Comparative Chemical Profiling and Antimicrobial/Anticancer Evaluation of Extracts from Farmed versus Wild Agelas oroides and Sarcotragus foetidus Sponges.

Marine sponges are highly efficient in removing organic pollutants and their cultivation, adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality. Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus, compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture, sponge farming can serve as a valuable resource of biomolecules, with promising potential in pharmaceutical and biomedical applications.

Therapeutic Potential of (-)-Agelamide D, a Diterpene Alkaloid from the Marine Sponge Agelas sp., as a Natural Radiosensitizer in Hepatocellular Carcinoma Models.

Radiation therapy (RT) is an effective local treatment for unresectable hepatocellular carcinoma (HCC), but there are currently no predictive biomarkers to guide treatment decision for RT or adjuvant systemic drugs to be combined with RT for HCC patients. Previously, we reported that extracts of the marine sponge Agelas sp. may contain a natural radiosensitizer for HCC treatment. In this study, we isolated (-)-agelamide D from Agelas extract and investigated the mechanism underlying its radiosensitization. (-)-Agelamide D enhanced radiation sensitivity of Hep3B cells with decreased clonogenic survival and increased apoptotic cell death. Furthermore, (-)-agelamide D increased the expression of protein kinase RNA-like endoplasmic reticulum kinase/inositol-requiring enzyme 1α/activating transcription factor 4 (PERK/eIF2α/ATF4), a key pathway of the unfolded protein response (UPR) in multiple HCC cell lines, and augmented radiation-induced UPR signaling. In vivo xenograft experiments confirmed that (-)-agelamide D enhanced tumor growth inhibition by radiation without systemic toxicity. Immunohistochemistry results showed that (-)-agelamide D further increased radiation-induced ATF4 expression and apoptotic cell death, which was consistent with our in vitro finding. Collectively, our results provide preclinical evidence that the use of UPR inducers such as (-)-agelamide D may enhance the efficacy of RT in HCC management.

Agesasines A and B, Bromopyrrole Alkaloids from Marine Sponges Agelas spp.

Exploration for specialized metabolites of Okinawan marine sponges Agelas spp. resulted in the isolation of five new bromopyrrole alkaloids, agesasines A (1) and B (2), 9-hydroxydihydrodispacamide (3), 9-hydroxydihydrooroidin (4), and 9E-keramadine (5). Their structures were elucidated on the basis of spectroscopic analyses. Agesasines A (1) and B (2) were assigned as rare bromopyrrole alkaloids lacking an aminoimidazole moiety, while 3-5 were elucidated to be linear bromopyrrole alkaloids with either aminoimidazolone, aminoimidazole, or N-methylated aminoimidazole moieties.

Bromopyrrole Alkaloids from the Sponge Agelas kosrae.

Two new sceptrin derivatives (1,2) and eight structurally-related known bromopyrrole-bearing alkaloids were isolated from the tropical sponge Agelas kosrae. By a combination of spectroscopic methods, the new compounds, designated dioxysceptrin (1) and ageleste C (2), were determined to be structural analogs of each other that differ at the imidazole moiety. Dioxysceptrin was also found to exist as a mixture of α-amido epimers. The sceptrin alkaloids exhibited weak cytotoxicity against cancer cells. Compounds 1 and 2 also moderately exhibited anti-angiogenic and isocitrate lyase-inhibitory activities, respectively.

A bromopyrrole-containing diterpene alkaloid from the Okinawan marine sponge Agelas nakamurai activates the insulin pathway in Huh-7 human hepatoma cells by inhibiting protein tyrosine phosphatase 1B.

Agelasine G (1), a known bromine-containing diterpene alkaloid, was isolated as a new type of protein tyrosine phosphatase (PTP) 1B inhibitor together with ageline B (2), an inactive debromo-derivative of 1, from the marine sponge Agelas nakamurai collected at Iriomote Island in Okinawa, Japan. Further biological evaluations revealed that compound 1 exhibited selective inhibitory activity against PTP1B over T-cell PTP and CD45 phosphatase. Compound 1 also enhanced the insulin-stimulated phosphorylation levels of Akt in Huh-7 cells more strongly than compound 2. The results obtained in this study suggest that compound 1 activates the insulin signaling pathway by inhibiting PTP1B activity.

Pyrrole Derivatives and Diterpene Alkaloids from the South China Sea Sponge Agelas nakamurai.

Two pairs of new non-brominated racematic pyrrole derivatives, (±)-nakamurine D (1) and (±)-nakamurine E (2), two new diterpene alkaloids, isoagelasine C (16) and isoagelasidine B (21), together with 13 known pyrrole derivatives ((±)-3 - 15), five known diterpene alkaloids (17 - 20, 22) were isolated from the South China Sea sponge Agelas nakamurai. The racemic mixtures, compounds 1 - 4, were resolved into four pairs of enantiomers, (+)-1 and (-)-1, (+)-2 and (-)-2, (+)-3 and (-)-3, and (+)-4 and (-)-4, by chiral HPLC. The structures and absolute configurations were elucidated on the basis of comprehensive spectroscopic analyses, quantum chemical calculations, quantitative measurements of molar rotations, application of van't Hoff's principle of optical superposition, and comparison with the literature data. The NMR and MS data of compound 3 are reported for the first time, as the structure was listed in SciFinder Scholar with no associated reference. These non-brominated pyrrole derivatives were found in this species for the first time. Compound 18 showed valuable cytotoxicities against HL-60, K562, and HCT-116 cell lines with IC50 values of 12.4, 16.0, and 19.8 µm, respectively. Compounds 16 - 19, 21, and 22 showed potent antifungal activities against Candida albicans with MIC values ranging from 0.59 to 4.69 µg/ml. Compounds 16 - 19 exhibited moderate antibacterial activities against Proteusbacillus vulgaris (MIC values ranging from 9.38 to 18.75 µg/ml).

Structures and Biological Evaluations of Agelasines Isolated from the Okinawan Marine Sponge Agelas nakamurai.

Three new N-methyladenine-containing diterpenes, 2-oxoagelasines A (1) and F (2) and 10-hydro-9-hydroxyagelasine F (3), were isolated from the Okinawan marine sponge Agelas nakamurai Hoshino together with eight known agelasine derivatives, 2-oxoagelasine B (4), agelasines A (5), B (6), D (7), E (8), F (9), and G (10), and ageline B (11). The structures of 1-3 were assigned on the basis of their spectroscopic data and their comparison with those of the literature. Compounds 3 and 5-11 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 10, 14, 15, 18, 14, 20, 12, and 12 mm at 20 µg/disc, respectively. All compounds were inactive (IC50 > 10 µM) against Huh-7 (hepatoma) and EJ-1 (bladder carcinoma) human cancer cell lines. Three 2-oxo derivatives (1, 2, and 4) exhibited markedly reduced biological activity against M. smegmatis. Moreover, compound 10 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 15 µM.

Agelamadins A and B, dimeric bromopyrrole alkaloids from a marine sponge Agelas sp.

Two structurally unique dimeric bromopyrrole alkaloids, agelamadins A (1) and B (2), were isolated from a marine sponge Agelas sp. Agelamadins A (1) and B (2) have a structure consisting of an agelastatin-like tetracyclic moiety and an oroidin-like linear moiety in common. The structures of 1 and 2 were elucidated on the basis of spectroscopic analysis. The antimicrobial activity and cytotoxicity of agelamadins A (1) and B (2) were evaluated.

Bromopyrrole alkaloids isolated from the Patagonian bryozoan Aspidostoma giganteum.

Nine new bromopyrrole alkaloids, aspidostomides A-H and aspidazide A (1-9), were isolated from the Patagonian bryozoan Aspidostoma giganteum. Aspidostomides A-H have dibromotyrosine- or bromotryptophan-derived moieties forming either linear amides or pyrroloketopiperazine-type lactams with a bromopyrrole carboxylic acid as a common structural motif. On the other hand, aspidazide A is a rare asymmetric acyl azide formed by an N-N link of two different pyrroloketopiperazine lactams and is the first isolated compound of this class from marine invertebrates. This work is the first report of secondary metabolites isolated from a bryozoan from the Patagonian region. The structures of compounds 1-9 were elucidated by spectroscopic methods and chemical transformations. One of these compounds, aspidostomide E (5), was moderately active against the 786-O renal carcinoma cell line.

Isolation and structures of axistatins 1-3 from the Republic of Palau marine sponge Agelas axifera Hentschel .

An investigation begun in 1979 directed at the Republic of Palau marine sponge Agelas axifera Hentschel for cancer cell growth inhibitory constituents subsequently led to the isolation of three new pyrimidine diterpenes designated axistatins 1 (1), 2 (2), and 3 (3), together with the previously reported formamides 4, 5, and agelasine F (6). The structures were elucidated by analysis of 2D-NMR spectra and by HRMS. All of the isolated compounds were found to be moderate inhibitors of cancer cell growth. Axistatins 1-3 (1-3), formamide 4, and agelasine F (6) also exhibited antimicrobial activity.

The marine sponge toxin agelasine B increases the intracellular Ca(2+) concentration and induces apoptosis in human breast cancer cells (MCF-7).

PURPOSE: In search for new drugs derived from natural products for the possible treatment of cancer, we studied the action of agelasine B, a compound purified from a marine sponge Agelas clathrodes. METHODS: Agelasine B was purified from a marine sponge Agelas clathrodes and assayed for cytotoxicity by MTT on two human breast cancer cells (MCF-7 and SKBr3), on a prostate cancer cells (PC-3) and on human fibroblasts. Changes in the intracellular Ca(2+) concentrations were assessed with FURA 2 and by confocal microscopy. Determination of Ca(2+)-ATPase activity was followed by Pi measurements. Changes in the mitochondria electrochemical potential was followed with Rhodamine 123. Apoptosis and DNA fragmentation were determined by TUNEL experiments. RESULTS: Upon agelasine B treatment, cell viability of both human breast cancer cell lines was one order of magnitude lower as compared with fibroblasts (IC(50) for MCF-7 = 2.99 µM; SKBr3: IC(50) = 3.22 µM vs. fibroblasts: IC(50) = 32.91 µM), while the IC(50) for PC-3 IC(50) = 6.86 µM. Agelasine B induced a large increase in the intracellular Ca(2+) concentration in MCF-7, SKBr3, and PC-3 cells. By the use of confocal microscopy coupled to a perfusion system, we could observe that this toxin releases Ca(2+) from the endoplasmic reticulum (ER). We also demonstrated that agelasine B produces a potent inhibition of the ER Ca(2+)-ATPase (SERCA), and that this compound induced the fragmentation of DNA. Accordingly, agelasine B reduced the expression of the anti-apoptotic protein Bcl-2 and was able to activate caspase 8, without affecting the activity of caspase 7. CONCLUSIONS: Agelasine B in MCF-7 cells induce the activation of apoptosis in response to a sustained increase in the [Ca(2+)]( i ) after blocking the SERCA activity. The reproduction of the effects of agelasine B on cell viability and on the [Ca(2+)]( I ) obtained on SKBr3 and PC-3 cancer cells strongly suggests the generality of the mechanism of action of this toxin.

Efecto de esfingolípidos y Agelosina B en la movilización de Ca2+ y la señalización celular en células de cáncer de mama (MCF-7) y su relación con la apoptosis / Effect of sphingolipids and Agelosin B on Ca2 + mobilization and cell signaling in breast cancer cells (MCF-7) and its relationship with apoptosis

Los esfingolípidos, como la ceramida (Cer), la ceramida-1-fosfato (C-1-P), la esfingosina (Sph) y la esfingosina-1-fosfato (S-1P) estan relacionados con la señalización intracelular en procesos como crecimiento celular, movilización intracelular de Ca+2 y apoptósis. En este trabajo se evaluó el efecto de estos esfingolípidos en la homeostasis de Ca+2 intracelular y en la apoptósis en células de cáncer de mama MCF-7. Se utilizaron fluoróforos específicos para el Ca+2 y microscopía confocal. Se demostró que en estas células, la Sph (20 uM), la Cer (10uM), la S-P (2uM) y la C--P (uM) aumentaron la concentración intracelular ce Ca+2, induciendo su liberación desde el retículo endoplasmático (RE). Además, se observo que la esfingosina abrioun canal de Ca2+ en la membrana plasmática. También se demostró que la Cer inhibe parcialmente la actividad de la Ca2+-ATPasa del RE (SERCA), de forma dosis dependiente, mientras que la ceramina, su análogo no hidrolisable la inhibe totalmente. La Sph también inhibe completamente la actividad de la SERCA, a la misma concentración que induce la liberación del Ca+2 del RE. Asimismo, se evaluó el efecto de estos esfingolípidos sobre la inducción de la apotósis en células MCF-7 evidenciando que el tratamiento con la Cer, la ceramida, la Sph inducen toxicidad. También se observo que mientras la ceramida activo la caspasa 7 y la caspasa 8, el esfingolipido natural, la Cer no tuvo ningún efecto. Por su parte, la Sph activa la caspasa 8 sin modificar la activdad de la caspasa 7. Tanto la Cer, como la ceramida y la Sph, disminuyeron la expresión de la proteína Bcl-2 amti-apoptótica, y también indujeron la fragmentación de ADN, visualizada mediante la técnica de TUNEL, demostrando que estos esfingolípidos inducen apoptósis en MCF-7. La agelasina B, toxina purificada a partir de la esponja marina Agelas clathrodes tiene un efecto citotóxico un orden de magnitud mayor en MCF-7, en comparación con fibroplastos humanos. La agelasina B induce la liberación del Ca+2 almacenado en el RE en celulas MCF-7, ademas de inhibir la actividad de la SERCA en un 100%. También se demostró que esta toxina induce apoptosis, ya que disminuye el potencial de membrana mitocondrial, activa la caspasa 8, disminuye la expresion de la proteina Bcl-2 e induce fragmentación del ADN de las células MCF-7. Este mecanismo es similar al efecto de la tapsigargina.

Bromopyrrole alkaloids as lead compounds against protozoan parasites.

In the present study, 13 bromopyrrole alkaloids, including the oroidin analogs hymenidin (2), dispacamide B (3) and dispacamide D (4), stevensine (5) and spongiacidin B (6), their derivatives lacking the imidazole ring bromoaldisin (7), longamide B (8) and longamide A (9), the dimeric oroidin derivatives sceptrin (10) and dibromopalau'amine (11), and the non-oroidin bromopyrrolohomoarginin (12), manzacidin A (13), and agelongine (14), obtained from marine sponges belonging to Axinella and Agelas genera have been screened in vitro against four parasitic protozoa, i.e., two Trypanosoma species (T. brucei rhodesiense and T. cruzi), Leishmania donovani and Plasmodium falciparum (K1 strain, a chloroquine resistant strain), responsible of human diseases with high morbidity and, in the case of malaria, high mortality. Our results indicate longamide B (8) and dibromopalau'amine (11) to be promising trypanocidal and antileishmanial agents, while dispacamide B (3) and spongiacidin B (6) emerge as antimalarial lead compounds. In addition, evaluation of the activity of the test alkaloids (2-14) against three different enzymes (PfFabI, PfFabG, PfFabZ) involved in the de novo fatty acid biosynthesis pathway of P. falciparum (PfFAS-II) identified bromopyrrolohomoarginin (12) as a potent inhibitor of PfFabZ. The structural similarity within the series of tested molecules allowed us to draw some preliminary structure-activity relationships. Tests against the mammalian L6 cells revealed important clues on therapeutic index of the metabolites. This is the first detailed study on the antiprotozoal potential of marine bromopyrrole alkaloids.

Agelastatin E, agelastatin F, and benzosceptrin C from the marine sponge Agelas dendromorpha.

The study of the n-butanol extract of the New Caledonian sponge Agelas dendromorpha led to the isolation and identification of three new pyrrole-2-aminoimidazole (P-2-AI) alkaloids, named agelastatins E (3) and F (4) and benzosceptrin C (5), together with 10 known metabolites, agelastatin A (1), agelastatin D (2), sceptrin (6), manzacidin A, tauroacidin A, taurodispacamide A, nortopsentin D, thymine, longamide, and 4,5-dibromopyrrole-2-carboxamide. Their structures were assigned by spectroscopic data interpretation. All the compounds were tested for cytotoxic activity.

From anti-fouling to biofilm inhibition: new cytotoxic secondary metabolites from two Indonesian Agelas sponges.

Chemical investigation of Indonesian marine sponges Agelas linnaei and A. nakamurai afforded 24 alkaloid derivatives representing either bromopyrrole or diterpene alkaloids. A. linnaei yielded 16 bromopyrrole alkaloids including 11 new natural products with the latter exhibiting unusual functionalities. The new compounds include the first iodinated tyramine-unit bearing pyrrole alkaloids, agelanesins A-D. These compounds exhibited cytotoxic activity against L5178Y mouse lymphoma cells with IC(50) values between 9.25 and 16.76 muM. Further new compounds include taurine acid substituted bromopyrrole alkaloids and a new dibromophakellin derivative. A. nakamurai yielded eight alkaloids among them are three new natural products. The latter include the diterpene alkaloids (-)-agelasine D and its oxime derivative and the new bromopyrrole alkaloid longamide C. (-)-Agelasine D and its oxime derivative exhibited cytotoxicity against L5178Y mouse lymphoma cells (IC(50) 4.03 and 12.5 microM, respectively). Furthermore, both agelasine derivatives inhibited settling of larvae of Balanus improvisus in an anti-fouling bioassay and proved to be toxic to the larvae. (-)-Agelasine D inhibited the growth of planktonic forms of biofilm forming bacteria S. epidermidis (MIC<0.0877 microM) but did not inhibit biofilm formation whereas the oxime derivative showed the opposite activity profile and inhibited only biofilm formation but not bacterial growth. The structures of the isolated secondary metabolites were elucidated based on extensive spectroscopic analysis involving one- and two-dimensional NMR as well as mass spectrometry and comparison with literature data.

Sceptrin, a marine natural compound, inhibits cell motility in a variety of cancer cell lines.

Sceptrin, a natural compound produced by various marine sponges, was tested for its effect on cell motility. We report for the first time that sceptrin inhibits cell motility in several cancer cell lines. The compound shows no toxicity at concentrations that are double the amount of sceptrin required for maximal inhibitory effect. Both random and factor-induced migration were impaired, suggesting that sceptrin targets a central process of cell motility machinery. Activity of de novo synthesized sceptrin was indistinguishable from sceptrin purified from Agelas nakamurai, and the inhibitory activity was found to be, at least partially, due to sceptrin's capability to inhibit cell contractility. Additionally, sceptrin was found to bind to monomeric actin, further suggesting a mechanism involving the actin cytoskeleton. Close analogues of sceptrin were synthesized, tested for their effect on cell motility, and found to be either equimolar or less potent compared to the parental compound. Inadvertent cell motility is a key contributing factor in various human diseases, including cancer and chronic inflammation. Marine compounds isolated from sponges have been proven to be an excellent source of metabolites that show biological activities. Given the recently achieved total synthesis of sceptrin in multigram quantities, sceptrin could prove to be an attractive lead molecule for further preclinical testing and development for therapeutic purposes, as well as a useful research tool to elucidate the mechanisms involved in cell motility.

Total synthesis and biological evaluation of the marine bromopyrrole alkaloid dispyrin: elucidation of discrete molecular targets with therapeutic potential.

The first total synthesis of dispyrin, a recently reported bromopyrrole alkaloid from Agelas dispar with an unprecedented bromopyrrole tyramine motif, was achieved in three steps on a gram scale (68.4% overall). No biological activity was reported for dispyrin, so we evaluated synthetic dispyrin against>200 discrete molecular targets in radioligand binding and functional assays. Unlike most marine natural products, dispyrin (1) possesses no antibacterial or anticancer activity, but was found to be a potent ligand and antagonist of several therapeutically relevant GPCRs, the alpha1D and alpha2A adrenergic receptors and the H2 and H3 histamine receptors.

Ageladine A, a pyrrole-imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye.

The alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.

Synthesis, antimicrobial and antineoplastic activities for agelasine and agelasimine analogs with a beta-cyclocitral derived substituent.

Agelasines and agelasimines are antimicrobial and cytotoxic purine derivatives isolated from marine sponges (Agelas sp.). We have synthesized structurally simplified analogs of these natural products starting from beta-cyclocitral. The novel compounds were found to be strong inhibitors of a wide variety of pathogenic microorganisms (incl. Mycobacterium tuberculosis) as well as cancer cell lines. The biological activities were generally in the same range as those previously found for the structurally more complex agelasines and agelasimines isolated in small amounts from natural sources. We also report for the first time that agelasine and agelasimine analogs inhibit growth of protozoa (Acanthamoeba castellanii and Acanthamoeba polyphaga). Acanthamoeba keratitis is an increasingly common and severe corneal infection, closely associated with contact lens wear.

Effects of Agelas oroides and Petrosia ficiformis crude extracts on human neuroblastoma cell survival.

Among marine sessile organisms, sponges (Porifera) are the major producers of bioactive secondary metabolites that defend them against predators and competitors and are used to interfere with the pathogenesis of many human diseases. Some of these biological active metabolites are able to influence cell survival and death, modifying the activity of several enzymes involved in these cellular processes. These natural compounds show a potential anticancer activity but the mechanism of this action is largely unknown. In this study, we investigated the effects of two Mediterranean sponges, Agelas oroides and Petrosia ficiformis on the viability of human neuroblastoma cells. Upon treatment with the methanolic extract of Petrosia ficiformis, a marked cytotoxic effect was observed at any concentration or time of exposure. In contrast, a time- and dose-dependent effect was monitored for Agelas oroides that induced the development of apoptotic features and ROS production in LAN5 cells. These events were suppressed by calpeptin or zVAD and by vitamin C suggesting that the cell death caused by Agelas oroides was calpain- and caspase-dependent and of oxidative nature. Comet assay showed that this methanolic extract was not able to produce a genotoxic effect. Future studies will be applied to investigate the effect of isolated bioactive compounds from crude extract of this sponge which are potentially useful for cancer therapeutics.