Cytotoxic Compounds from the Saudi Red Sea Sponge Xestospongia testudinaria

Bioassay-guided fractionation of the organic extract of the Red Sea sponge Xestospongia testudinaria led to the isolation of 13 compounds including two new sterol esters, xestosterol palmitate (2) and xestosterol ester of l6′-bromo-(7′E,11′E,l5′E)-hexadeca-7′,11′,l5′-triene-5′,13′-diynoic acid (4), together with eleven known compounds: xestosterol (1), xestosterol ester of 18′-bromooctadeca-7′E,9′E-diene-7′,15′-diynoic acid (3), and the brominated acetylenic fatty acid derivatives, (5E,11E,15E,19E)-20-bromoeicosa-5,11,15,19-tetraene-9,17-diynoic acid (5), 18,18-dibromo-(9E)-octadeca-9,17-diene-5,7-diynoic acid (6), 18-bromooctadeca-(9E,17E)-diene-7,15-diynoic acid (7), 18-bromooctadeca-(9E,13E,17E)-triene-7,15-diynoic acid (8), l6-bromo (7E,11E,l5E)hexadeca-7,11,l5-triene-5,13-diynoic acid (9), 2-methylmaleimide-5-oxime (10), maleimide-5-oxime (11), tetillapyrone (12), and nortetillapyrone (13). The chemical structures of the isolated compounds were accomplished using one- and two-dimensional NMR, infrared and high-resolution electron impact mass spectroscopy (1D, 2D NMR, IR and HREIMS), and by comparison with the data of the known compounds. The total alcoholic and n-hexane extracts showed remarkable cytotoxic activity against human cervical cancer (HeLa), human hepatocellular carcinoma (HepG-2), and human medulloblastoma (Daoy) cancer cell lines. Interestingly, the dibrominated C18-acetylenic fatty acid (6) exhibited the most potent growth inhibitory activity against these cancer cell lines followed by Compounds 7 and 9. Apparently, the dibromination of the terminal olefinic moiety has an enhanced effect on the cytotoxic activity.


Introduction
Marine sponges (phylum Porifera) are among the oldest and simplest animals, which grow in every ocean and have a great capacity to withstand extreme temperatures and pressures [1]. They are filter feeders, and their bodies are full of pores and channels that allows water to circulate through them [2,3]. Moreover, they are well-known for their production of secondary metabolites that constitute an effective defense mechanism against foreign predators [2,3]. So far, about 8000 species of Porifera, inhabiting different marine and freshwater ecosystems, have been described. Since the beginning of the exploration of marine natural products in the 1970s, the investigation of the secondary metabolites of Xestospongia sponges (family Petrosiidae), commonly known as barrel sponges, have been carried out successively in several regions around the world [4]. They have been recognized as rich sources of different chemical classes, such as isoquinoline, macrocyclic quinolizidines, pyridoacridine alkaloids, quinones, sterols, brominated polyacetylenic acids, and esters [4]. Previous investigations on the chemistry and pharmacology of genus Xestospongia have shown that its crude extracts and isolated compounds displayed remarkable bioactivities, such as anti-inflammatory, antioxidant, immunomodulatory, cytotoxicity, antimicrobial, insecticidal, HIV protease inhibition, cardiotonic, vasodilatation, and antiplasmodial activities [5][6][7][8][9]. The currently investigated species, Xestospongia testudinaria, has been the source of indole alkaloids, sterols, sterol esters, and brominated polyunsaturated fatty acids (BPUFAs) [4,10,11].
As part of our continued interest in identifying potential marine-derived bioactive compounds for treatment of contemporary diseases such as cancer and infectious diseases [12][13][14][15], we thoroughly investigated the chemical constituents and cytotoxic activity of the Red Sea sponge Xestospongia testudinaria. In this paper, we report about the purification and identification of thirteen compounds including two new xestersterol esters (2 and 4) with eleven known compounds. In addition, the cytotoxic activities of the compounds against three cancer cell lines will be evaluated.
The isolated compounds were evaluated for their antitumor activity against three cancer cell lines-human hepatocellular carcinoma (HepG-2), human medulloblastoma (Daoy), and human cervical cancer (HeLa) cells-using an MTT assay, as described previously [25], using dasatinib as a positive reference drug. Five concentrations (0-50 µg/mL) of each compound were prepared and incubated with each cell line, and the survival fraction curves were obtained to calculate the concentration that produced 50% cell growth inhibition (IC 50 ).
As shown in Table 3, the total ethanolic extract of X. testudinaria, n-hexane fraction, and Compound 6 exhibited broad spectrum inhibition of all tested cancer cell lines. In this context, the potency can be arranged in the following descending order: Compound 6 > n-hexane fraction > the total ethanolic extract on human cervical cancer (HeLa) and Daoy cells, while on HepG-2 cells, 6 > the total ethanolic extract > n-hexane fraction. Compounds 7 and 9 showed moderate selectivity against HeLa and Daoy cells. Compound 7 was more potent than 9, since the IC 50 of 7 was 30.38 and 23.1 µg/mL on HeLa and Daoy cells, respectively, compared to 9, which exhibited IC 50 values of 44.41 and 24.57 µg/mL on HeLa and Daoy cells, respectively. With regard to the sensitivity of cancer cells to the compounds, the Daoy cell line appeared to be the most sensitive towards the tested compounds, followed by HepG-2 and HeLa cell lines. Structure-activity relationships are proposed by comparing the IC 50 values of the isolated brominated polyacetylenic fatty acids. The results suggest that the cytotoxic activity was dramatically enhanced by the presence of an extra bromine atom as in Compound 6. With regard to the brominated compounds (5, 6, 7 and 9), the dibrominated compound (6) showed stronger cytotoxic activity than the monobrominated ones. On the other hand, the presence of a conjugated triple bond in Compound 6 may have contributed to the improved activity.

General Experimental Procedure
The IR spectra were recorded on JASCO 320-A spectrometers, while the 1 H and 13 C NMR spectra were recorded at the NMR Unit at the College of Pharmacy, Prince Sattam Bin Abdulaziz University, on an Ultra Shield Plus 500 MHz (Bruker) spectrometer operating at 500 MHz for proton and 125 MHz for carbon, respectively. The chemical shift values are reported in δ (ppm) relative to the TMS as an internal standard. 2D-NMR experiments (COSY, HSQC, HMBC, and NOESY) were obtained using a standard Bruker program. A HR-EI-MS, JEOL JMS-700, was used for accurate mass determination. Electron impact mode of ionization was used, keeping ionization energy at 70 ev. Resolution was set up to 10 k. A direct probe was used with a temperature ramp setting-an initial temperature of 50˝C, rising at a rate of 32˝C per minute, and a final temperature set up to 350˝C. Pre-coated silica gel TLC plates were used. The absorbance was read on a microplate reader (ELX 800, Bio-Tek Instruments, Winooski, VT, USA) at 549 nm. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was acquired from Sigma Aldrich (St Louis, MO, USA). DMEM/high glucose FBS and penicillin/streptomycin were purchased from Thermo Fisher Scientific. (Waltham, MA, USA).

Animal Material
The sponge was collected by hands using SCUBA diving from Ghurab Reef in the Red Sea at Jazan, Saudi Arabia between 15 and 30 meters in May 2013. The sponge was frozen immediately after collection and then freeze-dried to provide the dry material.
The sponge was identified as Xestospongia testudinaria by Prof. Rob van Soest at the Naturalis Biodiversity Center at Leiden in The Netherlands. A sample of the sponge is reserved in the collections of the Naturalis Biodiversity Center under the number RMNH Por. 9176. Another voucher specimen was placed in the Red Sea Marine Invertebrates Collection, Faculty of Pharmacy, King Abdulaziz University under the code No. DY-KSA-12. A complete description of the sponge has been previously published [5].

Extraction and Isolation
The freeze-dried sponge (750 g) was extracted with 96% ethanol (3ˆ2 L) at room temperature. The combined alcohol extracts were filtered and evaporated under reduced pressure using a rotatory evaporator at 38˝C to produce 115 g of the alcohol extract. When methanol (500 mL) was added to the dried ethanolic extract, a precipitate was formed and collected (26 g). Part of this precipitate was washed repeatedly with different organic solvents to yield a pure compound (1). The other part was applied over a silica gel column with n-hexane/acetone in gradient elution mode to yield Compounds 2-4. On the other hand, the methanol soluble part of the ethanolic extract (88 g) was suspended in 40% ethanol (1 L) and successively partitioned with 500-mL portions of n-hexane, dichloromethane, and n-butanol to afford the corresponding fractions.

Evaluation of the Antiproliferative Activity of the Compounds Using MTT Assay
The three utilized tumor cell lines were human cervical cancer (HeLa), human hepatocellular carcinoma (HepG-2) and human medulloblastoma (Daoy) cells. HeLa and HepG-2 cells were cultured in DMEM/high glucose supplemented with 10% FBS, 2 mM of L-glutamine, and 1% penicillin/streptomycin. Daoy cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mM of L-glutamine, and 1% penicillin/streptomycin. All isolated compounds were evaluated at the Cell Culture Laboratory, College of Pharmacy, King Saud University, in a three-cell line-one concentration (50 mg/mL) anticancer assay against the aforementioned cell lines, adapting the method described by Al-Salahi et al. 2014 [25].
The dose response curves of the compounds affecting ě50% inhibition in one-dose prescreening for each cell line were established with concentrations of 50, 25, 12.5, 6.25, 3.125, and 1.56 µg/mL, and the concentrations causing 50% cell growth inhibition (IC 50 ) were calculated. The cytotoxic activity of the anticancer drug dasatinib [26] against the tested cell lines was examined at the same concentrations of the tested compounds.

Conclusions
In conclusion, bioassay-directed fractionation of the active fractions of the organic extracts of the Red Sea sponge Xestospongia testudinaria afforded thirteen compounds including two new xestosterol esters (2 and 4) together with several known compounds (1, 2, 5-13). The identification of the compounds was achieved by analysis of the NMR and MS data of the compounds and by comparison with the literature. The compounds showed different cytotoxic activities towards the three tested cancer cell lines.