Claudio Ortenzi

The protozoan toxin climacostol inhibits growth and induces apoptosis of human tumor cell lines.

Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids, compounds that show antimicrobial, antiparasitic and antitumor activities. We investigated the cytotoxic activity of the chemically synthesized toxin on: (1) human tumor squamous carcinoma A431 cells, (2) human promyelocytic leukaemia HL60 cells, and (3) human non-tumor endothelial EA.hy926 cells. The results showed that climacostol effectively inhibited the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death, with non-tumor cells proving significantly more resistant to the toxin.

Purification and initial characterization of two pheromones from the marine Antarctic ciliate, Euplotes nobilii

Abstract Among a set of wild‐type strains of Euplotes nobilii, every one derived asexually from one specimen isolated from Terra Nova Bay (Ross Sea, Antarctica), two were found to be representative of different mating types mutually capable of inducing each other to form mating pairs through pheromones constitutively secreted into the extracellular environment. Pheromones of strain AC‐1 were purified to homogeneity and shown to be represented by two distinct proteins, that were denoted En‐1 and En‐2. En‐1, secreted in amounts three‐fold greater than En‐2, was determined to have a molecular weight of 5617 and an asparagine at the N‐ter‐minus of its amino acid sequence, while En‐2 has a molecular weight of 6290 and bears an asparctic acid at its N‐terminus. The fact that En‐1 and En‐2 are coreleased by genetically identical cells of the same strain was taken to imply that they carry a het‐erozygotic combination of allelic pheromone genes and that these genes are regulated by relationships of co‐dominance.

The cell type-specific signal proteins (Pheromones) of protozoan ciliates

In association with their mechanisms of self/non-self recognition (known as mating type systems), ciliates synthesize and constitutively secrete cell type-specific proteins into their extracellular medium. These proteins, designated as pheromones, have been isolated from species of Euplotes and shown to be members of families of structurally homologous molecules, all rich in intra-chain disulfide bonds and organized exclusively in helical conformation. Due to their similar architectures, they can interact with their membrane receptors in competition with one another and bind effectively to their cells of origin in autocrine fashion, or to other co-specific cells in paracrine fashion. In the former case, they promote the vegetative cell growth; in the latter, they induce cells to temporarily arrest their growth stage and shift to a mating (sexual) stage. These varied, context-dependent activities of ciliate pheromones imply an early evolution of basic properties of animal growth factors and cytokines in the unicellular eukaryotes.

Chemical offense by means of toxicysts in the freshwater ciliate, Coleps hirtus.

Coleps hirtus is a small common freshwater ciliate belonging to the protostomatid group, its body covered by calcified plates assembled to form an armor. Coleps feeds on bacteria, algae, flagellates, living and dead ciliates, animal and plant tissues. To assist its carnivorous feeding the ciliate is equipped with offensive extrusomes (toxicysts), clustering mainly in and around its oral aperture. In this study, we isolated the discharge of the toxicysts from living cells, evaluating its cytotoxic effects against various ciliate species, and demonstrating that it is essential for the effectiveness of Coleps’ predatory behavior. The analysis of the toxicyst discharge performed by liquid chromatography‐electrospray‐mass spectrometry and gas chromatography‐mass spectrometry, revealed the presence of a mixture of 19 saturated, monounsaturated and polyunsaturated free fatty acids with the addition of a minor amount of a diterpenoid (phytanic acid).

Chemical defence by mono-prenyl hydroquinone in a freshwater ciliate, Spirostomum ambiguum

Several species of ciliates produce and accumulate low molecular weight toxic compounds in specialised membrane-bound ejectable organelles: extrusomes. These compounds can be used in predator–prey interaction for killing prey as well as for chemical defence. Here, we describe the isolation and characterisation of 2-(3-methylbut-2-enyl)benzene-1,4-diol(mono-prenyl hydroquinone), the extrusomal defensive toxin of the freshwater heterotrich ciliate Spirostomum ambiguum. The toxin was purified at homogeneity by RP-HPLC, and its structural characterisation was carried out through NMR and MS measurements. In vivo experiments involving S. ambiguum and Climacostomum virens in predator–prey interaction, and the analysis of cytotoxic activity of mono-prenyl hydroquinone on a panel of free-living freshwater ciliates, indicated that the toxin is very effective in S. ambiguum’s chemical defence.

The protozoan toxin climacostol and its derivatives: Cytotoxicity studies on 10 species of free-living ciliates

Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to resorcinolic lipids, a group of compounds that show antimicrobial, antiparasitic, and anticancer activities. We investigated the cytotoxic activity of the chemically synthesized toxin and its alkyl and alkynyl derivatives on C. virens and nine other common species of free-living freshwater ciliates. Our results show that the cytotoxic potency of climacostol can be modulated by the substitution of the double bond present in the aliphatic chain of the toxin with a single or a triple one that was previously obtained during the synthesis of the unsaturated and saturated derivatives of the parent molecule. We demonstrated that the cytotoxicity level of the molecules considered in this study appears to be inversely correlated to the unsaturation level of their aliphatic chains, and that the potency of their action is also related to the target organism.

Letter to the Editor: NMR structure of the pheromone Er-22 from Euplotes raikovi

Cellular recognition and signal transduction processes mediated by polypeptides or small proteins, e.g., growth factors and hormones, are an important feature of intercellular communication in higher eukaryotes (e.g., Bradshaw, 1996). The pheromone regulatory system in Euplotes raikovi (E. raikovi) represents a similar, simpler system in a unicellular eukaryote, which may represent an evolutionary predecessor of the more sophisticated regulatory mechanisms in multicellular species (Luporini et al., 1996). This makes the E. raikovi system an attractive paradigm for investigating the structural basis of ‘self’ and ‘nonself’ recognition processes that elicit different cellular responses. NMR structures of the pheromones Er-1 (Mronga et al., 1994), Er-2 (Ottiger et al., 1994), Er-10 (Brown et al., 1993) and Er-11 (Luginbuhl et al., 1996b), as well as an X-ray crystal structure of Er-1 (Weiss et al., 1995) have previously been determined. These four pheromones are all members of the ‘PR group’ (Luporini et al., 1995) and share a common architecture. Local structure variations confer specificity in receptor association (Luginbuhl et al., 1994) without interfering with the ability to compete for each other’s cell receptors in homologous (autocrine) reactions for cell growth stimulation, and in

Natural products from aquatic eukaryotic microorganisms for cancer therapy: Perspectives on anti-tumour properties of ciliate bioactive molecules

Several modern drugs, including those for cancer therapy, have been isolated from natural sources, are based on natural products and its derivatives, or mime natural products. Some of them are in clinical use, others in clinical trials. The success of natural products in drug discovery is related to their biochemical characteristics and to the technologic methods used to study their feature. Natural compounds may acts as chemo-preventive agents and as factors that increase therapeutic efficacy of existing drugs, thus overcoming cancer cell drug resistance that is the main factor determining the failure in conventional chemotherapy. Water environment, because of its physical and chemical conditions, shows an extraordinary collection of natural biological substances with an extensive structural and functional diversity. The isolation of bioactive molecules has been reported from a great variety of aquatic organisms; however, the therapeutic application of molecules from eukaryotic microorganisms remains inadequately investigated and underexploited on a systematic basis. Herein we describe the biological activities in mammalian cells of selected substances isolated from ciliates, free-living protozoa common almost everywhere there is water, focusing on their anti-tumour actions and their possible therapeutic activity. In particular, we unveil the cellular and molecular machine mediating the effects of cell type-specific signalling protein pheromone Er-1 and secondary metabolites, i.e. euplotin C and climacostol, in cancer cells. To support the feasibility of climacostol-based approaches, we also present novel findings and report additional mechanisms of action using both in vitro and in vivo models of mouse melanomas, with the scope of highlighting new frontiers that can be explored also in a therapeutic perspective. The high skeletal chemical difference of ciliate compounds, their sustainability and availability, also through the use of new organic synthesis/modifications processes, and the results obtained so far in biological studies provide a rationale to consider some of them a potential resource for the design of new anti-cancer drugs.

DNA binding and oxidative DNA damage induced by climacostol-copper(II) complexes: implications for anticancer properties.

Climacostol is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids. Climacostol exerts a potent antimicrobial activity against a panel of bacterial and fungal pathogens. In addition it inhibits the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death via intrinsic pathway. In this work, we investigated the possibility that climacostol exerts a prooxidant effect, inducing plasmid DNA strand breakage and eukaryotic DNA damage in presence of Cu(II) ions. Inhibition of DNA breakage using SOD, catalase and neocuproine confirmed the involvement of reactive oxygen species and Cu(I) ions in the DNA damage. UV-visible absorption changes and mass spectrometric analysis identified a product of reaction as a deprotonated form of climacostol. Study of the interaction with DNA, using fluorescence spectroscopic techniques, showed that climacostol binds with DNA. Given the structure-activity relationship of this compound and the mechanism of its prooxidant effect, we propose that the Cu(II)-mediated oxidative DNA damage by climacostol could explain its antimicrobial and antiproliferative activity.

Antimicrobial activity of the protozoan toxin climacostol and its derivatives

Climacostol is a defense toxin produced by the ciliated protozoan Climacostomum virens and belongs to resorcinolic lipids, a group of compounds that shows antimicrobial, antiparasitic, and cytotoxic activities. In this study we investigate the antimicrobial activity of climacostol and its alkyl and alkynyl derivatives against a panel of bacterial and fungal pathogens. Our results show a good and comparable antimicrobial activity of the three compounds, which have resulted effective against Gram-positive bacteria and Candida with MIC and MBC ranging from 8 to 32 mg L−1, whereas no significant effect against Gram-negative species has been observed. Taken as a whole, the experimental data reported in the current study suggest that differences in the saturation rate of the lateral chain of climacostol are not related to the activity of the molecule. Therefore, it is likely that the general structure of the two moieties, i.e., the di-hydroxy-phenyl group and the alkenyl chains, contributes to the overall antibiotic behaviour.