Prostaglandins in Marine Organisms: A Review

Prostaglandins (PGs) are lipid mediators belonging to the eicosanoid family. PGs were first discovered in mammals where they are key players in a great variety of physiological and pathological processes, for instance muscle and blood vessel tone regulation, inflammation, signaling, hemostasis, reproduction, and sleep-wake regulation. These molecules have successively been discovered in lower organisms, including marine invertebrates in which they play similar roles to those in mammals, being involved in the control of oogenesis and spermatogenesis, ion transport, and defense. Prostaglandins have also been found in some marine macroalgae of the genera Gracilaria and Laminaria and very recently the PGs pathway has been identified for the first time in some species of marine microalgae. In this review we report on the occurrence of prostaglandins in the marine environment and discuss the anti-inflammatory role of these molecules.


Introduction
Marine organisms have a great potential to produce a vast variety of bioactive molecules with high antibiotic, anti-proliferative, and anti-inflammatory activity [1]. The biodiversity hosted by the oceans is greater than in terrestrial environments [2] but nonetheless, marine bioresources are still underexplored, and many species await to be discovered.
The high probability to find new interesting bioactive molecules from marine organisms has fostered the effort of the scientific community to adopt new technologies and approaches to increase the success of biodiscovery from marine resources, with a main focus on products with antibiotic, antitumor and anti-inflammatory activities. Of particular interest is the search for new anti-inflammatory compounds since inflammation processes are often related to the onset of chronic pathologies and tumors.
Indeed, inflammation processes represent a fundamental way to restore the original equilibrium of a cell or tissue whose physiology has been impaired by damaging stimuli [3]. At the same time, if inflammation is not blocked it can stimulate a cascade of events that eventually lead to serious diseases such as cancer and autoimmune disorders [4]. The inflammation-resolution process can have different features depending on the type of tissue and injurious stimulus [5], therefore, specific types of pro-resolution stimuli or drugs may be necessary [6]. Both the onset and the resolution of the inflammation are active processes that involve a complex interplay of different molecules [7] like chemokines, cell adhesion molecules, proteolytic enzymes, eicosanoids [8], reactive oxygen species (ROS), and reactive nitrogen species (RNS) [9,10]. Among these, eicosanoids deriving from oxidation of polyunsaturated fatty acids (PUFA) through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play a pivotal role both in the onset and in the resolution of inflammation [9]. The main products of COX enzymes are prostaglandins (PGs), fatty acid derivatives with a molecular structure based on 20 carbon atoms that share a prostanoic acid skeleton. Their nomenclature comprises 10 specific molecular groups, identified by the letters A through J, that differ by variation in the functional groups attached to positions 9 and 11 of the cyclopentane ring. For PGF, the additional subscript "α" or "β" denotes the spatial configuration of the carbon 9 hydroxyl group. For additional details on the nomenclature of prostaglandins see Lands, 1979 [25].
PGs action is mediated by the interaction with specific receptors present on the plasma membrane. These are transmembrane G-protein coupled receptors (GPCR), named as prostaglandin EP receptor (EP), prostaglandin F 2α receptor (FP), prostaglandin DP receptor (DP), and prostacyclin I 2 receptor (IP) receptors, that are highly selective for PGE 2 PGF 2α , PGD 2 , and PGI 2 , respectively [26]. The EP family comprises four isoforms (EP [1][2][3][4] ) that play a relevant role in inflammation processes [27]. The downstream signaling of this receptor family is responsible for the pleiotropic ability of PGE 2 to activate different processes, including cell proliferation, apoptosis, angiogenesis, inflammation, and immune surveillance in different cell types [24].
Most prostaglandins display a marked structure-activity specificity mainly determined by substitutions in the cyclopentanone ring and the degree of unsaturation of the side chains. They exert their function once secreted into the extracellular medium, where they are rapidly metabolized by 15-hydroxyprostaglandin dehydrogenase . This enzyme selectively oxidizes the hydroxyl group at carbon 15 into a 15-keto derivative [28] accompanied by a substantial loss of biological activity.
Prostaglandins derive from the sequential actions of highly specific enzymes ( Figure 1). Their synthesis is initiated by phospholipases A 2 (PLA 2 ), a family of enzymes that hydrolyze membrane phospholipids at the sn-2 position, liberating free fatty acid precursors, mainly ARA [15]. These enzymes represent a key step in the PG biosynthetic pathway, being regulated by Ca 2+ binding and phosphorylation by mitogen-activated protein kinase (MAPK) in response to different stimuli. Membrane-released ARA is then rapidly converted through the cyclization and inclusion of molecular oxygen in the precursor by the action of cyclooxygenase (COXs) enzymes into the unstable metabolite PGG 2 , which is subsequently reduced to PGH 2 by the same enzyme [14]. Cyclooxygenases exist in a substrate-limiting environment; thus, liberation of fatty acids from esterified stores results in the prompt formation of the products. There are two major COX isoforms; COX-1 is constitutively active and present in most cells in the body; expression of the COX-2 isoform is inducible in many tissues by pro-inflammatory and mitogenic stimuli, such as cytokines [29]. The specific transformation of the first product PGH 2 to other PGs and thromboxanes (TXs) by downstream enzymes is complexly orchestrated and is cell specific, since each cell tends to form mainly one of these compounds as the major product. For example, in brain and mast cells, PGH 2 is converted to PGD 2 , whereas it is converted in PGF 2α in the uterus; from the same precursor, vascular endothelial cells produce PGI 2 (prostacyclin) and platelets release thromboxane A 2 (TXA 2 ).
The conversion to PGE 2 , the most widespread PG, is due to PGE synthase-1, also present in different isoforms in mammals: microsomal PGE synthase-1 (mPTGES-1), mPTGES-2, and cytosolic PGE synthase (cPTGES), the latter of particular interest since frequently associated to the tumorigenic activity of PGE 2 [30].
Prostaglandin E 2 synthesis is a key event for the development of the three principal signs of inflammation: swelling, redness, pain and fever. Moreover, PGE 2 also contributes to the amplification of the inflammatory response by enhancing and prolonging signals produced by pro-inflammatory agents, such as interleukin 1α bradykinin, histamine, neurokinins, and complement [31,32]. These signals, in turn, can increase COX-2 expression thus further increasing PGE 2 synthesis. This PG, however, can have a double, inverse role, being also able to act as an immunosuppressant, repressing the differentiation of T helper 1 cells and limiting cytokine release and further prostaglandin synthesis by activating a negative feedback on mPGES-1 (Figure 2a). PGD 2 -synthesizing enzymes exist as two distinct genes coding for hematopoietic-and lipocalin-type PGD synthases (H-PTGDS and L-PTGDS, respectively). H-PTGDS is generally localized in the cytosol of immune and inflammatory cells, whereas L-PTGDS has a tissue-based expression [33]. The activity of PGD 2 has been mainly associated with inflammatory conditions, being involved in immunologically relevant functions. Its action seems to be mediated by the non-enzymatic production of the PGJ 2 family, which occurs through a spontaneous dehydration in aqueous solutions. One of the most studied PGJ 2 metabolites, 15-deoxy-∆12-PGJ 2 (15dPGJ 2 ), showed anti-inflammatory properties based on interaction with the intracellular targets Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB), Activator Protein 1 (AP-1), and Peroxisome Proliferator-Activated Receptor gamma (PPAR-γ) [31]. 15dPGJ 2 also induces the reduction of neutrophil migration and inhibits the release of Interleukin 6 (IL-6), Interleukin 1β (IL-1β), Interleukin 12 (IL-12), and Tumor Necrosis Factor-α (TNF-α) from macrophages ( Figure 2b). PGD 2 can be further metabolized to PGF 2α , although the latter can also be synthesized from PGH 2 via PGF synthase. PGF 2α acts via FP receptors, resulting in the elevation of intracellular free calcium concentrations that regulates numerous important physiological functions related to reproduction linking multiple molecular mechanisms that are fine-tuned coordinated events in mammalian physiology [34] (Figure 2c). The emerging role of PGF 2α in acute and chronic inflammation has opened new opportunities for the design of novel anti-inflammatory drugs.
A non-enzymatic dehydration reaction is also responsible for the formation of the PGA series from the corresponding PGE. The PG series A and J contain an α,β-unsaturated carbonyl group within the cyclopentenone ring, which seem to contribute to the anti-inflammatory activity of these PGs [35].
The enzyme prostaglandin I synthase (PGIS), a member of the cytochrome P450 superfamily, specifically converts PGH 2 to PGI 2 [33]. This PG has anti-mitogenic activity and inhibits DNA synthesis through specific IP receptors. It mediates pro-inflammatory stimuli in non-allergic acute inflammation, while acting as an anti-inflammatory mediator in Th2-mediated allergic inflammatory responses. Soon after having exerted its action, PGI 2 is rapidly converted by non-enzymatic processes to the inactive product 6-keto-PGF 1α (Figure 2d).

Prostaglandins and Derivative Molecules in Marine Organisms
Similar to terrestrial vertebrates, in marine vertebrates prostaglandins are involved in reproduction, osmoregulation, regulation of oxygenation, and cardiovascular system [36]. Marine invertebrates, including sponges, corals, and molluscs, also contain a wide range of prostaglandins, many of which are of the conventional type (PGA 2 , PGE 2 , PGD 2 , PGF 2α ), with similar functions as in mammals (reproduction, ion transport) and are also probably used as defense compounds [17]. Interestingly, in some invertebrates, PGs are able to perform different actions based on the tissue or compartment localization [37].
Complex marine photosynthesizing organisms, like some genera in the brown, green, and red algal groups (respectively, Laminaria, Euglena, and Gracilaria species) express the cyclooxygenase gene synthesizing PGE 2 , PGF 2α , and other PGs whose functions are not yet known, but seem to be associated to a defensive role [17].

Corals
Among marine invertebrates, corals represent a very interesting group producing specific PGs that are not present in mammals. The Caribbean gorgonian Plexaura homomalla was indeed the species in which PGs were firstly identified in a marine organism, representing also a major source of these compounds in nature [38] (Figure 3a). Weinheimer A. and Spraggins R. were the first to perform PGs extraction in P. homomalla, identifying in its dry cortex 15-epi-PGA 2 and its methyl ester acetate (respectively 0.2% and 1.3%) [15] in an R-configuration on their C-15 asymmetric center, a configuration not present in mammals. In addition to (15R)-epi-PGA 2 , also (15R)-PGE 2 , its methyl ester and a complex mixture of other prostaglandins were subsequently found by Light R. and Samuelsson B. [39]. However, these molecules were found also in S configurations [40] and the occurrence of one of the two seems to be related to the geographical distribution of P. homomalla [38].
The high PGs concentration in these invertebrates stimulated the attempt to use them as precursors for the chemical synthesis of the biologically active prostaglandins A 2 , E 2 , and F 2α , since their chemical synthesis in large-scale for medical and pharmaceutical purposes was complicated by the necessity of 16 different chemical reactions [41].
The observation that P. homomalla present in coral reefs is not commonly eaten by fishes, led Gerhart [42] to hypothesize that the high amount of PGs they contain could be used as chemical defense against potential predators, since it is known that in mammals they can cause vomiting and nausea when administered orally. Indeed, oral doses of both (15R)-PGA 2 and (15S)-PGA 2 caused vomiting in a test with fishes, while the PGA 2 present in the surrounding water did not cause any effect [42]. However, the hypothesized defensive role seems not to be realistic since PGA 2 is stored in coral tissues only as acetoxy methyl esters, whose conversion to (15R)-PGA 2 (the compound tested by Gerhart) needs about 24 hours [40]. Moreover, while (15R)-PGA 2 inhibits fishes from feeding, the acetoxy methyl ester form, orally delivered, does not show any repellent effect. For this reason, as the process of production of the active (15R)-PGA 2 seems too slow to provide the coral with an effective defense mechanism, the function of these molecules in corals is still an open question.
In addition to the identification of PGs molecules in corals, also the enzyme responsible for their synthesis, the cyclooxygenase, was isolated and characterized for the first time from P. homomalla and G. fruticosa [59].

Other Marine Invertebrates
One of the first studies on marine invertebrates, excluding corals, was a comparative analysis done by Christ E. and Van Dorp D., on representative species in the Mollusca (Mytilus), Crustacea (Homarus), and Cnidaria (Cyanea) phyla versus terrestrial animals [60]. The authors were able to find PGs, particularly PGF 1α but only at very low levels. In some cases, the arachidonic acid precursor was also not detectable making it difficult to assert the existence of PGs and their functional role [60]. These results were confirmed in extended studies including more species in the Chordata, Mollusca, Cnidaria, and Crustacea phyla [16,61,62].
With the improvement of instrument sensitivity, it was possible, more than 10 years later, to conduct functional studies on PGs in molluscs ( Figure 4a) and crustaceans (Figure 4b). Results obtained highlighted different roles for PGs in marine invertebrates, like reproduction, regulation of ion flux, and thermoregulation and fever, mediated respectively by PGF 2α , PGE 2 , and PGE 1 (  TXB2

Oncorhynchus mykiss
Vasodilator agent Thomson et al., 1998 [70] In the mollusc Modiolus demissus Dillwyn, 1817 the uptake and binding of prostaglandins by gills was investigated, considering that bivalves can both synthetize and accumulate PGs from the surrounding medium [71]. The study revealed the presence of tissue specific, time and pH dependent, PGs binding sites with higher affinity to PGA 2 with respect to PGE 2 or PGF 2α [71].
Moreover, three prostaglandin-lactones (PGE 2 -1,15-lactone, PGE 3 -1,15-lactone, and PGE 3 -1,15 -lactone-11-acetate) of the E and F series were identified in the mantle and body, respectively, of the nudibranch mollusc Tethys fimbria Linnaeus, 1767 (Figure 4a). High quantities of a complex mixture of PGF 2α and PGF 3α 1,15-lactones fatty acid esters (PLFE) were found in its eggs, particularly in mature ovotestis but not in immature ones suggesting a role for PLFE in mollusc reproduction [67]. Altogether, these results led to hypothesize a multiple biological role of prostaglandin-lactones, precursors of PGEs, as defense allormones, and involved in the control of smooth muscle contraction, and egg production and fertilization, depending on their body localization ( Table 2) [37].
Crustaceans (Figure 4b) also use PGs for physiological functions. Indeed, PGE 2 and PGF 2α were identified in the ovary of the prawn Marsupenaeus japonicus Spence Bate, 1888 where they participate in ovarian maturation (Table 2) [64] whereas in the crab Carcinus maenas Linnaeus, 1758 they are produced in blood cells following a stimulus induced with a calcium ionophore, in the presence of exogenous fatty acids (FA) [72].
Echinoderms (Figure 4c) also represent a source of PGs. Among all the echinoderms analyzed, the starfish Patinia pectinifera Muller and Troschel, 1842 had the highest amount of prostaglandins [73]. PGE 2 and PGF 2α have been identified from the sea cucumber Stichopus japonicus Selenka, 1867 using TLC [73]. A study conducted with PGs-3H evidenced that the sea urchin Arbacia punctulata Lamarck, 1816 could accumulate PGs from the surrounding water into its gut or stomach [74], and to accumulate PGA 1 both in fertilized and unfertilized eggs, with fertilized eggs accumulating more PGs than unfertilized eggs [74].
Strongylocentrotus nudus A. Agassiz, 1864 and S. intermedius A. Agassiz, 1864 were also reported to have a PGs-like activity in their inner organs [74].

Marine Vertebrates
As already mentioned, in fish PGs are involved in several processes, such as ovulation, spawning, osmoregulation, regulation of branchial ion fluxes and of the cardiovascular system [36]. Christ and Van Dorp in their comparative studies on PGs considered not only invertebrates, but also fish [60]. They identified moderate yields of PGE 1 in freshwater fish and lower yields in homogenates of gills of Salmo sp. [60]. Successively, PGE 2 was identified for the first time in the testis of the flounder Paralichthys olivaceus Temminck & Schlegel, 1846, PGF 1α in the semen of the salmon Oncorhynchus keta Walbaum, 1792 and PGE 2 and PGF 2α in the testis of the tuna Thunnus thynnus Linnaeus, 1758 [65] (Figure 5a).
In order to explain the reason why prostaglandins are present in these marine animals, although they are oviparous, it was suggested that the identified prostaglandins might be used for the contraction of smooth muscle during ejaculation and for the metabolism of testis in lower animals as in mammals (Table 2) [65]. After this study, PGE 2 was isolated and subsequently identified in the gastrointestinal tract and in the skin of the shark Triakis scyllia Müller & Henle, 1839 [16,75] (Figure 5b).
In the brook trout Salvelinus fontinalis PGE 2 and PGF 2α (Figure 5a) are synthetized in the follicle wall of mature oocytes, but the highest quantity of prostaglandins was found in the extra-follicular tissue [76]. PGE 2 was also found in the skin of Pleuronectes platessa Linnaeus, 1758 (Figure 5a) in response to the fungal extract of Epydermophyton floccosum (Harz) Langeron & Miloch, 1930 known be an inducer of erythema [77].

Red Macroalgae
Marine red algae are rich in C20 polyunsaturated fatty acids that, in animals, are precursors of prostaglandins, thromboxane, and other eicosanoids.
Although the presence of the prostaglandin-endoperoxide pathway has been demonstrated in non-mammal marine vertebrates and invertebrates, for a long time less was known about these enzymes in non-animal organisms. The first report about the presence of prostaglandins in macroalgae ( Figure 6) described the identification of PGE 2 and PGF 2α in Gracilaria lichenoides Greville, 1830 [80]. The genus Gracilaria, that comprises algae used in the food and cosmetic industry, is very rich in ARA, varying from 45.9% and 62.0% of the total FA depending on the season. Among these, G. vermiculophylla (Ohmi) Papenfuss, 1967 is one of the algae with the highest content of ARA [81]. This, with other Gracilaria species such as G. asiatica and G. chorda Holmes, 1896, seem to be responsible for a gastrointestinal disorder known as "onogori" poisoning in Japan when it is eaten raw [18]. The possible reason for this poisoning seems to be the fact that the COX contained in raw seaweeds use the highly unsaturated fatty acids to produce great amounts of PGE 2 in the stomach of victims in a short lapse of time [81]. G. vermiculophylla is a source of different types of prostaglandins besides PGE 2 , such as PGA 2 , PGF 2 , 15-keto-derivatives of prostaglandins, as well as other eicosanoids [82], whereas the congeneric species G. asiatica produces only 15-keto-PGE 2 [83]. G. vermiculophylla use PGA 2 , PGE 2 , 15-keto-PGE 2 and other eicosanoids as wounding-activated chemical defense molecules ( Table 2) [66], and recently a COX gene producing PGF 2α was cloned from this alga and heterologously expressed [84].
Other evidence of prostaglandins as defense molecules in red algae is the fact that gametophytes of the alga Chondrus crispus Stackhouse, 1797, when challenged with pathogens, metabolize C20 and C18 PUFAs not only into the corresponding hydroperoxides and derivatives, but also into molecules with mass fragmentation patterns very similar to prostaglandin B 1 and B 2 [85]. Furthermore, when the crude extract of this alga was treated with 50-100 µM methyl jasmonate for 6 h, PGA 2 and 15-keto-PGE 2 were identified [86].

Brown Macroalgae
Much less is known about the presence of prostaglandins in brown algae, and most studies have mainly focused on the brown algal kelp Laminaria digitata. Ritter et al. [19] showed that this brown alga uses the generation of oxylipins as a protective mechanism against stress conditions induced by an excess in copper (Table 2). Indeed, high concentrations of copper led to oxygen reactive species (ROS) accumulation in L. digitata cells, and consequently to a cascade of cellular responses. One of these responses was a significant release of PUFAs after 24 h of treatment, followed by the generation of oxylipins. Among complex oxylipins, also PGE 1 and PGD 1 , deriving from ETrA and PGJ 2 , PGA 2 , 15-keto-PGE 2 and PGB 2 , deriving from ARA were identified for the first time in brown algae [19]. Successively, the same authors showed that PGA 2 does not simply modulate but also triggers an oxidative response in L. digitata that, differently from the response induced by other molecules like methyljasmonate and lipopolysaccharides, occurs in seconds after treatment and in a dose-response-like manner. The authors hypothesized that PGA 2 can activate the generation of two different sources of ROS that can be used by the brown alga as defense molecules as in other marine organisms [68] (Figure 7).

Microalgae
PGs were discovered for the first time in diatoms, phytoplanktonic marine microalgae, only in 2017 [23]. Diatoms are a rich source of polyunsaturated fatty acids (PUFA), that, in some species, are precursors of polyunsaturated aldehydes (PUAs), i.e., oxylipins with pro-apoptotic and anti-proliferative activity, with a defensive role against predator (copepods) grazing [87].
Di Dato et al. [23]   More specifically, whereas cyclooxygenase-1 (COX-1) and microsomal prostaglandin E synthase 1 (mPTGES) were found in both species; prostaglandin H 2 D-isomerase (PTGDS) was found only in S. marinoi, while prostaglandin F synthase (PTGFS) was only detectable in T. rotula. The authors demonstrated the functioning of the pathway by measuring enzyme expression and molecule production by quantitative real time PCR (qPCR) and liquid chromatography/mass spectrometry (LC/MS) analysis, respectively. Interestingly, a wide set of PGs was revealed with representative molecules of each of the three series. In addition, a differential expression and concentration of molecules was reported among different species and strains of the same species, during different phases of growth and nutrient conditions [23]. However, the absence of PTGDS in T. rotula and of PTGFS in S. marinoi could be considered as a "potential absence." Indeed, the lack of a transcript in a transcriptome annotation can be due to a technical shortcoming and a limitation of the sequencing technology used, as in the case of very low expression levels of a target gene, that may fall under the detection limit.
It is interesting to note that, although the PGs pathway has been experimentally confirmed only in two diatom species, in silico analysis of transcriptomes from different species in different growth conditions, suggests the presence of PGs in many other diatom species [23].
Prostaglandins were identified also in the unicellular green alga Euglena gracilis G. A. Klebs, 1883 (Figure 8c), in which PGE 2 and PGF 2α were found at levels three times higher in cells grown in the dark than those grown in the light [88].
The presence of PGA, PGE, and PGF series and their esters have also been documented in the cyanobacteria of the taxa Oscillatoria and Microcystidaceae [89]. Indeed, PGF 2α was identified in Microcystis aeruginosa (Kützing) Kützing, 1846 [90] (Figure 8d).
Currently, to the best of our knowledge, no other marine microalga has been explored for the presence of PGs, and their role in diatoms is still under investigation. Di Dato and co-authors hypothesized that they can be used by diatoms for intercellular signaling and communication [23], in agreement with the role of these molecules in other organisms.
Microorganisms, and in particular microalgae such as diatoms, could represent a potential source of bioactive PGs, as alternative to their chemical synthesis to produce adequate amounts for pharmacological purposes as anti-inflammatory compounds. The biotechnological production of PGs from microalgae may also be convenient due to the natural presence of the full PGs pathway and the possibility to easily grow microalgae on a large scale in bioreactors under controlled culture conditions.

Marine Cyclopentenone Prostaglandins
Cyclopentenone prostaglandins are a sub-group of PGs characterized by the presence of a cyclopentenone moiety (CP) with a α,β-unsaturated ketone group in their cyclopentane ring. Their chemical reactivity is responsible for their anti-inflammatory, anti-tumoral and anti-viral activities [91], and the presence of the CP fragment in molecules with anticancer activity, can add more potency to these molecules. For instance, methyl-jasmonate increases its strength of action as an anti-tumor agent when a CP group is inserted in the molecule [92].
The group of cyclopentenone prostaglandins (CPPGs) includes canonical PGs of the A and J series, clavulones, and punaglandins.
Differently from the conventional PGs, that require receptor interaction to trigger their signal, cyclopentenone prostaglandins are actively transported inside the cells [91], where the CP group can interact with a wide variety of target molecules [92] including nuclear factors such as the heat shock protein 70 (HSP70) transcription factor, cyclin-dependent protein kinase (CDK), and NF-kB [35] and still not well defined mitochondrial factors [92].

Clavulones and Related Molecules
Clavulones (Figure 9) are acetoxy derivatives of PGA [91] characterized by a cyclopentenone fragment, a 12-S acetoxy function and two chains of different length: the αand the ω-chain with respectively seven and eight carbon atoms [93]. These are anti-tumor marine prostanoids isolated from the soft coral Clavularia viridis Quoy & Gaimard, 1833, a single Clavularia genus that inhabits Okinawa bay in Japan [44,94,95].
Clavulones exists in three isoforms, I, II, and III and two stereoisomers at the C-4 and C-12 positions. These molecules possess a significant anti-inflammatory effect at a concentration of 30 µg/mL in a fertilized chicken egg assay (Table 1) [44,95]. The effects of clavulones on the growth of human cancer cells were first studied using clavulone I, the form that is more abundant in C. viridis. At concentration of 4.0 µM there was an impairment of DNA synthesis in human HL-60 leukemic cells after 1 h incubation, causing irreversible cytotoxic changes after 3 h of exposition [45]. In a more recent study, the anti-cancer activity of clavulone II on HL-60 cells has been deeply studied [46] revealing that low concentrations of clavulone II induce anti-proliferative effects by inducing the down-regulation of cyclin D1 with consequent arrest of the cell cycle in G1. On the contrary, higher concentrations of clavulone II induces apoptosis through the disruption of mitochondrial membrane potential and the activation of caspase-9, -8, and -3 and of B-cell lymphoma 2 (Bcl2)-family proteins (Table 1) [46]. It was suggested that corals might use clavulones as repellent and toxic substances against other marine organisms (Table 2) [45]. Clavulones, similarly to PGAs, also possess antiviral activity. In particular, clavulone II inhibits replication of the vesicular stomatitis virus (VSV) in infected mouse L929 fibroblasts, by blocking the transcription of viral RNA, and consequently the viral protein (Table 1) [47].
Interestingly, both the anti-proliferative and antiviral activities of clavulones are stronger than those of PGAs [47].
Other than clavulone I, II, and III, from the same stolonifer coral, 20-acetoxi-claviridenone b and 20-acetoxi-claviridenone c were discovered [96]. In addition to these, also four halogenated analogues, called chlorovulones I-IV were identified and, among these, chlorovulone I was shown to have anti-proliferative and cytotoxic activities (Table 1) [48]. The structure of an epoxy prostanoid with anti-proliferative activity was also identified in the same soft coral [49] together with bromovulone I and iodovulone I. Both had anti-proliferative and cytotoxic activities even though slightly lower than those of chlorovulone I [50]. All these molecules showed a stronger anti-tumor activity against HL-60 cells in vitro with respect to clavulone I (Table 1) [48][49][50].
Through an assay-guided fractionation of a dichloromethane (CH 2 Cl 2 ) extract of C. viridis, three new cytotoxic molecules were isolated: claviridenone E, claviridenone F, and claviridenone G. In particular, claviridenone F showed a significant cytotoxicity against human lung adenocarcinoma (A549), HT-29, and mouse lymphocytic leukemia cells (P-388), while claviridenone G exhibited a high cytotoxic activity only against A549 (Table 1) [52]. In addition to these molecules, there are clavulone-related oxylipins from C. viridis, named clavirins I and II, for which it was proposed a derivation from clavulone III and I, respectively, having growth-inhibitory activity against human cervix carcinoma cell line (HeLa S3) ( Table 1) [53].
Other clavulone-related molecules that have been discovered are tricycloclavulone and clavubicyclone that may be derived from cycloaddition and electrocyclization of clavulone III, respectively. The latter molecule showed a moderate growth-inhibition activity against breast carcinoma (MCF-7) and ovarian carcinoma cells (OVCAR-3) in vitro (Table 1) [54]. Preclavulone lactone I and II and two minor chemical congeners, 17,18-dehydroclavulone I and clavulolactone I, were also isolated and characterized from C. viridis. Among these, 17,18-dehydroclavulone I has a (14Z,17Z)-double bond in the ω side chain, and this suggests that, differently from the other clavulones, it can derive from EPA instead of ARA [97].
Although there are structural similarities between prostaglandins and clavulones, the latter are not generated by a variant of the endoperoxide pathway, but from the lipoxygenase pathway by which arachidonic acid is converted into 8-(R)HPETE and then to preclavulone A [98]. Preclavulone A was identified also in an unrelated Caribbean coral, Pseudoplexaura porosa Houttuyn, 1772, suggesting that this molecule is widespread in corals [99]. In another study, the same authors isolated and defined the structures of 15 new halogenated iodo-, bromo-, and chlorovulones in C. viridis as minor constituents [100], but overall about 50 congeners of clavulones were identified in this soft coral [101].
At the moment of their discovery, there was great interest in the study of punaglandins, because of their anti-inflammatory and potent antitumor activities [104]. Indeed, these molecules, and in particular punaglandin 3, was shown to inhibit L1210 leukemia cell proliferation (IC 50 = 0.04 µM) (Table 1) [102] with an activity 15-fold higher compared to the corresponding clavulone [55]. This cytotoxic activity is approximately equal to the one of vincristine and doxorubicin that are among the most effective anticancer molecules used today [103].

Marine Thromboxane
Thromboxanes (TXs) are closely relate to PGs that bind specific receptors, called thromboxane receptors (TP) and act as strong promoters of platelet aggregation and vaso-and broncho-constriction in mammals [106]. They are labile and biologically active molecules deriving from arachidonic acid in the cyclooxygenase pathway in human platelets. Thromboxane A 2 (TXA 2 ) has a short half-life (30 s in human blood) being rapidly converted to a stable B form (TXB 2 ), which is used as a marker for the presence of TXA 2 [69].
Other than in mammals, the presence of TXs is documented also in marine organisms. TXB 2 , along with prostaglandins, has been reported in aqueous homogenates of the mollusc A. californica J. G. Cooper, 1863 (Figure 4a) [74] using radioimmunoassay. In the marine bivalve M. edulis Linnaeus, 1758 (Figure 4a), arachidonic acid metabolism, in addition to PGs, leads to the production of TXs in the gills, mantle, and adductor tissues [107]. In the polychaetae Arenicola marina Linnaeus, 1758 small amounts of TXB 2 are detectable in the digestive and reproductive tracts [107].
Different species of fish also produce TXs [69]. In the plasma of the elasmobranch Dasyatis sabina Lesueur, 1824 (Figure 5b), the presence of an immunoreactive TXB 2 (iTXB 2 ) has been detected at concentrations of 0.57 ± 0.03 ng/mL. The level of iTXB 2 increased to 3.0 ± 0.27 ng/mL when the plasma clots, but decreased to 1.5 ± 0.17 ng/mL in the presence of indomethacin, a cyclooxygenase inhibitor. These findings suggest that TXA 2 , the active thromboxane form, may be involved in blood clotting and may be generated by a cyclooxygenase-like enzyme as in mammals (Table 2) [69]. The binding of agonist (radiolabeled [125I]-BOP) and antagonist (L-657925 and L-657926) ligands of mammalian TP receptors to those of D. sabina, also demonstrates the similarity between this elasmobranch and mammalian TP receptors [69]. Three different species of shark (Figure 5b) (Chiloscyllium griseum Müller & Henle, 1838, Carcharhinus plumbeus Nardo, 1827 and Carcharhinus melanopterus Quoy & Gaimard, 1824) were also shown to produce TXB 2 as a major product of arachidonic acid metabolism suggesting that TXB 2 may be a biologically active prostanoid in these species [70].
Rowley et al. [78] tested the ability of leucocytes extracted from the blood of the dogfish Scyliorhinus canicula (Figure 5b) to produce and release PGs and TXs during their degranulation induced by the calcium ionophore A23187. While PGF 2α , PGE 2 , and PGD 2 levels were always high after the stimulus, the levels of TXB 2 were low and detectable in the supernatant only 5 min after the start of the stimulus, with levels increasing after 10 and 15 min [78].
Another direct evidence of the generation of TXB 2 in fish was demonstrated by the ability of washed whole blood cells from a variety of fishes of the Arabian Gulf to produce prostanoids from exogenous 14C-arachidonic acid in vitro [70]. In addition, rainbow trout thrombocytes, clotting cells similar to mammalian platelets, were shown to convert arachidonic acid mainly to TXB 2 [108,109]. These findings again suggest a role also for TXB 2 , in rainbow trout as a vasodilator (Table 2), while in mammals TXB 2 has no significant biological activity [70]. However, not all fish thrombocytes have the ability to produce thromboxane, possibly suggesting that these molecules are not essential for thrombocyte aggregation in certain species [110].

Conclusions
Prostaglandins and their derivatives, although first identified in terrestrial organisms, are also widespread in the marine environment testifying the great importance of these molecules. Nevertheless, the eco-physiological role of marine PGs remains mostly unclear because of contrasting and often old data available in the literature, although it has been suggested that they have a possible role as defensive molecules against predators.
Interestingly, many marine organisms have been shown to produce classical PGs along with new derivatives that are not found in terrestrial animals, e.g., corals that are able to produce punaglandins and clavulones, which are specific of the marine environment and have shown potent pharmacological activity against inflammation, tumors, and viruses. These findings, together with the discovery of PGs in unicellular eukaryotic microalgae, provides new stimuli to pursue the search for new marine PG-derived molecules with anti-inflammatory activity. Funding: This research was funded by Ministero dell'ambiente e della tutela del territorio e del mare, Italia, project name: "Genomics for a Blue Economy," grant number PGR00765.