Synthesis of cyclic ß-1,6-oligosaccharides from glucosamine monomers by electrochemical polyglycosylation.

The synthesis of protected precursors of cyclic ß-1,6-oligoglucosamines from thioglycosides as monomers is performed by electrochemical polyglycosylation. The monomer with a 2,3-oxazolidinone protecting group afforded the cyclic disaccharide exclusively. Cyclic oligosaccharides up to the trisaccharide were obtained using the monomer with a 2-azido-2-deoxy group.

Two-Dimensional Living Supramolecular Polymerization: Improvement in Edge Roughness of Supramolecular Nanosheets by Using a Dummy Monomer.

Supramolecular polymers are formed through nucleation (i. e., initiation) and polymerization processes, and kinetic control over the nucleation process has recently led to the realization of living supramolecular polymerization. Changing the viewpoint, herein we focus on controlling the polymerization process, which we expect to pave the way to further developments in controlled supramolecular polymerization. In our previous study, two-dimensional living supramolecular polymerization was used to produce supramolecular nanosheets with a controlled area; however, these had rough edges. In this study, the growth of the nanosheets was controlled by using a 'dummy' monomer to produce supramolecular nanosheets with smoothed edges.

Electrochemical synthesis of the protected cyclic (1,3;1,6)-ß-glucan dodecasaccharide.

Automated electrochemical assembly is an electrochemical method to synthesise middle-sized molecules, including linear oligosaccharides, and some linear oligosaccharides can be electrochemically converted into the corresponding cyclic oligosaccharides effectively. In this study, the target cyclic oligosaccharide is a protected cyclic (1,3;1,6)-ß-glucan dodecasaccharide, which consists of two types of glucose trisaccharides with ß-(1,3)- and ß-(1,6)-glycosidic linkages. The formation of the protected cyclic dodecasaccharide was confirmed by the electrochemical one-pot dimerisation-cyclisation of the semi-circular hexasaccharide. The yield of the protected cyclic dodecasaccharide was improved by using a stepwise synthesis via the linear dodecasaccharide.

Multistep, site-selective noncovalent synthesis of two-dimensional block supramolecular polymers.

Although the principles of noncovalent bonding are well understood and form the basis for the syntheses of many intricate supramolecular structures, supramolecular noncovalent synthesis cannot yet achieve the levels of precision and complexity that are attainable in organic and/or macromolecular covalent synthesis. Here we show the stepwise synthesis of block supramolecular polymers from metal-porphyrin derivatives (in which the metal centre is Zn, Cu or Ni) functionalized with fluorinated alkyl chains. These monomers first undergo a one-dimensional supramolecular polymerization and cyclization process to form a toroidal structure. Subsequently, successive secondary nucleation, elongation and cyclization steps result in two-dimensional assemblies with concentric toroidal morphologies. The site selectivity endowed by the fluorinated chains, reminiscent of regioselectivity in covalent synthesis, enables the precise control of the compositions and sequences of the supramolecular structures, as demonstrated by the synthesis of several triblock supramolecular terpolymers.

Spatiotemporal changes of tissue glycans depending on localization in cardiac aging.

Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of cells. METHODS: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. RESULTS: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. CONCLUSIONS: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.

Mechanistic insights into cancer drug resistance through optogenetic PI3K signaling hyperactivation.

Hyperactivation of phosphatidylinositol 3-kinase (PI3K) signaling is a prominent feature in cancer cells. However, the mechanism underlying malignant behaviors in the state remains unknown. Here, we describe a mechanism of cancer drug resistance through the protein synthesis pathway, downstream of PI3K signaling. An optogenetic tool (named PPAP2) controlling PI3K signaling was developed. Melanoma cells stably expressing PPAP2 (A375-PPAP2) acquired resistance to a cancer drug in the hyperactivation state. Proteome analyses revealed that expression of the antiapoptotic factor tumor necrosis factor alpha-induced protein 8 (TNFAIP8) was upregulated. TNFAIP8 upregulation was mediated by protein translation from preexisting mRNA. These results suggest that cancer cells escape death via upregulation of TNFAIP8 expression from preexisting mRNA even though alkylating cancer drugs damage DNA.

PRC2-dependent regulation of ganglioside expression during dedifferentiation contributes to the proliferation and migration of vascular smooth muscle cells.

Phenotypic switching between contractile (differentiated state) and proliferative (dedifferentiated state) vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling that contributes to atherosclerotic diseases. Gangliosides, a group of glycosphingolipids, have been detected in atherosclerotic lesions and are suspected to contribute to the disease process. However, the underlying mechanism, specifically with respect to their role in VSMC phenotype switching, is not clear. In this study, we sought to reveal the endogenous expression of gangliosides and their functional significance in VSMCs during atherosclerosis. We found that switching from the contractile to proliferative phenotype was accompanied by upregulation of a- and b-series gangliosides, which in turn, were regulated by polycomb repressor complex 2 (PRC2). Downregulation of ganglioside expression using an siRNA targeting ST3GAL5, which is required for the synthesis of a- and b-series gangliosides, attenuated the proliferation and migration of dedifferentiated VSMCs. Therefore, we concluded that the increased expression of a- and b-series gangliosides via PRC2 activity during dedifferentiation is involved in the proliferation and migration of VSMCs. Gangliosides may be an effective target in VSMCs for atherosclerosis prevention and treatment.

Synthesis of protected precursors of chitin oligosaccharides by electrochemical polyglycosylation of thioglycosides.

The synthesis of protected precursors of chitin oligosaccharides by electrochemical polyglycosylation of thioglycosides as monomer is described. Oligosaccharides up to the hexasaccharide were synthesized under optimized reaction conditions. Further, a modified method enabled the synthesis of oligosaccharides up to the octasaccharide by repeating electrolysis with additional monomers. The mechanism of the electrochemical polyglycosylation is also discussed, based on the oxidation potential of the monomer and oligosaccharides.

Epithelial and Mesenchymal Features of Pancreatic Ductal Adenocarcinoma Cell Lines in Two- and Three-Dimensional Cultures.

Pancreatic ductal adenocarcinoma (PDAC) is an intractable cancer that is difficult to diagnose early, and there is no cure other than surgery. PDAC is classified as an adenocarcinoma that has limited effective anticancer drug and molecular-targeted therapies compared to adenocarcinoma found in other organs. A large number of cancer cell lines have been established from patients with PDAC that have different genetic abnormalities, including four driver genes; however, little is known about the differences in biological behaviors among these cell lines. Recent studies have shown that PDAC cell lines can be divided into epithelial and mesenchymal cell lines. In 3D cultures, morphological and functional differences between epithelial and mesenchymal PDAC cell lines were observed as well as the drug effects of different anticancer drugs. These effects included gemcitabine causing an increased growth inhibition of epithelial PDAC cells, while nab-paclitaxel caused greater mesenchymal PDAC cell inhibition. Thus, examining the characteristics of epithelial or mesenchymal PDAC cells with stromal cells using a 3D co-culture may lead to the development of new anticancer drugs.

Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression.

Signaling pathways involving signal transducer and activator of transcription 3 (STAT3) play key roles in the aggressiveness of pancreatic ductal adenocarcinoma (PDAC), including their tumorigenesis, invasion, and metastasis. Cancer stem cells (CSCs) have been correlated with PDAC aggressiveness, and activation of STAT3 is involved in the regulation of CSC properties. Here, we investigated the involvement of interleukin-6 (IL-6) or the leukemia inhibitory factor (LIF)/glycoprotein 130 (gp130)/STAT3 pathway and their role in pancreatic CSCs. In PDAC CSC-like cells formed by culturing on a low attachment plate, autocrine/paracrine IL-6 or LIF contributes to gp130/STAT3 pathway activation. Using a gp130 inhibitor, we determined that the gp130/STAT3 pathway contributes to the maintenance of stemness features, the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP), and the invasion of PDAC CSC-like cells. The gp130/STAT3 pathway also modulates the transforming growth factor (TGF)-ß1/Smad pathway required for epithelial-mesenchymal transition induction through regulation of TGFß-RII expression in PDAC CSC-like cells. Furthermore, chromatin immunoprecipitation assays revealed that p-STAT3 can access the active promoter region of H19 to influence this metastasis-related long non-coding RNA and contribute to its transcription in PDAC CSC-like cells. Therefore, the autocrine/paracrine IL-6 or LIF/gp130/STAT3 pathway in PDAC CSC-like cells may eventually facilitate invasion and metastasis, two hallmarks of malignancy. We propose that inhibition of the gp130/STAT3 pathway provides a promising strategy for targeting CSCs for the treatment of PDAC.

TGF-ß1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-ß1) and whether EMT could be induced through TGF-ß1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-ß1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-ß1. Analysis of EMT markers showed that mRNA levels changed with TGF-ß1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-ß1 treatment. Invasion assays showed that TGF-ß1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-ß1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.

A New Anorectal Melanoma Cell Line Derived from a Primary Human Rectal Tumor.

BACKGROUND: Anorectal melanoma is a rare disease with a poor prognosis. Symptoms are often nonspecific, which complicates preoperative diagnosis. Here, we describe the establishment of MELS, a new anorectal melanoma cell line derived from resection of a rectal tumor in a 40-year-old Japanese man. METHODS: Histological, electron microscopic, and immunohistochemical features of S-100, HMB-45, Melan-A, and NSE positivity of the tumor were typical of surgically resected anorectal melanoma. RESULTS: MELS cells are round or oval and have sharp thorn-like protrusions on some or all cell membranes. The cells form irregular attached colonies with numerous floating cells in two-dimensional culture. Transmission electron microscopy revealed that some MELS cells have cytoplasmic melanosomes. Immunocytochemically, MELS cells and surgical tissues had the same staining pattern. MELS cells had lower growth rates than Caco-2 (a colon adenocarcinoma cell line) and A375 (a cutaneous melanoma cell line) cells. Oxaliplatin and irinotecan were more effective in MELS cells than in Caco-2 and A375 cells. CONCLUSIONS: No previous report provided detailed clinical information on an anorectal melanoma cell line. Thus, MELS cells should improve our understanding of the biological characteristics of anorectal melanoma and provide a novel platform for examining the effects of therapies for anorectal melanoma.

Cell Surface and Functional Features of Cortical Bone Stem Cells.

The newly established mouse cortical-bone-derived stem cells (mCBSCs) are unique stem cells compared to mouse mesenchymal stem cells (mMSCs). The mCBSC-treated hearts after myocardial infarction have been reported to have greater improvement in myocardial structure and functions. In this study, we examined the stemness features, cell surface glycan profiles, and paracrine functions of mCBSCs compared with mMSCs. The stemness analysis revealed that the self-renewing capacity of mCBSCs was greater than mMSCs; however, the differentiation capacity of mCBSCs was limited to the chondrogenic lineage among three types of cells (adipocyte, osteoblast, chondrocyte). The cell surface glycan profiles by lectin array analysis revealed that α2-6sialic acid is expressed at very low levels on the cell surface of mCBSCs compared with that on mMSCs. In contrast, the lactosamine (Galß1-4GlcNAc) structure, poly lactosamine- or poly N-acetylglucosamine structure, and α2-3sialic acid on both N- and O-glycans were more highly expressed in mCBSCs. Moreover, we found that mCBSCs secrete a greater amount of TGF-ß1 compared to mMSCs, and that the TGF-ß1 contributed to the self-migration of mCBSCs and activation of fibroblasts. Together, these results suggest that unique characteristics in mCBSCs compared to mMSCs may lead to advanced utility of mCBSCs for cardiac and noncardiac repair.

Glycan characteristics of human heart constituent cells maintaining organ function: relatively stable glycan profiles in cellular senescence.

Cell surface glycoproteins, which are good indicators of cellular types and biological function; are suited for cell evaluation. Tissue remodeling using various cells is a key feature of regenerative therapy. For artificial heart remodeling, a mixture of heart constituent cells has been investigated for organ assembly, however, the cellular characteristics remain unclear. In this study, the glycan profiles of human cardiomyocytes (HCMs), human cardiac fibroblasts (HCFs), and human vascular endothelial cells (ECs) were analyzed using evanescent-field lectin microarray analysis, a tool of glycan profiling, to clarify the required cellular characteristics. We found that ECs had more "α1-2fucose" and "core α1-6fucose" residues than other cells, and that "α2-6sialic acid" residue was more abundant in ECs and HCMs than in HCFs. HCFs showed higher abundance of "ß-galactose" and "ß-N-acetylgalactosamine" residues on N-glycan and O-glycan, respectively, compared to other cells. Interestingly, cardiac glycan profiles were insignificantly changed with cellular senescence. The residues identified in this study may participate in organ maintenance by contributing to the preservation of glycan components. Therefore, future studies should investigate the roles of glycans in optimal tissue remodeling since identifying cellular characteristics is important for the development of regenerative therapies.

Gangliosides as Signaling Regulators in Cancer.

At the plasma membrane, gangliosides, a group of glycosphingolipids, are expressed along with glycosphingolipids, phospholipids, and cholesterol in so-called lipid rafts that interact with signaling receptors and related molecules. Most cancers present abnormalities in the intracellular signal transduction system involved in tumor growth, invasion, and metastasis. To date, the roles of gangliosides as regulators of signal transduction have been reported in several cancer types. Gangliosides can be expressed by the exogenous ganglioside addition, with their endogenous expression regulated at the enzymatic level by targeting specific glycosyltransferases. Accordingly, the relationship between changes in the composition of cell surface gangliosides and signal transduction has been investigated by controlling ganglioside expression. In cancer cells, several types of signaling molecules are positively or negatively regulated by ganglioside expression levels, promoting malignant properties. Moreover, antibodies against gangliosides have been shown to possess cytotoxic effects on ganglioside-expressing cancer cells. In the present review, we highlight the involvement of gangliosides in the regulation of cancer cell signaling, and we explore possible therapies targeting ganglioside-expressing cancer.

Morphofunctional analysis of human pancreatic cancer cell lines in 2- and 3-dimensional cultures.

Genetic, transcriptional, and morphological differences have been reported in pancreatic ductal adenocarcinoma (PDAC) cases. We recently found that epithelial or mesenchymal features were enhanced in three-dimensional (3D) cultures compared to two-dimensional (2D) cultures. In this study, we examined the differences in the morphological and functional characteristics of eight PDAC cell lines in 2D and 3D cultures. Most PDAC cells showed similar pleomorphic morphologies in 2D culture. Under 3D culture, PDAC cells with high E-cadherin and low vimentin expression levels (epithelial) formed small round spheres encircled with flat lining cells, whereas those with high vimentin and low E-cadherin expression levels (mesenchymal) formed large grape-like spheres without lining cells and were highly proliferative. In 3D culture, gemcitabine was more effective for the spheres formed by PDAC cells with epithelial features, while abraxane was more effective on those with mesenchymal features. The expression levels of drug transporters were highest PDAC cells with high vimentin expression levels. These findings indicate that PDAC cells possess various levels of epithelial and mesenchymal characteristics. The 3D-culture method is useful for investigating the diversity of PDAC cell lines and may play important roles in the development of personalized early diagnostic methods and anticancer drugs for PDAC.

Polyvinyl alcohol increased growth, migration, invasion, and sphere size in the PK-8 pancreatic ductal adenocarcinoma cell line.

Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer used in eye drops, embolization particles, and artificial cartilage. It has also been shown to cause expansion of functional multipotent self-renewing hematopoietic stem cells under serum-free conditions. In this study, we examined the effects of PVA on human pancreatic ductal adenocarcinoma (PDAC) cell lines using 2-dimensional (2D) and 3D-cultures with serum-free medium. In the 2D-culture, PVA-treatment induced an aggregated colony-like appearance in PDAC cells. It increased the growth of PK-8 cells in a dose-dependent manner as well as significantly increasing migration and invasion abilities. qRT-PCR showed an increase in α2 integrin and a decrease in matrix metalloprotease levels in PVA-treated PK-8 cells. Through qRT-PCR analysis, ß1 integrin expression at the mRNA level was found to be decreased; however, it was unaltered at the protein level when assessed using FACS analysis. PVA further induced mesenchymal to epithelial transition-like alterations, including increased E-cadherin and decreased Vimentin and N-cadherin expression. Four cancer stem cell (CSC) markers were higher in PVA-treated PK-8 cells compared to controls. In 3D-culture, PVA-treated PK-8 cells showed a rod-like appearance with larger sphere size and higher growth ability. qRT-PCR showed that CSC markers did not increase and 2 of 4 drug transporters had decreased in PVA-treated PK-8 cells. These findings suggest that PVA increases the growth, migration, invasion, and sphere size of PK-8 cells, but does not increase the proportion of pancreatic CSCs under 3D-culture conditions with serum-free medium.

FGFR4 Inhibitor BLU9931 Attenuates Pancreatic Cancer Cell Proliferation and Invasion While Inducing Senescence: Evidence for Senolytic Therapy Potential in Pancreatic Cancer.

Fibroblast growth factor receptor 4 (FGFR4), one of four tyrosine kinase receptors for FGFs, is involved in diverse cellular processes. Activation of FGF19/FGFR4 signaling is closely associated with cancer development and progression. In this study, we examined the expression and roles of FGF19/FGFR4 signaling in human pancreatic ductal adenocarcinoma (PDAC). In human PDAC cases, FGFR4 expression positively correlated with larger primary tumors and more advanced stages. Among eight PDAC cell lines, FGFR4 was expressed at the highest levels in PK-1 cells, in which single-nucleotide polymorphism G388R in FGFR4 was detected. For inhibition of autocrine/paracrine FGF19/FGFR4 signaling, we used BLU9931, a highly selective FGFR4 inhibitor. Inhibition of signal transduction through ERK, AKT, and STAT3 pathways by BLU9931 reduced proliferation in FGF19/FGFR4 signaling-activated PDAC cells. By contrast, BLU9931 did not alter stemness features, including stemness marker expression, anticancer drug resistance, and sphere-forming ability. However, BLU9931 inhibited cell invasion, in part, by downregulating membrane-type matrix metalloproteinase-1 in FGF19/FGFR4 signaling-activated PDAC cells. Furthermore, downregulation of SIRT1 and SIRT6 by BLU9931 contributed to senescence induction, priming these cells for quercetin-induced death, a process termed senolysis. Thus, we propose that BLU9931 is a promising therapeutic agent in FGFR4-positive PDAC, especially when combined with senolysis (195/200).

Supramolecular double-stranded Archimedean spirals and concentric toroids.

Connecting molecular-level phenomena to larger scales and, ultimately, to sophisticated molecular systems that resemble living systems remains a considerable challenge in supramolecular chemistry. To this end, molecular self-assembly at higher hierarchical levels has to be understood and controlled. Here, we report unusual self-assembled structures formed from a simple porphyrin derivative. Unexpectedly, this formed a one-dimensional (1D) supramolecular polymer that coiled to give an Archimedean spiral. Our analysis of the supramolecular polymerization by using mass-balance models suggested that the Archimedean spiral is formed at high concentrations of the monomer, whereas other aggregation types might form at low concentrations. Gratifyingly, we discovered that our porphyrin-based monomer formed supramolecular concentric toroids at low concentrations. Moreover, a mechanistic insight into the self-assembly process permitted a controlled synthesis of these concentric toroids. This study both illustrates the richness of self-assembled structures at higher levels of hierarchy and demonstrates a topological effect in noncovalent synthesis.

Rapamycin promotes endothelial-mesenchymal transition during stress-induced premature senescence through the activation of autophagy.

BACKGROUND: Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). Therefore, it is highly expected to represent an anti-aging drug. Its anti-aging effect has been demonstrated at the mouse individual level. However, there are not many clinical findings with respect to its activity in humans. Here, we aimed to clarify the effect of rapamycin on human endothelial cells (ECs) as an in vitro model of human blood vessels. METHODS: Over the course of oxidative stress-induced senescence using hydrogen peroxide, we examined the effect of rapamycin on human coronary artery ECs (HCAECs). Senescence was evaluated by detecting senescence-associated ß-galactosidase (SA-ß-Gal) activity and the real-time PCR analysis of p16INK4a. Furthermore, expression levels of SASP factors were examined by real-time PCR and the expression of senescence-related antigens, such as intercellular adhesion molecule-1 (ICAM-1) and ganglioside GM1, were examined by fluorescence-activated cell sorting analysis and immunostaining. The inhibitory effect of rapamycin on mTOR signaling was examined by immunoblotting. The adhesion of leukocytes to HCAECs was evaluated by adhesion assays. Endothelial-mesenchymal transition (EndMT) induced by rapamycin treatment was evaluated by real-time PCR analysis and immunostaining for EndMT markers. Finally, we checked the activation of autophagy by immunoblotting and examined its contribution to EndMT by using a specific inhibitor. Furthermore, we examined how the activation of autophagy influences TGF-ß signaling by immunoblotting for Smad2/3 and Smad7. RESULTS: A decrease in SA-ß-Gal activity and the suppression of SASP factors were observed in HCAECs undergoing stress-induced premature senescence (SIPS) after rapamycin treatment. In contrast, ICAM-1 and ganglioside GM1 were upregulated by rapamycin treatment. In addition, leukocyte adhesion to HCAECs was promoted by this treatment. In rapamycin-treated HCAECs, morphological changes and the promotion of EndMT were also observed. Furthermore, we found that autophagy activation induced by rapamycin treatment, which led to activation of the TGF-ß pathway, contributed to EndMT induction. CONCLUSIONS: We revealed that although rapamycin functions to inhibit senescence and suppress SASP in HCAECs undergoing SIPS, EndMT is induced due to the activation of autophagy. Video abstract.