Yasunori Kushi

Cytotoxic and Apoptosis‐Inducing Activities of Steviol and Isosteviol Derivatives against Human Cancer Cell Lines

Seventeen steviol derivatives, i.e., 2–18, and 19 isosteviol derivatives, i.e., 19–37, were prepared from a diterpenoid glycoside, stevioside (1). Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines, nine steviol derivatives, i.e., 5–9 and 11–14, and five isosteviol derivatives, i.e., 28–32, exhibited activities with single‐digit micromolar IC50 values against one or more cell lines. All of these active compounds possess C(19)‐O‐acyl group, and among which, ent‐kaur‐16‐ene‐13,19‐diol 19‐O‐4′,4′,4′‐trifluorocrotonate (14) exhibited potent cytotoxicities against four cell lines with IC50 values in the range of 1.2–4.1 μM. Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis. These results suggested that acylation of the 19‐OH group of kaurane‐ and beyerane‐type diterpenoids might be useful for enhancement of their cytotoxicities with apoptosis‐inducing activity.

Cytotoxic activities of amino acid-conjugate derivatives of abietane-type diterpenoids against human cancer cell lines.

Nine amino acid conjugate derivatives, each 2–10 and 12–20, were prepared from abietic acid (1) and dehydroabietic acid (11), respectively, and they were evaluated for their cytotoxicities against four human cancer cell lines, i.e., leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3). All compounds showed cytotoxicities against HL60 with IC50 values in the range of 2.3–37.3 μM. In addition, most of the derivatives exhibited moderate cytotoxicities against the other cancer cell lines. Among the derivatives, methyl N‐[18‐oxoabieta‐8,11,13‐trien‐18‐yl]‐L‐tyrosinate (19) exhibited potent cytotoxic activities against four cancer cell lines with IC50 values of 2.3 (HL60), 7.1 (A549), 3.9 (AZ521), and 8.1 μM (SK‐BR‐3). Furthermore, all derivatives were shown to possess high selective cytotoxic activities for leukemia cells, since they exhibited only weak cytotoxicities against normal lymphocyte cell line RPMI1788.

Production and characterization of monoclonal antibodies specific to lactotriaosylceramide

We have established hybridoma cell lines producing monoclonal antibodies (mAbs) directed to N-acetylglucosaminylβ1-3galactose (GlcNAcβ1-3Gal) residue by immunizing BALB/c mice with lactotriaosylceramide (Lc(3)Cer). These obtained hybridoma cells, specific to Lc(3)Cer, were dual immunoglobulin (Ig)-producing cells which secreted both IgM and IgG molecules as antibodies. The established mAbs are able to react with not only Lc(3)Cer but also GlcNAcβ1-3-terminal glycosphingolipids (GSLs) despite branching or lactosamine chain lengths and human transferrin with terminal GlcNAc residues. Comparison of the variable regions of the cloned IgM and IgG by reversed transcription-polymerase chain reaction analysis confirmed that the variable regions determine the specificity, the other amino acids are conserved, and these mAbs are encoded by J558 and Vκ-21family genes. Furthermore, we have analyzed the expression of GSLs with GlcNAcβ1-3 epitope in acute leukemia cell lines and mouse fetal tissues using these mAbs, in which antigens were distributed comparatively. These mAbs are useful for studying the precise distribution of GlcNAcβ1-3Gal-terminating GSL expression in tissues as well as for detecting GSLs carrying terminal GlcNAcβ1-3Gal carbohydrate structure.

Role of Intracellular Lipid Logistics in the Preferential Usage of Very Long Chain-Ceramides in Glucosylceramide

Ceramide is a common precursor of sphingomyelin (SM) and glycosphingolipids (GSLs) in mammalian cells. Ceramide synthase 2 (CERS2), one of the six ceramide synthase isoforms, is responsible for the synthesis of very long chain fatty acid (C20–26 fatty acids) (VLC)-containing ceramides (VLC-Cer). It is known that the proportion of VLC species in GSLs is higher than that in SM. To address the mechanism of the VLC-preference of GSLs, we used genome editing to establish three HeLa cell mutants that expressed different amounts of CERS2 and compared the acyl chain lengths of SM and GSLs by metabolic labeling experiments. VLC-sphingolipid expression was increased along with that of CERS2, and the proportion of VLC species in glucosylceramide (GlcCer) was higher than that in SM for all expression levels of CERS2. This higher proportion was still maintained even when the proportion of C16-Cer to the total ceramides was increased by disrupting the ceramide transport protein (CERT)-dependent C16-Cer delivery pathway for SM synthesis. On the other hand, merging the Golgi apparatus and the endoplasmic reticulum (ER) by Brefeldin A decreased the proportion of VLC species in GlcCer probably due to higher accessibility of UDP-glucose ceramide glucosyltransferase (UGCG) to C16-rich ceramides. These results suggest the existence of a yet-to-be-identified mechanism rendering VLC-Cer more accessible than C16-Cer to UGCG, which is independent of CERT.

Unique gangliosides synthesized in vitro by sialyltransferases from marine bacteria and their characterization: ganglioside synthesis by bacterial sialyltransferases.

On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer and NeuAcα2-6Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.

Purification of Pyridylaminated Oligosaccharides Using 1,2-Dichloroethane Extraction

Fluorescence derivatization of the oligosaccharides released from glycoconjugates is widely used for precise structural characterization. To ensure labeling of the oligosaccharides, a large excess of fluorescence reagents is usually added to the reaction tube. Therefore, any excess reagents and by-products of the labeling reaction should be removed by several column chromatographies, including using a cellulose cartridge or spin columns. However, these purification steps are often time-consuming, expensive, and laborious. In this study, we found that 1,2-dichloroethane extraction could effectively and easily purify pyridylaminated oligosaccharides with a high recovery rate.

Adjustment of Matrix-Assisted Laser Desorption/Ionization for Glycolipids

Gangliosides isolated from biological sources are usually detected as sodium/potassium adduct ions and these ions are easily fragmented by dissociation of labile glycosidic bonds in the positive ion mode Mass Spectrometry (MS). A large number of conditions for fragment suppression of these acidic glycolipids by using non-acidic matrices or changing metal additions have been demonstrated. Compared to sodium/potassium adduct ions, the cesium adduct ions suppress the fragmentation of these acidic glycolipids. On the contrary, lithium adduct ions induce the fragmentation, but generate more informative fragment ions of glycolipids than other alkali metal adduct ions in post-source decay, MS/MS as well as MS spectra. To suppress the fragmentation of labile glycosidic bond and generate more informative fragmentation, we have examined and established the optimal condition for the detection of [M+Li]+ of gangliosides (GM3 and GM2) using the matrix-assisted laser desorption/ionization time-of-flight MS by adjusting matrix and alkali metal salt combinations and concentrations.

Simple and rapid removal of the interference in gangliosides extracted from HPTLC spot on MALDI-TOF MS analysis

Extraction of thin-layer chromatography (TLC) spot with organic solvents is one of the convenient methods for glycolipid isolation from crude samples, but other background contaminants are also co-extracted during this process. Calcium sulfate (gypsum), starch and synthetic polymers are commonly used as binders in TLC plates, and the co-extracted synthetic polymers usually interfere with further structural analyses, especially in analysis by mass spectrometry (MS). Several column chromatographic processes are therefore used to purify these extracts. However, the purification processes are time consuming and suffer from sample loss. In our previous study, we established a method for detergent removal from glycolipids in a detergent-resistant membrane microdomain (DRM). The procedure is based on selective detergent extraction, in which the sample is dried in a glass tube, followed by washing with non-polar organic solvents, 1,2-dichloroethane (DCE). In this study, we have attempted to apply this method for the background removal from gangliosides and glycosylinositolphosphoceramides extracted from high performance TLC (HPTLC) spots. After DCE washing, these glycolipids were detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, and it was found that DCE washing could effectively remove HPTLC backgrounds.