ABSTRACT
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.
Subject(s)
Immunologic Factors/pharmacology , Saponins/pharmacology , Spleen/drug effects , Animals , Cell Proliferation/drug effects , Drug Evaluation, Preclinical , Female , Lipopolysaccharides/pharmacology , Mice, Inbred BALB C , Spleen/cytology , Spleen/immunologyABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents , Macrophages/metabolism , Peptides , Sea Anemones , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Cell Line , Histamine/metabolism , Lipopolysaccharides/pharmacology , Macrophages/pathology , Mice , Nitric Oxide/metabolism , Peptides/chemistry , Peptides/genetics , Peptides/pharmacology , Sea Anemones/chemistry , Sea Anemones/geneticsABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Survival/drug effects , Saponins/pharmacology , Sea Cucumbers/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Carcinoma, Ehrlich Tumor/drug therapy , Carcinoma, Ehrlich Tumor/mortality , Carcinoma, Ehrlich Tumor/pathology , Cell Line, Tumor , DNA/metabolism , Humans , Kaplan-Meier Estimate , Mice , S Phase Cell Cycle Checkpoints/drug effects , Saponins/chemistry , Saponins/therapeutic use , Sea Cucumbers/metabolism , Transplantation, Homologous , Tumor Suppressor Protein p53/metabolismABSTRACT
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.
Subject(s)
Saponins/pharmacokinetics , Spleen/metabolism , Animals , Female , Mass Spectrometry/methods , Mice , Mice, Inbred BALB C , Saponins/administration & dosage , Spectrum Analysis/methods , TritiumABSTRACT
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.
