alpha-Neup5Ac-(2--3)-beta-D-Galp-(1--4)-[alpha-L-Fucp-(1--3)]-D-GlcpNAc and Neoplasm-Metastasis

Aberrant expression of glycoconjugates occurs during malignant transformation of cancer cells. Overexpression of sialoglycoconjugates in particular may play an important role in the progression, i.e., invasion or metastasis, of cancer. Various types of sialoglycoconjugates have been investigated to clarify their biological significance and clinical utility in diagnosing and treating colorectal cancer. This review focuses specifically on expression of mucin (MUC) 1 and it suggests that MUC1 with the specific structure of a sialo-oligosaccharide has biological significance in determining the metastatic potential of colorectal cancer cells and clinicopathological utility in evaluating the effectiveness of treatments and the prognosis for patients with colorectal cancer. Further studies are expected to contribute to the expanded use of cancer-associated sialoglycoconjugates in cancer diagnosis and therapy.

Topics: Antigens, Tumor-Associated, Carbohydrate; CA-19-9 Antigen; Colorectal Neoplasms; Diagnostic Imaging; Gene Expression Regulation, Neoplastic; Glycoconjugates; Humans; Mucin-1; Neoplasm Invasiveness; Neoplasm Metastasis; Oligosaccharides; Prognosis; Sialic Acids; Sialyl Lewis X Antigen

Tumor cells exhibit striking changes in cell surface glycosylation as a consequence of dysregulated glycosyltransferases and glycosidases. In particular, an increase in the expression of certain sialylated glycans is a prominent feature of many transformed cells. Altered sialylation has long been associated with metastatic cell behaviors including invasion and enhanced cell survival; however, there is limited information regarding the molecular details of how distinct sialylated structures or sialylated carrier proteins regulate cell signaling to control responses such as adhesion/migration or resistance to specific apoptotic pathways. The goal of this review is to highlight selected examples of sialylated glycans for which there is some knowledge of molecular mechanisms linking aberrant sialylation to critical processes involved in metastasis.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Cell Movement; Glycosylation; Humans; Integrins; Lewis X Antigen; N-Acetylneuraminic Acid; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Phenotype; Polysaccharides; Sialyl Lewis X Antigen

Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewisa and sialyl Lewisx. The sialyl Lewisa and sialyl Lewisx determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewisa and sialyl Lewisx are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewis(a/x) determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system.

Topics: Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; CA-19-9 Antigen; Carbohydrate Metabolism; Cell Adhesion; Cell Transformation, Neoplastic; Endothelium; Gangliosides; Humans; Lewis Blood Group Antigens; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Oligosaccharides; Selectins; Sialyl Lewis X Antigen

The carbohydrate determinants, sialyl Lewis A and sialyl Lewis X, which are frequently expressed on human cancer cells, serve as ligands for a cell adhesion molecule of the selectin family, E-selectin, which is expressed on vascular endothelial cells. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelium and thus contribute to hematogenous metastasis of cancer. The initial adhesion mediated by these molecules triggers activation of integrin molecules through the action of several cytokines and leads to the extravasation of cancer cells. Cancer cells also produce humoral factors that facilitate E-selectin expression on endothelial cells. The degree of expression of the carbohydrate ligands at the surface of cancer cells is well correlated with the frequency of hematogenous metastasis and prognostic outcome of patients with cancers. The alteration of glycosyltransferase activities that leads to the enhanced expression of these carbohydrate ligands on cancer cell surface are currently being investigated.

Topics: Animals; Biomarkers, Tumor; Cell Adhesion; E-Selectin; Glycosyltransferases; Humans; Lewis X Antigen; Models, Biological; Neoplasm Metastasis; Neoplasms; Oligosaccharides; P-Selectin; Prognosis; Sialyl Lewis X Antigen

Cimetidine has been shown to have beneficial effects in colorectal cancer patients. In this study, a total of 64 colorectal cancer patients who received curative operation were examined for the effects of cimetidine treatment on survival and recurrence. The cimetidine group was given 800 mg day(-1) of cimetidine orally together with 200 mg day(-1) of 5-fluorouracil, while the control group received 5-fluorouracil alone. The treatment was initiated 2 weeks after the operation and terminated after 1 year. Robust beneficial effects of cimetidine were noted: the 10-year survival rate of the cimetidine group was 84.6% whereas that of control group was 49.8% (P<0.0001). According to our previous observations that cimetidine blocked the expression of E-selectin on vascular endothelium and inhibited the adhesion of cancer cells to the endothelium, we have further stratified the patients according to the expression levels of sialyl Lewis antigens X (sL(x)) and A (sL(a)). We found that cimetidine treatment was particularly effective in patients whose tumour had higher sL(x) and sL(a) antigen levels. For example, the 10-year cumulative survival rate of the cimetidine group with higher CSLEX staining, recognizing sL(x), of tumours was 95.5%, whereas that of control group was 35.1% (P=0.0001). In contrast, in the group of patients with no or low levels CSLEX staining, cimetidine did not show significant beneficial effect (the 10-year survival rate of the cimetidine group was 70.0% and that of control group was 85.7% (P=n.s.)). These results clearly indicate that cimetidine treatment dramatically improved survival in colorectal cancer patients with tumour cells expressing high levels of sL(x) and sL(a).

Topics: Administration, Oral; Adult; Aged; Antimetabolites, Antineoplastic; Biomarkers, Tumor; CA-19-9 Antigen; Cell Adhesion; Chemotherapy, Adjuvant; Cimetidine; Colorectal Neoplasms; Female; Fluorouracil; Gangliosides; Histamine H2 Antagonists; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Oligosaccharides; Sialyl Lewis X Antigen; Survival Analysis; Treatment Outcome

Nuclear texture reflects the overall structures of the chromatin organization. We recently reported the principles and prognostic importance of image analysis of nuclei from metastatic prostate cancer. Immunohistochemical up regulation of the adhesion molecule sialyl Lewis(x) is also reported to be a prognostic parameter. Presently we analyzed statistically the prognostic impact of these 2 new parameters compared to well-known clinical parameters in metastatic prostate cancer.. Prognostic factors, such as sedimentation rate, alkaline and acid phosphatases, hemoglobin, testosterone, performance status, pain due to metastasis, T category, histological grade and patient age, were included in a multivariate Cox proportional hazards regression analysis based on 262 patients from the Scandinavian Prostatic Cancer Group Study-2. Extent of bone lesions, deoxyribonucleic acid ploidy, texture analysis and sialyl Lewis(x) molecules based on subsets of these 262 patients were also analyzed in the same multivariate model.. This test identified chromatin texture as the most important factor (p < 0.001), followed by reaction of the oligosaccharide sialyl Lewis(x) (p < 0.01). Among the routine clinical and laboratory data, sedimentation rate, alkaline phosphatase and hemoglobin (p < 0.05) showed prognostic importance. Performance status, pain due to metastasis and extent of bone lesions showed prognostic value in the univariate analysis (p < 0.05).. These data indicate that computerized nuclear texture analysis as well as up regulation of sialyl Lewis(x) molecules may be new important prognostic factors in metastatic prostate cancer. Furthermore the prognostic importance of sedimentation rate, alkaline phosphatase and hemoglobin was confirmed.

Topics: Aged; Humans; Lewis X Antigen; Male; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Staging; Oligosaccharides; Prognosis; Proportional Hazards Models; Prospective Studies; Prostatic Neoplasms; Retrospective Studies; Sialyl Lewis X Antigen; Survival Rate

Expression of sialyl-Lewisx (sLex) antigen was studied immunohistochemically in 110 resected human gastric carcinomas using an anti-sLex monoclonal antibody. Lymph node, liver, and peritoneal metastases were clearly more prevalent in tumors expressing high levels of sLex than in those with no or low-level sLex expression. No correlation was found between sLex expression and histologic grade or histologic type of the Lauren classification. Among the tumors with lymph node metastasis, 44% expressed high levels of sLex in both the primary tumor and involved lymph nodes, and 14% of the metastatic lesions demonstrated increased sLex expression. The 5-year survival rate of the patients undergoing complete (R0) gastric resections was 60% in the sLex high-expression group, which was significantly lower than that of the sLex low-expression group (81%) and of the no-expression group (87%) (p < 0.05). These results suggest that high-level sLex expression is related to both an increased risk of metastasis and poor prognosis in gastric cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Female; Gastrectomy; Humans; Immunohistochemistry; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Metastasis; Oligosaccharides; Peritoneal Neoplasms; Prognosis; Risk; Sialyl Lewis X Antigen; Stomach Neoplasms; Survival Rate

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.

Topics: Animals; Bortezomib; CA-19-9 Antigen; Cell Adhesion; E-Selectin; Humans; Ligands; Lung Neoplasms; Mice; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

The formation of distant metastasis resulting from vascular dissemination is one of the leading causes of mortality in non‑small cell lung cancer (NSCLC). This metastatic dissemination initiates with the adhesion of circulating cancer cells to the endothelium. The minimal requirement for the binding of leukocytes to endothelial E‑selectins and subsequent transmigration is the epitope of the fucosylated glycan, sialyl Lewis x (sLex), attached to specific cell surface glycoproteins. sLex and its isomer sialyl Lewis a (sLea) have been described in NSCLC, but their functional role in cancer cell adhesion to endothelium is still poorly understood. In this study, it was hypothesised that, similarly to leukocytes, sLe glycans play a role in NSCLC cell adhesion to E‑selectins. To assess this, paired tumour and normal lung tissue samples from 18 NSCLC patients were analyzed. Immunoblotting and immunohistochemistry assays demonstrated that tumour tissues exhibited significantly stronger reactivity with anti‑sLex/sLea antibody and E‑selectin chimera than normal tissues (2.2‑ and 1.8‑fold higher, respectively), as well as a higher immunoreactive score. High sLex/sLea expression was associated with bone metastasis. The overall α1,3‑fucosyltransferase (FUT) activity was increased in tumour tissues, along with the mRNA levels of FUT3, FUT6 and FUT7, whereas FUT4 mRNA expression was decreased. The expression of E‑selectin ligands exhibited a weak but significant correlation with the FUT3/FUT4 and FUT7/FUT4 ratios. Additionally, carcinoembryonic antigen (CEA) was identified in only 8 of the 18 tumour tissues; CEA‑positive tissues exhibited significantly increased sLex/sLea expression. Tumour tissue areas expressing CEA also expressed sLex/sLea and showed reactivity to E‑selectin. Blot rolling assays further demonstrated that CEA immunoprecipitates exhibited sustained adhesive interactions with E‑selectin‑expressing cells, suggesting CEA acts as a functional protein scaffold for E‑selectin ligands in NSCLC. In conclusion, this work provides the first demonstration that sLex/sLea are increased in primary NSCLC due to increased α1,3‑FUT activity. sLex/sLea is carried by CEA and confers the ability for NSCLC cells to bind E‑selectins, and is potentially associated with bone metastasis. This study contributes to identifying potential future diagnostic/prognostic biomarkers and therapeutic targets for lung cancer.

Topics: Aged; Carcinoembryonic Antigen; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; E-Selectin; Female; Fucosyltransferases; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Polysaccharides; Sialyl Lewis X Antigen

Organ-specific colonization suggests that specific cell-cell recognition is essential. Yet, very little is known about this particular interaction. Moreover, tumor cell lodgement requires binding under shear stress, but not static, conditions. Here, we successfully isolate the metastatic populations of cancer stem/tumor-initiating cells (M-CSCs). We show that the M-CSCs tether more and roll slower than the non-metastatic (NM)-CSCs, thus resulting in the preferential binding to the peritoneal mesothelium under ascitic fluid shear stress. Mechanistically, this interaction is mediated by P-selectin expressed by the peritoneal mesothelium. Insulin-like growth factor receptor-1 carrying an uncommon non-sulfated sialyl-Lewis

Topics: Animals; Ascitic Fluid; Carcinoma; Cell Adhesion; Cell Line, Tumor; Epithelium; Female; Fucosyltransferases; HEK293 Cells; Humans; Hydrodynamics; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Stem Cells; Oligosaccharides; Ovarian Neoplasms; P-Selectin; Peritoneal Neoplasms; Peritoneum; Receptor, IGF Type 1; Sialyl Lewis X Antigen; Stress, Mechanical

Topics: Animals; Cell Line, Tumor; Dextrans; Dynamic Light Scattering; E-Selectin; Female; Lymphatic Metastasis; Magnetic Resonance Imaging; Magnetite Nanoparticles; Male; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Oligosaccharides; Polyethylene Glycols; Sialyl Lewis X Antigen; Static Electricity; Thermodynamics

Distant metastases account for the majority of cancer-related deaths in breast cancer. The rate and site of metastasis differ between estrogen receptor (ER)-negative and ER-positive tumours, and metastatic fate can be very diverse even within the ER-negative group. Characterisation of new pro-metastatic markers may help to identify patients with higher risk and improve their care accordingly. Selectin ligands aberrantly expressed by cancer cells promote metastasis by enabling interaction between circulating tumour cells and endothelial cells in distant organs. These ligands consist in carbohydrate molecules, such as sialyl-Lewis x antigen (sLex), borne by glycoproteins or glycolipids on the cancer cell surface. We have previously demonstrated that the molecular scaffold presenting sLex to selectins (e.g. glycolipid vs. glycoproteins) was crucial for these interactions to occur. Moreover, we reported that detection of sLex alone in breast carcinomas was only of limited prognostic value. However, since sLex was found to be carried by several glycoproteins in cancer cells, we hypothesized that the combination of the carbohydrate with its carriers could be more relevant than each marker independently. In this study, we addressed this question by analysing sLex expression together with two glycoproteins (BST-2 and LGALS3BP), shown to interact with E-selectin in a carbohydrate-dependent manner, in a cohort of 249 invasive breast cancers. We found both glycoproteins to be associated with distant metastasis risk and poorer survival. Importantly, concomitant high expression of BST-2 with sLex defined a sub-group of patients with ER-negative tumours displaying higher risks of liver and brain metastasis and a 3-fold decreased survival rate.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Disease-Free Survival; E-Selectin; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; GPI-Linked Proteins; Humans; Lewis X Antigen; Ligands; Middle Aged; Neoplasm Metastasis; Prognosis; Sialyl Lewis X Antigen

Oral squamous cell carcinoma (OSCC) is an aggressive malignancy with high mortality rates. Major challenges for OSCC management include development of resistance to therapy and early formation of distant metastases. Cancer stem cells (CSCs) have emerged as important players in both pathologic mechanisms. Increased fucosylation activity and increased expression of fucosylated polysaccharides, such as Sialyl Lewis X (SLex), are associated with invasion and metastasis. However, the role of fucosylation in CSCs has not been elucidated yet. We used the spheroid culture technique to obtain a CSC-enriched population and compared orospheres with adherent cells. We found that orospheres expressed markers of CSCs and metastasis at higher levels, were more invasive and tumorigenic, and were more resistant to cisplatin/radiation than adherent counterparts. We found fucosyltransferases FUT3 and FUT6 highly up-regulated, increased SLex expression and increased adhesion by shear flow assays in orospheres. Inhibition of fucosylation negatively affected orospheres formation and invasion of oral CSCs. These results confirm that orospheres are enriched in CSCs and that fucosylation is of paramount importance for CSC invasion. In addition, SLex may play a key role in CSC metastasis. Thus, inhibition of fucosylation may be used to block CSCs and metastatic spread.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Cell Adhesion; Cisplatin; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Fucose; Head and Neck Neoplasms; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Oligosaccharides; Shear Strength; Sialyl Lewis X Antigen; Stress, Mechanical

The metastasis of hematogenous cancer cells is associated with abnormal glycosylation such as sialyl lewis antigens. Although the hepatitis B virus X protein (HBx) plays important role in liver disease, the precise function of HBx on aberrant glycosylation for metastasis remains unclear.. The human hepatocellular carcinoma tissues, HBx transgenic mice and HBx-transfected cells were used to check the correlation of expressions between HBx and Sialyl lewis antigen for cancer metastasis. To investigate whether expression levels of glycosyltransferases induced in HBx-transfected cells are specifically associated with sialyl lewis A (SLA) synthesis, which enhances metastasis by interaction of liver cancer cells with endothelial cells, ShRNA and siRNAs targeting specific glycosyltransferases were used.. HBx expression in liver cancer region of HCC is associated with the specific synthesis of SLA. Furthermore, the SLA was specifically induced both in liver tissues from HBx-transgenic mice and in in vitro HBx-transfected cells. HBx increased transcription levels and activities of α2-3 sialyltransferases (ST3Gal III), α1-3/4 fucosyltransferases III and VII (FUT III and VII) genes, which were specific for SLA synthesis, allowing dramatic cell-cell adhesion for metastatic potential. Interestingly, HBx specifically induced expression of N-acetylglucosamine-β1-3 galactosyltransferase V (β1-3GalT 5) gene associated with the initial synthesis of sialyl lewis A, but not β1-4GalT I. The β1-3GalT 5 shRNA suppressed SLA expression by HBx, blocking the adhesion of HBx-transfected cells to the endothelial cells. Moreover, β1-3GalT 5 silencing suppressed lung metastasis of HBx-transfected cells in in vivo lung metastasis system.. HBx targets the specific glycosyltransferases for the SLA synthesis and this process regulates hematogenous cancer cell adhesion to endothelial cells for cancer metastasis.

Topics: Adult; Animals; CA-19-9 Antigen; Carcinoma, Hepatocellular; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Glycosylation; Glycosyltransferases; Hepatitis B virus; Human Umbilical Vein Endothelial Cells; Humans; Lewis X Antigen; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Trans-Activators; Viral Regulatory and Accessory Proteins

Sialyltransferases have received much attention recently as they are frequently up-regulated in cancer cells. However, the role played by each sialyltransferase in tumour progression is still unknown. α2,3-Sialyltransferases ST3Gal III and ST3Gal IV are involved in sialyl-Lewis(x) (SLe(x)) synthesis. Given that the role of ST3Gal III in pancreatic adenocarcinoma cells has been previously reported, in this study we have focused on investigating the role of ST3Gal IV in the acquisition of adhesive, migratory and metastatic capabilities and, secondly, in analyzing the expression of ST3Gal III and ST3Gal IV in pancreatic adenocarcinoma tissues versus control tissues. ST3Gal IV overexpressing pancreatic adenocarcinoma MDAPanc-28 cell lines were generated. They showed a heterogeneous increase in SLe(x), and enhanced E-selectin adhesion and migration. Furthermore, when injected into nude mice, increased metastasis and decreased survival were found in comparison with controls. The behaviour of MDAPanc-28 ST3Gal IV overexpressing cells in these processes was similar to the already reported MDAPanc-28 ST3Gal III overexpressing cells. Furthermore, pancreatic adenocarcinoma tissues tended to express high levels of ST3Gal III and ST3Gal IV together with other fucosyltransferase genes FUT3 and FUT6, all involved in the last steps of sialyl-Lewis(x) biosynthesis. In conclusion, both α2,3-sialyltransferases are involved in key steps of pancreatic tumour progression processes and are highly expressed in most pancreatic adenocarcinoma tissues.

Topics: Adenocarcinoma; Aged; Animals; beta-Galactoside alpha-2,3-Sialyltransferase; Cell Membrane; Cell Movement; E-Selectin; Female; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Mice; Mice, Nude; Middle Aged; N-Acetylneuraminic Acid; Neoplasm Metastasis; Oligosaccharides; Pancreatic Neoplasms; Protein Binding; RNA, Messenger; Sialyl Lewis X Antigen; Sialyltransferases

Radical segmentectomy has been performed for small-sized non-small cell lung cancer (NSCLC). However, underestimation of mediastinal lymph node metastasis in the absence of hilar or interlobar metastasis (skip N2) affects surgical strategy. Our aim was to investigate preoperative and intraoperative predictors of skip N2 in clinical stage (c-stage) IA NSCLC.. From 1998 to 2011, 279 patients (155 men and 124 women) with c-stage IA NSCLC (230 pN0, 17 pN1, 12 skip N2, 20 non-skip N2) underwent systematic lobectomy (R0 resection) at our institute. We compared preoperative serum concentrations of carcinoembryonic antigen, cytokeratin 19 fragment, sialyl Lewis X (SLX), and pre- and intraoperative clinicopathological features of pN0 and skip N2 patients. Receiver operator characteristic (ROC) curve analysis was performed to distinguish between the two patient groups.. The 5-year survival rate of skip N2 patients was 78.6%, higher than that of non-skip N2 patients (44.9%), and not significantly different than that of pN0 (86.7%) or pN1 patients (82.4%). The mean serum SLX concentration in skip N2 patients (28.0 U/ml) was elevated compared to that in pN0 patients (22.9 U/ml). In ROC analysis of SLX, the area under the curve was 0.710, and the optimal cut-off value was 21.4 U/ml (sensitivity, 91.7%; specificity, 51.7%). In multivariate analysis, SLX was an independent predictor of skip N2 in patients with c-stage IA NSCLC (odds ratio, 9.43; p = 0.006).. Skip N2 metastasis is common in patients with c-stage IA NSCLC with high serum SLX, and lobectomy with complete dissection of hilar and mediastinal lymph nodes should remain the standard surgical procedure for these cases.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Non-Small-Cell Lung; Female; Follow-Up Studies; Humans; Keratin-19; Lung Neoplasms; Male; Neoplasm Metastasis; Neoplasm Staging; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen; Survival Rate

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLe(x) expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of 'triple-negative' breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments.

Topics: Anti-Inflammatory Agents; Breast Neoplasms; Cell Adhesion; Cell Aggregation; Cell Line, Tumor; Cell Proliferation; Cytokines; E-Selectin; Endothelium; Female; Humans; Hyaluronan Receptors; Inflammation Mediators; Interleukin-6; Metformin; Neoplasm Metastasis; Neoplastic Cells, Circulating; Oligosaccharides; Phenotype; Sialyl Lewis X Antigen; Spheroids, Cellular; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Sialyl Lewis x (sLex) is a selectin ligand whose overexpression in epithelial cancers mediates metastasis formation. The molecular basis of sLex biosynthesis in colon cancer tissues is still unclear. The prerequisite for therapeutic approaches aimed at sLex down-regulation in cancer, is the identification of rate-limiting steps in its biosynthesis. We have studied the role of α1,3-fucosyltransferases (Fuc-Ts) potentially involved in sLex biosynthesis in specimens of normal and cancer colon as well as in experimental systems. We found that: (i) in colon cancer, but not in normal mucosa where the antigen was poorly expressed, sLex correlated with a Fuc-T which, like Fuc-TVI, was active on 3'sialyllactosamine at a low concentration (Fuc-T(SLN)); (ii) competitive RT-PCR analysis revealed that the level of Fuc-T mRNA expression in both normal and cancer colon was Fuc-TVI>Fuc-TIII>Fuc-TIV; Fuc-TV and Fuc-TVII expression was negligible; (iii) sLex was expressed only by the gastrointestinal cell lines displaying both Fuc-TVI mRNA and Fuc-T(SLN) activity, but not by those expressing only Fuc-TIII mRNA; (iv) transfection with Fuc-TVI cDNA, but not with Fuc-TIII cDNA, induced sLex expression in gastrointestinal cell lines; (v) Fuc-TVI knock-down with specific siRNA induced down-regulation of Fuc-TVI mRNA and Fuc-T(SLN) activity and a dramatic inhibition of sLex expression. These data indicate that in colon cancer tissues Fuc-TVI is a key regulator of sLex biosynthesis which can be the target of RNA-interference-based gene knock-down approaches.

Topics: Base Sequence; Colorectal Neoplasms; DNA, Complementary; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycosylation; Humans; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; RNA Interference; Selectins; Sialyl Lewis X Antigen; Transfection; Tumor Cells, Cultured

Metastatic breast cancer (MBC) is currently an incurable condition that is primarily treated with palliative measures. Isolation of circulating tumor cells (CTCs) from the peripheral blood of these patients provides a predictive prognostic indicator, independent of the type of therapy, site of occurrence and biological characteristics of the primary disease. It has been well established that glycosylation processing pathways are disturbed in cancer, leading to alterations in the glycan content of glycoproteins.. The bi-, tri- and tetraantennary glycans containing sialyl Lewis x (sLe(x)) epitopes (A2F1G1, A3F1G1, A4F1G1 and A4F2G2) were quantified using normal phase high-performance liquid chromatography in combination with exoglycosidase array digestions in the glycan pools released from sera of 27 patients with advanced breast cancer (16 with CTCs <5/7.5 ml and 11 with CTCs ≥5/7.5 ml) and 13 healthy women.. The levels of all these glycans were significantly higher in patients with CTCs ≥5/7.5 ml compared with patients with CTCs <5/7.5 ml.. As high levels of glycans containing sLe(x) epitopes were associated with CTCs, their measurement may provide a new noninvasive approach for determining prognosis in women with MBC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Case-Control Studies; Cell Count; Chromatography, High Pressure Liquid; Female; Humans; Middle Aged; Neoplasm Metastasis; Neoplastic Cells, Circulating; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen

Cimetidine has been shown to have anti-metastatic activity and improves the survival of patients with colorectal cancer. One hypothesis is its modulation of the expression of the cell adhesion molecule by target organ endothelial cells. Because of the inconclusive results in clinical trials of gastric cancer, we investigated the effects of cimetidine on the adhesion of gastric cancer cells to activated endothelial cells and on the expression of some cell adhesion molecules. Human endothelial cells were pre-incubated with cimetidine for 6 h, incubated with the cytokine tumor necrosis factor for 4 h, and the endothelial surface expression of E-selectin was evaluated by flow cytometry, immunostaining and ELISA. Further, we investigated E-selectin mRNA expression by RT-PCR. Three gastric cancer cell lines (SGC-7901, MGC-803, BGC-823) and a normal gastric epithelial cell line, GES-1, were studied for the surface expression of sialyl Lewis x by flow cytometry and immunostaining. Adherence of CFSE-labeled gastric cancer cells and GES-1 cells to endothelial cell monolayers was determined. Cimetidine significantly reduced E-selectin expression of activated endothelial cells, but did not influence E-selectin expression at the mRNA level. Three gastric cancer cell lines expressed high levels of sialyl Lewis x, whereas GES-1 did not. Cimetidine also significantly decreased gastric cancer cell adherence to stimulated endothelial cells. The inhibition of E-selectin expression corresponded to the reduction of tumor cell adherence. The effects of cimetidine on tumor adhesion were almost nullified by pre-incubation with E-selectin and sialyl Lewis x antibody. Furthermore, there was no significant change of GES-1 adherence to endothelial cells by TNF-α, cimetidine, E-selectin and sialyl Lewis x antibody. The inhibiton of gastric cancer cell adherence to cytokine-stimulated endothelial cells treated with cimetidine appears to result from blocking endothelial E-selectin expression. These data support the hypothesis that cimetidine may exert its anti-metastatic effects in gastric cancer, in part, by inhibiting E-selectin/sialyl Lewis x-mediated adherence of gastric cancer cells to endothelial cells in the metastasis target organs.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cimetidine; E-Selectin; Endothelial Cells; Enzyme Inhibitors; Female; Humans; Liver; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Oligosaccharides; Random Allocation; Sialyl Lewis X Antigen; Stomach Neoplasms; Tumor Necrosis Factor-alpha

The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers. We found that genes involved in the synthesis of sialyl-Lewis x (sLe(x); FUT3, FUT4, and ST3GAL6) are significantly increased in estrogen receptor alpha-negative (ER-negative) tumors compared with ER-positive ones. SLe(x) expression had no influence on the survival of patients whether they had ER-negative or ER-positive tumors. However, high expression of sLe(x) in ER-positive tumors was correlated with metastasis to the bone where sLe(x) receptor E-selectin is constitutively expressed. The ER-positive ZR-75-1 and the ER-negative BT20 cell lines both express sLe(x) but only ZR-75-1 cells could adhere to activated endothelial cells under dynamic flow conditions in a sLe(x) and E-selectin-dependent manner. Moreover, L/P-selectins bound strongly to ER-negative MDA-MB-231 and BT-20 cell lines in a heparan sulfate (HS)-dependent manner that was independent of sLe(x) expression. Expression of glycosylation genes involved in heparan biosynthesis (EXT1 and HS3ST1) was increased in ER-negative tumors. Taken together, our results suggest that the context of sLe(x) expression is important in determining its functional significance and that selectins may promote metastasis in breast cancer through protein-associated sLe(x) and HS glycosaminoglycans.

Topics: beta-Galactoside alpha-2,3-Sialyltransferase; Blotting, Western; Breast Neoplasms; Cell Adhesion; Cell Line; Cell Line, Tumor; E-Selectin; Female; Fucosyltransferases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycomics; Heparitin Sulfate; Human Umbilical Vein Endothelial Cells; Humans; Lewis X Antigen; N-Acetylglucosaminyltransferases; Neoplasm Metastasis; Neoplasms, Hormone-Dependent; Oligonucleotide Array Sequence Analysis; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; Sialyl Lewis X Antigen; Sialyltransferases; Sulfotransferases

How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that alpha1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that alpha1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.

Topics: Bone Marrow; Bone Marrow Neoplasms; Carcinoembryonic Antigen; CD146 Antigen; Cell Adhesion; Cell Movement; E-Selectin; Fucosyltransferases; Humans; Hyaluronan Receptors; Liver; Liver Neoplasms; Male; Neoplasm Metastasis; Oligosaccharides; Prostatic Neoplasms; Sialoglycoproteins; Sialyl Lewis X Antigen

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.

Topics: CA-19-9 Antigen; CD18 Antigens; Cell Adhesion; Cell Line, Tumor; Cell Movement; Endothelial Cells; Humans; Integrin alpha4beta1; Melanoma; Neoplasm Metastasis; Neutrophils; Oligosaccharides; Selectins; Sialyl Lewis X Antigen; Stress, Mechanical; Vascular Cell Adhesion Molecule-1

The sialyl Lewis x tetrasaccharide with a propylamine aglycon was assembled by chemoselective glycosylation from a p-tolyl thioglycosyl donor obtained from an enzymatically synthesized sialodisaccharide. Combining the advantages of highly efficient enzymatic synthesis of sialoside building blocks, and diverse chemical glycosylation, this chemoenzymatic approach is practical for obtaining complex sialosides and their analogues.

Topics: Humans; Inflammation; Models, Molecular; Molecular Conformation; Neoplasm Metastasis; Oligosaccharides; Selectins; Sialic Acids; Sialyl Lewis X Antigen

Prostate cancer (PCa) cell tethering and rolling on microvascular endothelium has been proposed to promote the extravasation of PCa cells. We have shown that these adhesive events are mediated through binding interactions between endothelial (E)-selectin and Lewis carbohydrates on PCa cells. Prior data indicate that E-selectin-mediated rolling of bone-metastatic PCa MDA PCa 2b (MDA) cells is dependent on sialyl Lewis X (sLe(X))-bearing glycoproteins. To explore the molecular basis of sLe(X) synthesis and E-selectin ligand (ESL) activity on PCa cells, we compared and contrasted the expression level of glycosyltransferases, characteristically involved in sLe(X) and ESL synthesis, in ESL(+) MDA cells among other ESL(-) metastatic PCa cell lines. We also created and examined ESL(hi) and ESL(lo) variants of MDA cells to provide a direct comparison of the glycosyltransferase expression level. We found that normal prostate tissue and all metastatic PCa cell lines expressed glycosyltransferases required for sialo-lactosamine synthesis, including N-acetylglucosaminyl-, galactosyl-, and sialyltransferases. However, compared with expression in normal prostate tissue, ESL(+) MDA cells expressed a 31- and 10-fold higher level of alpha1,3 fucosyltransferases (FT) 3 and 6, respectively. Moreover, FT3 and FT6 were expressed at 2- to 354-fold lower levels in ESL(-) PCa cell lines. Consistent with these findings, ESL(hi) MDA cells expressed a 131- and 51-fold higher level of FT3 and FT6, respectively, compared with expression in ESL(lo) MDA cells. We also noted that alpha1,3 FT7 was expressed at a 5-fold greater level in ESL(hi) MDA cells. Furthermore, ESL(lo) MDA cells did not display sLe(X) on glycoproteins capable of bearing sLe(X), notably P-selectin glycoprotein ligand-1. These results implicate the importance of alpha1,3 FT3, FT6, and/or FT7 in sLe(X) and ESL synthesis on metastatic PCa cells.

Topics: Cell Line, Tumor; E-Selectin; Endothelial Cells; Glycosyltransferases; Humans; Ligands; Male; Microcirculation; Neoplasm Metastasis; Oligosaccharides; Prostatic Neoplasms; Protein Binding; Sialyl Lewis X Antigen

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLe(x)). SLe(x) oligosaccharide on tumor cells can be recognized by E- and P-selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E-selectin reactivity, which was sLe(x) dependent, P-selectin reactivity with this cell line was sLe(x)-independent. The sLe(x)-Neg variant of the 4T1 cell line with markedly diminished expression of sLe(x) and lack of sLe(a), provided a unique opportunity to characterize P-selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E-selectin binding. We observed that P-selectin binding was Ca(2+)-independent and sulfation-dependent. We found that P-selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P-selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P-selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P-selectin-reactive ligands on the surface of human MDA-MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease.

Topics: Animals; Breast Neoplasms; Calcium; Cell Line, Tumor; Cell Membrane; Chondroitin Sulfates; Fucosyltransferases; Glycosaminoglycans; Heparin; Humans; Ligands; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; P-Selectin; Proteoglycans; Sialyl Lewis X Antigen; Transfection

Lewis antigens are terminal fucosylated oligosaccharides synthesized by the sequential action of several glycosyltransferases. The fucosyltransferases are the enzymes responsible for the addition of terminal fucose to precursor oligosaccharides attached to proteins or lipids. These oligosaccharides, defined as cell surface markers, have been implicated in different types of intercellular interactions and in adhesion and invasion processes. Transfection of HT-29/M3 colon cancer cells with the full length of human fucosyltransferase (FUT1), induces the synthesis of H type 2 and Lewis y antigens, associated with a decrease of sialyl-Lewis x. The capacity to develop primary tumors when cells were injected intrasplenically was similar in parental and FUT1-transfected cells, but the capacity to colonize the liver after spleen removal was significantly reduced in M3/FUT1 transfected cells. These results indicate that the expression of FUT1 induces changes in the metastatic capacity of HT-29/M3 colon cancer cells, as a consequence of the altered expression pattern of type 2 Lewis antigens. Also, an association between MUC5AC expression and the degree of gland differentiation in both primary splenic tumors and hepatic metastases was detected.

Topics: Adenocarcinoma; Colonic Neoplasms; E-Selectin; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; HT29 Cells; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Oligosaccharides; Protein Binding; Sialyl Lewis X Antigen; Transfection

Serum CA15-3 has been one of the most reliable tumor markers used in monitoring breast cancer patients; however, its sensitivity in detecting metastases is limited. To increase its sensitivity, the combined measurement of other tumor markers with CA15-3 was investigated.. Serum CA15-3, carcinoembryonic antigen (CEA) and sialyl Lewis X (CSLEX) were simultaneously measured in a prospective series of 455 postoperative breast cancer patients with or without metastasis. The diagnostic parameters sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for detecting metastases were compared. The correlation of values between pairs of tumor markers was analyzed. The efficacy of combined measurement of two different tumor markers was also evaluated.. The sensitivity for detecting metastases was 61.5, 56.9 and 52.3%; specificity was 97.2, 93.6 and 96.2%; PPV was 78.4, 59.7 and 69.4%; NPV was 93.8, 92.9 and 92.4%; and accuracy was 92.1, 88.8 and 89.9% for CA15-3, CEA and CSLEX, respectively. The values for CA15-3 were significantly correlated with those for CEA (P < 0.001) but not those for CSLEX. The combined measurement of CSLEX and CA15-3 increased the sensitivity by 17.0% but that of CEA and CA15-3 increased the sensitivity by only 10.8%. All diagnostic parameters for the combined measurement of CSLEX and CA15-3 were higher than those for the combined measurement of CEA and CA15-3.. These findings suggest that CSLEX may be more useful than CEA in combination with CA15-3 in monitoring breast cancer patients. The results of this study suggest that CSLEX may be more useful than CEA in combination with CA15-3 in monitoring breast cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Carcinoembryonic Antigen; Female; Humans; Middle Aged; Mucin-1; Neoplasm Metastasis; Oligosaccharides; Prospective Studies; Reproducibility of Results; Sensitivity and Specificity; Sialyl Lewis X Antigen

Interaction between tumor cells and endothelium plays a major role in cancer invasion and metastasis. Among various cell adhesion molecules, the cognate interaction between sialyl Lewis antigen expressed in the tumor cell surface and E-selectin expressed on endothelial cells is considered to be crucial for the tumor cell adhesion to the endothelium.. The sialyl Lewis X (sL(X)) expression in 45 specimens from renal cell carcinoma patients was examined using immunohistochemistry.. In this study, we demonstrate that the immunoreactivity for sL(X) in renal cell carcinoma specimens not only correlates with conventional histopathologic parameters but also serves as a useful indicator for the prognosis of renal cell carcinoma.. Since beneficial effect of cimetidine has been reported and ascribed to its inhibitory action on the expression of E-selectin, a ligand molecule of sialyl Lewis antigen, cimetidine may also show inhibitory effect on the tumor recurrence and metastasis of renal cell carcinoma with high level of sL(X) expression.

Topics: Adjuvants, Immunologic; Carcinoma, Renal Cell; Cimetidine; Humans; Immunohistochemistry; Kidney Neoplasms; Lewis Blood Group Antigens; Neoplasm Invasiveness; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

Expression of sialyl Lewis(x) (sLe(x)) and sLe(a) on tumor cells is thought to facilitate metastasis by promoting cell adhesion to selectins on vascular endothelial cells. Experiments supporting this concept usually bypass the early steps of the metastatic process by employing tumor cells that are injected directly into the blood. We investigated the relative role of sLe(x) oligosaccharide in the dissemination of breast carcinoma, employing a spontaneous murine metastasis model. An sLe(x) deficient subpopulation of the 4T1 mammary carcinoma cell line was produced by negative selection using the sLe(x)-reactive KM93 MAb. This subpopulation was negative for E-selectin binding but retained P-selectin binding. Both sLe(x)-negative and -positive cells grew at the same rate; however, sLe(x)-negative cells spread more efficiently on plates and had greater motility in wound-scratch assays. Mice inoculated in the mammary fat pad with sLe(x)-negative and -positive variants produced lung metastases. However, the number of lung metastases was significantly increased in the group inoculated with the sLe(x)-negative variant (p = 0.0031), indicating that negative selection for the sLe(x) epitope resulted in enrichment for a subpopulation of cells with a high metastatic phenotype. Cell variants demonstrated significant differences in cellular morphology and pattern of tumor growth in primary and secondary tumor sites. These results strongly suggest that loss of sLe(x) may facilitate the metastatic process by contributing to escape from the primary tumor mass.

Topics: Animals; E-Selectin; Female; Ligands; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

End-products of glycolysis as well as phospholipid precursors and catabolites have been suggested as metabolic indicators of tumor progression. To test the hypothesis that increased levels of such indicators can distinguish metastatic phenotypes, we determined a limited cellular 1H-NMR metabolic profile of subpopulations of murine mammary 4T1 cells that differ in their metastatic potential. Subpopulations with differing metastatic phenotypes were identified by sorting for the expression of the cell surface adhesion oligosaccharide sialylated Lewis x (sLeX). The sLeX-negative subpopulation metastasizes to the lung of syngeneic mice more rapidly than the sLeX-positive subpopulations. The metabolic profile of the sLeX-negative subpopulation indicated higher levels of lactate and total choline metabolites than the sLeX-positive subpopulation, suggesting that altered metabolism is a critical component of the malignant phenotype. Analysis of shed cellular material from the sLeX-negative subpopulation displayed an increased ratio of phosphocholine to glycerophosphocholine when compared to the parental line and sLeX-positive subpopulation. Serum obtained from mice inoculated with either sLeX-negative or sLeX-positive tumor cells contained broader methylene resonances (P = 0.0002; P = 0.0003) and narrower methyl resonances (P = 0.0013; P < 0.0001) when compared to serum of naive mice. However, line widths of methylene and methyl resonances were not useful for distinguishing between the two tumor phenotypes. Results of this study further support the notion that metabolic indicators of malignancy can correlate with in vivo metastatic behavior.

Topics: Animals; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; Cell Adhesion; Cell Proliferation; Cell Separation; Flow Cytometry; Glycolysis; Humans; Hydrocarbons; Magnetic Resonance Spectroscopy; Mammary Neoplasms, Animal; Methane; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; Phenotype; Phosphorylcholine; Prognosis; Sialyl Lewis X Antigen; Time Factors

Metastasis is still the most serious reason for the high mortality of cancer patients. It is a complex process in which platelets play a crucial role. Several attempts have been performed to inhibit the metastatic process, some of these using modified liposomes. The aggregation behaviour of human platelets and HT29 colon carcinoma cells in the presence of liposomes with a modified surface has been investigated in the present study. Liposomes (PC/CH/DMPE) were unmodified, sterically stabilized by polyethylene glycol (PEG-DSPE), or equipped with the carbohydrate ligand sialyl Lewis(X) (conjugated to PEG-DMPE or DMPE as anchor) intended to specifically compete with ligands expressed by HT29 cells. We found in vitro that an addition of surface modified liposomes to human platelets in plasma caused an up to 2.9-fold increase in platelet aggregation. In addition, when HT29 tumor cells were mixed with platelets and surface modified liposomes, the number of tumor cells found in aggregates increased significantly from 8.3 % (only tumor cells) to 30.2 %. This result was supported by fluorescence micrographs demonstrating a strong association of platelets and liposomes around the tumor cells. In addition, a clear decrease in number and a change in the distribution of metastases after intravenous injection of HT29 cells in combination with liposomes was observed in vivo. While in control mice metastases in lung, liver and in intestine were prevailing, liposomal treatment resulted in a new localization of metastases in muscles. Taking together, the ability of surface modified liposomes to enhance aggregate formation of platelets and tumor cells has been demonstrated for the first time. The capability of these vesicles to interfere with the metastatic process might have implications for the use of such liposomes for therapeutic applications.

Topics: Blood Platelets; Cell Line, Tumor; Cell Separation; Drug Carriers; Drug Delivery Systems; Flow Cytometry; Humans; Ligands; Liposomes; Microscopy, Fluorescence; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Oligosaccharides; Phosphatidylcholines; Phosphatidylethanolamines; Platelet Aggregation; Polyethylene Glycols; Protein Binding; Sialyl Lewis X Antigen; Surface Properties; Time Factors

Unfractionated heparin reduces metastasis in many murine models. Multiple mechanisms are proposed, particularly anticoagulation and/or inhibition of P-selectin and L-selectin. However, the doses used are not clinically tolerable and other heparins are now commonly used. We studied metastasis inhibition by clinically relevant levels of various heparins and investigated the structural basis for selectin inhibition differences.. Five clinically approved heparins were evaluated for inhibition of P-selectin and L-selectin binding to carcinoma cells. Pharmacokinetic studies determined optimal dosing for clinically relevant anticoagulant levels in mice. Experimental metastasis assays using carcinoma and melanoma cells investigated effects of a single injection of various heparins. Heparins were compared for structural relationships to selectin inhibition.. One (Tinzaparin) of three low molecular weight heparins showed increased selectin inhibitory activity, and the synthetic pentasaccharide, Fondaparinux, showed none when normalized to anticoagulant activity. Experimental metastasis models showed attenuation with unfractionated heparin and Tinzaparin, but not Fondaparinux, at clinically relevant anticoagulation levels. Tinzaparin has a small population of high molecular weight fragments not present in other low molecular weight heparins, enriched for selectin inhibitory activity.. Heparin can attenuate metastasis at clinically relevant doses, likely by inhibiting selectins. Equivalent anticoagulation alone with Fondaparinux is ineffective. Clinically approved heparins have differing abilities to inhibit selectins, likely explained by size distribution. It should be possible to size fractionate heparins and inhibit selectins at concentrations that do not have a large effect on coagulation. Caution is also raised about the current preference for smaller heparins. Despite equivalent anticoagulation, hitherto unsuspected benefits of selectin inhibition in various clinical circumstances may be unwittingly discarded.

Topics: Animals; Anticoagulants; Cell Line, Tumor; Disaccharides; Factor Xa; Fibrinolytic Agents; Fondaparinux; Heparin; Heparin, Low-Molecular-Weight; Kinetics; L-Selectin; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Oligosaccharides; P-Selectin; Polysaccharides; Protein Binding; Selectins; Sialyl Lewis X Antigen; Temperature; Thrombosis; Time Factors; Tinzaparin

Clustered presentation of sialyl Lewis X (sLe(X)) on tumor cell mucins is thought to facilitate metastasis through binding to selectin adhesion receptors expressed on platelets, leukocytes and endothelial cells. Thus, interfering with sLe(X) assembly might provide a chemotherapeutic method for treating metastatic disease. Prior studies have shown that peracetylated disaccharides can act in cells as substrates for the assembly of oligosaccharides related to sLe(X) synthesis, and the assembly of oligosaccharides on the disaccharides diverts the assembly of sLe(X) from endogenous cell surface glycoconjugates. Here, we show that treatment of cultured human adenocarcinoma cells with micromolar concentrations of the peracetylated disaccharide, (Ac)(6)GlcNAcbeta1,3Galbeta-O-naphthalenemethanol (AcGnG-NM) reduces the expression of sLe(X) and diminishes binding in vitro to selectin-coated dishes, thrombin-activated platelets, and tumor necrosis factor alpha-activated endothelial cells. Altering glycosylation in this way significantly reduced the ability of tumor cells to distribute to the lungs of wild-type mice over a 3-h period after i.v. injection. No significant difference in biodistribution was noted after the injection of AcGnG-NM-treated tumor cells into P-selectin deficient mice, although the extent of lung seeding was reduced compared with that in wild-type mice. In vitro, we demonstrate that normal mouse platelets, but not P-selectin-deficient platelets, bound to control tumor cells and protected them from leukocyte-mediated cytolysis. Conversely, treatment of tumor cells with disaccharide markedly reduced the ability of normal platelets to protect them from cytolysis. Finally, in an experimental metastasis model, we show that treatment of tumor cells with the disaccharide markedly reduced their lung colonization potential after injection into severe combined immunodeficient mice. These findings suggest that this compound may represent a novel class of chemotherapeutic agents for prevention and treatment of metastatic disease.

Topics: Adenocarcinoma; Blood Platelets; CA-19-9 Antigen; Carbohydrate Sequence; Cell Adhesion; Colonic Neoplasms; Disaccharides; Endothelium, Vascular; Gangliosides; Glycosylation; Humans; Lung Neoplasms; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Tissue Distribution; Tumor Cells, Cultured

Sialyl Lewis(x) (sLe(x)) is one ligand for E selectin (CD62E), a glycoprotein that is expressed on activated endothelial cells. Adhesion mediated by endothelial E selectin and sLe(x) expressed on human tumor cells could be relevant for the development of metastases. The aim of this study was to investigate whether or not a correlation exists between pre-operative levels of ganglioside serum sLe(x) and disease-free interval and survival in colorectal cancer patients.. Thirty Duke's B and 52 Duke's C patients undergoing resection for colorectal cancer were studied. The median follow-up time was 34.8 months. A pool of 57 sera from normal subjects was used as an Internal Normal Standard (INS). sLe(x) analyses were performed by a thin layer chromatography (TLC) immunostaining technique. Results were expressed as the ratio (R) between bands of INS and bands from each neoplastic serum.. The median R value was 0.80 in normal subjects, 0.70 in Duke's B patients and 1.0 in Duke's C patients. No significant differences were detected between sLe(x) concentrations in sera from normal and neoplastic subjects (p = 0.1). Using an arbitrary cutoff of R = 0.9, the mean disease-free interval in the whole series was 47.4 months for R <0.9 and 126.0 months for R > or = 0.9 (p = 0.04). The survival time was 76.8 months for patients with R values <0.9 and 156.3 months for patients with R values > or =0.9 (p = 0.1).. High serum levels of ganglioside sLe(x) significantly correlate with a favorable prognosis and with a lower occurrence of metastases. It is conceivable that soluble sLe(x) may compete with membrane-bound sLe(x) in the course of interactions between activated endothelium and tumor cells that have reached the circulation.

Topics: Aged; Biomarkers, Tumor; Case-Control Studies; Chromatography, Thin Layer; Colorectal Neoplasms; Disease-Free Survival; Female; Humans; Italy; Lewis X Antigen; Male; Neoplasm Metastasis; Oligosaccharides; Prognosis; Sialyl Lewis X Antigen; Survival Analysis

Topics: Animals; Carbohydrate Metabolism; Diagnostic Imaging; Disaccharides; Glycosylation; Humans; Magnetic Resonance Imaging; Mice; Neoplasm Metastasis; Neoplasms; Oligosaccharides; Selectins; Sialic Acids; Sialyl Lewis X Antigen

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLe(x)), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLe(x) played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLe(x) and alpha-1,3-fucosyltransferase-VII (alpha-1,3 Fuc T-VII, enzyme for SLe(x) synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLe(x) and alpha-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLe(x) expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLe(x) and alpha-1,3 FucT-VII.

Topics: Antibodies, Monoclonal; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Cells, Cultured; Chromones; DNA, Complementary; Endothelium, Vascular; Fucosyltransferases; Humans; Insulin; Laminin; Liver Neoplasms; Morpholines; Neoplasm Metastasis; Oligosaccharides; Phenotype; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sialyl Lewis X Antigen; Signal Transduction; Transfection; Up-Regulation

Forskolin (FSK) is known as an up-regulator of intracellular cAMP and inhibitor of cancer growth and metastasis. The effects of FSK on the metastasis potential and its mechanisms were studied using a human hepatocarcinoma cell line, H7721. It was found that FSK stimulated cell growth, increased cAMP in the cells, and enhanced the metastasis-related phenotypes, including adhesion to laminin (Ln) and human umbilical vein epithelial cells (HUVEC), chemotactic migration and invasion. These effects were supposed to result from the increase of the SLex expression induced by FSK, since only the monoclonal antibody of SLex showed a significant attenuation of the enhanced metastasis-associated phenotypes. Using H7721 cells transfected with the sense or antisense cDNA of protein kinase B (PKB) and some inhibitors of signal transduction, it was discovered that FSK up-regulated the expression of SLex via PKB, but it was independent of phosphotidylinositide-3-kinase (PI-3K). A subtype of atypical protein kinase C (a-PKC) might also participate in the up-regulation of SLex expression by FSK, and cAMP/PKA pathway is a negative regulator of SLex expression on H7721 cells. It can be concluded that FSK shows a metastasis-promoting effect ex vivo.

Topics: Antibodies, Monoclonal; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Division; Cell Line; Cell Line, Tumor; Cell Movement; Cells, Cultured; Chemotaxis; Colforsin; Cyclic AMP; DNA, Complementary; Endothelium, Vascular; Enzyme Inhibitors; Flow Cytometry; Humans; Laminin; Neoplasm Metastasis; Oligosaccharides; Phenotype; Phosphatidylinositol 3-Kinases; Protein Kinase C; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sialyl Lewis X Antigen; Signal Transduction; Transfection; Up-Regulation

The roles of terminal sialyl and fucosyl residues in cell surface glycans in the metastatic potential of H7721 cells, a human hepatocarcinoma cell line, were studied.. Neuraminidase and alpha-L-fucosidase were used to remove the sialyl and fucosyl residues, respectively. Cell adhesion to fibronectin (Fn), laminin (Ln), and human umbilical vein epithelial cell (HUVEC), as well as chemotactic cell migration and invasion, were selected as the parameters of metastatic potential ex vivo.. Sialyl residue is not essential for cell adhesion to Fn, but is important in cell adhesion to Ln and invasion, and is crucial in cell adhesion to HUVEC and migration. In contrast, fucosyl residue contributes more than sialyl residue to cell adhesion to Fn and Ln, but less to adhesion to HUVEC, and is not essential in chemotactic cell migration and invasion. Cell adhesion to HUVEC, migration, and invasion were inhibited by the monoclonal antibody of sialyl Lewis X, but not by the antibody of non-sialyl Lewis X.. Terminal sialyl residues on cell surface glycans are more important than fucosyl residues in mediating cell adhesion to HUVEC and cell migration/invasion, but the reverse is true in cell adhesion to Fn and Ln.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Chemotaxis; Epithelial Cells; Fibronectins; Fucose; Humans; Laminin; Lewis Blood Group Antigens; Liver Neoplasms; Membrane Glycoproteins; Neoplasm Metastasis; Oligosaccharides; Sialic Acids; Sialyl Lewis X Antigen; Tumor Cells, Cultured; Umbilical Veins

Macrosphelide B (MSB), a 16-membered macrolide from Microsphaeropsis sp. FO-5050, inhibits adhesion of sialyl Lewis(x) (sLe(x))-expressing HL-60 cells to LPS-activated (E-selectin-expressing) human umbilical vein endothelial cells (HUVECs) in vitro. This study examines MSB effects on metastasis of B16/BL6 mouse melanoma cells (B16/BL6 cells) and L5178Y-ML mouse lymphoma cells in vivo and analyzes the MSB antimetastatic activity mechanism. When administered MSB at 20 mg/kg/day, lung metastatic nodules of B16/BL6 cells were significantly decreased (T/C = 45%). However, no inhibition of metastasis of L5178Y-ML cells to the spleen and liver was observed. Flow cytometry analysis showed that B16/BL6 cells expressed high levels of sLe(x) antigen, whereas expression on L5178Y-ML cells was low. Under in vitro conditions, B16/BL6 cells demonstrated a greater degree of adhesion to LPS-activated HUVECs than L5178Y-ML cells, but adhesion was significantly inhibited by MSB and sLe(x) antibody. Combined therapy of MSB and cisplatin (CDDP) induced remarkable lung metastasis inhibition without adverse effects of CDDP to the host. All these findings suggest that MSB suppresses lung metastasis of B16/BL6 cells by inhibiting cell adhesion to endothelial cells through the sLe(x) molecule.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Adhesion; Cell Adhesion Molecules; Cell Division; Cells, Cultured; Cisplatin; Dose-Response Relationship, Drug; E-Selectin; Endothelium, Vascular; Heterocyclic Compounds; HL-60 Cells; Humans; Lymphoma; Melanoma, Experimental; Mice; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

Carbohydrate expression of cancer cells is closely related to the metastatic nature of colorectal cancer. In the present study we investigated the relevance of carbohydrate expression profiles of colorectal cancer cells in the primary lesion to metastatic distribution patterns as well as prognosis in 134 cases. Carbohydrate expression was estimated by histochemistry with 17 kinds of lectins and 3 kinds of Lewis-related monoclonal antibodies (MAbs), and correlations between the staining and clinicopathological parameters were examined. The results showed that lymphatic invasion, lymph node metastasis, and peritoneal metastasis correlated with staining with lectins that bind galactose/N-acetylgalactosamine residues (Gal/GalNAc) such as Maclura pomifera (MPA), Arachis hypogaea (PNA), Helix pomatia (HPA), and Vicia villosa (VVA). In contrast, hepatic metastasis correlated with staining with Anguilla anguilla lectin (AAA), anti-LewisX (LEX-2), anti-sialyl Lewisa (NS 19-9), and anti-sialyl-dimeric LewisX (FH-6) MAbs, all of which bind preferentially to fucosylated carbohydrate chains. The five-year survival rate of patients was related to the staining of cancers with MPA, HPA, FH-6 or NS19-9, and MPA- and FH-6 staining were independent prognostic factors. We conclude that carbohydrate expression profiles of cancer cells are relevant to the route of tumor cell dissemination, metastatic pattern as well as prognosis of colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Carbohydrate Metabolism; Carbohydrate Sequence; Carbohydrates; Colorectal Neoplasms; Female; Humans; Lectins; Lewis X Antigen; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Oligosaccharides; Peritoneal Neoplasms; Prognosis; Prospective Studies; Sialyl Lewis X Antigen; Staining and Labeling

The expressions of Lewis antigens, alpha1,3 fucosyltransferase (alpha1,3 FucT)-VII, and the metastatic potential of the 7721 human hepatocarcinoma cell line after the transfection of the cDNA of nm23-H1, a known metastasis-suppressive gene, were studied using mock cells as the control, which were transfected with the pcDNA3 vectors.. Cell adhesion to human umbilical vein epithelial cells (HUVEC), chemotaxic cell migration through transwells, and invasion through matrigel were selected as the metastasis-related phenotypes to assess the metastatic potential at the cell level.. The results showed that the expression of SLe(x) was high, while the expression of Le(x), SDLe(x), and SLe(a) were very low on the surface of the mock cells. After transfection of nm23-H1, the expressions of SLe(x), alpha1,3 FucT-VII, and the cell adhesion to HUVEC, as well as cell migration and invasion were simultaneously decreased in all three clones of nm23-H1-transfected cells. Among different clones, the decreased expressions of SLe(x) and alpha1,3 FucT-VII were roughly correlated to each other, and also, in general, proportional to the ability of cell adhesion to HUVEC, cell migration, and invasion. The expressions of these metastasis-related phenotypes were lowest in clone 3 and highest in clone 4. Only the specific monoclonal antibody to SLe(x) (KM93) significantly abolished the cell adhesion, migration, and invasion, while other monoclonal antibodies against SDLe(x) or Le(x) and SLe(a) only slightly inhibited or entirely failed to inhibit the above-mentioned phenotypes. However, the rate of cell growth was not changed after the transfection of nm23-H1, and the ability of colony formation on the soft agar was only decreased in one clone.. These findings reveal that the down-regulation of alpha1,3 FucT-VII and its product, SLe(x), is one of the mechanisms to explain the metastasis-suppressive effect of the nm23-H1 gene.

Topics: Blotting, Northern; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Chemotaxis; DNA, Complementary; Down-Regulation; Epithelial Cells; Flow Cytometry; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Oligosaccharides; Phenotype; Sialyl Lewis X Antigen; Transcription Factors; Transfection; Tumor Cells, Cultured; Umbilical Cord

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of cells in the host connective tissue. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly involved in the adhesion of colon carcinoma cells to the endothelium of target organ. Interaction of T84 colon cancer cells to purified E-selectin in vitro caused an increase in the tyrosine phosphorylation of a number of proteins as well as the modulation of cellular properties correlated to the metastatic phenotype. Specifically, E-selectin-stimulated actin reorganization, increased collagenase secretion, and induced cell migration. Treatment of T84 cells with herbimycin A inhibited cell adhesion as well as selectin-induced increase of cell migration, and cytoskeleton assembly. Our data demonstrate that binding of cancer cells to E-selectin starts signal transduction pathways which may affect the tumor metastatic abilities.

Topics: Benzoquinones; Carcinoma; Cell Adhesion; Cell Movement; Cells, Cultured; Coculture Techniques; Colonic Neoplasms; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Humans; Lactams, Macrocyclic; Matrix Metalloproteinase 2; Neoplasm Metastasis; Oligosaccharides; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rifabutin; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Topics: Animals; Carbohydrate Metabolism; Carcinoma, Lewis Lung; Cell Adhesion; Cells, Cultured; CHO Cells; Cricetinae; E-Selectin; Endothelium, Vascular; Humans; Intercellular Adhesion Molecule-1; Ligands; Liposomes; Lung Neoplasms; Neoplasm Metastasis; Oligosaccharides; Phosphatidylethanolamines; Phosphorylcholine; Polyethylene Glycols; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Carcinoembryonic antigen (CEA) has been reported to promote the metastatic potential in some experimental tumors. Adhesion molecules are known to play an important role in the process of metastasis. Cytokines, including interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), which are produced by Kupffer cells, induce endothelial cells to express adhesion molecules. As a result, the present study was designed to investigate whether the interaction between CEA and Kupffer cells accelerated the metastatic potential of tumors in the liver. Kupffer cells isolated from the liver of male BALB/c mice were cultured with CEA, either with or without the addition of a cytokine inhibitor. The levels of IL-1beta and TNF-alpha were examined in a culture medium. An adhesion assay of colon cancer cell lines to human umbilical vein endothelial cells was also performed. When CEA was added to the Kupffer cell culture medium, cytokines were produced. Elevated levels of cytokines appeared to lead to increased rates of adhesion of cancer cells to endothelial cells. However, these phenomena were blocked by the addition of cytokine inhibitors. CEA stimulated Kupffer cells to produce cytokines. An elevated number of cytokines have been proven to promote the expression of adhesion molecules in endothelial cells. These processes are therefore considered to contribute to the metastasis of malignant cells to the liver. These results suggest that cytokine inhibitors may therefore play an important role in the inhibition of hepatic metastasis.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; CA-19-9 Antigen; Carcinoembryonic Antigen; Cell Adhesion; Cell Communication; Colonic Neoplasms; Culture Media; Disease Progression; Dose-Response Relationship, Drug; E-Selectin; Endothelium, Vascular; Gangliosides; Humans; Interleukin-1; Kupffer Cells; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Oligosaccharides; Pyrazoles; Pyridines; Sialyl Lewis X Antigen; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The pCMV4 plasmid containing the cancer-promoting gene, c-erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the 'neo' selectable marker. Several clones showing stable expression of c-erbB2/neu were established and characterized by determination of c-erbB2/neu mRNA and its encoded protein p185. Expression of Lewis antigens and alpha1,3-fucosyltransferases and the biological behavior of 7721 cells after c-erbB2/neu transfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLe(x) expression on the surface of mock cells was high, whereas expression of SDLe(x), Lex and SLe(a) was absent or negligible. This is compatible with the abundant expression of alpha1,3-fucosyltransferase VII, very low expression of alphafucosyltransferase III/VI, and almost absent expression of alpha1,3-fucosyltransferase IV in the mock cells. After transfection of c-erbB2/neu, expression of SLe(x) and alpha1,3-fucosyltransferase VII were simultaneously elevated, but that of alphafucosyltransferase III/VI was not altered. The expression of both SLe(x) and alpha1,3-fucosyltransferase VII correlated positively with the expression of c-erbB2/neu in different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P-selectin, as well as cell migration and invasion, were increased in c-erbB2/neu-transfected cells. These increases also correlated positively with the expression intensities of c-erbB2/neu, SLe(x) and alpha1,3-fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLe(x) (KM93) and SDLe(x) (FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P-selectin and cell migration and invasion. mAbs to SDLe(x) and SLe(a) did not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of alpha1,3-fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c-erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of alpha1,3-fucosyltransferase VII and its specific product, SLe(x), and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the c-erb

Topics: Antibodies; Carcinoma, Hepatocellular; Cell Adhesion; Cells, Cultured; DNA, Complementary; Endothelium, Vascular; Fibronectins; Fucosyltransferases; Genes, erbB-2; Humans; Liver Neoplasms; Neoplasm Metastasis; Oligosaccharides; P-Selectin; Phenotype; RNA, Messenger; Sialyl Lewis X Antigen; Transfection; Tumor Cells, Cultured; Up-Regulation

Serum from patients with different malignancies contain an abnormal concentration of a a1-acidic-glycoprotein (AAG) and also, increased levels of AAG are associated with the presence of tumor mass. In the present report, serum levels of AAG were measured by radial immunodiffusion in squamous cell carcinoma of the head and neck (SCCHN) patients taking into account disease status parameters such as tumor localization, stage and extension of disease. Immunohistochemical methods, SDS-PAGE and Western-blotting were employed to study the expression of AAG and a carbohydrate related antigen (sialyl Lewis x) in tumor tissues and derived fractions. AAG showed abnormal levels in 7/15 oral cavity tumor patients sera, 2/5 oropharynx and 5/10 larynx tumors; increased AAG serum levels belonged to patients with disseminated disease. On the other hand, the presence of AAG and sialyl Lewis x were demonstrated in carcinoma cells and in derived fractions from tumor tissues belonging to patients with elevated AAG serum levels. In the present study, we have found elevated levels of AAG in serum samples from SCCHN patients; these neoplastic cells are capable to express AAG.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Colorectal Neoplasms; Epithelial Cells; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Staging; Oligosaccharides; Organ Specificity; Orosomucoid; Sialyl Lewis X Antigen

Selectins bind to carbohydrate ligands in a calcium-dependent manner and play critical roles in host defense and possibly in tumor metastasis. To isolate peptides that mimic E-selectin ligands, we screened a phage peptide library using E-selectin as a target molecule. This attempt unexpectedly failed, probably because the binding affinity of E-selectin to its ligand is low. We then took an approach that is analogous to the isolation of anti-idiotype antibodies and were able to isolate peptides that bound to anticarbohydrate antibodies recognizing E-selectin ligands. These peptides, enriched for their binding to anti-Lewis A antibody, were found to bind to E-, P- and L-selectins in a calcium-dependent manner. Phage harboring the identified peptide IELLQAR and synthetic peptides having the same sequence inhibited the binding of sialyl Lewis X or sialyl Lewis A oligosaccharides to E-selectin. The adhesion of HL-60 and B16 melanoma cells expressing sialyl Lewis X to E-selectin was also inhibited by the phage-displaying IELLQAR peptide. Moreover, i.v. injected IELLQAR peptide inhibited the lung colonization of mouse B16 melanoma and human lung tumor cells expressing sialyl Lewis X. These results demonstrate that it is possible to isolate peptides mimicking carbohydrate ligands by screening the peptides for binding to anticarbohydrate antibodies and then using them to inhibit carbohydrate-dependent experimental tumor metastasis.

Topics: Animals; Brevican; Cell Adhesion; Chondroitin Sulfate Proteoglycans; E-Selectin; HL-60 Cells; Humans; Lectins, C-Type; Lewis Blood Group Antigens; Lung; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Nerve Tissue Proteins; Oligopeptides; Oligosaccharides; Peptide Fragments; Recombinant Fusion Proteins; Sialyl Lewis X Antigen; Transfection

Previous studies have shown that sialyl Lewis a (SLea) and sialyl Lewis x (SLex) correlated to hematogenous metastasis of human cancers. Although SLea/SLex and E-selectin act as a set of adhesion molecules in vitro, it is not clear whether the in vivo correlation is exclusively mediated by the adhesion function. To address this issue, we investigated whether or not the role of SLea/SLex antigens on hematogenous metastasis to the liver in SCID mice was exclusively mediated by adhesion by using antibodies for these antigens and SLea/SLEx-negative, human pancreas adenocarcinoma cell line PCI-6. The absence of SLea/SLex expression was supported by the absent flow cytometric detection of the antigens as well as by the absent attachment augmentation to activated endothelial cells. PCI-6 cells are xenotransplantable to nude and SCID mice and produce vascular endothelial cell growth factor (VEGF) in a significant amount. PCI-6 cells, 1 x 10(6), were injected into the spleens of SCID mice, and resultant liver metastases were evaluated six weeks later. We observed an inhibitory effect on the establishment and growth of metastatic colonies when anti-SLea or anti-SLex antibody was administered. This indicates that SLea/x antigens have an important in vivo role, even in the metastasis of SLea/SLex-negative tumor cells. This implies that there may be an in vivo function of SLea/x antigens other than that of the attachment between tumor and endothelial cells.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Line; Endothelial Growth Factors; Endothelium, Vascular; Flow Cytometry; Humans; Interleukin-1; Lewis Blood Group Antigens; Lymphokines; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Oligosaccharides; Pancreatic Neoplasms; Sialyl Lewis X Antigen; Specific Pathogen-Free Organisms; Transplantation, Heterologous; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

In this retrospective study we describe the immunohistochemical expression pattern of sLe(x) epitopes in endothelial and epithelial cells of 59 squamous carcinomas of the tongue, and relate this to the relative survival rates of the patients. Endothelial sLe(x) expression was significantly elevated in malignant lesions compared to normal tissues, but did not have any prognostic value for the relative survival rate. In contrast, epithelial sLe(x) expression was decreased in carcinomas compared to normal tongue. The patients whose carcinoma showed only moderate epithelial HECA-452 reactivity had a significantly better relative survival rate than the patients with tumor specimens with neglible or very high HECA-452 reactivity. The epithelial staining with the two other anti-sLe(x) antibodies (CSLEX-1 and 2F3) did not correlate with the survival rates of tongue carcinoma patients.

Topics: Antibodies, Monoclonal; Carbohydrate Sequence; Carcinoma, Squamous Cell; Endothelium; Epithelium; Female; Humans; Immunohistochemistry; Male; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Prognosis; Retrospective Studies; Sialyl Lewis X Antigen; Survival Rate; Tongue Neoplasms

To study the significance of E-selectin and its ligand-sLeX in the metastasis of hepatocellular carcinoma (HCC).. Flow cytometry and immunohistochemistry were used to detect the expression of E-selectin and its ligand-sLeX in HCC cell lines and in human HCC tissues.. The positive rate of E-selectin in vascular endothelial cells adjacent to cancer nest in tumors was 67.9% (19/28). In tumors accompanied with emboli or satellite foci, it was significantly higher than that without emboli or satellite foci (P < 0.05). The positive rate of E-selectin was not related to tumor size, tumor capsule, AFP, and the degree of differentiation. The positive expressions of sLeX in SMMU-7721, PLC/PRF/5 and HepGII cell lines were 7.03%, 63.35% and 97.29% respectively. The positive cells of sLeX were mainly distributed in the margin of tumors; the positive expression of sLeX in HCC cells in emboli or invasive tumor tissues was much higher than that in primary foci.. E-selectin and its ligand-sLeX are closely correlated with the metastasis of HCC.

Topics: Adult; Carcinoma, Hepatocellular; Cell Line, Tumor; E-Selectin; Female; Flow Cytometry; Humans; Immunohistochemistry; Ligands; Liver Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

To study the correlation of sialyl Lewis-X (SLeX) antigen expression with the metastatic potential of human colorectal cancer.. The expression of biosynthetic enzyme of SLeX antigen--alpha1, 3Fuc-T mRNA in various cell lines of colorectal cancer possessed with different metastatic potentials (LoVo and HT(29)) was investigated by in situ hybridization. The expression of SLeX antigen was studied using immunohistochemistry and fluorescence-activated flow cytometry qualitatively and quantitatively.. The expression level of alpha1, 3Fuc-T mRNA and SLeX antigen in highly metastatic LoVo cells was high (21.2 +/- 7.7, 32.8 +/- 10.9, P < 0.05), and the expression level was low in HT(29) cells (10.8 +/- 5.2, 21.9 +/- 8.8) which are known possessed with a lower metastatic potential.. The expression of SLeX antigen is correlated to the metastatic potentiality of human colorectal cancer.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Fucosyltransferases; HT29 Cells; Humans; Immunohistochemistry; Lewis X Antigen; Neoplasm Metastasis; Oligosaccharides; RNA, Messenger; Sialyl Lewis X Antigen

Aberrant expression of cell surface carbohydrates such as sialyl Lewis X is associated with tumor formation and metastasis. In order to determine the roles of sialyl Lewis X in tumor metastasis, mouse melanoma B16-F1 cells were stably transfected with alpha1, 3-fucosyltransferase III to express sialyl Lewis X structures. The transfected B16-F1 cells, B16-FTIII, were separated by cell sorting into three different groups based on the expression levels of sialyl Lewis X. When these transfected cells were injected into tail veins of C57BL/6 mice, B16-FTIII.M cells expressing moderate amounts of sialyl Lewis X in poly-N-acetyllactosamines produced large numbers of lung tumor nodules. Surprisingly, B16-FTIII.H cells expressing the highest amount of sialyl Lewis X in shorter N-glycans died in lung blood vessels, producing as few lung nodules as B16-FTIII.N cells which lack sialyl Lewis X. In contrast, B16-FIII.H cells formed more tumors in beige mice and NK cell-depleted C57BL/6 mice than did B16-FTIII.M cells. B16-FTIII.H cells bound to E-selectin better than did B16-FTIII.M cells, but both cells grew at the same rate. These results indicate that excessive expression of sialyl Lewis X in tumor cells leads to rejection by NK cells rather than tumor formation facilitated by attachment to endothelial cells.

Topics: Animals; Cell Division; Cytotoxicity, Immunologic; E-Selectin; Flow Cytometry; Fucosyltransferases; Graft Rejection; Killer Cells, Natural; Lewis X Antigen; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Oligosaccharides; Recombinant Proteins; Sialyl Lewis X Antigen; Transfection; Tumor Cells, Cultured

Alpha(1,3)- and alpha(1,4)-fucosylated oligosaccharides such as sialyl-Lewis(x) (sialyl-Le(x)) and sialyl-Lewis(a) (sialyl-Le(a)) have been reported to participate in tumor cell adhesion to activated endothelium. We examined by cytofluorometry the expression of Le(x), sialyl-Le(x), sialyl-Le(x) dimeric, Le(a), and sialyl-Le(a) on the surface of two human lung adenocarcinoma cell lines with different lung colonization potential. High expression levels of all of these antigens were detected in the metastatic HAL-8Luc cells, whereas the closely related nonmetastatic HAL-24Luc cells only expressed the sialyl-Le(a) and sialyl-Le(x) dimeric antigens, both at lower level than in HAL-8Luc cells. Five alpha(1,3)-fucosyltransferases (alpha(1,3)-Fuc-T) controlling the synthesis of these molecules have been identified to date (Fuc-TIII-Fuc-IVII). The expression of these five genes was also higher in the metastatic cells than in the nonmetastatic counterparts as was shown by Northern blot analysis. In vitro adhesion assays showed that only the metastatic cell line adheres significantly to E-selectin-expressing human endothelial cells. Moreover, monoclonal antibody (mAb) blockade of E-selectin completely abolished tumor cell binding. Adhesion inhibition experiments using mAbs against sialylated fucosylated oligosaccharides expressed on tumor cells indicated that these antigens are involved in the binding. Anti-sialyl-Lex(x) mAb (CSLEX-1) inhibited adhesion by 85%; it had the most pronounced inhibitory effect. These findings suggest that the overexpression of alpha(1,3)-Fuc-T genes in the metastatic HAL-8Luc cells, compared with HAL-24Luc cells, results in an enhanced surface display of fucosylated oligosaccharides, which contributes to the adhesive capacity of these cells to the activated endothelium and correlates with their lung colonization potential.

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; Antigens, Surface; Blotting, Northern; CA-19-9 Antigen; Cell Adhesion; E-Selectin; Endothelium; Flow Cytometry; Fucosyltransferases; Gangliosides; Gene Expression; Humans; Lewis X Antigen; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Sialyl Lewis-x (sLx) is a cellular adhesion molecule (CAM) that has been implicated in the inflammatory reaction and cancer metastasis. The sLx is the carbohydrate ligand of endothelial-selectin (E-selectin), an inducible vascular endothelial CAM. The role of sLx has been investigated in several cancers, and its presence has been correlated with advanced disease stage, decreased disease-free survival, and greater metastatic potential. A recent study has found that cultured head and neck (HN) squamous cell carcinoma (SCC) cell lines express sLx and that binding of these cells to cytokine activated endothelium correlates with the endothelial expression of E-selectin. The purpose of this study was to identify sLx in the tumors of patients with HNSCC and to see if the presence of sLx correlated with disease status.. We performed immunohistochemical (IHC) staining to detect the sLx antigen using the monoclonal antibody (MAb) KM-93. Eighty-two specimens of HNSCC that were obtained at the time of resection or biopsy were analyzed for sLx staining patterns and intensities. The clinical outcomes of survival, disease-free interval, and incidence of distant metastasis were then assessed to determine whether there was a correlation with sLx tumor expression. In addition, we analyzed specimens from metastatic HNSCC sites for expression of sLx.. The sLx expression was identified in 62% of the primary tumor specimens and 87% of the metastatic tumor specimens analyzed by IHC. The staining pattern in the HNSCC tumors differed from that seen in normal squamous epithelium but was variable in both intensity and distribution. The sLx expression in the metastatic sites was higher than in the primary sites in 67% of the specimens (10 of 15). There was no correlation between sLx staining and disease status.. The results of this study demonstrate that sLx is present in HNSCC and supports the data that show that sLx may play a role in the metastasis of HNSCC. Future studies are warranted to evaluate the role of sLx, E-selectin, and other CAMs in HNSCC.

Topics: Adolescent; Aged; Aged, 80 and over; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lewis X Antigen; Male; Middle Aged; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

The expression of blood group antigens A, B, and H, as well as sialylated and nonsialylated forms of Lewis(a) and Lewis(x), was studied using immunohistochemical methods in normal and tumor tissues in the following cohort of patients: 51 patients with primary breast carcinoma, 13 with metastatic lymph node lesions, and 16 with benign tumors of the breast. As a control, normal tissue was obtained from a similar group of 22 patients with breast cancer. The noncancerous tissues expressed the same A/B/H antigens as the patients' red blood cells and also usually expressed Lewis-related antigens. Seventy-six percent of primary carcinomas failed to express the appropriate A/B/H antigens, and in one blood group A patient the tumor tissue expressed B antigen. In the metastatic lesions, Lewis(a)/sialyl Lewis(a) expression was reduced when compared with the primary tumors, but Lewis(x)/sialyl Lewis(x) antigens were still expressed. These results suggest a possible relationship between the metastatic behavior of the tumor and expression of the blood group antigens.

Topics: Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Breast Neoplasms; CA-19-9 Antigen; Carbohydrate Sequence; Gangliosides; Humans; Immunohistochemistry; Lewis Blood Group Antigens; Lewis X Antigen; Lymph Nodes; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen

We have previously reported that colon cancer cells metastasized to the liver expressed an increased amount of sialyl Lewis X (SLeX) antigen compared to their corresponding primary lesions. It is now well known that SLeX antigen and sialyl Lewis A (SLeA) antigen are ligands for the selectins expressed on the endothelial cells. Therefore, it is assumed that SLeX-rich colon cancer cells could be easily adhered to the endothelial cells that express selectins. In this report we have tried to induce selectin expression on the human liver sinusoidal endothelial cells and have examined the adhesion of SLeX-high or -low expressing colon cancer cells to the interleukin-1beta (IL-1beta)-treated liver specimens using Stamper-Woodruff assay. These human colon cancer cells are termed KM12HX or KM12LX cells, respectively. A significantly increased number of KM12HX cells adhered to the IL-1beta-treated liver specimens compared to KM12LX cells. The adhesion of KM12HX cells was inhibited by the pretreatment of tumor cells with anti-SLeX antibody or by the pretreatment of liver specimens with anti-selectin antibodies. Selectin expression on the liver sinusoidal endothelial cells and endothelial cells of blood vessels after IL-1beta treatment was confirmed by immunohistochemically using anti-selectin monoclonal antibodies (MAbs). These findings strongly suggest that SLeX-expressing cancer cells could adhere to the sinusoidal endothelial cells via an SLeX-selectin interaction system and this could be a first step for colon cancer cells that metastasize to the liver. The mechanism by which these selectins can be induced in vivo is the next problem to be considered.

Topics: Cells, Cultured; Colonic Neoplasms; Endothelium; Humans; Interleukin-1; Ligands; Liver; Liver Neoplasms; Neoplasm Metastasis; Oligosaccharides; Selectins; Sialyl Lewis X Antigen

To elucidate the early events of blood-borne metastasis under actual blood flow, real-time trafficking of RAW117 large cell lymphoma cells, namely parental RAW117-P and liver-metastatic RAW117-H10 cells, was investigated using positron emission tomography (PET). Both types of cells accumulated in the liver immediately after injection via the portal vein, and were eliminated from the liver time-dependently. The elimination rate of RAW117-H10 cells, however, was slower than that of RAW117-P cells, suggesting that RAW117-H10 cells interact more strongly with hepatic sinusoidal endothelium than the parental cells. This result correlated with the metastatic potential of these cells: RAW117-H10 cells metastasized in the liver to a greater extent than RAW117-P cells after injection via this route. To investigate the role of sialylglycoconjugates in the interaction of RAW117-H10 cells with the hepatic endothelium after injection via the portal vein, the trafficking of RAW117-H10 cells was examined after the cells had been treated with sialidase. The elimination rate of RAW117-H10 cells from liver was observed to be greatly accelerated by sialidase treatment. To elucidate what kind of sialylglycoconjugates is related to this phenomenon, we analyzed the distribution of sialyl Lewis A and sialyl Lewis X antigens of both sublines of RAW117 by using flow cytometry. RAW117-H10 cells were found to express a much higher level of sialyl Lewis A than RAW117-P cells, whereas the amount of sialyl Lewis X did not differ significantly. These findings suggest that some sialylglycoconjugates, perhaps sialyl Lewis A in particular, play an important role in the initial interaction of RAW117-H10 cells with the hepatic endothelium, leading to metastasis.

Topics: Animals; Antigens, Neoplasm; CA-19-9 Antigen; Cell Adhesion; Cell Movement; Female; Flow Cytometry; G(M1) Ganglioside; Gangliosides; Injections, Intravenous; Liver Neoplasms; Lung Neoplasms; Lymphoma, Large B-Cell, Diffuse; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Cells, Circulating; Neuraminidase; Portal Vein; Sialyl Lewis X Antigen; Tomography, Emission-Computed; Tumor Cells, Cultured

Topics: Animals; CA-19-9 Antigen; Cell Adhesion; Cell Membrane; E-Selectin; Female; Galactosides; Galectin 1; Gangliosides; Glycoconjugates; Hemagglutinins; Humans; Hyaluronan Receptors; Hyaluronic Acid; Lectins; Lewis Blood Group Antigens; Neoplasm Metastasis; Neoplasms; Oligosaccharides; Ovarian Neoplasms; Sialyl Lewis X Antigen

A human colon cancer cell line, OCUC-LM1(LM), was established from a liver metastasis in our laboratory. Intrasplenic injection of LM into nude mice was repeated three and five times, and the daughter cell lines were designated as LM-H3 and LM-H5 respectively. The level of sialyl Lewis A (SLA) in the supernatant of LM-H3 and LM-H5 was 3 and 4.5 times higher than that of LM respectively. Flow cytometric analysis of SLA expression showed that the peak channel for LM was 113; for LM-H3, 126; and for LM-H5, 146. The mean fluorescence intensity of LM was 102.3 +/- 43.5; for LM-H3, 126.2 +/- 28.4; and for LM-H5, 144.8 +/- 23.4. In endothelial cell adhesion assays, the percentages of adherent LM-H3 and LM-H5 cells were significantly higher than for LM. The activity of alpha1-->4 fucosyltransferase was higher in LM-H3 and LM-H5 than in LM, but there was no difference in alpha2-->3 sialyltransferase activities for type 1 chain among the cell lines. Our results suggest that SLA expression is associated with acquisition of a high capacity for liver metastasis of colon cancer; increased SLA expression is due mainly to increased fucosyltransferase activity.

Topics: Animals; Antibodies; beta-Galactoside alpha-2,3-Sialyltransferase; Carbohydrate Conformation; Carbohydrate Sequence; Cell Adhesion; Cells, Cultured; Colonic Neoplasms; E-Selectin; Endothelium, Vascular; Flow Cytometry; Fucosyltransferases; Humans; Lewis Blood Group Antigens; Liver Neoplasms; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Sialyltransferases; Transplantation, Heterologous; Tumor Cells, Cultured; Umbilical Veins

To evaluate the correlation between serum levels of sialyl Lewis X-i antigen and distant metastasis and survival in patients with non-small cell lung cancer (NSCLC), we measured the serum levels of the tumor marker in 371 patients with untreated NSCLC. The sialyl Lewis X-i antigen level was measured using a RIA kit. In patients with adenocarcinoma or other NSCLC subtypes, there was a correlation between serum sialyl Lewis X-i antigen and stage of the disease (P = 0.0001 and P = 0.0015, respectively). Levels of the marker varied significantly depending on the number of metastatic organs in adenocarcinoma (P = 0.0089) and in other NSCLC subtypes (P = 0.002). Univariate analysis showed that survival of NSCLC patients with high (more than 100 units/ml) sialyl Lewis X-i antigen levels was significantly poorer than that of patients with low antigen levels (P = 0.0001). Multivariate analysis using Cox's proportional hazard model showed that high sialyl Lewis X-i antigen levels correlated significantly with poor survival (P = 0.004). Our data suggest that a high serum level of sialyl Lewis X-i antigen seems to be an indicator of the presence of metastasis and might indicate the need for a careful investigation of all putative metastatic sites. The serum levels of sialyl Lewis X-i antigen may reflect the extension of metastasis and would be helpful in considering treatment options.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Female; Humans; Lewis Blood Group Antigens; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Staging; Oligosaccharides; Sialyl Lewis X Antigen; Survival Analysis; Time Factors

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.

Topics: Animals; Base Sequence; CA-19-9 Antigen; E-Selectin; Fucosyltransferases; Gangliosides; Liver Neoplasms; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Transgenic; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Peptide Fragments; Recombinant Fusion Proteins; Sialyl Lewis X Antigen

Metastatic prostate cancer has an unpredictable long-term prognosis. At present, there are few specific predictors to indicate the outcome for the individual patient. We have studied immunoreactivity for type-2 carbohydrate structures, known to be involved in various cell adhesion processes, in patients with metastatic prostate cancer. One group of patients (n = 26) did not progress within 3 years after orchiectomy, while another group of patients (n = 33) progressed within 1 year following castration and survived less than 2 years. Among the parameters studied, sialyl LewisX carbohydrate up-regulation was the only variable showing significant association with poor prognosis (P < 0.01). Sialyl LewisX discriminated between these two outcome groups with 71% predictability and 96% specificity. Our results indicate that up-regulation of sialyl LewisX is associated with hormonal-resistant, aggressive disease. This prognostic marker may, therefore, have an important role in selecting proper treatment for patients with metastatic prostate cancer.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Carbohydrate Sequence; Humans; Immunohistochemistry; Lewis X Antigen; Male; Middle Aged; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Predictive Value of Tests; Prognosis; Prostatic Neoplasms; Sialyl Lewis X Antigen; Up-Regulation

This report concerns the stepwise biosynthesis in vitro of Sialyl Lewis X, (SA-Le(x)), a carcinoembryonic antigen, in human colon carcinoma KM12 cells exhibiting different metastatic behaviors. The significance of SA-Le(x) has become even more apparent since the detection of its terminal epitope NeuAc(alpha 2-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, as the binding ligand of the selectin family member ELAM-1. The activity level of galactosyltransferase GalT-4 which catalyzes the formation of core nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) is very high in all the metastatic lines tested with highly metastatic lines (KM12-SM) exhibiting the highest activity. The same activity pattern for galactosyltransferase is also observed when tested with iLcOse5Cer (GlcNAc beta 1-3nLcOse4Cer), the precursor for polylactosamine glycolipid. Sialyltransferase SAT-3 which catalyzes the formation of LM1 (NeuAc alpha 2-3nLcOse4Cer), the precursor for SA-Le(x), is also present in all the metastatic cell lines although the activity levels are much lower compared to galactosyltransferase. The fucosyltransferase FucT-3, which catalyzes the formation of R'-Gal-Fuc(alpha 1-3)GlcNAc-R linkage, is active with both nonsialylated substrate, nLcOse4Cer, and sialylated substrate, LM1 (NeuAc alpha 2-3nLcOse4Cer) with the formation of either Le(x) (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc-Cer) or SA-Le(x) (NeuAc alpha 2-3nLcOse4Cer). However, the sialylated substrate LM1 is preferred to enzymatic activity since it exhibited lower Km (46 microM) than that of nLcOse4Cer (67 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Colonic Neoplasms; Fucosyltransferases; Galactosyltransferases; Humans; Kinetics; Lewis X Antigen; Molecular Sequence Data; Neoplasm Metastasis; Oligosaccharides; Sialyl Lewis X Antigen; Tumor Cells, Cultured