Cytotoxic Compounds of Two Demosponges (Aplysina aerophoba and Spongia sp.) from the Aegean Sea

The class of demosponges is the biggest and most diverse of all described sponge species and it is reported to produce a plethora of chemically different metabolites with interesting biological activities. The focus of the present study was to investigate the chemical composition of two Mediterranean demosponges, targeting their brominated compounds and prenylated hydroquinones, compounds with interesting cytotoxic and anti-microbial properties. In order to gain a deeper insight into the chemical diversity of their metabolites and their activities, 20 pure secondary metabolites including new natural products were isolated from two different species (Aplysina aerophoba and Spongia sp.) using various chromatographic techniques. Their structures were confirmed by NMR and HRMS, revealing molecules with various chemical scaffolds, mainly prenylated hydroquinones from Spongia sp. and halogenated compounds from Aplysina aerophoba, including 5 novel natural products. The isolated compounds were investigated for their cytotoxic properties using 9 different cell lines, and especially one compound, 2,6-dibromo-4-hydroxy-4-methoxycarbonylmethylcyclohexa-2,5-dien-1-one showed good activities in all tested models.


Introduction
Sponges are a prolific source of bioactive natural compounds with unique structural features unprecedented in the terrestrial environment [1]. Demospongiae is their biggest and most diverse class [2], and they are known to produce a variety of chemically different metabolites, including terpenes, alkaloids, macrolides, peptides, betaines, ceramides, lipids, and halogenated compounds with potential interest regarding industrial and medical applications including antiviral, antitumor, antimicrobial, or generally cytotoxic properties. Therefore, they are of considerable biotechnological interest [3,4]. These compounds often possess multiple ecological functions, primarily the protection against predators, competitors for space, biofoulers, or opportunistic pathogenic microorganisms [5].
The sponge Aplysina aerophoba Schmidt, 1862 (Demospongiae, Verongida, Aplysinidae) is a common Mediterranean, photophilic species growing on stable substrates like rocks or Figure 1. The chemical structures of compounds 1 to 20 as elucidated by NMR and MS; compounds 1-5 are novel natural products isolated from A. aerophoba, compounds 6-15 were isolated from A. aerophoba and they have been previously described in the literature, compounds 16-20 were isolated from Spongia sp. and they are known compounds.

Biological Material
Spongia sp. was collected at Heraklion, Crete, Greece in April 2018, and Aplysina aerophoba at the same place in June 2018. Both were morphologically identified by Dimitris Poursanidis (Postdoc fellow, IACM-FORTH, Foundation for Research and Technology-Heraklion, Crete, Greece-CEO of terraSolutions marine environment research). The sponges were cut into small pieces and immediately stored in EtOH 96% until further processing. Voucher samples are deposited at the Institute of Pharmacy, Pharmacognosy, University of Innsbruck, Austria.

Instrumentation
Optical rotations were measured with a polarimeter P-2000 (JASCO, Tokyo, Japan) using a 10.0 cm tube and CHCl 3 as the solvent. IR spectra were obtained on a Platinum ATR FTIR spectrometer (Bruker, MA, USA), and ECD experiments conducted on a J-1500 spectrophotometer (JASCO, Tokyo, Japan). NMR experiments were performed on two spectrometers from Bruker, Bruker Avance II 600 (600 MHz for 1 H, 150 MHz for 13 C) and Avance III HD (400 MHz for 1 H, 100 MHz for 13 C). The isolated compounds were dissolved in MeOD or chloroform using tetramethylsilane (TMS) as internal standard. Highresolution mass spectra were measured with aQ-Exactive HF-X Orbitrap mass spectrometer (Thermo, MA, USA) and a micrOTOF-Q II mass spectrometer (Bruker-Daltonics, Bremen, Germany) whereas low-resolution mass spectra were recorded on an Agilent InfinityLab LC/MSD System. It comprised of an Agilent 1260 HPLC, equipped with binary pump, autosampler, column oven, and photodiode array detector (Santa Clara, CA, USA). For the purification of compounds, a Reveleris ® X2 iES flash chromatography system (Büchi, Flawil, Switzerland) and a semi-preparative UltiMate 3000 HPLC from Dionex (Thermo, Waltham, MA, USA), comprising a P580 pump, an ASI 100 automated sample injector, an UVD 170 U detector, and a fraction collector, were used. Sephadex LH-20 material was purchased from Sigma-Aldrich (St. Louis, MI, USA). Analytical HPLC experiments were performed on a LC-20AD XR System (Shimadzu, Tokyo, Japan).

Chemicals and Reagents
All solvents required for extraction and isolation were purchased from VWR International (Vienna, Austria) and petroleum ether (PE), dichloromethane, acetone, and ethyl acetate (EtOAc) were distilled before use. Solvents for analytical experiments had pro analysis (p.a.) quality at least and were obtained from Merck (Darmstadt, Germany). Deuterated solvents were supplied by Euriso-Top (Saint-Aubin, France). Ultrapure water was produced by a Sartorius arium ® 611 UV (Göttingen, Germany) purification system. Silica gel 40-63 µm and pre-packed cartridges for flash chromatography were purchased from Merck (Darmstadt, Germany) and Büchi (Flawil, Switzerland), respectively.

Extraction and Isolation
Aplysina aerophoba (approximately 1 kg) was extracted five times in an ultrasonic bath (Bandelin Sonorex 35 KHz, Berlin, Germany) for 15 min each using EtOH 96%. Afterwards, the ethanolic extract (89 g) was partitioned successively between PE, EtOAc, BuOH, and H 2 O (3 times each × 500 mL). HPLC analysis of the fractions indicated that the PE (6.2 g), EtOAc (14.0 g), and BuOH (8.7 g) fractions contained brominated compounds, therefore, they were combined and used for further fractionation, while the water fraction (58.0 g) was dismissed.

Computational Methods
The 3D structures of the molecules were drawn and subjected to conformational analysis in MacroModel v. 9 (Schrödinger LLC, New York, NY, USA) using OPLS-3 forcefield in chloroform. The conformers obtained in an energy window of 10 Kcal/mol were further submitted to geometrical optimization at B3LYP/6-31G(d,p) level for compounds 3 and 14, and B3LYP/6-31++(d,p) for compound 5. Further NMR chemical shift calculation was performed by using mPW1PW91/6-31G+(d,p)/CPCM/methanol for compounds 3 and 14 (data are not shown), and specific rotation calculation of compound 5 was conducted at B3LYP/6-311G++(d,p)/CPCM level in chloroform by considering the sodium D line frequency in the calculation. The obtained specific rotation values were Boltzmann-averaged and utilized for comparison with the experimentally obtained value in chloroform.
SH-SY5Y human neuroblastoma cell line was kindly provided by Dr. Obexer (Tyrolean Cancer Research Institute); human colon adenocarcinoma cell lines DLD-1, SW-480, and LOVO were purchased from DSMZ (Braunschweig, Germany). Human myeloma cell lines NCI-H929, OPM-2, and U266 were also purchased from DSMZ (Braunschweig, Germany). Cell lines were routinely fingerprinted and tested for mycoplasma negativity. Primary human foreskin fibroblasts were purchased from Promocell, Heidelberg, Germany.

Determination of Cell Viability of T24 and AGS Cell Lines (MTT Assay)
To determine the influence of the isolated substances on cell viability towards T24 bladder and AGS stomach cells, MTT assay was performed (Mosmann, 1983). T24 cells were seeded into 96-well plates with 2.0 × 10 4 cells per well (100 µL), incubated for 24 h at 37 • C with 5% CO 2 , and washed with 200 µL/well of PBS. Incubation of the cells with 100 µL of extract or pure substances at different concentrations (100 to 0.1 µM for pure compounds and 500-100 µg/mL for extracts in DMEM without additives) was performed for 24 h at 37 • C/5% CO 2 . Subsequently, the supernatant was removed and cells were washed twice with PBS (200 µL/well). In addition, 50 µL of MTT reagent were added to each well and after an incubation period of 24 h at 37 • C/5% CO 2 , the MTT reagent was removed and replaced by 50 µL DMSO per well to dissolve the formed insoluble formazan crystals. After 10 min, the amount of formazan was quantified spectrophotometrically in a plate reader at λ = 595 nm, with λ = 690 nm as a reference wavelength. Medium + 10% FCS served as a positive control, while the respective medium used for the sample preparation served as an untreated control. As a negative control, 10% DMSO was used. For AGS, a cell density of 5 × 10 4 was used and substances were diluted in RPMI medium. At least three analyses in triplicates were performed for each cell line and each concentration of the compounds tested and a solvent control was always included. Data are shown as mean percentage of viable cells and standard error of the mean (SEM) (error bars).

Cytotoxicity Assays Using FACS Analysis
The induction of apoptosis was measured in cancer cell lines and in fibroblasts/peripheral blood mononuclear cells of healthy donors using established protocols [19]. Adherent cell lines (neuroblastoma cell line SH-SY5Y, colon carcinoma cell lines DLD-1, SW-480, and LOVO, as well as primary foreskin fibroblasts) were seeded at a concentration of 1 × 10 5 cells/mL in 96-well plates the day before treatment in order to allow attachment. This medium was then replaced by media containing the respective compounds at different concentrations and cells were incubated for 24 h. Solvent controls were always included. Supernatants were collected and pooled with the trypsinized cells of the respective wells. These samples were then stained with AnnexinV-FITC (MabTag GmbH, Friesoythe, Germany) and propidium iodide (Merck, Darmstadt, Germany) and processed by flow cytometry using FACS Canto II and Diva software (Becton Dickinson, San Jose, CA, USA) Biosciences). Data were further analyzed using GraphPad Prism 5.0 software.
Myeloma cell lines NCI-H929, OPM-2, and U266 as well as peripheral blood mononuclear cells (PBMCs) of healthy donors (5 × 10 5 cells/mL) were incubated with the same compounds for 24 h and subjected to FACS analysis as described above. Etoposide (Merck, Darmstadt, Germany) was used as positive control for adherent cancer cell lines and Bortezomib (Eubio, Vienna, Austria) was used as positive control for myeloma cell lines. The extent of non-apoptotic cells (AnnexinV/propidium iodide negativity) was calculated as percentage of control (untreated) and mean percentage of viable cells and standard deviation (error bars) are shown.

Structure Elucidation
Five out of fifteen compounds isolated from A. aerophoba were novel natural products and their structure elucidation is described below.

Compound 3
Compound 3 was assigned to the molecular formula C 22 H 21 Br 4 N 3 O 7 , determined by HR-ESI-MS (753.8063 calcd. for [M-H] -, found 753.8059). The NMR data revealed a new compound bearing the substructure of the right half of fistularin 1 [21], including a 2oxazolidone ring joined directly to 2,6-dibromophenol, and a N-(2-hydroxypropyl)formamide moiety. Characteristic NMR chemical shifts indicated the presence of an additional 2,6dibromo-4-methylene-phenol group, which was methylated at position 1, as indicated by the HMBC correlation of the methoxy group (δ H 3.83) to C-1 (δ C 153.0) (Figure 4). Furthermore, the protons of the methylene group H-7 (δ H 3.89) showed an HMBC correlation to the carbon of an oxime group, C-8 (δ C 152.6), and a further correlation with the carbon of the amide group of the first substructure, C-9 (δ C 163.5). The IR spectrum also showed characteristic vibrational frequencies of N-O and C=N bonds of oxime group at 939 and 1662 cm −1 . Compound 3 was finally identified as (Z)-N-(3-(2,6-dibromo-4-(2-oxooxazolidin-5-yl)phenoxy)-2-hydroxypropyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide, a new natural product with the trivial name aeroplysinin-5. In order to establish the absolute configuration of compounds 3 (and also its known derivative, 14), one needs to decipher the relative stereochemistry prior to the absolute one. Compound 3 has chiral centers at C-11 and C-19. Due to the lack of NOEs between the protons of the respective carbon atoms, arising from being in two distant tails of the molecule, it was not possible to deduce any conclusion regarding their relative configuration. In an attempt to solve this issue, NMR chemical shift calculation along with computing of the DP4+ probability were applied on all the generated conformers of the possible stereoisomers of compound 3. However, the obtained results failed to establish the relative configurations of the chiral centers, which is possibly arising from (i) the lack of interaction or effect between the two chiral centers, (ii) the high flexibility of the molecule which makes it difficult for proper conformational sampling. The process was similarly applied for the other known derivate (compound 14), but no results could be concluded regarding its relative or absolute configuration, too.

Compound 2
Compound 2 was assigned to the molecular formula C 21

Compound 5
Compound 5 was assigned the molecular formula C 12 [22]. Additionally, the absence of the NOESY correlation of the methine H-4 to the -OH group of position 3a indicated a different stereochemistry at these positions. Collectively, the relative configuration of compound 5 was deduced as 6aR,3aS,4R. This compound revealed a very week ECD spectrum, which could not be used for the establishment of its absolute stereochemistry. Therefore, optical rotation calculation was implemented to establish its absolute configuration. Briefly, after conformational analysis of compound 5 in chloroform by using MMFF forcefield, 13 conformers were obtained, which were subjected to geometry optimization at B3LYP/6-c31++G(d,p)/CPCM/chloroform and optical rotation at B3LYP/aug-pVTZ/CPCM/chloroform level and by considering the frequency of sodium D line for the calculation. The results demonstrated a Boltzmann-averaged specific rotation value of −59.68 (589.3 nm, sodium D line) for isomer 6aR,3aS,4R. Considering the sign of the calculated specific value, the absolute configuration of compound 5 could be deciphered accordingly. The deviation of the calculated numerical value and the experimental one could be possibly emanated from the conformational analysis and overestimation of the calculation methods. Therefore, compound 5 is a new natural stereoisomer of subereatensin [21]. It was identified as (6aR,3aS,4R)-ethyl 4-ethoxy-3a-hydroxy-2-oxo-1,2,3,3a,4,6ahexahydrocyclopenta[b]pyrrole-6-carboxylate and given the trivial name iso-subereatensin.

Cytotoxic Properties
Cytotoxic properties of almost all compounds isolated from Spongia sp. and Aplysina aerophoba to T24 bladder and AGS stomach tumor cells was determined using the common MTT assay by Mossman et al. Table 4 displays the respective, expressed as IC 50 values, as the concentration (µM) of each test compound causing 50% effect in the respective assays. For less active compounds, the % viability at 100 µM concentration is given. Additionally, the two crude extracts were investigated (500 µg/mL). Spongia sp. did not show any toxicity effects whereas the Aplysina aerophoba extract showed a cell viability of only 25.3% after a 24 h treatment. Compounds 13, 16, 17, and 19 showed strong cytotoxic effects towards AGS cells with compound 17 being the most active compound with an IC 50 value of 0.99 µM. Compounds 12 and 18 indicated moderate cytotoxic activity towards AGS cells. However only one compound showed significant toxicity towards T24 cells, i.e., for compound 13, an IC 50 of 12.42 µM was determined. Compounds 7, 14, 17, and 18 exerted moderate effects. In addition, the same substances (compounds 5-20) were investigated for a potential apoptotic activity towards the neuroblastoma cell line SH-SY5Y in three concentrations (25 to 100 µM) using a fluorescence activated cell sorting readout (FACS). Significant cytotoxicity was observed for compound 13 in this assay (cell viability was measured below 20% for all concentrations) ( Figure 6). Subsequently, the five most promising compounds according to the initial screening in the above-mentioned cell lines (compounds 13, 16, 17, 18, and 19) were chosen for further investigations. They were additionally investigated in three colon cancer cell lines (DLD-1, SW-480 and Lovo) for potential cytotoxic effects. Again, compound 13 revealed activity in all cell lines (Figure 7), and decreased cell viability. Compound 18 and 19 showed moderate activity in the highest concentration (100 µM), with a remaining cell viability of around 60% for SW-480 and Lovo cell lines. Compound 13 was found to be cytotoxic against these colon carcinoma cell lines even at lower concentrations ranging from 1-25 µM ( Figure 8A). For SH-SY5Y cells, compound 13 was titrated from 25 to 0.63 µM and the IC 50 value was determined with 1.78 [CI 95 1.34-2.38] ( Figure 8B). In comparison, etoposide, a standard cytotoxic compound used in clinical practice for neuroblastoma treatment, killed 50% of SH-SY5Y cells at a concentration of 340 nM (data not shown). We also utilized human primary fibroblasts for testing in order to delineate a general cytotoxicity of this compound. These non-cancerous cells were significantly less sensitive to 13 supporting a potential anti-cancer effect of this compound with a certain degree of selectivity ( Figure 8C    Encouraged by the positive results, compound 13 was further investigated in a hematological cancer model, i.e., multiple myeloma. Again, cytotoxicity was found at low concentrations (concentration range 1 to 25 µM), showing a decreased viability of NCI-H929, OPM-2, and U266 cell lines of 4%, 54%, and 34%, respectively, after 24 h incubation time at a concentration of 5 µM. In non-cancerous blood cells, i.e., peripheral blood mononuclear cells (PBMCs) of healthy donors, compound 13 induced significantly lower levels of cell death, again suggesting that cancer cells might be more susceptible (PBMC 1-3, Figure 9).

Discussion and Conclusions
Marine sponges are gaining the attention of the scientific community because of their unique secondary metabolites with a diversity of biological activities. Some of them are being tested in clinical trials against various diseases, mainly with the focus on anti-cancer drugs [29,30].
A. aerophoba and Spongia sp. were selected for this study because initial screening experiments already indicated that they contain interesting and possibly new metabolites.
Apart from the commonly studied bromotyrosines from A. aerophoba which are compounds with interesting antibacterial and antitumor activities [13,31], attention has been focused on chitin which has further applications, e.g., as scaffold in tissue engineering, pharmacological applications, and regenerative medicine [13,32]. Furthermore, the farming of Spongia sp. for commercial purposes as bath sponges has been going on for more than a century [33]. These sponges have a high commercial value and they are successfully employed in experimental aquaculture experiences [34]. The enormous filtering capacity of sponges has led to the suggestion that they be farmed for bioremediation purposes, e.g., to reduce the high bacterial loads resulting from sewage discharges and areas subjected to aquaculture activities [33,34]. Additionally, Spongia sp. have recently been studied as an alternative source of collagen which is found in their extracellular skeletal matrix [17].
In our study, twenty metabolites, mainly halogenated compounds and prenylated hydroquinones, were isolated using a purification protocol including liquid-liquid extraction, fractionation on silica gel column and Sephadex LH-20 columns, and a final clean-up step with semipreparative HPLC or flash chromatography. Four brominated metabolites and an isomer of subereatensin isolated from A. aerophoba have been identified as novel natural products and two additional brominated compounds were reported from A. aerophoba for the first time.
When evaluating the possible cytotoxic effects of the isolated metabolites, only a few of them showed activities in the investigated cell lines. For AGS stomach tumor cells, compound 17 was found to be the most toxic, while compound 13 showed the highest cytotoxic activity in all other cell lines tested. Therefore, this compound seems to be interesting for further studies, especially because our data indicated selectivity towards cancer cells.
Sponges often contain diverse and abundant microbial communities in their tissues and in many cases, the associated bacterial communities account for over 40% of the biomass of their hosts [30,34]. In the past few years, more and more evidence has accumulated in which a part of the isolated metabolites are not produced by the sponges themselves, but instead, they are products of the metabolic activities of bacteria living in the sponge tissue [34]. This also could be the case for the compounds isolated within this study. Our results suggest that A. aerophoba and Spongia sp., among other demosponges which currently attract increased interest in the scientific community, are a rich source of interesting compounds. This refers to unique chemical structures as well as promising bioactivities.