Ekaterina S. Menchinskaya

Anticancer Activity of Sea Cucumber Triterpene Glycosides

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.

The marine triterpene glycoside frondoside A exhibits activity in vitro and in vivo in prostate cancer.

Despite recent advances in the treatment of metastatic castration‐resistant prostate cancer (CRPC), outcome of patients remains poor due to the development of drug resistance. Thus, new drugs are urgently needed. We investigated efficacy, toxicity and mechanism of action of marine triterpene glycoside frondoside A (FrA) using CRPC cell lines in vitro and in vivo. FrA revealed high efficacy in human prostate cancer cells, while non‐malignant cells were less sensitive. Remarkably, proliferation and colony formation of cells resistant to enzalutamide and abiraterone (due to the androgen receptor splice variant AR‐V7) were also significantly inhibited by FrA. The marine compound caused cell type specific cell cycle arrest and induction of caspase‐dependent or ‐independent apoptosis. Up‐regulation or induction of several pro‐apoptotic proteins (Bax, Bad, PTEN), cleavage of PARP and caspase‐3 and down‐regulation of anti‐apoptotic proteins (survivin and Bcl‐2) were detected in treated cells. Global proteome analysis revealed regulation of proteins involved in formation of metastases, tumor cell invasion, and apoptosis, like keratin 81, CrkII, IL‐1β and cathepsin B. Inhibition of pro‐survival autophagy was observed following FrA exposure. In vivo, FrA inhibited tumor growth of PC‐3 and DU145 cells with a notable reduction of lung metastasis, as well as circulating tumor cells in the peripheral blood. Increased lymphocyte counts of treated animals might indicate an immune modulating effect of FrA. In conclusion, our results suggest that FrA is a promising new drug for the treatment of mCRPC. Induction of apoptosis, inhibition of pro‐survival autophagy, and immune modulatory effects are suspected modes of actions.

Structure of cucumarioside I2 from the sea cucumber Eupentacta fraudatrix (Djakonov et Baranova) and cytotoxic and immunostimulatory activities of this saponin and relative compounds

A new triterpene glycoside cucumarioside I2 (1) has been isolated from holothurian Eupentacta fraudatrix. The structure of 1 was elucidated using extensive NMR spectroscopy (1H and 13C NMR, 1H–1H COSY, 1D TOCSY, HSQC, H2BC, HMBC and NOESY) and MALDI-TOF-MS. Glycoside 1 is a disulfated branched pentaoside having rare 3-O-methyl-D-xylose. Cytotoxic activity of the glycoside 1 and known cucumariosides H (2), A5 (3), A6 (4), B2 (5) and B1 (6) against mouse Ehrlich carcinoma cells and their influence on lysosomal activity of mouse peritoneal macrophages have been studied. Glycosides 1 and 5 possessed low cytotoxicities, glycoside 6 was not cytotoxic while compounds 2, 3 and 4 possessed moderate cytotoxicities. Glycosides 1, 3 and 5 increased the lysosomal activity of macrophages on 15–17% at doses of 1–5 μg/mL. Hence lysosomal activity depends on structures of both aglycone and carbohydrate chain and does not have a direct correlation with cytotoxicities of the glycosides.

Cyclic Steroid Glycosides from the Starfish Echinaster luzonicus: Structures and Immunomodulatory Activities

Five new steroid glycosides, luzonicosides B-E (2-5), belonging to a rare structure group of marine glycosides, containing carbohydrate moieties incorporated into a macrocycle, and a related open carbohydrate chain steroid glycoside, luzonicoside F (6), were isolated from the starfish Echinaster luzonicus along with the previously known cyclic steroid glycoside luzonicoside A (1). The structures of compounds 2-6 were established by extensive NMR and ESIMS techniques as well as chemical transformations. Luzonicoside A (1) at concentrations of 0.01-0.1 μM was shown to be potent in lysosomal activity stimulation, intracellular ROS level elevation, and NO synthesis up-regulation in RAW 264.7 murine macrophages. Luzonicoside D (4) was less active in these biotests.

Chapter 3 - Immunomodulatory and Anticancer Activity of Sea Cucumber Triterpene Glycosides

Abstract The triterpene glycosides are composed of a carbohydrate chain and triterpene aglycone and are widely distributed in sea cucumbers ( Holothurioidea , Echinodermata ). Most aglycones have 18(20)-lactones and belong to the holostane type. Carbohydrate chains of sea cucumber glycosides have from two to six monosaccharide residues including xylose, quinovose, glucose, and 3- O -methylglucose and sometimes 3- O -methylxylose, 3- O -methylquinovose, 3- O -methylglucuronic acid, and 6- O -acetylglucose. They may contain one, two, or three sulfate groups. At the milli- and micromolar concentrations, sea cucumber glycosides show hemolytic, cytotoxic, antifungal, and other biological activities caused by membranotropic action. The basis of membranotropic action of the glycosides is their ability to attach to cell biomembranes and form nonselective ion-conducting complexes with 5(6)-nonsaturated sterol components of cell membranes, preferably with cholesterol. Such sterol/saponin interactions result in an efflux of some ions, nucleotides, and peptides, disrupting ion homeostasis and osmolarity followed by lysis and cell death. Some sea cucumber glycosides show an immunostimulatory effect at subtoxic nanomolar concentrations. Incubation of immune cells with the glycosides induces their activation resulting in an increase of immune cell adhesion on an extracellular matrix, enhancement of cell spreading and motility, increase of macrophage lysosomal activity, ROS formation, and phagocytic activity. The most effective immunostimulants are monosulfated glycosides, whereas di- and trisulfated glycosides are immunodepressants. Injection of subtoxic doses of some glycosides induces an increase in the number of antibody-producing plaque-forming cells in mouse spleens, an increase in the number, size and acidity of lysosomes of peritoneal macrophages, and increase of phagocytic index, resulting in heightened resistance by host animals against bacterial infections. Proteomic methods have demonstrated that the mechanism of immunomodulatory action of some sea cucumber glycosides on mouse splenocytes includes regulation of the expression of some proteins involved in the formation of cellular immune response. These glycosides regulate the expression of proteins associated with lysosome maturation, activation and merging, phagocytosis, cytoskeletal reorganization, cell adhesion, mobility, and proliferation of immune cells. It was shown that glycosides moderately induce production of some cytokines, restore the level of some CD markers of lymphocytes, increase bactericidal activity of leukocytes, and induce a significant increase in mouse resistance to lethal doses of some pathogenic microorganisms and radiation. 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. These studies have shown the triterpene glycosides block egg cleavage and development of sea urchin embryos, suppress the proliferation of various human tumor cell lines in vitro , possess antiangiogenic effect, and cause cancer cell cycle arrest. Several sea cucumber glycosides are reported to induce tumor cell apoptosis in vitro and more importantly, IP administration in rodents of solutions of some sea cucumber triterpene glycosides show significant reduction of both tumor burden and metastasis. Recently, it was found that the new immunomodulatory lead compound, Cumaside, based on the holothurian triterpene glycoside, cucumarioside A 2 -2, demonstrates inhibition of tumor initiation and proliferation, in vivo and exhibits significant synergy with 5-fluorouracil.

Polyoxygenated steroids from the gorgonian Menella woodin with capabilities to modulate ROS levels in macrophages at response to LPS.

Four new polyoxygenated sterol derivatives (1-4) along with the compounds (5-7) previously known from other biological sources were isolated from the gorgonian Menella woodin, collected from the Vietnamese waters. Structures of 1-4 were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses as well as comparison with those reported in literature data. Compounds 1, 4, and 6 decrease the production of reactive oxygen species (ROS) by the murine macrophages of RAW 264.7 line at induction by endotoxic lipopolysaccharide (LPS) from Escherichia coli.

Hsp70 Induction and Anticancer Activity of U-133, the AcetylatedTrisglucosydic Derivative of Echinochrome

Acetylated trisglucosydic derivative of 2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoqinone (natural sea urchin pigment echinochrome), compound U-133, was synthesized. The effect of U-133 upon mouse Ehrlich ascitic carcinoma(EAC) cells in vitro and its antitumor activity in vivo was investigated. The synthesized U-133 did not display the pronounced cytotoxicity against EAC cells in the concentrations up to 80 μM. In non-cytotoxic concentration of 5 μM U-133 significantly induced Hsp70 expression in cancer cells and enhanced cell-mediated cytotoxicity of mouse splenocytes against EAC cells. The administration of U-133 in dosage of 100 mg/kg in carcinoma bearing mice resulted in significant inhibition of tumor growth and increase in mouse lifespan.

Four New Steroidal Glycosides, Protolinckiosides A - D, from the Starfish Protoreaster lincki.

Four new steroidal glycosides, protolinckiosides A – D (1 – 4, resp.), were isolated along with four previously known glycosides, 5 – 8, from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 – 4 were elucidated by extensive NMR and ESI‐MS techniques as (3β,4β,5α,6β,7α,15α,16β,25S)‐4,6,7,8,15,16,26‐heptahydroxycholestan‐3‐yl 2‐O‐methyl‐β‐d‐xylopyranoside (1), (3β,5α,6β,15α,24S)‐3,5,6,8,15‐pentahydroxycholestan‐24‐yl α‐l‐arabinofuranoside (2), sodium (3β,6β,15α,16β,24R)‐29‐(β‐d‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d‐xylopyranosyl)oxy]stigmast‐4‐en‐15‐yl sulfate (3), and sodium (3β,6β,15α,16β,22E,24R)‐28‐(β‐d‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d‐xylopyranosyl)oxy]ergosta‐4,22‐dien‐15‐yl sulfate (4). The unsubstituted β‐d‐galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 – 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli.

Sulfated O-polysaccharide with anticancer activity from the marine bacterium Poseidonocella sedimentorum KMM 9023T

The sulfated polysaccharides are of study interest due to their high structural diversity and broad spectrum of biological activity including antitumor properties. In this paper, we report on the structural analysis of sulfated O-specific polysaccharide (OPS) and in vitro anticancer activity of O-deacylated lipopolysaccharide (DPS) of the marine-derived bacterium Poseidonocella sedimentorum KMM 9023T achieved by a multidisciplinary approach (chemical analysis, NMR, MS, and bioassay). The OPS is shown to include two rare monosaccharide derivatives: 3-deoxy-9-O-methyl-d-glycero-d-galacto-non-2-ulosonic acid (Kdn9Me) and 3-O-acetyl-2-O-sulfate-d-glucuronic acid (D-GlcA2S3Ac). The structure of polysaccharide moiety of a previously unknown carbohydrate-containing biopolymer is established: →4)-α-Kdnp9Me-(2→4)-α-d-GlcpA2S3Ac-(1→. From a biological point of view, we demonstrate that DPS of the P. sedimentorum KMM 9023T has no cytotoxicity and inhibits colony formation of human HT-29, MCF-7 and SK-MEL-5 cells in a dose-dependent manner. The investigated polysaccharide is the second glycan isolated from the bacteria of the genus Poseidonocella: previously we studied the OPS of P. pacifica KMM 9010T (Kokoulin et al., 2017). Both polysaccharides are sulfated and contain rare residues of ulosonic acids. Thus, obtained findings provide a new knowledge about kinds and antitumor properties of sulfated polysaccharides and can be a starting point for further investigations of mechanisms of anticancer action of carbohydrate-containing biopolymers from marine Gram-negative bacteria.