Antitumor activity of cucumarioside A2-2.

BACKGROUND: The cytotoxic activity of sea cucumber glycosides against different types of cells and cell lines, including human tumor cell lines, has been studied for many years. However, the molecular mechanism(s) of the antitumor action of triterpene glycosides on cancer cells remain unclear. This article reports a continuation of investigations of triterpene glycoside cucumarioside A2-2 isolated from the Far-Eastern sea cucumber Cucumaria japonica. It describes a study of glycoside anticancer activity in vivo and glycoside interaction with mouse Ehrlich carcinoma cells in vitro. METHODS: The cytotoxicity of cucumarioside A2-2 and its effect on apoptosis, the cell cycle, DNA biosynthesis and p53 activity, and glycoside anticancer action against Ehrlich carcinoma cells were studied. RESULTS: Cucumarioside A2-2 influences tumor cell viability at micromolar concentrations. The EC50 for glycoside estimated by nonspecific esterase assay and MTT assay was 2.1 and 2.7 µM, respectively. Cucumarioside A2-2 at a subcytotoxic range of concentrations exhibits a cytostatic effect by blocking cell proliferation and DNA biosynthesis in the S phase. It may induce apoptosis in tumor cells in a caspase-dependent way, bypassing the activation of the p53-dependent segment. CONCLUSION: The anticancer and proapoptotic properties of cucumarioside A2-2 may be due to direct interaction of the glycoside with tumor cells. The in vivo anticancer effect of cucumarioside A2-2 may be associated with the ability of the drug to arrest the cell cycle in the synthetic phase and induce programmed tumor cell death.

[Elaboration of immune stimulating lipid-saponin subunit antigen carrier based on glycolipid monogalactosyldiacylglycerol from sea macrophytes and triterpene glycosides from Cucumaria japonica].

The ability of some triterpene glycosides of holothurians: holotoxin A1 from Apostichopus japonicus and a mixture of monosulphated triterpene glycosides from Cucumaria japonica called cucumarioside (CD) to form supramolecular complexes with cholesterol (Chol) and monogalactosyldiacylglycerol (MGDG) or phosphatidylcholine (PC) was studied. A transmission electron microscopy method was used to observe supramolecular lipid-saponin complexes formed by holotoxin A1 and CD with cholesterol in the presence of membrane lipids. The observed supramolecular complexes are tubular nanoparticles with a length of 100-300 nm, an external diameter of 10-16 nm and an internal diameter of 2-6 nm. The formation of tubular nanoparticles was more effective in the presence of MGDG than with PC. Nanoparticles forming in the presence of MGDG are shaped as a tubule, have a constant diameter and a strongly pronounced internal channel. In contrast, PC has no such properties; this lipid is unable to fully integrate in tubular nanoparticles. Based on electron-microscopy data the range of weight ratio of MGDG-Chol-CD was determined as a 1-10:2:3 that provided most effective formation of tubular nanoparticles. Different methods of incorporation of model antigens in complex MGDG-Chol-CD were studied. Influenza haemagglutinin and neuraminidase from commercial vaccine "Influvac" and pore forming protein YompF from Yersinia pseudotuberculosis were used as model antigens. From 54 to 72% of protein of "Influvac" vaccine and 88-92% of YompF were incorporated in supramolecular complexes depending on the method of incorporation. The loss of functional activity of haemagglutinin of vaccine "Influvac" was the result of applying ultrasonic disintegration for incorporation of this protein in complex MGDG-Chol-CD. YompF incorporation in MGDG-Chol-CD complex led to the increased diameter of tubular particles, in the same time incorporation of vaccine "Influvac" antigens produced the "cap" formation at the end of tubules. The possibility of a described supramolecular complex MGDG-Chol-CD to be a carrier for subunit bacterial and viral antigens is shown.

[Toxicological characteristics of cumazid].

Cumazid, a new prophylactic immunotherapy preparation, has been created based on the total glycoside fraction isolated from Cucumaria japonica sea cucumber species. The acute and chronic toxicity of the preparation has been characterized according to the pharmacopoeieal requirements. With respect to the acute toxicity upon intragastric administration, cumazid belongs to class IV (weakly dangerous) drugs. In 3-month chronic tests, cumazid administered in doses within 1 - 100 microg/kg did not produce any significant toxic action on laboratory animals.

Antitumor activity of the immunomodulatory lead Cumaside.

A new immunomodulatory lead Cumaside that is a complex of monosulfated triterpene glycosides from the sea cucumber Cucumaria japonica and cholesterol possesses significantly less cytotoxic activity against sea urchin embryos and Ehrlich carcinoma cells than the corresponding glycosides. Nevertheless Cumaside has an antitumor activity against different forms of experimental mouse Ehrlich carcinoma in vivo both independently and in combination with cytostatics. The highest effect occurs at a treatment once a day for 7 days before the tumor inoculation followed by Cumaside treatment once a day for 7 days. Prophylactic treatment with Cumaside and subsequent therapeutic application of 5-fluorouracil suppressed the tumor growth by 43%.

[Complexation between triterpene glycosides of holothurians and cholesterol is the basis of lipid-saponin carriers of subunit protein antigens].

The ability of some triterpene glycosides of holothurians: cucumarioside A2-2 from Cucumaria japonica, cucumarioside G1 from Cucumaria fraudatrix, frondoside A from Cucumaria frondosa, and holotoxin A1 from A postichopus japonicus to form lipid-saponin supramolecular complexes was studied. The formation of supramolecular cholesterol-glycosides complexes between cholesterol and these glycosides in water medium was observed by transmission electron microscopy. These complexes were considered as nanoparticles with different structure. Complexes formed by cholesterol with cucumarioside A2-2, holotoxin A1, and frondoside A are tubular nanoparticles. In contrast, complexes between cholesterol and cucumarioside G1 have different structured. The structure of nanoparticles formed in the presence of cucumarioside A2-2, holotoxin A1, and cucumarioside G1 was dependent on the ratio of cholesterol in the lipid-saponin system. On the other hand, frondoside A did not shown this tendency. In lipid-saponin systems with a similar molar ratio cholesterol-glycoside, the ordering of the supramolecular structure decreases in the following order: cucumarioside A2-2, holotoxin A1, frondoside A. A comparative analysis of the morphology of the supramolecular complexes and the peculiarities of the molecular structure of triterpene glycosides studied, demonstrated that the structure of supramolecular complexes formed depends on the branching and length of the glycoside carbohydrate chain. On the other hand, the formation of monomeric cholesterol-glycosides complexes depends on the peculiarities of the structure of aglycone. Thus, the possibility of the formation of a new type of antigen carries on the basis of marine triterpene glycosides was proved.

Immunomodulatory properties of frondoside A, a major triterpene glycoside from the North Atlantic commercially harvested sea cucumber Cucumaria frondosa.

Frondoside A, a major triterpene glycoside from North Atlantic commercially harvested sea cucumber Cucumaria frondosa, possesses strong immunomodulatory properties in subtoxic doses. Frondoside A stimulates lysosomal activity of mouse macrophages in vivo at a maximal effective stimulatory dose of 0.2 microg per mouse and is maintained over 10 days. This glycoside also shows a 30% stimulation of lysosomal activity in mouse macrophages in vitro at concentrations of 0.1-0.38 microg/mL. Frondoside A enhances macrophage phagocytosis of the bacterium Staphylococcus aureus in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates reactive oxygen species formation in macrophages in vitro at a maximal effective concentration of 0.001 microg/mL. Frondoside A stimulates an increase in the number of antibody plaque-forming cells (normally B-cells in spleen) in vivo with a maximal stimulatory effect at a concentration of 0.2 microg per mouse (stimulatory index, 1.86). Frondoside A has a weak effect upon immunoglobulin (Ig) M production after immunization with sheep erythrocytes in mice. Frondoside A does not stimulate Ig production in mice and does not significantly enhance the ovalbumin-stimulated IgM and IgG antibody levels in ovalbumin-immunized mice. Hence frondoside A is an immunostimulant of cell-based immunity including phagocytosis without a significant effect on amplification of humoral immune activity or adjuvant properties. Therefore, frondoside A may provide curative and/or preventive treatment options against diseases wherein a depleted immune status contributes to the pathological processes.

[Monosulfated triterpene glycosides from Cucumaria okhotensis Levin et Stepanov, a new species of sea cucumbers from Sea of Okhotsk].

Three compounds were isolated from the fraction of monosulfated triterpene glycosides from Cucumaria okhotensis, a new sea cucumber species, and their structures were elucidated. First of them, okhotoside A1-1, is a new glycoside containing tetrasaccharide sugar moiety; the second, okhotoside A2-1, is a new pentaoside with a glucose residue in the second position of sugar moiety (such a structural peculiarity has been found in holothurians of the genus Cucumaria for the first time); and the third is a previously known pentaoside cucumarioside A0-1 from C. japonica. The species-specificity of the triterpene glycosides from C. okhotensis was revealed, which justifies the description of this sea cucumber as a new species.

Estrogenic activity of triterpene glycosides in yeast two-hybrid assay.

Estrogenic potency of six triterpene glycosides, Holothurin A, Holotoxin A1, Frondoside A, Cucumarioside A2-2 and Cauloside C, that are natural products and semi-synthesized Ginsenoside-Rh2, were examined with yeast two-hybrid system, including expressed genes of human estrogen receptor, hERalpha, the co-activator TIF2 and lacZ as a reporter gene. Only Ginsenoside-Rh2 exhibited significant moderate estrogenic activity in the concentration range of 10(-7) to 10(-6)M. Its effect was approximately 30% of the activity of 17beta-estradiol applied at half-effective concentration. This indicates Ginsenosides-Rh2 is a weak phytoestrogen. The sea cucumber triterpene glycosides, Holothurin A, Holotoxin A1, Cucumarioside A2-2 and Frondoside A, and plant glycoside Cauloside C had no appreciable estrogenic activity. Data obtained by yeast two-hybrid assay reflect structure-activity relationship between tested compounds and 17beta-estradiol. Only Ginsenoside-Rh2 has some similarity in chemical structure with 17beta-estradiol that might explain affinity of this glycoside to the hERalpha receptor.

Triterpene glycosides from the far eastern sea cucumber Pentamera calcigera II: disulfated glycosides.

Three new triterpene glycosides, calcigerosides D(1) (1), D(2) (2), and E (3), have been isolated from the sea cucumber Pentamera calcigera. Their structures have been deduced from extensive spectral analysis (NMR and MS) and chemical evidence. All the compounds are disulfated pentaosides differing in aglycon structure and position of sulfate group, which were determined by the measurement of NT(1) values in the cases of glycosides 1 and 2. Glycoside 1 is a nonholostane derivative, that is, it lacks an 18(20)-lactone, which is very rare among the sea cucumber glycosides.

System-theoretical (Holistic) approach to the modelling of structural-functional relationships of biomolecules and their evolution: an example of triterpene glycosides from sea cucumbers (Echinodermata, holothurioidea).

Triterpene glycosides from sea cucumbers (Holothurioidea, Echinodermata) are used as a model for studying the biochemical evolution for correlation between the glycoside membranolytic activity and biological functions. Concepts of evolutionary morphology are applied at the molecular level. The concept of Van-der-Klaauve's- Dullemeijer's system-theoretical (holistic) approach is used for the model of structural-functional relationships of the glycosides. Network diagrams of structural-functional relationships have been prepared for many glycosides. The diagrams correlate well with experimental data and show a very complex and flexible action of the natural selection on the structural fragments of the glycosides. The diagrams also show overlapping in the functional components that provide stability to the general structural plan of glycosides during evolution. The method may be applied to other biomolecules.

Triterpene glycosides from the Far-Eastern sea cucumber Pentamera calcigera. 1. Monosulfated glycosides and cytotoxicity of their unsulfated derivatives.

Three new monosulfated triterpene glycosides, calcigerosides B (2), C(1) (3), and C(2) (4), along with the known cucumarioside G(2) (1), have been isolated from the sea cucumber Pentamera calcigera. Their structures have been deduced from extensive spectral analysis (NMR and MS) and chemical evidence. Compounds 2-4 present a novel pentasacharide chain never reported before in sea cucumber triterpene glycosides. The desulfated derivatives of calcigerosides B, C(1), and C(2) (5, 7, and 9, respectively) showed moderate cytotoxicity (IC(50) = 5 microg/mL) against a selection of four human and mouse tumor cell lines.

Toxins from sea cucumbers (holothuroids): chemical structures, properties, taxonomic distribution, biosynthesis and evolution.

Studies on structures, biological activities, chemical properties, taxonomic distribution, biosynthesis, and evolution of toxins from sea cucumbers (the phylum Echinodermata, the class Holothurioidea) were reviewed with special emphasis on recent results from our laboratory. These toxins are triterpene oligoglycosides having very often one or several sulfate groups in carbohydrate moieties. Their aglycones belong to lanostane derivatives and sometimes contain shortened side chains. Many aglycones are labile in the acid medium. There is a relationship between structures of the glycosides and taxonomic positions of corresponding animals, producers of these toxins. Toxins from sea cucumbers act on delta 5-sterol-containing biological membranes with the formation of glycoside-sterol complexes followed by the disturbance of membrane permeability and inhibition of activities of some membrane enzymes. The presence of the toxins causes the alterations in membrane sterol compositions of toxic sea cucumbers in comparison with non-toxic species. These alterations include the change of delta 5-sterols for those having 7(8)- and 9(11)-double bonds as well as biotransformation of a part of free sterol fractions into sterol sulfates and sterol xylosides.

Two different modes of inhibition of the rat brain Na+, K(+)-ATPase by triterpene glycosides, psolusosides A and B from the holothurian Psolus fabricii.

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na+, K(+)-ATPase (Na, K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na+, K(+)-ATPase with I50 values of 1 x 10(-4) M and 3 x 10(-4) M, respectively. PsA significantly stimulated [3H]ATP binding to Na+, K(+)-ATPase, weakly increased [3H]ouabain binding to the enzyme, and inhibited K(+)-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [3H]ATP binding to Na+, K(+)-ATPase, and had no effect on [3H]ouabain binding to the enzyme. K(+)-Phosphatase activity was inhibited by PsB in parallel with Na+, K(+)-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na+, K(+)-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.

Koreoside A, a new nonholostane triterpene glycoside from the sea cucumber Cucumaria koraiensis.

The new triterpene glycoside koreoside A (1) has been isolated from the sea cucumber Cucumaria koraiensis. Koreoside A (1) is the first glycoside reported from holothurians that presents a delta(7) nonholostane aglycon without a lactone group and with a shortened side chain. Its structure has been elucidated by 13C and 1H NMR as well as FABMS studies.

Structure of eximisoside A, a novel triterpene glycoside from the Far-Eastern sea cucumber psolus eximius.

The new triterpene saponin eximisoside A (1) has been isolated from the Far-Eastern sea cucumber Psolus eximius and its structure elucidated by 1D and 2D NMR (13C, 1H, 1H-1H COSY, HMQC, and NOESY spectra), and FABMS studies. The structural features of eximisoside A (1) are the rare presence of a 23 double bond in the aglycon and the absence of quinovose, typically found in the oligosaccharide chain of holothurian glycosides.

Application of morphological trends of evolution to phylogenetic interpretation of chemotaxonomic data.

Data on the membranotropic action of triterpene glycosides from sea cucumbers (Holothurioidea, Echinodermata) are used as a model for studying the evolution of biochemical adaptations. Conceptions such as the theory of modes of organogenesis, and the theory of phylembryogeneses developed by the Russian school of evolutionary morphology, and other morphological trends of evolutionary transformations of anatomical structures are applicable to the phylogenetic interpretation of chemotaxonomic data. The processes of ontogenesis and biosynthesis of secondary metabolites are compared within the framework of the theory of phylembryogeneses. The deep inner similarity of these processes are shown. Concurrence of the modes of phyembryogenesis noted by Severtsov and the principles of micromolecular evolution noted by Gottlieb and Harborne are also shown. The biological significance of presence or absence of substances, (or some features of these substances) for the whole organism that produced them is the main criterion we propose as an approach for polarization of a biochemical phylogenetic series. The application of methods of comparative anatomy to the molecular level for purposes of investigation on evolution of biomolecules and their functions could be called "chemical morphology".

Hemolytic activities of triterpene glycosides from the holothurian order Dendrochirotida: some trends in the evolution of this group of toxins.

Hemolysis and K+ loss from mouse erythrocytes, induced by triterpene glycosides and their derivatives from this order of sea cucumbers were studied. Sulfate groups, attached to position 4 of the first xylose residue and to position 6 of the third glucose residue of the branched pentaosides, having 3-O-methyl-groups in terminal monosaccharide moieties increase K+ loss. A sulfate group at C-4 of the first xylose residue increases the hemolytic activity while a sulfate at C-6 of the third monosaccharide unit decreases it. A sulfate group at C-6 of terminal 3-O-methylglucose drastically decreases the hemolytic activity and rate of K+ loss. The presence of a sulfate group at the first xylose residue in glycosides having no 3-O-methyl group at the terminal monosaccharide decreases hemolytic activity and rate of K+ loss. The presence of the 16-ketone group in aglycones having the 7(8)-double bond significantly decreases activity. These results correlate with the previously proposed trends in evolution of sea cucumber glycosides from substances having sulfate groups at C-6 of glucose and 3-O-methylglucose units to substances sulfated at C-4 of the first xylose or having no sulfate groups, and from substances with aglycone 16-ketone to substances having no oxygen functions in this position.

Structure of cucumarioside G2, a novel nonholostane glycoside from the sea cucumber Eupentacta fraudatrix.

The new triterpene glycoside cucumarioside G2 [1] has been isolated from the sea cucumber Eupentacta fraudatrix. Glycoside 1 is the first triterpene glycoside with the 23,24,25,26,27-pentanorlanostane type of aglycone. Its structure has been established by chemical transformations as well as 13C- and 1H-nmr, eims, and liquid sims studies.