Kinase-Based Screening of Marine Natural Extracts Leads to the Identification of a Cytotoxic High Molecular Weight Metabolite from the Mediterranean Sponge Crambe tailliezi.

Regulated cell death (RCD) results from the activation of one or more signal transduction modules both in physiological or pathological conditions. It is now established that RCD is involved in numerous human diseases, including cancer. As regulated cell death processes can be modulated by pharmacological tools, the research reported here aims to characterize new marine compounds acting as RCD modulators. Protein kinases (PKs) are key signaling actors in various RCDs notably through the control of either mitosis (e.g., the PKs Aurora A and B) or necroptosis (e.g., RIPK1 and RIPK3). From the primary screening of 27 various extracts of marine organisms collected in the Mediterranean Sea, an extract and subsequently a purified high molecular weight compound dubbed P3, were isolated from the marine sponge Crambe tailliezi and characterized as a selective inhibitor of PKs Aurora A and B. Furthermore, P3 was shown to induce apoptosis and to decrease proliferation and mitotic index of human osteosarcoma U-2 OS cells.

Batzella, Crambe and Monanchora: Highly Prolific Marine Sponge Genera Yielding Compounds with Potential Applications for Cancer and Other Therapeutic Areas.

Pyrroloquinoline and guanidine-derived alkaloids present distinct groups of marine secondary metabolites with structural diversity that displayed potentialities in biological research. A considerable number of these molecular architectures had been recorded from marine sponges belonging to different marine genera, including Batzella, Crambe, Monanchora, Clathria, Ptilocaulis and New Caledonian starfishes Fromia monilis and Celerina heffernani. In this review, we aim to comprehensively cover the chemodiversity and the bioactivities landmarks centered around the chemical constituents exclusively isolated from these three marine genera including Batzella, Crambe and Monanchora over the period 1981-2017, paying a special attention to the polycyclic guanidinic compounds and their proposed biomimetic landmarks. It is concluded that these marine sponge genera represent a rich source of novel compounds with potential applications for cancer and other therapeutic areas.

Crambescin C1 Exerts a Cytoprotective Effect on HepG2 Cells through Metallothionein Induction.

The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities.

A single betaproteobacterium dominates the microbial community of the crambescidine-containing sponge Crambe crambe.

Crambe crambe is a marine sponge that produces high concentrations of the pharmacologically significant pentacyclic guanidine alkaloids (PGAs), Crambescines and Crambescidines. Although bio-mimetic chemical synthesis of PGAs suggests involvement of microorganisms in their biosynthesis, there are conflicting reports on whether bacteria are associated with this sponge or not. Using 16S rRNA gene pyrosequencing we show that the associated bacterial community of C. crambe is dominated by a single bacterial species affiliated to the Betaproteobacteria. Microscopy analysis of sponge tissue sections using a specific probe and in situ hybridization confirmed its dominance in the sponge mesohyl and a single microbial morphology was observed by transmission electron microscopy. If confirmed the presence of a simple bacteria community in C. crambe makes this association a very pertinent model to study sponge-bacteria interactions and should allow further research into the possible implication of bacteria in PGA biosynthesis.

A bioreaction-diffusion model for growth of marine sponge explants in bioreactors.

Marine sponges are sources of high-value bioactives. Engineering aspects of in vitro culture of sponges from cuttings (explants) are poorly understood. This work develops a diffusion-controlled growth model for sponge explants. The model assumes that the explant growth is controlled by diffusive transport of at least some nutrients from the surrounding medium into the explant that generally has a poorly developed aquiferous system for internal irrigation during early stages of growth. Growth is assumed to obey Monod-type kinetics. The model is shown to satisfactorily explain the measured growth behavior of the marine sponge Crambe crambe in two different growth media. In addition, the model is generally consistent with published data for growth of explants of the sponges Disidea avara and Hemimycale columella. The model predicted that nutrient concentration profiles for nutrients, such as dissolved oxygen within the explant, are consistent with data published by independent researchers. In view of the proposed model's ability to explain available data for growth of several species of sponge explants, diffusive transport does play a controlling role in explant growth at least until a fully developed aquiferous system has become established. According to the model and experimental observations, the instantaneous growth rate depends on the size of the explant and all those factors that influence the diffusion of critical nutrients within the explant. Growth follows a hyperbolic profile that is consistent with the Monod kinetics.

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640.

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.