Search

Breast cancer is the most prevalent form of cancer in women worldwide. Triple-negative breast cancer is the most unfavorable for patients, but it is also the most sensitive to chemotherapy. Triterpene glycosides from sea cucumbers possess a high therapeutic potential as anticancer agents. This study aimed to identify the pathways triggered and regulated in MDA-MB-231 cells (triple-negative breast cancer cell line) by the glycosides cucumarioside A0-1 (Cuc A0-1) and djakonovioside A (Dj A), isolated from the sea cucumber Cucumaria djakonovi. Using flow cytometry, fluorescence microscopy, immunoblotting, and ELISA, the effects of micromolar concentrations of the compounds on cell cycle arrest, induction of apoptosis, the level of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and expression of anti- and pro-apoptotic proteins were investigated. The glycosides caused cell cycle arrest, stimulated an increase in ROS production, and decreased Δψm in MDA-MB-231 cells. The depolarization of the mitochondrial membrane caused by cucumarioside A0-1 and djakonovioside A led to an increase in the levels of APAF-1 and cytochrome C. This, in turn, resulted in the activation of caspase-9 and caspase-3 and an increase in the level of their cleaved forms. Glycosides also affected the expression of Bax and Bcl-2 proteins, which are associated with mitochondria-mediated apoptosis in MDA-MB-231 cells. These results indicate that cucumarioside A0-1 and djakonovioside A activate the intrinsic apoptotic pathway in triple-negative breast cancer cells. Additionally, it was found that treatment with Cuc A0-1 resulted in in vivo inhibition of tumor growth and metastasis of murine solid Ehrlich adenocarcinoma.

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), treatment is inevitably hampered by the development of drug resistance. Thus, new drugs are urgently needed. We investigated the efficacy, toxicity, and mechanism of action of the marine triterpene glycoside cucumarioside A2-2 (CA2-2) using an in vitro CRPC model. CA2-2 induced a G2/M-phase cell cycle arrest in human prostate cancer PC-3 cells and caspase-dependent apoptosis executed via an intrinsic pathway. Additionally, the drug inhibited the formation and growth of CRPC cell colonies at low micromolar concentrations. A global proteome analysis performed using the 2D-PAGE technique, followed by MALDI-MS and bioinformatical evaluation, revealed alterations in the proteins involved in cellular processes such as metastatic potential, invasion, and apoptosis. Among others, the regulation of keratin 81, CrkII, IL-1β, and cathepsin B could be identified by our proteomics approach. The effects were validated on the protein level by a 2D Western blotting analysis. Our results demonstrate the promising anticancer activity of CA2-2 in a prostate cancer model and provide insights on the underlying mode of action.

Four new mono- and trisulfated triterpene penta- and tetraosides, djakonoviosides C1 (1), D1 (2), E1 (3), and F1 (4) were isolated from the Far Eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida), along with six known glycosides found earlier in other Cucumaria species. The structures of unreported compounds were established on the basis of extensive analysis of 1D and 2D NMR spectra as well as by HR-ESI-MS data. The set of compounds contains six different types of carbohydrate chains including two new ones. Thus, djakonovioside C1 (1) is characterized by xylose as the second residue, that was a branchpoint in the pentasaccharide chain. Meanwhile, only quinovose and rarely glucose have been found earlier in pentasaccharide chains branched at C-2 of the second sugar unit. Djakonovioside E1 (3) is characterized by a tetrasaccharide trisulfated chain, with glucose as the second residue. So, in the series of isolated glycosides, three types of sugars in the second position were presented: the most common, quinovose—in six compounds; glucose—in three substances; and the rare xylose—in one glycoside. The set of aglycones was composed of holostane- and non-holostane-type polycyclic systems; the latter comprised normal and reduced side chains. Noticeably, isokoreoside A (9), isolated from C. djakonovi, was a single glycoside having a 9(11)-double bond, indicating two oxidosqualenecyclases are operating in the process of the biosynthesis of aglycones. Some of the glycosides from C. djakonovi, which were characterized by pentasaccharide branched chains containing one to three sulfate groups, are chemotaxonomic features of the representatives of the genus Cucumaria. The assortment of sugar parts of Cucumaria’s glycosides was broadened with previously undescribed penta- and tetrasaccharide moieties. The metabolic network of sugar parts and aglycones is constructed based on biogenetic relationships. The cytotoxic action of compounds 1–10, isolated from C. djakonovi, against human breast cancer cell lines was investigated along with the hemolytic activity. Erythrocytes were, as usual, more sensitive to the membranolytic action of the glycosides than cancer cells. The triple-negative breast cancer MDA-MB-231 cell line was more vulnerable to the action of glycosides in comparison with the other tested cancer cells, while the MCF-7 cell line was less susceptible to cytotoxic action. Djakonovioside E1 (3) demonstrated selective action against ER-positive MCF-7 and triple-negative MDA-MB-231 cell lines, while the toxic effect in relation to normal mammary epithelial cells (MCF-10A) was absent. Cucumarioside A2-5 (6) inhibited the formation and growth of colonies of cancer cells to 44% and tumor cell migration to 85% of the control. Quantitative structure–activity relationships (QSAR) were calculated on the basis of the correlational analysis of the physicochemical properties and structural features of the glycosidic molecules and their membranolytic activity. QSAR revealed the extremely complex nature of such relationships, but these calculations correlated well with the observed SAR.

Two fucosylated chondroitin sulfates were isolated from the sea cucumbers Psolus peronii and Holothuria nobilis using a conventional extraction procedure in the presence of papain, followed by anion-exchange chromatography on DEAE-Sephacel. Their composition was characterized in terms of quantitative monosaccharide and sulfate content, and structures were mainly elucidated using 1D- and 2D-NMR spectroscopy. As revealed by the data of the NMR spectra, both polysaccharides along with the usual fucosyl branches contained rare disaccharide branches α-D-GalNAc4S6R-(1→2)-α-L-Fuc3S4R → attached to O-3 of the GlcA of the backbone (R = H or SO3−). The polysaccharides were studied as stimulators of hematopoiesis in vitro using mice bone marrow cells as the model. The studied polysaccharides were shown to be able to directly stimulate the proliferation of various progenitors of myelocytes and megakaryocytes as well as lymphocytes and mesenchymal cells in vitro. Therefore, the new fucosylated chondroitin sulfates can be regarded as prototype structures for the further design of GMP-compatible synthetic analogs for the development of new-generation hematopoiesis stimulators.

Seven new monosulfated triterpene glycosides, djakonoviosides A (1), A1 (2), A2 (3), and B1–B4 (4–7), along with three known glycosides found earlier in the other Cucumaria species, namely okhotoside A1-1, cucumarioside A0-1, and frondoside D, have been isolated from the far eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida). The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds of groups A and B differ from each other in their carbohydrate chains, namely monosulfated tetrasaccharide chains are inherent to group A and pentasaccharide chains with one sulfate group, branched by C-2 Qui2, are characteristic of group B. The aglycones of djakonoviosides A2 (3), B2 (5), and B4 (7) are characterized by a unique structural feature, a 23,16-hemiketal fragment found first in the sea cucumbers’ glycosides. The biosynthetic pathway of its formation is discussed. The set of aglycones of C. djakonovi glycosides was species specific because of the presence of new aglycones. At the same time, the finding in C. djakonovi of the known glycosides isolated earlier from the other species of Cucumaria, as well as the set of carbohydrate chains characteristic of the glycosides of all investigated representatives of the genus Cucumaria, demonstrated the significance of these glycosides as chemotaxonomic markers. The membranolytic actions of compounds 1–7 and known glycosides okhotoside A1-1, cucumarioside A0-1, and frondoside D, isolated from C. djakonovi against human cell lines, including erythrocytes and breast cancer cells (MCF-7, T-47D, and triple negative MDA-MB-231), as well as leukemia HL-60 and the embryonic kidney HEK-293 cell line, have been studied. Okhotoside A1-1 was the most active compound from the series because of the presence of a tetrasaccharide linear chain and holostane aglycone with a 7(8)-double bond and 16β-O-acetoxy group, cucumarioside A0-1, having the same aglycone, was slightly less active because of the presence of branching xylose residue at C-2 Qui2. Generally, the activity of the djakonoviosides of group A was higher than that of the djakonoviosides of group B containing the same aglycones, indicating the significance of a linear chain containing four monosaccharide residues for the demonstration of membranolytic action by the glycosides. All the compounds containing hemiketal fragments, djakonovioside A2 (3), B2 (5), and B4 (7), were almost inactive. The most aggressive triple-negative MDA-MB-231 breast cancer cell line was the most sensitive to the glycosides action when compared with the other cancer cells. Okhotoside A1-1 and cucumarioside A0-1 demonstrated promising effects against MDA-MB-231 cells, significantly inhibiting the migration, as well as the formation and growth, of colonies.

Sea cucumber triterpene glycosides are a class of secondary metabolites that possess distinctive chemical structures and exhibit a variety of biological and pharmacological activities. The application of MS-based approaches for the study of triterpene glycosides allows rapid evaluation of the structural diversity of metabolites in complex mixtures. However, the identification of the detected triterpene glycosides can be challenging. The objective of this study is to establish the first spectral library containing the mass spectra of sea cucumber triterpene glycosides using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. The library contains the electrospray ionization tandem mass spectra and retention times of 191 triterpene glycosides previously isolated from 15 sea cucumber species and one starfish at the Laboratory of the Chemistry of Marine Natural Products of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry. In addition, the chromatographic behavior and some structure-related neutral losses in tandem MS are discussed. The obtained data will accelerate the accurate dereplication of known triterpene glycosides and the annotation of novel compounds, as we demonstrated by the processing of LC-MS/MS data of Eupentacta fraudatrix extract.

Three new tetrasulfated triterpene glycosides, chilensosides E (1), F (2), and G (3), have been isolated from the Far-Eastern sea cucumber Paracaudina chilensis (Caudinidae, Molpadida). The structures were established based on extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds differ in their carbohydrate chains, namely in the number of monosaccharide residues (five or six) and in the positions of sulfate groups. Chilensosides E (1) and F (2) are tetrasulfated pentaosides with the position of one of the sulfate groups at C-3 Glc3, and chilensoside G (3) is a tetrasulfated hexaoside. The biogenetic analysis of the glycosides of P. chilensis has revealed that the structures form a network due to the attachment of sulfate groups to almost all possible positions. The upper semi-chain is sulfated earlier in the biosynthetic process than the lower one. Noticeably, the presence of a sulfate group at C-3 Glc3—a terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2—excludes the possibility of this sugar chain’s further elongation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, which is a characteristic feature of the mosaic type of biosynthesis. The hemolytic action of compounds 1–3 against human erythrocytes and cytotoxic activities against five human cancer cell lines were tested. The compounds showed moderate hemolytic activity but were inactive against cancer cells, probably because of their structural peculiarities, such as the combination of positions of four sulfate groups.

Five new triterpene (4,4,14-trimethylsterol) di-, tri- and tetrasulfated pentaosides, chilensosides A (1), A1 (2), B (3), C (4), and D (5) were isolated from the Far-Eastern sea cucumber Paracaudina chilensis. The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The structural variability of the glycosides concerned the pentasaccharide chains. Their architecture was characterized by the upper semi-chain consisting of three sugar units and the bottom semi-chain of two sugars. Carbohydrate chains of compounds 2–5 differed in the quantity and positions of sulfate groups. The interesting structural features of the glycosides were: the presence of two sulfate groups at C-4 and C-6 of the same glucose residue in the upper semi-chain of 1, 2, 4, and 5 and the sulfation at C-3 of terminal glucose residue in the bottom semi-chain of 4 that makes its further elongation impossible. Chilensoside D (5) was the sixth tetrasulfated glycoside found in sea cucumbers. The architecture of the sugar chains of chilensosides A–D (1–5), the positions of sulfation, the quantity of sulfate groups, as well as the aglycone structures, demonstrate their similarity to the glycosides of the representatives of the order Dendrochirotida, confirming the phylogenetic closeness of the orders Molpadida and Dendrochirotida. The cytotoxic activities of the compounds 1–5 against human erythrocytes and some cancer cell lines are presented. Disulfated chilensosides A1 (2) and B (3) and trisulfated chilensoside C (4) showed significant cytotoxic activity against human cancer cells.

Five new triterpene di-, tri- and tetrasulfated hexaosides (chitonoidosides I (1), J (2), K (3), K1 (4) and L (5)) were isolated from the Far-Eastern sea cucumber Psolus chitonoides, collected near Bering Island (Commander Islands) from a depth of 100–150 m. The structural variability of the glycosides concerned both the aglycones (with 7(8)- or 9(11)-double bonds) and carbohydrate chains differing from each other by the third sugar residue (Xyl or sulfated by C-6 Glc) and/or by the fourth—terminal in the bottom semi-chain—residue (Glc or sulfated by C-6 MeGlc) as well as by the positions of a sulfate group at C-4 or C-6 in the sixth—terminal in the upper semi-chain—residue (MeGlc). Hemolytic activities of these compounds 1–5 against human erythrocytes as well as cytotoxicity against human cancer cell lines, HeLa, DLD-1 and HL-60, were studied. The hexaosides, chitonoidosides K (3) and L (5) with four sulfate groups, were the most active against tumor cells in all the tests. Noticeably, the sulfate group at C-4 of MeGlc6 did not decrease the membranolytic effect of 5 as compared with 3, having the sulfate group at C-6 of MeGlc6. Erythrocytes were, as usual, more sensitive to the action of the studied glycosides than cancer cells, although the sensitivity of leukemia promyeloblast HL-60 cells was higher than that of other tumor cells. The glycosides 1 and 2 demonstrated some weaker action in relation to DLD-1 cells than against other tumor cell lines. Chitonoidoside K1 (4) with a hydroxyl at C 25 of the aglycone was not active in all the tests. The metabolic network formed by the carbohydrate chains of all the glycosides isolated from P. chitonoides as well as the aglycones biosynthetic transformations during their biosynthesis are discussed and illustrated with schemes.

Four new triterpene disulfated glycosides, chitonoidosides E1 (1), F (2), G (3), and H (4), were isolated from the Far-Eastern sea cucumber Psolus chitonoides and collected near Bering Island (Commander Islands) at depths of 100–150 m. Among them there are two hexaosides (1 and 3), differing from each other by the terminal (sixth) sugar residue, one pentaoside (4) and one tetraoside (2), characterized by a glycoside architecture of oligosaccharide chains with shortened bottom semi-chains, which is uncommon for sea cucumbers. Some additional distinctive structural features inherent in 1–4 were also found: the aglycone of a recently discovered new type, with 18(20)-ether bond and lacking a lactone in chitonoidoside G (3), glycoside 3-O-methylxylose residue in chitonoidoside E1 (1), which is rarely detected in sea cucumbers, and sulfated by uncommon position 4 terminal 3-O-methylglucose in chitonoidosides F (2) and H (4). The hemolytic activities of compounds 1–4 and chitonoidoside E against human erythrocytes and their cytotoxic action against the human cancer cell lines, adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and monocytes THP-1, were studied. The glycoside with hexasaccharide chains (1, 3 and chitonoidoside E) were the most active against erythrocytes. A similar tendency was observed for the cytotoxicity against adenocarcinoma HeLa cells, but the demonstrated effects were moderate. The monocyte THP-1 cell line and erythrocytes were comparably sensitive to the action of the glycosides, but the activity of chitonoidosides E and E1 (1) significantly differed from that of 3 in relation to THP-1 cells. A tetraoside with a shortened bottom semi-chain, chitonoidoside F (2), displayed the weakest membranolytic effect in the series.

The article describes the structure-activity relationships (SAR) for a broad series of sea cucumber glycosides on different tumor cell lines and erythrocytes, and an in silico modulation of the interaction of selected glycosides from the sea cucumber Eupentacta fraudatrix with model erythrocyte membranes using full-atom molecular dynamics (MD) simulations. The in silico approach revealed that the glycosides bound to the membrane surface mainly through hydrophobic interactions and hydrogen bonds. The mode of such interactions depends on the aglycone structure, including the side chain structural peculiarities, and varies to a great extent. Two different mechanisms of glycoside/membrane interactions were discovered. The first one was realized through the pore formation (by cucumariosides A1 (40) and A8 (44)), preceded by bonding of the glycosides with membrane sphingomyelin, phospholipids, and cholesterol. Noncovalent intermolecular interactions inside multimolecular membrane complexes and their stoichiometry differed for 40 and 44. The second mechanism was realized by cucumarioside A2 (59) through the formation of phospholipid and cholesterol clusters in the outer and inner membrane leaflets, correspondingly. Noticeably, the glycoside/phospholipid interactions were more favorable compared to the glycoside/cholesterol interactions, but the glycoside possessed an agglomerating action towards the cholesterol molecules from the inner membrane leaflet. In silico simulations of the interactions of cucumarioside A7 (45) with model membrane demonstrated only slight interactions with phospholipid polar heads and the absence of glycoside/cholesterol interactions. This fact correlated well with very low experimental hemolytic activity of this substance. The observed peculiarities of membranotropic action are in good agreement with the corresponding experimental data on hemolytic activity of the investigated compounds in vitro.

Six new triterpene tetra-, penta- and hexaosides, chitonoidosides A (1), A1 (2), B (3), C (4), D (5), and E (6), containing one or two sulfate groups, have been isolated from the Far-Eastern sea cucumber Psolus chitonoides, collected near Bering Island (Commander Islands) from the depth of 100–150 m. Three of the isolated compounds (1, 3 and 6) are characterized by the unusual aglycone of new type having 18(20)-ether bond and lacking a lactone in contrast with wide spread holostane derivatives. Another unexpected finding is 3-O-methylxylose residue as a terminal unit in the carbohydrate chains of chitonoidosides B (3), C (4), and E (6), which has never been found before in the glycosides from holothurians belonging to the Psolidae family. Moreover, this monosaccharide is sulfated in the compound 4 into unprecedented 3-O-methylxylose 4-O-sulfate residue. Chitonoidoside C (4) is characterized by tetrasaccharide moiety lacking a part of the bottom semi-chain, but having disaccharide fragment attached to C-4 of Xyl1. Such architecture is not common in sea cucumber glycosides. Cytotoxic activities of the compounds 1–5 against mouse and human erythrocytes and human cancer cell lines: adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and leukemia promyeloblast HL-60 cells were studied. The cytotoxic effect of chitonoidoside d (5) was the most significant in this series due to the presence of pentasaccharide disulfated sugar chain in combination with holostane aglycone. Surprisingly, the glycosides 1 and 3, comprising the new aglycone without γ-lactone, demonstrated similar activity to the known compounds with holostane aglycones. Chitonoidoside C (4) was less cytotoxic due to the different architecture of the carbohydrate chain compared to the other glycosides and probably due to the presence of a sulfate group at C-4 in 3-O-MeXyl4.

Nine new mono-, di-, and trisulfated triterpene penta- and hexaosides, kurilosides A3 (1), D1 (2), G (3), H (4), I (5), I1 (6), J (7), K (8), and K1 (9) and two desulfated derivatives, DS-kuriloside L (10), having a trisaccharide branched chain, and DS-kuriloside M (11), having hexa-nor-lanostane aglycone with a 7(8)-double bond, have been isolated from the Far-Eastern deep-water sea cucumber Thyonidium (=Duasmodactyla) kurilensis (Levin) and their structures were elucidated based on 2D NMR spectroscopy and HR-ESI mass-spectrometry. Five earlier unknown carbohydrate chains and two aglycones (having a 16β,(20S)-dihydroxy-fragment and a 16β-acetoxy,(20S)-hydroxy fragment) were found in these glycosides. All the glycosides 1–9 have a sulfate group at C-6 Glc, attached to C-4 Xyl1, while the positions of the other sulfate groups vary in different groups of kurilosides. The analysis of the structural features of the aglycones and the carbohydrate chains of all the glycosides of T. kurilensis showed their biogenetic relationships. Cytotoxic activities of the compounds 1–9 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells, and erythrocytes were studied. The highest cytotoxicity in the series was demonstrated by trisulfated hexaoside kuriloside H (4), having acetoxy-groups at C(16) and C(20), the latter one obviously compensated the absence of a side chain, essential for the membranolytic action of the glycosides. Kuriloside I1 (6), differing from 4 in the lacking of a terminal glucose residue in the bottom semi-chain, was slightly less active. The compounds 1–3, 5, and 8 did not demonstrate cytotoxic activity due to the presence of hydroxyl groups in their aglycones.

Six new monosulfated triterpene tetra-, penta- and hexaosides, namely, the kurilosides A1 (1), A2 (2), C1 (3), D (4), E (5) and F (6), as well as the known earlier kuriloside A (7), having unusual non-holostane aglycones without lactone, have been isolated from the sea cucumber Thyonidium (= Duasmodactyla) kurilensis (Levin) (Cucumariidae, Dendrochirotida), collected in the Sea of Okhotsk near Onekotan Island from a depth of 100 m. Structures of the glycosides were established by 2D NMR spectroscopy and HR-ESI mass spectrometry. Kurilosides of the groups A and E contain carbohydrate moieties with a rare architecture (a pentasaccharide branched by C(4) Xyl1), differing from each other in the second monosaccharide residue (quinovose or glucose, correspondingly); kurilosides of the group C are characterized by a unique tetrasaccharide branched by a C(4) Xyl1 sugar chain; and kurilosides of the groups D and F are hexaosides differing from each other in the presence of an O-methyl group in the fourth (terminal) sugar unit. All these glycosides contain a sulfate group at C-6 of the glucose residue attached to C-4 Xyl1 and the non-holostane aglycones have a 9(11) double bond and lack γ-lactone. The cytotoxic activities of compounds 1–7 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells and erythrocytes were studied. Kuriloside A1 (1) was the most active compound in the series, demonstrating strong cytotoxicity against the erythrocytes and JB-6 cells and a moderate effect against Neuro 2a cells.

Thirteen new mono-, di-, and trisulfated triterpene glycosides, quadrangularisosides A–D4 (1−13) have been isolated from the sea cucumber Colochirus quadrangularis, which was collected in Vietnamese waters. The structures of these glycosides were established by 2D NMR spectroscopy and HR-ESI (High Resolution Electrospray Ionization) mass spectrometry. The novel carbohydrate moieties of quadrangularisosides D–D4 (8−12), belonging to the group D, and quadrangularisoside E (13) contain three sulfate groups, with one of them occupying an unusual position—at C(4) of terminal 3-O-methylglucose residue. Quadrangularisosides A (1) and D3 (11) as well as quadrangularisosides A1 (2) and D4 (12) are characterized by the new aglycones having 25-hydroperoxyl or 24-hydroperoxyl groups in their side chains, respectively. The cytotoxic activities of compounds 1–13 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells, erythrocytes, and human colorectal adenocarcinoma HT-29 cells were studied. All the compounds were rather strong hemolytics. The structural features that most affect the bioactivity of the glycosides are the presence of hydroperoxy groups in the side chains and the quantity of sulfate groups. The membranolytic activity of monosulfated quadrangularisosides of group A (1, 2) against Neuro 2a, JB-6 cells, and erythrocytes was relatively weak due to the availability of the hydroperoxyl group, whereas trisulfated quadrangularisosides D3 (11) and D4 (12) with the same aglycones as 1, 2 were the least active compounds in the series due to the combination of these two structural peculiarities. The erythrocytes were more sensitive to the action of the glycosides than Neuro 2a or JB-6 cells, but the structure–activity relationships observed for glycosides 1–13 were similar in the three cell lines investigated. The compounds 3−5, 8, and 9 effectively suppressed the cell viability of HT-29 cells. Quadrangularisosides A1 (2), C (6), C1 (7), and E (13) possessed strong inhibitory activity on colony formation in HT-29 cells. Due to the synergic effects of these glycosides (0.02 μM) and radioactive irradiation (1 Gy), a decreasing of number of colonies was detected. Glycosides 1, 3, and 9 enhanced the effect of radiation by about 30%.

Triterpene glycosides are characteristic secondary metabolites of sea cucumbers (Holothurioidea, Echinodermata). They have hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. These natural products suppress the proliferation of various human tumor cell lines in vitro and, more importantly, intraperitoneal administration in rodents of solutions of some sea cucumber triterpene glycosides significantly reduces both tumor burden and metastasis. The anticancer molecular mechanisms include the induction of tumor cell apoptosis through the activation of intracellular caspase cell death pathways, arrest of the cell cycle at S or G2/M phases, influence on nuclear factors, NF-κB, and up-down regulation of certain cellular receptors and enzymes participating in cancerogenesis, such as EGFR (epidermal growth factor receptor), Akt (protein kinase B), ERK (extracellular signal-regulated kinases), FAK (focal adhesion kinase), MMP-9 (matrix metalloproteinase-9) and others. Administration of some glycosides leads to a reduction of cancer cell adhesion, suppression of cell migration and tube formation in those cells, suppression of angiogenesis, inhibition of cell proliferation, colony formation and tumor invasion. As a result, marked growth inhibition of tumors occurs in vitro and in vivo. Some holothurian triterpene glycosides have the potential to be used as P-gp mediated MDR reversal agents in combined therapy with standard cytostatics.

Ten new di-, tri- and tetrasulfated triterpene glycosides, psolusosides B1 (1), B2 (2), J (3), K (4), L (5), M (6), N (7), O (8), P (9), and Q (10), were isolated from the sea cucumber Psolus fabricii collected in the Sea of Okhotsk near the Kurile Islands. Structures of these glycosides were established by two-dimensional (2D) NMR spectroscopy and HR-ESI mass-spectrometry. It is particularly interesting that highly polar compounds 9 and 10 contain four sulfate groups in their carbohydrate moieties, including two sulfates in the same terminal glucose residue. Glycoside 2 has an unusual non-holostane aglycone with 18(16)-lactone and a unique 7,8-epoxy fragment. Cytotoxic activities of compounds 1−10 against several mouse cell lines such as Ehrlich ascites carcinoma cells, neuroblastoma Neuro 2A, normal epithelial JB-6 cells, and erythrocytes were quite different depending both on structural peculiarities of these glycosides and the type of cells subjected to their actions. Psolusoside L (5), pentaoside, with three sulfate groups at C-6 of two glucose and one 3-O-methylglucose residue and holostane aglycone, is the most active compound in the series. The presence of a sulfate group at C-2 of the terminal glucose residue attached to C-4 of the first (xylose) residue significantly decreases activities of the corresponding glycosides. Psolusosides of group B (1, 2, and known psolusoside B) are inactive in all tests due to the presence of non-holostane aglycones and tetrasaccharide-branched sugar chains sulfated by C-2 of Glc4.

Seven sulfated triterpene glycosides, psolusosides B (1), E (2), F (3), G (4), H (5), H1 (6), and I (7), along with earlier known psolusoside A and colochiroside D have been isolated from the sea cucumber Psolus fabricii collected in the Sea of Okhotsk. Herein, the structure of psolusoside B (1), elucidated by us in 1989 as a monosulfated tetraoside, has been revised with application of modern NMR and particularly MS data and proved to be a disulfated tetraoside. The structures of other glycosides were elucidated by 2D NMR spectroscopy and HR-ESI mass-spectrometry. Psolusosides E (2), F (3), and G (4) contain holostane aglycones identical to each other and differ in their sugar compositions and the quantity and position of sulfate groups in linear tetrasaccharide carbohydrate moieties. Psolusosides H (5) and H1 (6) are characterized by an unusual sulfated trisaccharide carbohydrate moiety with the glucose as the second sugar unit. Psolusoside I (7) has an unprecedented branched tetrasaccharide disulfated carbohydrate moiety with the xylose unit in the second position of the chain. The cytotoxic activities of the compounds 2−7 against several mouse cell lines—ascite form of Ehrlich carcinoma, neuroblastoma Neuro 2A, normal epithelial JB-6 cells, and erythrocytes—were quite different, at that hemolytic effects of the tested compounds were higher than their cytotoxicity against other cells, especially against the ascites of Ehrlich carcinoma. Interestingly, psolusoside G (4) was not cytotoxic against normal JB-6 cells but demonstrated high activity against Neuro 2A cells. The cytotoxic activity against human colorectal adenocarcinoma HT-29 cells and the influence on the colony formation and growth of HT-29 cells of compounds 1−3, 5−7 and psolusoside A was checked. The highest inhibitory activities were demonstrated by psolusosides E (2) and F (3).

The immunomodulatory effect of triterpene glycoside cucumarioside A2-2 (CA2-2), isolated from the Far Eastern sea cucumber Cucumaria japonica, was compared with lipopolysaccharide (LPS) on mouse spleen. It has been shown that the intraperitoneal (i.p.) glycoside administration leads to increased spleen macrophage activating markers iba-1, IL-1β, iNOs, ROS and NO formation, with additional change of macrophage phenotype to M1. The mass spectrometry profiles of peptide/protein were obtained using MALDI-TOF-MS on the different parts of spleen sections isolated by laser mircodissection techniques. It was found that i.p. stimulation of animals with CA2-2 leads to marked changes in the intensity of the characteristic peaks of spleen peptides/proteins, primarily in red pulp.

The Far Eastern sea cucumber Eupentacta fraudatrix is an inhabitant of shallow waters of the south part of the Sea of Japan. This animal is an interesting and rich source of triterpene glycosides with unique chemical structures and various biological activities. The objective of this study was to investigate composition and distribution in various body components of triterpene glycosides of the sea cucumber E. fraudatrix. We applied LC-ESI MS (liquid chromatography–electrospray mass spectrometry) of whole body extract and extracts of various body components for metabolic profiling and structure elucidation of triterpene glycosides from the E. fraudatrix. Totally, 54 compounds, including 26 sulfated, 18 non-sulfated and 10 disulfated glycosides were detected and described. Triterpene glycosides from the body walls, gonads, aquapharyngeal bulbs, guts and respiratory trees were extracted separately and the distributions of the detected compounds in various body components were analyzed. Series of new glycosides with unusual structural features were described in E. fraudatrix, which allow clarifying the biosynthesis of these compounds. Comparison of the triterpene glycosides contents from the five different body components revealed that the profiles of triterpene glycosides were qualitatively similar, and only some quantitative variabilities for minor compounds were observed.

Nine new sulfated triterpene glycosides, magnumosides A1 (1), A2 (2), A3 (3), A4 (4), B1 (5), B2 (6), C1 (7), C2 (8) and C4 (9) as well as a known colochiroside B2 (10) have been isolated from the tropical Indo-West Pacific sea cucumber Neothynidium (=Massinium) magnum (Phyllophoridae, Dendrochirotida) collected in the Vietnamese shallow waters. The structures of new glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. All the isolated new glycosides were characterized by the non-holostane type lanostane aglycones having 18(16)-lactone and 7(8)-double bond and differed from each other by the side chains and carbohydrate moieties structures. Magnumoside A1 (1) has unprecedented 20(24)-epoxy-group in the aglycone side chain. Magnumosides of the group A (1–4) contained disaccharide monosulfated carbohydrate moieties, of the group B (5, 6)—tetrasaccharide monosulfated carbohydrate moieties and, finally, of the group C (7–9)—tetrasaccharide disulfated carbohydrate moieties. The cytotoxic activities of the compounds 1–9 against mouse spleen lymphocytes, the ascites form of mouse Ehrlich carcinoma cells, human colorectal carcinoma DLD-1 cells as well as their hemolytic effects have been studied. Interestingly, the erythrocytes were more sensitive to the glycosides action than spleenocytes and cancer cells tested. The compounds 3 and 7 significantly inhibited the colony formation and decreased the size of colonies of DLD-1 cancer cells at non-cytotoxic concentrations. Moreover, the synergism of effects of radioactive irradiation and compounds 3 and 7–9 at subtoxic doses on proliferation of DLD-1 cells was demonstrated.

Four new trisulfated triterpene glycosides, fallaxosides D4 (1), D5 (2), D6 (3) and D7 (4) have been isolated from the sea cucumber Cucumaria fallax (Cucumariidae, Dendrochirotida). The structures of the glycosides have been elucidated by 2D NMR spectroscopy and HRESIMS. All the glycosides have the lanostane aglycones of a rare non-holostane type with 7(8)-, 8(9)- or 9(11)-double bonds, one or two hydroxyl groups occupying unusual positions in the polycyclic nucleus and shortened or normal side chains. The pentasaccharide carbohydrate moieties of 1–4 have three sulfate groups. The cytotoxic activity of glycosides 1–4 against the ascite form of mouse Ehrlich carcinoma cells and mouse spleen lymphocytes and hemolytic activity against mouse erythrocytes have been studied.