Synthesis Of sLeX Research Articles

Essential functions, syntheses and detection of sialyl Lewis X on glycoproteins

It is widely acknowledged that sialyl Lewis X (sLeX), the composition and linkage of which are N-acetylneuraminic acid (Neu5Ac) α2-3 galactose (Gal) β1-4 [fucose (Fuc) α1-3] N-acetylglucosamine, is usually attached to the cell surface. It presents as a terminal structure on either glycoproteins or glycolipids and has been demonstrated to be related to various biological processes, such as fertilization and selectin binding. Due to the vital role of sLeX, its synthesis as well as its determination approaches have attracted considerable attention from many researchers. In this review, the focus is sLeX on glycoproteins. The biological importance of sLeX in fertilization and development, immunity, cancers, and other aspects will be first introduced. Then the chemical and enzymatic synthesis of sLeX including the contributions from more than 15 international research groups will be described, followed by a brief view of the sLeX detection focusing on monosaccharides and linkages. This review is valuable for those readers who are interested in the chemistry and biology of sLeX.

Bisimidazolium Salt Glycosyltransferase Inhibitors Suppress Hepatocellular Carcinoma Progression In Vitro and In Vivo.

Hepatocellular carcinoma is a leading cause of cancer death, and the disease progression has been related to glycophenotype modifications. Previously synthesized bisimidazolium salts (C20 and C22) have been shown to selectively inhibit the activity of glycosyltransferases in cultured cancer cell homogenates. The current study investigated the anticancer effects of C20/C22 and the possible pathways through which these effects are achieved. The therapeutic value of C20/C22 in terms of inhibiting cancer cell proliferation, metastasis, and angiogenesis, as well as inducing apoptosis, were examined with hepatic cancer cell line HepG2 and a xenograft mouse model. C20/C22 treatment downregulated the synthesis of SLex and Ley sugar epitopes and suppressed selectin-mediated cancer cell metastasis. C20/C22 inhibited HepG2 proliferation, induced cell-cycle arrest, increased intracellular ROS level, led to ER stress, and eventually induced apoptosis through the intrinsic pathway. Furthermore, C20/C22 upregulated the expressions of death receptors DR4 and DR5, substantially increasing the sensitivity of HepG2 to TRAIL-triggered apoptosis. In vivo, C20/C22 effectively inhibited tumor growth and angiogenesis in the xenograft mouse model without adverse effects on major organs. In summary, C20 and C22 are new promising anti-hepatic cancer agents with multiple mechanisms in controlling cancer cell growth, metastasis, and apoptosis, and they merit further development into anticancer drugs.

L1CAM as an E-selectin Ligand in Colon Cancer.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.

α-1,3-Fucosyltransferase-VII siRNA inhibits the expression of SLex and hepatocarcinoma cell proliferation.

The increased expression of sialyl-Lewisx (SLex) epitope on the surface of tumor cells has been known for decades. However, genetic manipulation of the expression of SLex and the role of SLex in cancer cell proliferation remains to be fully elucidated. The present study suggested that the monoclonal antibody of SLex (KM93) significantly inhibited the proliferation of human hepatocarcinoma (HCC) cells. The expression levels of three sialyl-Lewis oligosaccharide antigens, SLex, SLea and dimeric SLex (SDLex), were determined on the cell surface of the MHCC97 human HCC cell line. The expression of SLex was markedly higher in MHCC97 cells than in normal liver cells. The expression of SDLex was also relatively high, however, no significant difference was observed between normal liver cells and HCC cells. The expression of SLea was only detected in trace quantities. Fucosyltransferase (FUT) is the key enzyme of the fucosylation step in the biosynthesis of sialyl-Lewis oligosaccharide antigens. Therefore, the present study investigated the expression of FUTs. It was found that the mRNA and protein expression levels of FUT7 were high in the MHCC97 HCC cell line compared with levels in normal liver cells. FUT6 was also expressed at a high level, although the difference was not statistically significant between MHCC97 cells and normal liver cells. No expression of FUT3 was detected. The results were consistent with the change insialyl-Lewis antigens. The effects of FUT7 small interfering (si)RNA transfection on the expression of FUT7, expression of SLex and MHCC97 cell proliferation were also examined. Following FUT7 siRNA transfection, the expression of FUT7 was markedly downregulated, as determined by western blot and reverse transcription-quantitative polymerase chain reaction methods. The results from flow cytometry showed that the synthesis of SLex was also inhibited, which was consistent with the downregulated expression of FUT7. MHCC97 cell proliferation was also significantly inhibited following FUT7 siRNA transfection, which was correlated with suppression of the S-phase in cell cycle progression. By using inhibitors of various signaling pathways, it was found that the knockdown of FUT7 inhibited the activation of phospholipase Cγ (PLCγ) by inhibiting the translocation and phosphorylation of PLCγ. In conclusion, the results suggested that FUT7 has animportant functional role in human HCC cell proliferation by controlling cell cycle progression via the PLCγ/extracellular signal-regulated kinase signaling pathway. The inhibition of SLex and FUT7 siRNA transfection may provide a novel therapeutic methodology to treat tumors that express SLex glycoconjugates.

Abstract 5885: Sialyl Lewis X-P-selectin connection between ovarian tumor-mesothelium in early stage metastasis

Abstract Metastasis is one of the major challenges for the treatment of ovarian cancer. Unlike other solid tumors, ovarian cancer primarily disseminates within the peritoneal cavity. A crucial step in metastasis formation is the adhesion of ovarian cancer cells onto the peritoneal mesothelium under ascitic shear flow. However, the adhesion mechanisms engaged in this tumor-mesothelium interaction remain elusive due to a lack of physiologically relevant model to manipulate and investigate this dynamic process. In this study, using a 3D microfluidic platform, we found that the metastatic population of cancer stem cells (M-CSCs) exhibited slower rolling velocity and higher binding ability to the peritoneal mesothelium than non-metastatic (NM)-CSCs under flow condition. This adhesion cascade was mediated by P-selectin which expressed on the peritoneal mesothelium. The key carbohydrate determinant on M-CSCs was a glycoprotein, but not a glycolipid, with its recognition as sialyl-Lewis X (sLeX) in a sialic acid- and fucose-dependent manner. Moreover, several glycosyltransferase genes including B4GALT4, ST3GAL3, ST3GAL4 and FUT-5 involved the synthesis of sLeX were upregulated in M-CSCs. Knocking down FUT-5 significantly inhibited ovarian tumor cell adhesion on the mesothelium in vitro and reduced the metastatic potential in vivo. Taken together, our findings revealed that a distinct sLeX-P-selectin axis of ovarian tumor-mesothelium interaction in early metastasis and may offer the possibility of new therapeutic targets. (This work is supported by RGC grant 17122014) Citation Format: Shan-Shan Li, Carman K. M. Ip, Ayon A. Hassan, Matthew Y. H. Tang, Susan Yung, Tak-Mao Chan, Ho Cheung Shum, Alice S. T. Wong. Sialyl Lewis X-P-selectin connection between ovarian tumor-mesothelium in early stage metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5885. doi:10.1158/1538-7445.AM2017-5885

Human chorionic gonadotropin (hCG) inhibits onset of preeclampsia in a “humanized” mouse model and restores uterine angiogenesis and regulatory T cells

During inflammation, E- and P-selectins appear on activated endothelial cells to interact with leukocytes through sialyl-Lewis x and sialyl-Lewis a antigens (sLex/a). These selectins can also interact with tumor cells in a sialyl-Lewis-dependent manner and for this reason, they are thought to play a key role in metastasis. Diverting the biosynthesis of sialyl-Lewis antigens toward nonadhesive structures is an attractive gene therapy for preventing the hematogenous metastatic spread of cancers. We have previously shown that transfection of α(1,2)-fucosyltransferase-I (FUT1) in Chinese hamster ovary (CHO) cells had a slight effect on the overall sialylation while the synthesis of sLEx was dramatically prevented. We herein delivered the gene of FUT1 by a human immunodeficiency virus-derived lentiviral vector to three human cancer cell lines including pancreatic (BxPC3), hepatic (HepG2), and colonic (HT-29) cancer cells. We found that on FUT1 transduction, all cells exhibited a dramatic decrease in sLex synthesis with a concomitant increase in Ley and Leb expression, without any detectable effect on the level of cell surface sLea antigens. In parallel, FUT1-transduced HT-29 and HepG2 cells, but not BxPC3 cells, failed to interact with E-selectin as assessed by E-selectin-binding assay or dynamic adhesion to activated endothelial cells. We show also that transduced FUT1 efficiently fucosylates the P-selectin ligand PSGL-1 without altering P-selectin binding. These results have important implications for understanding cell-specific reactions underlying the synthesis of selectin ligands in cancer cells and may provide a basis for the development of anti-metastatic gene therapy.

The Effect of Overexpression of α (1, 3)- FucosyltransferaseVII on Adhesive Capability of Human Colon Carcinoma HT-29 Cells to HUVECs

There are sufficient evidences that Lewis antigens are tumor-associated molecules and correlated to high grade and poor prognosis tumors. In this study, we investigated the effect of (α1,3)-fucosyltransferase VII overexpression on the synthesis of sLex and adhesive capability of human colon carcinoma HT-29 cells to HUVECs.The pIRES2-EGFP-FucT VII eukaryotic expression vector were transiently transfected into HT-29 cells. The changes of FucT VII protein and mRNA expression were determined by flow cytomet- ry and Real-Time PCR; the effect of FucT VII overexpression on synthesis of its downstream product-sLex is detected by Flow cytometry; Rose-Bengal method is used to assay the capability of HT-29 cell adhesion to HUVECs. Results: Eukaryotic expression vector pIRES2-EGFP-FucT VII was successfully transfected into HT-29 cells and made FucT VII overexpressed; compared with that of control group, expression level of the sLeX on the surface of FucT VII transfected HT-29 cells was significantly higher; FucT VII overexpression could enhance the adhesive capability of HT-29 cells to HUVECs. Our data suggest that overexpression of FucT VII could strengthen adhesion of sLeX-mediated HT-29 cells to HUVECs through upregulating sLeX synthesis.

Neutrophil interactions with sialyl Lewis X on human nonsmall cell lung carcinoma cells regulate invasive behavior

The carbohydrate sialyl Lewis X (sLeX) is expressed on leukocytes and carcinoma cells and binds to selectins during inflammatory processes and early metastasis. Synthesis of sLeX depends on activity of enzymes, including α(1,3/1,4) fucosyltransferase (FucT-III). Tumor necrosis factor-α (TNF-α) up-regulates FucT-III, resulting in increased sLeX in the airways of patients with respiratory disease; however, the mechanisms that regulate sLeX in the inflammatory tumor microenvironment are not well understood. The authors stably transfected human lung carcinoma cell lines with the FucT-III gene and exposed them to TNF-α to investigate its role in regulation of sLeX expression and selectin-binding ability using semiquantitative real-time polymerase chain reaction and flow cytometry. Cytokine expression was examined in transfected cells using chemiluminescent arrays and enzyme-linked immunosorbent assays, and invasion was studied using Matrigel assays and alterations in morphology. Human lung tissue arrays were analyzed for immunohistochemical detection of sLeX and neutrophils. Stimulation of FucT-III-transfected cells with recombinant human (rh) TNF-α up-regulated sLeX expression and increased E-selectin binding. Transfected cells secreted high levels of interleukin 8, growth-regulated oncogene-α, and mast cell proteinase-1. Cells exposed to rhTNF-α, neutrophil-conditioned media, and cultures with a 5:1 ratio of neutrophils to cancer cells had significantly increased sLeX expression and invasiveness and underwent nonadherent morphologic changes. In lung carcinomas, but not in normal lung tissues, 71% of tumors were highly positive for sLeX expression in areas of increased neutrophil infiltration. The current results indicated that neutrophils may be recruited to areas of FucT-III activity and sLeX expression in lung carcinomas to enhance the invasive and metastatic potential of lung cancer cells.

Abstract 2339: Stimulation of sialyl Lewis X carbohydrate expression in lung carcinoma cells by neutrophils: Implications for metastasis

Abstract Sialyl Lewis X (sLeX) is a carbohydrate epitope expressed on leukocytes and carcinoma cells that promotes binding of both cell types to vascular endothelium by selectin mediated processes. These binding events are crucial for leukocyte trafficking to lymphoid tissues and sites of inflammation and metastasis of cancer cells. Increased sLeX expression is correlated with advanced disease and a poor prognosis for several types of carcinomas. Synthesis of sLeX is dependent on activity of the enzymes α-(1,3)-fucosyltransferase (FucT-III) and α2,3-sialyltransferase. Exposure to tumor necrosis factor alpha (TNF-α) has been described to upregulate FucT-III resulting in increased sLeX in the airways of patients with respiratory disease, however, little is known about the molecular mechanisms involved in regulation of sLeX expression in the inflammatory tumor microenvironment. We stably transfected lung carcinoma cells with FucT-III to investigate the role of TNF-α in regulation of sLeX expression, selectin binding ability, and metastatic potential of lung carcinoma cells. Stimulation of FucT-III transfected but not mock-transfected H1299 and A549 lung carcinoma cells with recombinant TNF-α resulted in a concentration dependent upregulation of sLeX expression, increased binding of carcinoma cells to E-selectin, and increased migration of transfectants through Matrigel. Lung carcinoma cells transfected with FucT-III demonstrated high secretion of the neutrophil chemoattractant cytokines IL-8, GRO-α and MCP-1. Co-culture of lung carcinoma cells with neutrophil conditioned media and neutrophils isolated from blood at an optimal ratio of 1:5 resulted in significantly increased sLeX expression (p = 0.02 and p = 0.002 respectively). Exposure of transfected carcinoma cells to neutrophil conditioned media and neutrophils resulted in morphological changes consistent with increased invasive potential and a TNF-α dependent increased cellular migration through Matrigel. In patient lung carcinoma specimens but not in normal lung tissues, we observed areas of high sLeX expression that correlated with increased neutrophil infiltration in 80% of tumors. We conclude that FucT-III activity and sLeX expression in lung carcinomas may recruit neutrophils in the tumor microenvironment that enhance the metastatic potential of lung tumor cells. This work was supported in part by funds from the National Institutes of Health/National Cancer Institute grant K08 CA111829-04. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2339.

Activation of host antiviral RNA-sensing factors necessary for herpes simplex virus type 1-activated transcription of host cell fucosyltransferase genes FUT3, FUT5, and FUT6 and subsequent expression of sLex in virus-infected cells

Herpes simplex virus type 1 (HSV-1) induces expression of a selectin receptor, the carbohydrate epitope sialyl Lewis X (sLe(x)), at the surface of infected cells. The molecular background to this phenomenon is that a viral immediate early RNA interacts with as yet unidentified host factors, eventually resulting in transcription of three dormant host fucosyltransferase genes (FUT3, FUT5, and FUT6), whose gene products are rate-limiting for synthesis of sLe(x). The aim of the present study was to define the immediate targets for the viral RNA in this process. We found that the Protein Kinase R (PKR) inhibitors 2-aminopurine (2-AP) and C16 inhibited FUT3, FUT5, and FUT6 expression as well as HSV-1-induced expression of sLe(x), indicating a primary role of PKR as a viral RNA target. The PKR-dependent activation of the FUT genes seemed neither to involve PKR effects on translation nor to involve NF-kappaB- or JNK-dependent activation. IMD-0354, known as an inhibitor of the NF-kappaB-activating factor IKK-2, induced FUT transcription via a novel IKK-2-independent mechanism, irrespective of whether the cells were virus-infected or not. Altogether, the results suggested that PKR is the primary target for HSV-1 early RNA during induction of FUT3, FUT5, and FUT6, and that the subsequent steps in the transcriptional activation of these host genes involve a hitherto unknown IMD-0354, yet IKK-2-independent, pathway.

FUT7 antisense sequence inhibits the expression of FUT7/sLeX and adhesion between embryonic and uterine cells

Implantation is a complex developmental event that is initiated by recognition and adhesion of the embryo to the endometrial epithelium. sLeX is an oligosaccharide antigen acting as the ligand of L-selectin, and is stage-specifically expressed in the endometrial epithelium. The adhesion system mediated by L-selectin and sLeX oligosaccharide plays an important role in this process. FUT7 is a key enzyme for sLeX synthesis, and the regulation of sLeX through FUT7 may influence maternal-fetal recognition. In this study, we observed the effect of FUT7 antisense oligodeoxynucleotide on the expression of FUT7 and sLeX, as well as adhesion in an in vitro implantation model consisting of the human uterine epithelial cell line RL95-2 and the human embryonic cell line JAR. Results showed that the expression of FUT7 was significantly decreased, compared with controls, after FUT7 antisense oligodeoxynucleotide transfection into RL95-2 cells, as determined by RT-PCR, Western blotting, and indirect immunofluorescence. Synthesis of sLeX was also decreased, consistent with the FUT7 decrease, as shown by indirect immunofluorescence. The adhesion of embryonic cells to uterine epithelial cells was significantly reduced (P < 0.01) compared with the control. These data indicate that the use of a FUT7 antisense oligodeoxynucleotide can cause a significant reduction of both FUT7 and sLeX antigen, and thereby inhibit the adhesion of embryo cells to endometrium. This approach may provide a new way to regulate reproduction.

Butyrate induces sLex synthesis by stimulation of selective glycosyltransferase genes

Sialyl Lewisx (sLex) is an important tumor-associated carbohydrate antigen present on the cell surface glycoconjugates involved in leukocyte migration and cancer metastasis. We report the formation of sLex epitope in butyrate-treated human pancreatic adenocarcinoma cells expressing MUC1 and core 2 N-acetylglucosaminyltransferase (C2GnT). Butyrate treatment stimulates not only the transgene but also a group of endogenous glycosyltransferase genes involved in the synthesis of sLex. Current finding raises a concern about the proposed use of butyrate as a cancer therapeutic agent.

Airway epithelial wound repair: role of carbohydrate sialyl Lewisx

Epithelial repair is a complex cellular and molecular process, the details of which are still not clearly understood. Plasma membrane glycoconjugates can modulate cell function by altering the function of protein and lipids. Sialyl Lewisx (sLex), a fucose-containing tetrasaccharide, decorates membrane-bound and secreted proteins and mediates cell-cell interaction. In the present study we investigated the role of sLex in airway epithelial repair. Using immunohistochemistry, we showed an increased expression of sLex in areas of damaged bronchial epithelium compared with intact regions. Confluent monolayers of airway epithelial cells were mechanically wounded and allowed to close. Wounded monolayers were photographed for wound closure kinetics, fixed for immunocytochemical studies, or subjected to RNA extraction. Examining the expression of different alpha1,3-fucosyltransferases (FucT), enzymes that mediate the final step in the synthesis of sLex, we found that FucT-IV was the common gene expressed in all cell lines and primary airway epithelial cells. We demonstrated an increased expression of sLex over time after mechanical injury. Blocking of sLex with an inhibitory antibody completely prevented epithelial repair. Our data suggest an essential functional role for sLex in epithelial repair. Further studies are necessary to explore the exact mechanism for sLex in mediating cell-cell interaction in bronchial epithelial cells to facilitate epithelial migration and repair.

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.

Effect of N-acetylglucosaminyltransferase V on the expressions of other glycosyltransferases

Transfection of sense cDNA of N-acetylglucosaminyltransferase V (GnTV) into H7721 human hepatocellular carcinoma cells resulted in the decreased expression of surface sialyl Lewis X (SLex), a sialylated fucose-containing antigen. The enzymatic mechanisms were speculated to be the concomitantly decreased expression of α1,3-fucosyltransferase (FucT)-III, -VI, -VII and the branching enzyme of O-glycans, core 2-β1,6-N-acetylglucosaminyltransferase (C2GnT)-I, -II. These two glycosyltransferase families were suggested to be the key enzymes in the synthesis of SLex. The expression of α2,3-sialyltransferase (ST3)-IV, but not ST3-I, -II and -III was elevated by sense GnTV. However, it did not cause the increase of SLex synthesis. Transfection of antisense GnTV into H7721 cells showed entirely opposite effects on the expression of above-mentioned SLex and glycosyltransferases as the sense GnTV.

Gene expression of fucosyl- and sialyl-transferases which synthesize sialyl Lewisx, the carbohydrate ligands for E-selectin, in human breast cancer.

The adhesion of circulating cancer cells to vascular endothelium is an important step in the hematogenous metastasis of cancer. Until recently, it has been believed that carbohydrate antigens are expressed on cancer cells, and E-selectin is expressed on endothelial cells to effect this adhesion. We investigated the gene expression of fucosyl-transferase (Fuc-T) and sialyltransferase (ST), which are involved in the synthesis of sialyl Lewisx (s-Lex) in breast cancer by using Northern blot analysis. The concentration of s-Lex in the cancerous portion was increased, compared to that in the adjacent non-cancerous portion. A correlation was found between the concentration of s-Lex and the amount of Fuc-T VI message in 9 cases of breast cancer tissue. Expression of the Fuc-T III message was found in only one case who expressed s-Lea. No expression of the Fuc-T V or VII message was observed. There was no relationship between the concentration of s-Lex and the amount of ST3N and ST4 transcripts. Similar findings were obtained from an analysis using cell lines derived from human breast cancer. When Fuc-T VI gene was transfected to MCF-7 cells, the expression of s-Lex was markedly induced on MCF-7 cells, and the attachment of cancer cells to endothelial cells was enhanced. These findings suggest that Fuc-T VI is chiefly involved in the synthesis of s-Lex on breast cancer cells.

セレクチン阻害薬の開発における効率的グリコシル化反応の設計と展開

Current progress on glycobiology and glycotechnology urge the development of various new drugs. As an example of the applicable studies based on the carbohydrate chemistry, there is the drug design of selectin antagonists which block the interaction between the selectin and its natural ligand, sialyl Lewis X (sLeX). The highly effective synthesis of sLeX is one of the important things for the investigation of selectin antagonists, for example, to establish a high-throughput screening (HTS) of active compounds. As a result, we succeeded the highly practical synthesis of sLeX pentasaccharide and discovered a potent selectin antagonist, GSC-150, using the HTS. In addition, in order to establish a large scale synthesis of GSC-150, a one-pot glycosylation characterized by “Armed-Disarmed Coupling” was developed. This glycosylation was an applicable method for the synthesis of various LeX analogs.

Glucose-transporter-type-I-gene amplification correlates with sialyl-Lewis-X synthesis and proliferation in lung cancer.

Increased glucose transport is a common characteristic of most tumors. To examine the role of elevated glucose uptake in lung cancer, we performed PCR amplification of 2 facilitative glucose transporter genes (GLUT1 and GLUT3) and immunohistochemical staining for GLUT1, proliferating cell nuclear antigen (PCNA), and sialyl Lewis x (sLe(x)) on tumor specimens from 327 patients with lung cancer who underwent surgical resection from 1980 to 1993. To evaluate the relationship between GLUT, alpha-2,3-sialyltransferase (ST), and alpha-1,3-fucosyltransferase (Fuc-T) genes, PCR amplification of the ST3N and Fuc-TVII also was performed. Amplification of GLUT1 was significantly greater than that of GLUT3. GLUT1 and GLUT3 amplification correlated with PCNA staining (p < 0.01). In addition, GLUT1 amplification correlated with the grading of sLe(x) staining as well as with the grading of GLUT1 staining (p < .03, p < 0.01). GLUT1 was co-amplified with ST3N and Fuc-TVII genes, which are involved in the synthesis of sLe(x) (p < 0.01). The survival of patients whose tumors showed GLUT1 amplification was significantly shorter than that of patients whose tumors did not (p < 0.01). In a multivariate analysis of survival, GLUT1 remained a statistically significant prognostic factor. Our results suggest that GLUT1 amplification may participate in sLe(x) synthesis as well as in proliferation, and may be of prognostic value in lung cancer.