An Unusual Pathway of Mitoptosis Found in Ehrlich Carcinoma Cells.

An integrated microscopic study of the destruction of mouse Ehrlich ascites carcinoma (EAC) cells under starvation conditions has been carried out. It has been found that, in addition to apoptosis, necrosis, and apoptotic necrosis, already known for EAC, cell destruction can also occur through mitochondrial autolysis, which is proposed to be considered a new kind of mitoptosis. A mitoptosis in EAC is characterized by the appearance of many autolyzing mitochondria, the fusion of which leads to rupture of the cell membrane and the ejection of the nucleus from the cell. It is assumed that the polymorphism of EAC destruction patterns is explained by the different physiological state of the cells, which determines the "choice" of the cell death mechanism. This situation poses a challenge for researchers to develop complex inducers with the ability to stimulate all possible types of cancer cell death.

Cucumarioside A2-2 causes changes in the morphology and proliferative activity in mouse spleen.

The immunomodulatory effect of triterpene glycoside cucumarioside A2-2 (CA2-2), isolated from the Far Eastern sea cucumber Cucumaria japonica, on the mouse spleen was investigated in comparison with lipopolysaccharide (LPS). It has been shown that the intraperitoneal (i.p.) glycoside administration did not influence on splenic weights, while the statistically significant increase in splenic weight was observed after LPS administration. Changes in the ratio of red to white pulp after CA2-2 or LPS administration were observed. The proportion of splenic white pulp after glycoside or LPS administration increased by up to 34% and 36%, respectively. A detailed study of the distribution of the РСNA (Proliferating Cell Nuclear Antigen) marker showed that the proliferative activity in the white pulp under CA2-2 and LPS influence increased 2.07 and 2.24 times, respectively. The localization of PCNA-positive nuclei in the white pulp region, as well as their dimensional characteristics, suggests that a large proportion of the proliferating cell population consisted of B cells. The mass spectrometry profiles of spleen peptide/protein homogenate were obtained using the MALDI-TOF-MS (Matrix -Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) approach. It was found that i.p. stimulation of animals with CA2-2 or LPS leads to marked changes in the intensity of revealed characteristic peaks of peptides/proteins after exposure to immunostimulants.

[Anti-Inflammatory Activity of the Polypeptide of the Sea Anemone, Heteractis crispa].

The anti-inflammatory effect of the recombinant polypeptide HCGS 1.20, a Kunitz-type serine protease inhibitor of the sea anemone Heteractis crispa, was investigated. It was shown that the polypeptide inhibits the increase of the concentration of calcium ions in mouse bone marrow derived macrophages elicited by histamine, and reduces the content of NO in lipopolysaccharide stimulated macrophages. A presumable mechanism of anti-inflammatory action of the polypeptide was being discussed.

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.

Determination of cucumarioside A2-2 in mouse spleen by radiospectroscopy, MALDI-MS and MALDI-IMS.

The distribution of triterpene glycoside cucumarioside A2-2, the main compound of medical lead Cumaside in immunodeficiency diseases, in mouse spleen was determined. For this purpose the stability and dynamics of glycoside content changes over time in Balb/c mouse spleen tissue homogenate as well as the study of the cucumarioside A2-2 spatial distribution in tissue sections were investigated using radiospectroscopy, MALDI-MS and MALDI Imaging Mass Spectrometry (IMS), correspondingly. Cucumarioside A2-2 is reliably detected by MALDI-MS in the mouse spleen tissue after single intraperitoneal (i.p.) injection at a dosage of 5 mg/kg. The glycoside is stable in the spleen and does not undergo metabolic transformation in either tissue homogenates or in the intact organ within 24 h after i.p. injection. The cucumarioside A2-2 was absorbed fairly rapidly: the glycoside maximum concentration (Cmax) in tissue homogenate was observed in the first 30 min after injection; the minimum values were registered in 3 h. These results are in agreement with those obtained in the pharmacokinetic study of (3)H-cucumarioside A2-2. It was established by MALDI-IMS that glycoside was mainly located in the tunica serosa part of the spleen and only a small amount was detected within the red and white pulp of the organ. MALDI MS images obtained 15-30 min post dosage clearly reflect high drug concentrations in the regions surrounding the organ followed by its decline in the surface part and a very slight redistribution to the internal part of the spleen.

Interaction of holothurian triterpene glycoside with biomembranes of mouse immune cells.

The in vitro interactions between triterpene glycoside, cucumarioside A(2)-2, isolated from the Far-Eastern holothurian Cucumaria japonica, and mouse splenocyte and peritoneal macrophage biomembranes were studied. Multiple experimental approaches were employed, including determination of biomembrane microviscosity, membrane potential and Ca(2+) signaling, and radioligand binding assays. Cucumarioside A(2)-2 exhibited strong cytotoxic effect in the micromolar range of concentrations and showed pronounced immunomodulatory activity in the nanomolar concentration range. It was established that the cucumarioside A(2)-2 effectively interacted with immune cells and increased the cellular biomembrane microviscosity. This interaction led to a dose-dependent reversible shift in cellular membrane potential and temporary biomembrane depolarization; and an increase in [Ca(2+)](i) in the cytoplasm. It is suggested that there are at least two binding sites for [(3)H]-cucumarioside A(2)-2 on cellular membranes corresponding to different biomembrane components: a low affinity site match to membrane cholesterol that is responsible for the cytotoxic properties, and a high affinity site corresponding to a hypothetical receptor that is responsible for immunostimulation.

Decreased ROS level and activation of antioxidant gene expression in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia.

Microbe-plant interactions often lead to a decrease in the reactive oxygen species (ROS) level of plant cells, which allows pathogen survival through the suppression of plant immune responses. In the present investigation, we tested whether transformation of Rubia cordifolia cells by Agrobacterium rhizogenes had a similar effect. We isolated partial cDNA sequences of ascorbate peroxidase, catalase and Cu/Zn superoxide dismutase genes (RcApx1, RcApx2, RcApx3, RcCAT1, RcCAT2, RcCSD1, RcCSD2 and RcCSD3) from plant tissues, as well as pRiA4-transformed and normal calli of Rubia cordifolia, and studied their expression by real-time PCR. Transcription profiling revealed that ascorbate peroxidase (RcApx1) and Cu/Zn superoxide dismutase (RcCSD1) were the most abundant transcripts present in both plant tissues and non-transformed calli. Catalase genes were weakly expressed in these samples. The pRiA4-transformed calli showed enhanced expression of several genes encoding ROS-detoxifying enzymes. Confocal microscopy imaging revealed decreased ROS level in pRiA4-transformed calli compared to the control. These results demonstrate that A. rhizogenes, like other plant pathogens, uses a strategy aimed at decreasing ROS levels in host cells through the general upregulation of its antioxidant genes.

[Polar steroids from Solaster endeca starfish and the physiological activity of polar steroids from three starfish species].

Four polyhydroxylated steroids, new (20R)-5alpha-cholestan-3beta,6alpha,8,15alpha,24,26-hexaol (I) and known (20R,25S)-5alpha-cholestan-3beta6alpha,8,15beta,16beta,26-hexaol, (20R,25S)-5alpha-cholestan-3beta,6alpha,15beta,16beta,26-pentaol, and marthasterone sulfate were isolated from the Solaster endeca starfish inhabiting the Sea of Okhotsk and characterized. Steroid (I) contains a 24,26-dihydroxylated side chain, which is uncommon for starfish polyols. The isolated steroids and related metabolites from two starfish species of the Evasterias genus (in total, 15 compounds) were weakly cytotoxic in a human HeLa cell culture and some of them were inhibitors of nonspecific esterase from mouse Ehrlich carcinoma. The effects of these compounds on the p53 protein activity were studied in a yeast two-hybrid test system and both inhibitors and stimulators of this activity were found among them.

[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%.

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.

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.

Immunomodulatory properties of Cumaside.

The medical lead, so-called Cumaside, was created on the basis of triterpene oligoglycosides from the Far-Eastern edible sea cucumber (holothurian) Cucumaria japonica and its immunomodulatory properties were studied. The haemolytic activity of Cumaside was significantly reduced in comparison with original glycosides due to the glycoside-cholesterol complex formation. The influence of Cumaside on mouse macrophages in low doses was accompanied by more then two-fold stimulation of lysosomal activity. This preparation was found to increase significantly the animal resistance against bacterial infections elicited by various pathogens. It stimulated phagocytosis, ROS formation, IL6 and TNF-alpha production in lymphocytes, increased the number of antibody producing cells and amplified the expression of several cell surface molecules (CD3, CD4, CD8) preliminary cultured with hydrocortisone. At the same time the preparation did not affect the delayed-type hypersensitivity, proliferative activity of lymphocytes, cytotoxic activity of NK-cells and cytokine IFNgamma and IL12p70 release. The mechanism of Cumaside action is discussed.

Immunomodulatory Properties of Cucumariosides from the Edible Far-Eastern Holothurian Cucumaria japonica.

Holothurian triterpene glycosides (cucumariosides) are known to possess multiple biological activities. Here we show that cucumariosides from the Far Eastern edible holothurian (sea cucumber), Cucumaria japonica, and their semisynthetic derivatives possess potent immunomodulatory properties. Intraperitoneal injection of cucumariosides (0.2-20 ng per mouse) induced macrophage lysosomal activity in a dose-dependent manner (up to 250% of control). The stimulatory effect was related to the chemical structure of cucumariosides and was especially influenced by the number and position of sulfate groups in the carbohydrate moiety of the molecules. In vitro, an inhibitory rather than a stimulatory effect of cucumariosides on phagocytosis and release of tumor necrosis factor-alpha (TNF-alpha) was found. Virtually all cucumariosides inhibited latex bead phagocytosis by human peripheral blood granulocytes in a dose-dependent manner. Also, the lipopolysaccharide-induced TNF-alpha production by immune cells in human blood diluted with RPMI-1640 was decreased, without a clear relation to the structure and dose of the compounds. The data are discussed in terms of possible mechanisms underlying immunomodulatory properties of triterpene glycosides from C. japonica.

The effect of pH on biological activity of plant cytotoxin cauloside C.

The effect of plant carboxyl-containing glycoside cauloside C upon eucaryotic cells has been studied. The glycoside interacts with cells as a pH-dependent cytotoxin and increases K+ leakage and Ca2+ uptake with strong action in acidic media Cell viability after glycoside action at acidic pH may be recovered by the shift of medium pH from 5.6 to 7.4. Directed transport of low molecular weight effectors such as cAMP and Ca2+ to human embryo fibroblasts under the action of cauloside C has been demonstrated. Calcium uptake is accompanied by about a twofold stimulation of fibroblast proliferation in serum-free medium. The manifestation of the effect depends on the strictly determined time of the 'open' state of the membrane permeability (2 min) and upon concentration of glycoside in the medium (1 ng/ml) Cauloside C-stimulated Ca-transport is not blocked by Ca-channel blockers such as verapamil, diltiasem, and nitrendipine (all at a concentration of 1 x 10(-6) M) but these blockers inhibit cauloside C-stimulated proliferation of fibroblasts. We conclude that stimulation of fibroblast proliferation is caused by activation of membrane associated Ca-channels at the expense of calcium, incorporated into cells with cauloside C. The use of cauloside C as a new biochemical tool for cell permeabilisation is suggested.

Stimulatory effects of starfish sapogenins (Asterias amurensis and Lethasterias nanimensis chelifera) on molluscan heart (Spisula sachalinensis).

Sapogenins from the starfish Asterias amurensis and Lethasterias nanimensis chelifera, 5 alpha-pregn-9(11)-ene-3 beta,6 alpha-diol-20-one, 5 alpha-cholest-9(11)-ene-3 beta,6 alpha-diol-23-one, 5 alpha-cholesta-9(11),24(25)-diene-3 beta,6 alpha-diol-23-one, (20E)-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one and 24 zeta-methyl-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one, stimulated the contractile force of the heart of the mollusk Spisula sachalinensis at concentration of 5 x 10(-5) M. Ouabain, a specific inhibitor of Na+,K(+)-ATPase, at concentration of 5 x 10(-5) M had no effect on this physiological model. Starfish sapogenins of the cholestane series moderately inhibited rat brain cortex Na+,K(+)-ATPase and decreased Ca2+ influx into Ehrlich carcinoma cells. In contrast, pregnane asterogenin asterone did not inhibit Na+,K(+)-ATPase and increased the influx of Ca2+ into cells. These effects were not the result of cell membrane damage, because none of the compounds tested have hemolytic activity.