3 specifically recognizing H disaccharide (Fucα1,2Galβ) structures, and anti-A, anti-B, YB-2, and anti–sialyl Lewis X (SLX) antibodies, together with the analyses of glycosyltransferases involved in the synthesis of ABH antigens in the same tissues.ResultsThe YB-3 antibody enabled us to detect colorectal tumors, particularly tumors in the distal large intestine and the rectum, with high sensitivity (74.3%) and specificity (100%). From immunohistochemical and enzymatic analyses of colorectal tissues, we found that once α1,2fucosylation had proceeded in tumor tissues, blood group A or B antigen was also synthesized in approximately half of the tissues of A or B blood type, but not in their normal tissues. A correlation of survival rate with immunostaining of tissues was found only by YB-3 antibody and not by anti-A, anti-B, or anti-SLX antibody.ConclusionsAs a predictor of postoperative prognosis of patients with colorectal cancer, immunodetection of α1,2fucosylated antigens with the YB-3 antibody seemed to be superior to blood groups A, B, or SLX antigen in colorectal tumor tissues.

Chemically synthesized sugar‐cholestanols possess a preferential anticancer activity involving promising therapeutic potential against human esophageal cancer

The understanding of the cell signaling pathways and the molecular events leading to cell death of cancer cells will provide in‐depth perspective into the identification and development of potent anticancer agents. A balance between cell proliferation and cell death has been raised as a rational target for the management of malignant tumors. In the present study, the authors demonstrated that chemically synthesized sugar‐cholestanols consisting of GlcNAcβ‐, Galβ‐ and GlcNAcβ1,3Galβ‐cholestanols exerted a strong inhibiting activity against cell proliferation of esophageal cancer cells, but cholestanol itself did not show such an activity against the same cancer cells at all. In addition to their predominant role as an antiproliferation agent, evidence based on the molecular analyses suggested that sugar‐cholestanols played a regulatory role in multiple signal transduction pathways inducing apoptosis through both the death signal‐extrinsic and the mitochondria‐intrinsic pathways. Sugar‐cholestanols seemed to be more susceptible to esophageal cancer cells than to non‐cancerous esophageal cells at the very early event of their exposure and, further, to suppress specifically the expression of vascular endothelial growth factor. Taken together, these novel functions of sugar‐cholestanols indicate that they could have promising therapeutic potential against human esophageal cancer. (Cancer Sci 2007; 98: 1358–1367)

Novel sugar-cholestanols as anticancer agents against peritoneal dissemination of tumor cells

Chemically synthesized sugar-cholestanols with mono-, di-, and tri-saccharides attached to cholestanol showed strong inhibiting activity against the proliferation of colorectal and gastric cancer cells. In contrast, cholestanol without sugar moieties was totally ineffective. Furthermore, when cancer cells were exposed to GlcNAcRβcholestanol (R = (−) or β1-3Gal), the compound was rapidly taken up via the lipid rafts/microdomains on the cell surface. The uptake of sugar-cholestanol in mitochondria increased gradually and was followed by the release of cytochrome c from mitochondria and the activation of apoptotic signals through the mitochondrial pathway and the caspase cascade, leading to apoptotic cell death, characterized by DNA ladder formation and nuclear fragmentation. Additionally, the examination of GlcNAcRβcholestanol in a mouse model of peritoneal dissemination showed a dramatic reduction of tumor growth (P < 0.003) and prolonged mouse survival time (P < 0.0001). Based on these observations, we believe that the sugar-cholestanols described here have clinical potential as novel anticancer agents.

Blood group substances as potential therapeutic agents for the prevention and treatment of infection with noroviruses proving novel binding patterns in human tissues.

Blood group-related glycans determining ABO and Lewis blood groups are known to function as attachment factors for most of the norovirus (NoV) strains. To identify binding specificity of each NoV, recombinant norovirus-like particles (VLPs) and human saliva samples with different ABO, Lewis phenotypes and secretor status have been commonly applied. When binding specificities of VLPs prepared from 16 different genotypes of NoVs in GI and GII genogroups were characterized in samples of human gastric mucosa compared to human saliva based on blood group phenotypes, considerable differences were observed for several strains. Novel binding specificities determined by an ELISA using preparations from human gastric mucosa were also ascertained by immunohistochemical analyses using human jejunal mucosa, widely believed to be susceptible to NoV infection. Further, A, B and O(H) blood group substances prepared from porcine and squid tissues were found to be effective for preventing ABO blood group-specific binding of VLPs to both saliva and mucosa samples. Therefore, these blood group substances might have potential for the prevention and treatment of NoV infection.

Expression of Carbohydrate Antigens in Human Esophageal Squamous Cell Carcinoma: Prognostic Application and Its Diagnostic Implications

BackgroundTumor markers whose antigenic determinants have been demonstrated to consist of carbohydrates are probably one of the most extensive tools that have been used in routine cancer diagnosis. In this study, the relevance of carbohydrate antigen expression profile was examined in esophageal squamous cell carcinoma together with prognosis in 130 patients.MethodsThe expression of carbohydrate antigens was estimated immunohistochemically by anti–sialyl Lewis a (sialyl Lea) and anti–sialyl Lewis x (sialyl Lex) monoclonal antibodies, and correlation between their staining and clinicopathological status was examined. In addition, the correlation of both carbohydrate antigens expression was evaluated with microvessel density (MVD).ResultsExpressions of sialyl Lewis antigens and MVD were associated with several clinicopathological features that reflect the tumor aggressiveness in esophageal cancer. The 5-year survival rate of patients was found to be associated with expression of sialyl Lea and sialyl Lex antigens and with MVD; thus, all of them were revealed to be independent prognostic factors.ConclusionsCombination of these factors offered a better prediction of prognosis of esophageal squamous cell carcinoma. Further, carbohydrate antigens represent a promising target for therapeutic approaches against the disease.

Quantitative evaluation of lectin-reactive glycoforms of α1-acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection

α1‐Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein‐labeled AGP using lectins with the aid of laser‐induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A‐reactive or Aleuria aurantia lectin‐reactive AGP. Labeled AGP was applied at the anodic end of a fused‐silica capillary (50 μm id, 360 μm od, 27 cm long) coated with linear polyacryloyl‐β‐alanyl‐β‐alanine, and electrophoresis was carried out for about 10 min in 60 mM 3‐morpholinopropane‐1‐sulfonic acid‐NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin‐reactive glycoform peaks from lectin‐non‐reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin‐reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5 mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro‐purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies.

Abstract A27: Comprehensive analysis of glycans on α1-acid glycoprotein from various cancer patients by MALDI-TOF-MS for assigning a prognostic predictor

α1-Acid glycoprotein (AGP) is well-known as an acute-phase protein, a major plasma glycoprotein with highly glycosylated, branched N-linked glycans and one of the markers for inflammation. In our previous study (Cancer 101:2825–2836, 2004), with the aid of crossed affinoimmunoelectrophoresis (CAIE), glycoforms of plasma AGP from various cancer patients could be easily determined based on the degrees of branching and extent of fucosylation characterized by reactivities with Con A lectin and Aleuria aurantia lectin, respectively. Results clearly indicated that patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated tri- and tetraantennary glycans for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms with no such changes were expected to have a good prognosis irrespective of their clinical stages. In this study, we purified AGP from plasma samples of the same patients and then analysed detailed structures of their glycans involving a new tumor marker using a high throughput glycosylation kit (BlotGlyco, Sumitomo Bakelite, Tokyo, Japan) combined with MALDI-TOF-MS analysis. More than two hundreds of glycan structures were assigned and relative abundance of each glycan was calculated. Follow-up studies of the relative abundance of glycans indicated that the abundance of fucosylated tri- and tetraantennary glycans in patients with a poor prognosis was quite high, and most of which were estimated to form sialyl-LeX structures (NeuAcα2,3Galβ1,4[Fucα1,3]GlcNAcβ) with the non-reducing end sialyl residues. Whereas, in those with a good prognosis, such an abundance was found to be as low as in healthy controls. These results indicated that no distinct difference was observed from our previous CAIE data and that more precise glycan structures instead of glycoforms could be easily assigned by the present method, providing an aberrant marker for predicting the fate of patients after surgery. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A27.