L-selectin ligands in rat high endothelium: multivalent sialyl Lewis x glycans are high-affinity inhibitors of lymphocyte adhesion

Lymphocyte homing is initiated by their tethering to and rolling on the high endothelium and is followed by extravasation into the lymph nodes. We show here that glycosylated cell adhesion molecule-1 (GlyCAM-1), CD34, and sialyl Lewis x (sLex) are present on rat lymph node high endothelium analyzed by using monoclonal antibodies. α(1,3)fucosyltransferase VII (Fuc-TVII), the last enzyme involved in the synthesis of the sLex sequence is also expressed on the rat lymph node high endothelium. We have synthesized a family of sLex-decorated oligosaccharide structures and used them to inhibit lymphocyte binding to high endothelium in the Stamper-Woodruff assay. Monovalent sLex, branched di- and tetravalent sLex, as well as a linear tetravalent sLex significantly reduce lymphocyte binding to endothelium. The branched and linear forms of tetravalent sLex were clearly superior inhibitors of the L-selectin-dependent lymphocyte adhesion, with IC50 values in low nanomolar range. In contrast, the fucose-free analogs having the same charge and approximately the same size as the corresponding sLex glycans had no effect on lymphocyte binding and served as negative controls. Taken together, these data show the crucial importance of sLex in the endothelial ligands for L-selectin. Furthermore, we suggest that L-selectin acts as an oligomer on the lymphocyte surface as it binds multivalent sLex glycans.     

Role of Sialylglycoconjugate(s) in the Initial Phase of Metastasis of Liver-metastatic RAW117 Lymphoma Cells

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.     

特异结合唾液酸化LewisX抗原DNA适配子抑制肝癌细胞株HepG2体外侵袭转移

目的 观察特异结合唾液酸化LewisX (SLeX)抗原DNA适配子抑制HepG2细胞与E-选择素黏附能力及其体外抑制HepG2细胞浸润转移能力.方法 采用黏附实验、Transwell体外侵袭实验,检测该适配子对HepG2细胞与E选择素黏附及对HepG2细胞体外侵袭的影响.结果 该DNA适配子可有效抑制HepG2细胞与E-选择素黏附,黏附细胞数随适配子浓度的增加而减少(P＜0.01),20 nmol浓度的适配子与单克隆抗体CSLEX-1效果相似;Transwell侵袭实验中,5、10、20nmol适配子组的侵袭细胞数分别为159.00±3.27、142.00±5.50、115.00±5.07,与对照组(178.00±4.64)比较,差异有统计学意义(P＜0.01).结论 特异结合唾液酸化LewisX抗原DNA适配子可以抑制HepG2细胞与E-选择素黏附,阻断Lewis-selectin途径,抑制HepG2细胞体外侵袭转移.     

基质金属蛋白酶-9和唾液酸化Lewis-X抗原表达与肝癌侵袭转移相关性研究

目的探讨基质金属蛋白酶-9(MMP-9)和唾液酸化Lewis-X抗原(sialyl Lewis-X,SLeX)与肝细胞性肝癌(HCC)侵袭转移的相关性。方法对45例HCC根治性切除病理组织切片进行苏木精-伊红染色,并用CD105,MMP-9,SLeX进行免疫组化染色,计数被CD105抗体染色的肿瘤血管内皮细胞数,以测定微血管密度(MVD),并对MVD与MMP-9、SLeX的关系进行分析。结果MMP-9、SLeX表达都呈阳性的肝癌组织微血管密度高于MMP-9和SLeX单阳性及二者均阴性的肝癌组织。结论MMP-9高表达有利于癌组织局部浸润,SLeX高表达有利于癌细胞血行转移。     

Correlation between nm23 Protein and Several Cell Adhesion Molecules in Human Gastric Carcinoma

The correlation between nm23 protein (nm23) expression and the expression of several cell adhesion molecules was studied immunohistochemically in 110 resected gastric carcinomas. Formalin-fixed and paraffin-embedded samples were serially sectioned and stained with antibodies against nm23, integrin β₁ subfamily members (α₂β₁, α₃β₁ and α₄β₁), LFA-1, ICAM-1, sialyl Lewis^x (sLe^x) and CD44h , -V3, and -V6. Primary carcinomas presenting with either lymph node involvement or liver metastasis expressed significantly reduced levels of nm23 compared to tumors without metastasis. The percent of tumors expressing each adhesion molecule was as follows: α₂β₁, 27.3%; α₃β₁, 20.0%; α₄β₁, 14.5%; LFA-1, 14.5%; ICAM-1, 12.7%; sLe^x, 67.3%; CD44h , 55.5%; CD44V3, 20.0%; and CD44V6, 4.5%. Expression of α₂β₁ integrin and high levels of sLe^x were significantly correlated with lymph node metastasis, and expression of α₃β₁ integrin and high levels of sLe^x were correlated with liver metastasis. Expression of ICAM-1 was inversely correlated with liver metastasis. Comparing the expression of each cell adhesion molecule with nm23 immunoreactivity, expression of sLe^x was significantly associated with nm23 expression. Of tumors expressing high levels of sLe^x, 75% showed reduced nm23 expression, compared to 52% of tumors with weak or no sLe^x expression (P < 0.05). A similar tendency was also observed in the metastasized secondary tumors. These results suggest that reduced nm23 expression may promote the metastatic properties of cancer cells in concert with increased sLe^x expression.     

CD4+ CD56+ neoplasia: clinical and biological features with emphasis on cytotoxic drug-induced apoptosis and expression of sialyl Lewis X

CD4+CD56+ neoplasia is a rare malignancy of unclarified origin. So far only 57 cases have been reported. We characterized in detail a case of CD4+ CD56+ malignancy with special emphasis on apoptosis induced by cytotoxic drugs and expression of sialyl Lewis X (CD15s). The disease was diagnosed in a 73-year-old female presenting with skin involvement, generalized lymphadenopathy and bone marrow infiltration. Treatment with cladribine/mitoxantrone induced a short-lasting partial response and the patient died 6 months after diagnosis. The neoplastic cells expressed CD4, CD56, HLA-DR, and CD15s. PCR for the T-cell receptor γchain revealed a polyclonal amplification product. In situ hybridization for Epstein-Barr Virus (EBV) was negative. Cytotoxic granule-associated proteins were not detected, consistent with the observation that the cells did not mediate cytotoxic activity against several target cells. Apoptosis of the tumor cells was inducible by anthracyclines and cladribine but not with gemcitabine. Combinations of cladribine or gemcitabine with anthracyclines however, resulted in synergistic effects on apoptosis. Expression of CD15s on the CD56+ cells was three times higher than on CD56+ cells from healthy controls. The results demonstrate that the features of the present case is in accordance with the diagnosis of CD4+ CD56+ malignancy. This is the first report demonstrating increased CD15s expression on a CD4+ CD56+ neoplasia, possibly explaining the frequent occurrence of the disease in the skin.     

上皮性卵巢肿瘤患者血浆中Sialyl Tn抗原水平的临床评估

目的对上皮性卵巢肿瘤患者血浆中STN水平进行评估.方法选用54例卵巢癌患者,以50例健康非孕妇女、12例妊娠妇女、76例其他妇科良性疾病妇女作为对照,通过放免方法对血清中STN水平进行检测.结果卵巢癌患者血清STN水平升高者为53.7%,在健康妇女中仅4%,在妇科良性疾病患者中为10.5%,妊娠妇女为0.在研究组和对照组之间存在着显著差异(P＜0.001).卵巢癌患者的STN水平为(109.2±146.4)×103U/L,并且随卵巢癌临床分期的增加,抗原水平及检出阳性率也增加.按组织学类型则阳性率最高的是黏液性囊腺癌(63.3%).结论由于STN抗原对卵巢癌的检测敏感性、特异性、阳性预测值、阴性预测值均较高,并且有较低的假阳性率,故STN抗原可作为卵巢癌检测的又一可靠指标.     

Expression of sialyl Lewis(a) relates to poor prognosis in cholangiocarcinoma

AIM: High levels of serum sialyl Lewisa (sLea) are frequently found in cholangiocarcinoma (CCA) patients and have been suggested to be a serum marker for CCA. However, the significance of this antigen in CCA is unknown. In this study, the clinical significance of sLea expression in CCA tissues and the possible role of sLea in vascular invasion in vitro were elucidated. METHODS: Expression of sLea in tumor tissues of 77 patients with mass-forming CCA and 33 with periductal infiltrating CCA was determined using immunohistochemistry. The in vitro assays on adhesion and transmigration of CCA cells to human umbilical vein endothelial cells were compared between CCA cell lines with and without sLea expression. RESULTS: sLea was aberrantly expressed in 60% of CCA tumor tissues. A significant relationship was found between the frequency of sLea expression and the mass-forming type CCA (P= 0.041), well differentiated histological grading (P=0.029), and vascular invasion (P=0.030). Patients with positive sLea expression had a significantly poorer prognosis (21.28 wk, 95% CI=16.75-25.81 wk) than those negative for sLea (37.30 wk, 95% CI=27.03-47.57 wk) (P<0.001). Multivariate analysis with adjustment for all covariates showed that patients positive for sLea possessed a 2.3-fold higher risk of death than patients negative for sLea (P<0.001). The role of sLea in vascular invasion was demonstrated using in vitro adhesion and transmigration assays. KKU-M213, a human CCA cell-line with a high expression of sLea, adhered and transmigrated to IL-1β-activated endothelial cells of the human umbilical vein more than KKU-100, the line without sLea expression (P<0.001). These processes were significantly diminished when the antibodies specific to either sLea or E-selectin were added to the assays (P<0.001) CONCLUSION: This study demonstrates the clinical significance of sLea expression in vascular invasion, and an unfavorable outcome in CCA. The role of sLea in vascular invasion which may lead to poor prognosis is supported by the in vitro adhesion and transmigration studies.     

宫颈癌组织SLeX、CD44v6和E-Cad表达及其临床意义

目的探讨细胞黏附分子唾液酸化路易斯-X(SLeX)抗原、CD44v6和E-钙黏附素(E-Cad)在浸润性宫颈鳞癌(ICC)中的表达及其临床意义。方法应用免疫组织化学方法,检测60例ICC组织和20例慢性宫颈炎(CCV)组织中SLeX、CD44v6和E-Cad蛋白的表达水平,并结合临床病理特征进行分析。结果ICC组织中SLeX、CD44v6和E-Cad蛋白阳性表达率分别为60.0%(36/60)、66.7%(40/60)和45.0%(27/60)。ICC组织SLeX及CD44v6阳性表达率均显著高于CCV组织(2χ=18.11、10.58,P<0.05),而E-Cad阳性表达率显著低于CCV组织(2χ=15.11,P<0.05)。ICC组织SLeX和CD44v6高表达及E-Cad低表达与ICC的分化程度和淋巴转移有关(χ2=4.15~14.61,P<0.05),但与临床分期无关(χ2=1.64~3.21,P>0.05);SLeX和CD44v6表达与E-Cad表达有关(2χ=7.87、7.58,P<0.05)。结论ICC侵袭转移过程中,SLeX、CD44v6和E-Cad表达可能具有正、负协同效应,联合检测三者表达可作为判断ICC预后的参考指标。     

Involvement of carbohydrate antigen sialyl Lewisx in colorectal cancer metastasis

PURPOSE: Recognition of metastatic tumor cells with distinct biochemical phenotypes predominant in the primary tumors should be useful not only for establishment of new therapeutic approaches but also for identification of highrisk or low-risk patients for relapse. We examined whether carbohydrate antigens, sialyl Lewis^x (sLe^x) and sialyl Lewis^a (sLe^a) are involved in colorectal cancer metastasis. METHODS: Metastatic abilities of human colon cancer cell variants that were selected for their high or low cell surface levels of sLe^x (KM12-HX and KM12-LX, respectively) were analyzed. Also, immunohistochemical expressions of sLe^x and sLe^a in 159 primary colorectal cancers were examined to determine the clinical significance of increased expression of these antigens. RESULTS: KM12-HX cells adhered more readily to tumor necrosis factor- activated endothelial cells than did KM12-LX cells. Increased adhesion of KM12-HX cells to activated endothelial cells was inhibited by antibodies against E-selectin and sLe^x and by modification of cell surface carbohydrates. KM12-HX cells showed more invasive ability in vitro and more metastatic potential in the liver of nude mice than KM12-LX cells. Although no difference was seen in the expression of six messenger ribonucleic acids corresponding to progression or metastasis of colorectal cancer, expression of fucosyltransferase was found to be responsible for the higher expression of sLe^x in KM12-HX cells. Clinical records of patients showed that disease-free survival rate of patients with sLe^x-positive tumors was significantly poorer than that of those with sLe^x-negative tumors. Cox's multivariate analysis revealed that the sLe^x status was an independent predictive factor for disease recurrence (P = 0.004), depth of invasion (P = 0.0005), and histologic type> (P = 0.037), but sLe^a status, age, gender, tumor location, N stage, and vessel invasion were not. CONCLUSION: Increased expression of sLe^x could be involved in establishment of colorectal cancer metastasis. It appears that examining sLe^x expression may serve as a potent marker of the recurrence in patients with colorectal cancer.Supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a New 10-Year Strategy for Cancer Control, Japan. Read at the meeting of The American Society of Colon and Rectal Surgeons, Seattle, Washington, June 9 to 14, 1996.