An organic extract from ascidian Ciona robusta induces cytotoxic autophagy in human malignant cell lines.

The last decades have seen an increase in the isolation and characterization of anticancer compounds derived from marine organisms, especially invertebrates, and their use in clinical trials. In this regard, ascidians, which are included in the subphylum Tunicata, represent successful examples with two drugs, Aplidine© and Yondelis© that reached the market as orphan drugs against several malignancies. Here, we report that an organic extract prepared from homogenized tissues of the Mediterranean ascidian Ciona robusta inhibited cell proliferation in HT-29, HepG2, and U2OS human cells with the former being the most sensitive to the extract (EC50 = 250 µg/mL). We demonstrated that the ascidian organic extract was not cytotoxic on HT-29 cells that were induced to differentiate with sodium butyrate, suggesting a preference for the mixture for the malignant phenotype. Finally, we report that cell death induced by the organic extract was mediated by the activation of a process of cytotoxic autophagy as a result of the increased expression of the LC3-II marker and number of autophagic vacuoles, which almost doubled in the treated HT-29 cells. In summary, although the detailed chemical composition of the Ciona robusta extract is still undetermined, our data suggest the presence of bioactive compounds possessing anticancer activity.

Oocyte quality assessment in marine invertebrates: a novel approach by fluorescence spectroscopy

BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

Spermiotoxicity of nickel nanoparticles in the marine invertebrate Ciona intestinalis (ascidians).

Nickel nanoparticles (Ni NPs) are increasingly used in modern industries as catalysts, sensors, and in electronic applications. Due to this large use, their inputs into marine environment have significantly increased; however, the potential ecotoxicological effects in marine environment have so far received little attention. In particular, little is known on the impact of NPs on gamete quality of marine organisms and on the consequences on fertility potential. The present study examines, for the first time, the impact of Ni NPs exposure on sperm quality of the marine invertebrate Ciona intestinalis (ascidian). Several parameters related with sperm status such as plasma membrane lipid peroxidation, mitochondrial membrane potential (MMP), intracellular pH, DNA integrity, and fertilizing ability were assessed as toxicity end points after exposure to different Ni NPs concentrations. Ni NPs generate oxidative stress that in turn induces lipid peroxidation and DNA fragmentation, and alters MMP and sperm morphology. Furthermore, sperm exposure to Ni NPs affects their fertilizing ability and causes developmental anomalies in the offspring. All together, these results reveal a spermiotoxicity of Ni NPs in ascidians suggesting that the application of these NPs should be carefully assessed as to their potential toxic effects on the health of marine organisms that, in turn, may influence the ecological system. This study shows that ascidian sperm represent a suitable and sensitive tool for the investigation of the toxicity of NPs entered into marine environment, for defining the mechanisms of toxic action and for the environmental monitoring purpose.

Role of cyclic AMP in the maturation of Ciona intestinalis oocytes.

Immature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidian Ciona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae from in vitro matured oocytes showed different morphology from those derived from in vivo mature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates.

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis.

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.

Cytotoxic and apoptogenic activity of a methanolic extract from the marine invertebrate Ciona intestinalis on malignant cell lines.

Marine invertebrates provide a series of natural products with different biological activities. Several of these compounds and their derivatives showed a potent anticancer effect. Tunicates represent an important source of bioactive agents, leading to the isolation of ecteinascidin-743 (ET-743), a compound isolated from the Caribbean sea squirt Ecteinascidia turbinata with a potent cytotoxic activity against a variety of tumours in vitro and in vivo. Current phase II clinical trials against soft tissue sarcomas in Europe and the United States indicate that ET-743 represents a highly promising anticancer agent. Another example is aplidine from the Mediterranean tunicate Aplidium albicans, with a broad spectrum activity against various types of cancers, such as colorectal, lymphoma, thyroid and renal cancers. In the present work, we reported, for the first time, that a partially purified methanolic extract prepared from the ascidian Ciona intestinalis inhibited cell proliferation in human cell lines of different origin, including Caco2, HPB-ALL, U-937 and HL-60 and induced early apoptotic events, such as caspase-3 activation and internucleosomal DNA degradation. We suggest the presence in the Ciona intestinalis extract of bioactive compounds possessing anticancer activity.

Calcium currents correlate with oocyte maturation during the reproductive cycle in Octopus vulgaris.

Using the whole-cell voltage clamp technique, we have studied the Ca2+ currents and the steady-state conductance during different oocyte growth stages and during the reproductive cycle of the female of Octopus vulgaris. Evidence is presented that L-type Ca2+ currents are high in small pre-vitellogenic oocytes (80-150 microm diameter) and significantly lower in early vitellogenic oocytes (180-300 microm diameter). Similarly, a significant decrease of the steady-state conductance occurred from the pre to early- vitellogenic oocytes. Octopus oocytes showed larger Ca2+ currents in the reproductive rather than non-reproductive periods. These data indicates that ion and L-type Ca2+ currents play a role in oocyte growth and cytoplasmic maturation, and possibly in preparing the plasma membrane to the interaction with the spermatozoon. By using fluorescent microscopy, we show that oocytes from 80 to 400 microm diameter have the large germinal vesicle characteristic of the immature oocytes. In subsequent stages of growth (up to 1000 microm diameter) the nucleus is no more visible and the metaphase spindle appears. These data demonstrate that Octopus vulgaris oocytes are arrested in the first meiotic prophase up to the early-vitellogenic stage and resume meiosis at this stage up to a second block presumably in metaphase I. We discuss a possible role for progesterone as the hormonal stimulus for the first prophase-metaphase meiotic transition.