E-selectin expression induced by pancreas-carcinoma-derived interleukin-1α results in enhanced adhesion of pancreas-carcinoma cells to endothelial cells

Cellular adhesion of sialyl-Lewis-a(SLe^a)-positive pancreas carcinoma to endothelial cells (EC) is augmented by activation of EC via up-regulated E-selectin expression on EC. Co-cultivation of pancreas-carcinoma cells, PCI-24, with human umbilical-vein endothelial cells (HUVEC) for 5 hr at the PCI-to-HUVEC ratio of 1:10 induced E-selectin expression on the endothelial-cell surface, augmenting SLe^a-positive pancreas-carcinoma cell attachment with HUVEC. Culture supernatants of 6 tested pancreas-carcinoma cell lines contained soluble, E-selectin-inducing factor(s). The E-selectin-inducing effect by the supernatants was blocked by the protein-kinase-C inhibitor, H7. Antibodies against SLe^a and E-selectin but not SLe^x or ICAM-I blocked the increased pancreas-carcinoma-to-endothelial attachment. Paraformaldehyde(PFA)-fixed PCI-24 cells also induced E-selectin on vascular endothelial cells upon direct contact with endothelial cells, indicating the presence of a membrane-bound form. The 6 pancreas-carcinoma lines all produced IL-1α mRNA and protein but not IL-1β or TNF-α protein and/or mRNA Absorption of IL-lα from the supernatants by IL-lα-specific antibody almost completely abolished E-selectin-inducing activity. Anti-IL-lα antibody also abolished the E-selectin-inducing activity of PFA-fixed PCI. IL-1α production by PCI cells was up-regulated by TNF-α. These observations suggest that substance(s) produced by pancreas-carcinoma cells, in this case, IL-1α, may contribute to pancreas-carcinoma-cell colonization in non-inflamed, distant locations in vivo, by activating vascular endothelial cells.     

Endothelial and epithelial expression of sialyl Lewisx and sialyl Lewisa in lesions of breast carcinoma

Tumor cells can invade and generate metastasis via either lymphatics or blood vessels. When tumor cells are circulating in the blood, they must adhere to the vessel wall, which is lined by endothelium, before they can extravasate and form new metastases. Several families of adhesion molecules have been identified to play a role in the extravasation cascade. Selectins and their sialyl Lewis^x and/or sialyl Lewis^a (sLe^x and sLe^a, respectively) containing ligands play an initiating role in this cascade; we have now analyzed their role in the generation of metastatic breast carcinoma lesions. We examined expression of endothelial E- and P-selectin, expression of epithelial and endothelial sLe^x and sLe^a normal tissues compared with primary and metastatic breast in carcinoma lesions within individual patients. While normal breast epithelial cells do not express sLe^x or sLe^a, epithelial expression of these oligosaccharide epitopes was enhanced in primary breast carcinoma lesions. Furthermore, epithelial expression levels of sLe^x and/or sLe^a were even higher in most patients (9 of 12) who had metastatic compared with primary lesions. We show that endothelia in primary lesions express more sLe^x than in normal tissue and that metastatic lesions express even higher amounts of sLe^x compared with primary lesions. The expression of P- and E-selectin was also greatly enhanced in tumor-bearing tissue compared with normal tissue. Our data support the hypothesis that while they are circulating in the blood, sLe^x- and/or sLe^a-expressing carcinoma cells have a higher probability for extravasation at sites where the endothelium expresses E- and P-selectin and for generation of new metastases. Int. J. Cancer 74:296-300, 1997. © 1997 Wiley-Liss, Inc.     

E-selectin binding by pancreatic tumor cells is inhibited by cancer sera

Tumor cells interact with endothelial cells during both intraand extravasation. Understanding how these interactions are modulated could lead to the development of ways to alter the metastatic potential of tumor cell. Three pancreatic cancer cell lines, SW1990, CAPAN-2 and PANC-1, were examined for their ability to bind to the endothelial cell adhesion molecule E-selectin (ELAM-1). SW1990 cells exhibited highest binding, highest surface expression of the carbohydrate antigens sialylated Lewis² (sLe²) and sialylated Lewis^x (sLe^x) and released the most high m. w. sLe² and sLe^x antigens. Expression of sLe² and sLe^x antigens and binding to E-selectin were reduced by pre-treatment of SW1990 cells with the O-linked glycosylation inhibitor benzyl-α-GaINAc but not with the N-linked glycosylation inhibitor tunicamycin. Expression of peptide epitopes associated with MUCI apomucins was increased by benzyl-α-GaINAc. Cell binding was greatly reduced by mucins purified from SW1990 xenografts and by an antibody against sLe². An antibody against sLe^x had a much less marked effect. Sera from pancreatic cancer patients reduced SW1990 cell binding to E-selectin but sera from normals did not. The degree of inhibition was related to the sLe^x level in the sample. When cancer serum was separated by column chromatography on Sephacryl S-400, the void volume fractions contained most of the sLe² and sLe^x antigens and most of the inhibitory activity to E-selectin binding. Differences in the relative availability of sLe² and sLe^x ligands on serum molecules and on the SW1990 cell surface may account for the differences between antibody and serum inhibition results. Thus SW1990 cell adhesion to E-selectin is mediated by ligands on mucinous glycoproteins, and adhesion can be inhibited by mucins, high blood levels of sLe^x and reduction of cellular O-linked glycosylation. © 1994 Wiley-Liss, Inc.     

Phenotypes correlating to metastatic properties of pancreas adenocarcinoma in vivo: The importance of surface sialyl Lewisa antigen

Metastasis to the liver often occurs in patients during the natural course of pancreatic cancer. Using carcinoma cell lines established from 9 such patients, we examined phenotypes of cell lines to search for correlations with their potential to metastasize to the liver. Anti-asialo GMI-treated nude mice were used. PCI-43, -55, -24 and -6, in this order, had frequent metastases, while PCI-10, -19, -35, -64, and -66 did not. In vitro doubling time, surface expression of sialyl Lewis ^a (SLe ^a), VLA-4/6, LFA-1/3, CEA, E-selectin, VCAM-I, NCAM, Mac-I, HLA-ABC/DR/DQ, ICAM-1/2, production of interleukin-1α, tumor necrosis factor-α, and matrix metalloproteinase, as well as susceptibility to cytotoxicity by natural killer cells, were all examined. Expression of surface SLe^a was significantly associated with metastasis; numbers of metastatic colonies of SLe ^a-positive and -negative cell lines were 21.6 ± 33.9 and 6.5 ± 14.3 (p < 0.01), respectively. Moreover, the intensity of surface SLe ^a expression of each PCI line correlated with the number of metastatic colonies in the liver. When anti-SLe^a monoclonal antibody (MAb) was administered, the development of liver metastasis by PCI-43 cells was significantly repressed, as compared with a control MAb. Although a reverse correlation between surface ICAM-1 expression and liver metastasis was noted, the species-restricted function of ICAM-1 makes interpretation difficult. Collective evidence indicates that expression of SLe ^a is an important positive mediator in the hematogenous metastasis of pancreas carcinoma. © 1996 Wiley-Liss, Inc.     

Adhesion of human breast cancer cells to vascular endothelium mediated by Sialyl Lewisx/E-selectin

The adhesion molecules involved in the attachment of breast cancer cells to endothelial cells were investigated in vitro. All six human breast cancer cell lines expressed sialyl Lewis &supx; antigen(s-Le &supx;). Only two cell lines expressed sialyl Lewis&supa; antigen. A correlation was found between the degree of s-Le&supx; expression and the attachment of cancer cells to human umbilical vein endothelial cells (HUVECs) activated by IL-1beta. Monoclonal antibodies against s-Le &supx; or E-selectin inhibited this adhesion. These findings suggest that s-Le &supx; on the surface of cancer cells and E-selectin on the surface of endothelial cells play roles in adheision of breast cancer cells to vascular endothelium.     

Expression of E-selectin on endothelial cells of small veins in human colorectal cancer

E-selectin is an adhesion molecule of endothelial cells that binds to cancer cells mediated by sialyl Lewis A (sLe^a) or sialyl Lewis X (sLe^x). It is suspected to be involved in hematogenous metastasis of tumors. Therefore, it is worth examining E-selectin expression in human colorectal cancer and its hepatic metastasis. In the present study, E-selectin was clearly revealed on the endothelial cells of small vessels adjacent to cancer nests both in primary and in metastatic nests in immunohistochemistry. In these tissues, E-selectin was observed on the endothelial cells lining the lumen of small vessels. Its expression adjacent to cancer nests appears to be induced through some stimuli by cancer cells, since its degree of expression is inversely correlated to the distance of the blood vessels from the cancer nests (p < 0.001). Endothelial cells adjacent to the metastatic lesion expressed E-selectin more extensively than those adjacent to the primary foci. This is also in line with the finding on serum E-selectin levels which were significantly elevated in the metastatic group as compared with the non-metastatic group. The serum E-selectin level may provide useful information in the diagnosis for hepatic metastasis of colorectal cancer, although the results are still tentative. © 1995 Wiley-Liss, Inc.     

Dimethyl sulfoxide (DMSO) increases expression of sialyl lewis X antigen and enhances adhesion of human gastric carcinoma (NUGC4) cells to activated endothelial cells

Dimethyl sulfoxide (DMSO) exerts a number of biological effects including the promotion of cell differentiation in cultured cells. In this study, we examined the effect of DMSO on the adhesion of tumor cells to endothelial cells. In vitro treatment of human gastric adenocarcinoma (NUGC4) cells with DMSO resulted in increased adhesion to interleukin-1 (IL-l)-activated human endothelial cells compared with DMSO-untreated NUGC4 cells. In flow cytometry, treating NUGC4 cells with DMSO enhanced the expression of sialyl Lewis x (sialyl Le^x) and sialyl dimeric Le^x antigens on their surface. Also, the binding of Limulus polyphemus agglutinin (LPA), which specifically binds to cell-surface sialic acids, was increased by DMSO. The adhesion of DMSO-treated NUGC4 cells to activated endothelial cells was blocked by neuraminidase pre-treatment of tumor cells or by antibody against either endothelial leukocyte adhesion molecule-1 (ELAM-I) or sialyl Le^x. Thus, it is suggested that enhanced adhesion following DMSO treatment is mediated by the interaction of sialyl Le^x expressed on NUGC4 cells with ELAM-I of endothelial cells. The modulation of sialyl Le^x antigen by DMSO provides a useful system for studying the regulatory mechanism of Lewis-related carbohydrate antigens and also for understanding the metastatic properties of cancer cells.     

Ratio of IgG: IgM antibodies to sialyl LewisX and GM3 correlates with tumor growth after immunization with melanoma-cell vaccine with different adjuvants in mice

Human melanoma cells (from biopsies and culture) express sialyl-Lewis^x and sialyl Lewis^a, the ligands for ECAM. These ligands may facilitate tumor progression and metastasis in human cancers. To test whether the antibodies to these ligands inhibit tumor progression, IgG and IgM responses to sLe^x and sLe^a were induced in C57BL/6j mice (n = 76) by immunization with human melanoma cells, with or without adjuvants (BCG, MPL, KLH). Control mice were treated with saline or BCG. Tumor growth and antigen expression were monitored after challenge with B16 mouse melanoma cells expressing sLe^x, sLe^a and the ganglioside GM₃. Tumor growth was reduced in mice immunized with BCG alone or cells with BCG or MPL, while tumors in mice receiving cells without adjuvants grew larger than in the control. Augmentation of IgM titers to sLe^x and GM₃ after immunization with BCG, or with cells with BCG or MPL correlated with retarded tumor growth, while increased IgG titers to sLe^x significantly correlated with aggressive tumor growth in mice immunized with cells without adjuvants. SLe^x, sLe^a and GM₃ were expressed in tumors from mice treated with saline or BCG. SLe^x expression, in particular, was lost in tumors growing in mice immunized with cells with or without adjuvants. Anti-sLe^x antibodies may promote or prevent tumor growth by antigenic modulation or by cytotoxic killing of tumor cells. Since early anti-sLe^x IgM correlated with tumor regression, in contrast to anti-sLe^x IgG, it may serve as a potential early endpoint for the effectiveness of melanoma vaccines expressing the antigens. Int. J. Cancer 75:117–124, 1998.© 1998 Wiley-Liss, Inc.     

Functional role of sialyl Lewis X and fibronectin-derived RGDS peptide analogue on tumor-cell arrest in lungs followed by extravasation

Our study demonstrates that synthetic sialyl Lewis X (SLe^x) as a ligand for selectins and fibronectin-derived RGDS peptide analogue[Ar(DRGDS)₃] inhibits lung metastases produced by i.v. co-injection of B16-BL6 melanoma cells. To investigate the inhibitory mechanisms in a living animal, we performed positron-emission tomography (PET) analysis after i.v. injection of [2-¹⁸F]2-fluoro-2-deoxy-D-glucose-labeled tumor cells with or without liposomal SLe^x or Ar(DRGDS)₃. The real-time PET measurement for the first 120 min, started immediately after injection, showed that tumor-cell arrest, i.e., accumulation in the target organ (lung) was remarkably inhibited by liposomal SLe^x, but not inhibited by Ar(DRGDS)₃ or liposomal Me-SLe^x, which is not recognized by selectins. In contrast, AR(DRGDS)₃ inhibited the invasion of B16-BL6 cells into reconstituted basement membrane (Matrigel) following tumor arrest, whereas SLe^x- or Me-SLe^x-entrapped liposomes did not affect tumor invasion. In the metastatic processes containing tumor-cell lodgement and arrest in the target organ followed by extravasation (invasion), SLe^x resulted in the inhibition of initial arrest of tumor cells, presumably tumor-endothelium interaction, while Ar(DRGDS)₃ achieved inhibition of tumor invasion into basement membrane at later steps of the cascade, consequently leading to inhibition of metastasis. Thus, tumor-cell arrest in lungs in the metastatic processes must be precisely and properly controlled by different adhesion molecules at different stages, which are similar to those observed in leukocyte-endothelium interaction. © 1996 Wiley-Liss, Inc.     

Lewis x Antigen Mediates Adhesion of Human Breast Carcinoma Cells to Activated Endothelium. Possible Involvement of the Endothelial Scavenger Receptor C-type Lectin

Lewis x (Le^x, CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galβ(1–4)Fucα(1–3)GlcNAc, which is expressed on glycoconjugates in human polymorphonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Le^x in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Le^x mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Le^x+-cells induced by anti-Le^x mAbs and complement when cells were adhered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF-7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Le^x-dependent PMN interaction with HUVEC. Although both anti-Le^x mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blotting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Le^x-trisaccharide, interacts with specific glycoproteins of M _r␣∼␣28 kD and 10 kD from MCF-7 cells. The interaction between Le^x+-cancer cells and vascular endothelium is a potential target for cancer treatment.     

Attachment of human colon cancer cells to vascular endothelium is enhanced by N-acetylglucosaminyltransferase V

Expression of N-acetylglucosaminyltransferase V (GnT-V) in colon cancer has been shown to be related to hematogenous metastasis and poor prognosis. To investigate the mechanism by which cancer cells expressing GnT-V metastasize to distant organs, we established GnT-V-overexpressing DLD-1 and WiDr cells (human colon cancer cell lines) by transfecting them with a GnT-V expression vector. Attachment to endothelial cells expressing E-selectin was studied, and expression of the E-selectin ligand, sialyl Lewis x, in colon cancer cells was investigated. Both of the cell lines showed reduced adhesion to fibronectin as compared with mock transfectants. In contrast, attachment to human umbilical vein endothelial cells expressing E-selectin was significantly enhanced by GnT-V expression (p < 0.01). Sialyl Lewis x is a ligand for E-selectin and a marker for poor prognosis of colon cancer. Its synthesis in cells has been shown to involve GnT-V. We demonstrated that expression of sialyl Lewis x in colon cancer cells was induced by GnT-V expression. These results suggest that GnT-V induces sialyl Lewis x expression and leads colon cancer cells to metastasize by enhancing their ability to attach to vascular endothelium in distant organs, such as liver or lung. Inhibition of GnT-V activity may prevent metastasis in colon cancer patients with high sialyl Lewis x expression.     

Increased level of circulating adhesion molecules in the sera of breast cancer patients with distant metastases

The adhesion of circulating cancer cells to the vascular endothelium is an important at step in the hematogenous metastasis of cancer. E-selectin expressed on endothelial cells and carbohydrate ligands expressed on cancer cells mediate this adhesion. We investigated the clinical significance of such cell adhesion molecules in breast cancer. The cytosol concentration of sialyl Lewis(x) was found more elevated in cancerous tissue than that in adjacent non-cancerous tissue. In the serum, sialyl Lewis(x) and soluble E-selectin were seen elevated in patients with advanced and recurrent breast cancer, especially in those with distant metastases. From the above, we have concluded that sialyl Lewis(x) and soluble E-selectin could be used as tumor markers with a close relationship to the metastasis of breast cancer.      

Production of interleukin-1β and interleukin-6 in hepatoblastoma

Thrombocytosis and fever are frequent symptoms in children with hepatoblastoma. Interleukin-6 (IL-6) has been shown to mediate thrombocytosis and an acute-phase reaction including fever. We therefore investigated samples from I4 untreated patients with hepatoblastoma for this cytokine and in addition for interleukin-I α (IL-Iα), interleukin-Iβ (IL-Iβ) and tumor necrosis factor-α (TNF-α), all of which are known to induce IL-6 production. High serum levels of IL-6 were only found in 3/I4 patients; the other cytokines were not detectable. In contrast, I2/I4 tumors produced substantial amounts of IL-6 in primary cell culture, while IL-Iβ was found in 3/I4 supernatants; IL-Iα and TNF-α were always negative. Immunoenzymatic staining of fresh tumors revealed that IL-6 is not produced by the tumor cells, but rather by surrounding fibroblasts and endothelial cells. In tumor cells only IL-Iβ, but neither IL-Iα, TNF-α nor IL-6, could be detected. In co-culture experiments with fibroblasts and endothelial cells, addition of hepatoblastoma cells enhanced IL-6 production. Including an IL-I receptor antagonist abolished this effect incompletely. Our results suggest that tumor cells in hepatoblastoma induce IL-6 production in surrounding fibroblasts and endothelial cells by virtue of their endogenous secretion of IL-Iβ and supposedly some other, as yet unidentified, mediator.     

Liver endothelial E-selectin mediates carcinoma cell adhesion and promotes liver metastasis

E-selectin is a cytokine-inducible endothelial cell adhesion receptor which is involved in the process of leukocyte rolling, the first in a cascade of interactions leading to leukocyte transmigration. Several studies have implicated this receptor in carcinoma cell adhesion to the endothelium, an interaction thought to be required for tumor extravasation during metastasis. To study the role of this receptor in the process of metastasis, we utilized a murine carcinoma line H-59 which is highly metastatic to the liver in vivo. When adhesion of H-59 cells to primary cultures of murine hepatic endothelial cells was measured, it was found that the tumor cells had a low which basall of adhesion to the sinusoidal endothelial cells, which could be significantly and specifically augmented by pre-activation of the endothelial cells with rTNFα. This incremental increase in adhesion to the activated endothelium could be completely and specifically abolished by a neutralizing monoclonal antibody to murine E-selectin (MAb 9A9). Similar results were obtained with 2 highly metastatic human colorectal carcinoma lines, HM 7 and CX-1, but not with a second murine subline, M-27, which is poorly metastatic to the liver. To assess the role of E-selectin in metastasis to the liver in vivo, the effect of MAb 9A9 on experimental liver metastasis was evaluated using the syngeneic H-59 model. We show here that this antibody caused a marked, specific and Fc-independent inhibition of experimental liver metastasis, reducing the median number of metastases by 97% relative to the control groups. Our results provide evidence that endothelial E-selectin is a mediator of carcinoma metastasis to the liver. Int. J. Cancer 71:612-619, 1997. © 1997 Wiley-Liss, Inc.     

E-选择素介导肝癌细胞HepG2和内皮细胞的早期黏附

目的　探讨sLeX 和E 选择素在肝癌细胞与内皮细胞早期黏附中的作用 ,并筛选有临床应用前景的抗黏附药物。方法　应用荧光活细胞染料BCECF -AM标记HepG2肝癌细胞。应用体外黏附实验检测肝癌细胞HepG2和人脐静脉内皮细胞 (HUVEC)的黏附 ,并测试不同药物对二者黏附的影响。结果　sLeX 和E 选择素是肝癌细胞与内皮细胞早期黏附的必要条件之一。HepG2条件培养液处理HUVEC可促进其与HepG2的黏附 ;地塞米松、氧化苯砷、左旋咪唑、放线菌素和sLeX 的类似物在低浓度下即有明显的抗黏附作用。结论　肝癌细胞可能分泌炎症因子样物质 ,促进靶组织内皮细胞的活化 ,地塞米松、氧化苯砷、左旋咪唑、放线菌素和sLeX 的类似物可能成为较有前景的、预防肝癌转移的抗黏附药物     

IL-1α gene-transfected human melanoma cells increase tumor-cell adhesion to endothelial cells and their retention in the lung of nude mice

The interleukin-1α (IL-I) gene was introduced by retroviral gene transfer into the A375P human melanoma cell line. Two hygromycin-resistant colonies, colony 3 and colony 6, which respectively do not and do express and release IL-I, were selected on the basis of Northern blot and ELISA. Both colonies adhered to resting human endothelial cells (EC) to the same extent. Pre-treatment of EC for 6 hr with conditioned medium (CM) from colony 6, but not from colony 3, increased the adhesion of A375P melanoma and HT-29 colon-carcinoma cells to EC. This increase was blocked by adding interleukin-I-receptor antagonist (IL-I ra) to the EC monolayer. Treatment of EC with colony-6-CM increased the expression of intercellular-adhesion molecule I (ICAM-I), vascular-cell-adhesion molecule I (VCAM-I) and E-selectin. Co-cultivation of colony-6 but not colony-3 melanoma cells with EC caused time-dependent increased expression of these adhesion proteins, reflecting their kinetics of expression on EC. Treating the EC with monoclonal antibodies to VCAM-I and E-selectin abolished the colony-6-CM-induced increase in adhesion respectively to A375P melanoma and HT-29 colon-carcinoma cells. In vivo, i.v. injection of colony-6 cells in nude mice increased the expression of VCAM-I on lung microvascular EC. The retention of radiolabeled A375P melanoma cells in the lung was increased in nude mice primed with colony-6 cells, but not with colony-3 cells, injected 6 hr earlier. These results demonstrate that IL-I produced constitutively by transformed A375P melanoma cells is functionally active, inducing adhesion molecules on EC that enhance their adhesiveness for tumor cells and increase tumor-cell retention in the lung of nude mice. © 1996 Wiley-Liss, Inc.     

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 β-1,6- N -acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

Background  

The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLe^x) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 N -acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLe^x (C2-O-sLe^x). Our goal was to determine the expression profiles of C2-O-sLe^x in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLe^x present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLe^x was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6- N -acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).