Cytotoxic Metabolites from the Okinawan Ascidian Diplosoma virens

The unstable isomeric compounds 5-hydroxy-7-prop-2-en-(E)-ylidene-7,7a-dihydro-2H-cyclopenta[b]pyran-6-one (1) and 5-hydroxy-7-prop-2-en-(Z)-ylidene-7,7a-dihydro-2H-cyclopenta[b]pyran-6-one (2), previously described as antimicrobial metabolites from the sponge Ulosa sp., were isolated and identified as major components of the ascidian Diplosoma virens. In this paper, full spectral data for 2 and complete 13C-NMR data for 1, based on 2D NMR measurements, are provided for the first time. Compounds 1 and 2 showed cytotoxity against HCT116 cells (human colorectal cancer cells) by triggering apoptotic cell death.

In this study, we confirmed the structure of 2 by interpreting its 2D-NMR data, and we provided full spectral data for 2 and 1. In addition, the compounds' cytotoxity against HCT116 cells (human colorectal cancer cells) via apoptotic cell death was demonstrated.

Results and Discussion
A specimen of Diplosoma virens (72 g, wet weight) was collected by hand from the coast of Hateruma Island, Okinawa, and extracted with acetone. The acetone extract was analyzed using 1 H-NMR, and two major compounds were observed. The acetone extract was partitioned between aqueous MeOH and hexanes. The aqueous MeOH phase was concentrated in vacuo and then partitioned between CH 2 Cl 2 and water. Purification of the CH 2 Cl 2 extract by silica gel column chromatography followed by reversed-phase (C8) HPLC (H 2 O/MeOH) yielded compounds 1 (0.13%) and 2 (0.086%). When a solution of 1 or 2 in CH 2 Cl 2 or MeOH was concentrated, a large portion of the resulting residue was no longer soluble in the organic solvents. Compound 1 was identified by performing comprehensive spectral analyses ( Figure 1 and Table 1) and by comparing resulting spectral data with those in the literature [15].
Analysis of the 13 C-NMR and HRFABMS data [m/z 191.0691 (M + H) + , Δ -1.7 mmu] for compound 2 provided a molecular formula of C 11 H 10 O 3 , which suggested seven degrees of unsaturation. The IR absorption bands at 1680 and 3250 cm -1 indicated the presence of carbonyl and hydroxyl groups. The spectral data of compound 2 showed close similarities to those of 1. The 1 H-and 13 C-NMR data analysis indicated the presence of a carbonyl carbon (δ C 187. 8  6.08 (br s) 6.12 (br s) a 1 H-NMR (400 MHz) and 13   The chemical shift of H-10 in 2 (δ H 7.76) was at lower field than in 1 (δ H 6.89) owing to the magnetic anisotropy effect of the carbonyl group and the chemical shift of H-9 in 2 (δ H 6.65) was at higher field than in 1 (δ H 7.04), suggesting a Z configuration for the C-3, 9 double bond of 2. This was confirmed by an NOEDS experiment, in which NOE was observed between H-9 and H-4 ( Figure 3).

Biological Activities
Compounds 1 and 2 showed cytotoxity against HCT116 cells (human colorectal cancer cells) in a dose dependent manner (Figure 4a). Necrosis is a form of traumatic cell death that results from acute cellular injury. In contrast, apoptosis is a form of programmed cell death involving a biochemical cascade that includes caspases and cysteine proteases. Caspase 3/7 exists downstream in the caspase cascade.
To examine the type of cell death induced by these compounds at 20 ppm, caspase 3/7 activity was measured in HCT116 cells in the presence of compounds 1 and 2. Caspase 3/7 activity in cells treated with compounds 1 and 2 was expressed as percent induced, compared to control cells not treated with the compound (Figure 4b). The caspase 3/7 induction associated with compounds 1 and 2 were 53.6% and 73.6%, respectively, indicating that these compounds induce apoptotic cell death by activating caspases through the mitochondrial/cytochrome C stress pathway that begins with the release of cytochrome C from mitochondria [16][17][18].

Conclusions
In this study we isolated compounds 1 and 2, constituents of the sponge Uloma sp. [15], as major components of the ascidian Diplosoma virens, and we confirmed the structure of 2 by interpreting its 2D-NMR and MS data, thus providing full spectral data for 2 and 1. Compounds 1 and 2 showed weak cytotoxity against HCT116 cells (human colorectal cancer cells) by triggering apoptotic cell death. C 11 cyclopentenones (didemnenones) [6] and the related compounds (nakienone and terpiodiene) [19,20] have been isolated from ascidians, cyanobacteria and a sponge, respectively. Isolation of a series of the C 11 compounds, including compounds 1 and 2, from unrelated marine organisms supports the potential microbial origin of these compounds. Studies on the origin of compounds 1 and 2 and their bioactivities are under way in our laboratory.

General
Optical rotations were measured on a JASCO P-1020 polarimeter. UV spectra of the methanol solutions were measured on a JASCO V-550 spectrophotometer. IR spectra were recorded as dry films on a JASCO FT/IR-300 spectrometer. The 1 H-, 13 C-, and 2D-NMR spectra were recorded on a JEOL lambda 400 or a JEOL α-500 spectrometer, and 1 H-and 13 C-chemical shifts were referenced to the solvent peaks [δ H 7.24 and δ C 77.0 in CDCl 3 ]. Mass spectra were measured on a Waters Quattro micro API triple quadruple mass analyzer. Open column chromatography was performed on Kieselgel 60 (70-230 mesh, Merck). Preparative HPLC was run on a Waters 600 multi solvent system using a reversed-phase column (YMC-Pack C8, 20 x 250 mm I. D., YMC). All solvents used were reagent grade.

Animal Collection
The small yellowish green ascidian was collected at low tide from the coast of Hateruma Island, Okinawa (Japan) in April 2006, and identified as Diplosoma virens by Professor Euichi Hirose, University of the Ryukyus, Japan. The identified ascidian was kept frozen at -80˚C until used. A voucher specimen was deposited at the University of the Ryukyus (Specimen no. 070222).
Cell viability: The MTT assay was used to examine the cell viability. Briefly, HCT116 cells were seeded at a density of 2.5 X 10 4 cells/mL on 96-well plates and cultured for 17 hrs with or without the test compound. After incubation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, 0.5 mg/mL) was added to each well, the samples were again incubated for 3 hrs, and then they were removed from suspension. Extraction with DMSO (100 μL), measured at 570 nm, provided the reference for readings at 630 nm with a microplate reader (BIO-RAD Model 550, BIO-RAD, USA).
Caspase activity: HCT116 cells were plated at a density of 2.5 X 10 4 cells on 96-well plates, which were incubated for 17 hrs with or without the test compound. Caspase activation in cultured cells was measured using the commercially available caspase-3/7 assay kit (Promega, USA), following the protocol supplied by the manufacturer. Each cultured cell in the well was incubated at room temperature for 2 hrs 30 min with 100 μL of proluminescent substrate containing Z-DEVD (Caspase-Glo TM 3/7), provided with the kit. Following caspase cleavage, a substrate reacts with luciferase and releases light in the presence of ATP and oxygen. The luminescence of the reaction products was measured with a CL-detector (CLD-110, Tohoku Electronic Co.).