Cytotoxic Effect of the Genus Sinularia Extracts on Human SCC25 and HaCaT Cells

Soft corals of the genus Sinularia are being increasingly adopted to treat a wide variety of disease processes. However, the mechanism underlying its activity against human oral cancer cells is poorly understood. This study evaluates the cyototoxicity effects of the genus Sinularia extracts (S. grandilobata, S. parva, S. triangula, S. scabra, S. nanolobata and S. gibberosa) by SCC25 and HaCaT cells. The cell adhesion assay indicates that extracts reduce the cell attachment. Extracts exhibit a dose-dependent cytotoxic effect using MTS assay.Treatment of extracts to observe the morphological alterations in cells, membrane blebbing, nuclear condensation, and apoptotic bodies is demonstrated. Flow cytometry shows that extracts sensitized the cells in the G0/G1 and G2/M phases with a concomitant significantly increased sub-G1 fraction, suggesting cell death by apoptosis. Extracts of the genus Sinularia thus apparently cause apoptosis of SCC25 and HaCaT cells, and warrant further research investigating the possible antioral cancer compounds in these soft corals.


Introduction
Medicinal marine organisms are most appropriate for pharmacological research and drug development, since their constituents can be employed not only as therapeutic agents, but also as starting materials or models for synthesis of drugs of pharmacologically active compounds. Many efforts have recently been made to identify new therapeutic drugs against cancer, especially using novel biologically active compounds from natural marine organisms [1].
Coral growths are a few hundred million years old. Pressure from the natural selection has led corals to develop a delicate chemical balance for self protection. Soft corals (coelenterata, octocorallia, alcyonaceae) are a rich source of steroids and terpenoids [2,3], and most isolated diterpenes are cembranolides [4]. Hence, such chemical toxins may exhibit various biological activities, such as antitumor, antimicrobial, and HIV-inhibitory activity. The authors have previously reported various bioactive metabolites from marine organisms, including sesquiterpenoids, diterpenoids, and steroids [5][6][7][8]. Many of these metabolites have been found to be cytotoxic, or to possess other biological activities [9][10][11]. However, little thorough cytotoxicity research has been performed on these soft corals. Therefore, this study investigates the cytotoxic mechanism of the organic extracts of six Taiwanese soft corals, namely Sinularia grandilobata, S. parva, S. triangula, S. scabra, S. nanolobata, and S. gibberosa.
Oral cancer is a significant global public health problem, causing high morbidity and mortality that have not improved in decades [12]. Squamous cell carcinomas (SCCs) are the most common type of oral cancer. Although new operative techniques and adjuvant measures including chemotherapy and radiotherapy against oral SCCs have progressed, patients with advanced oral SCCs still have a 2 Journal of Toxicology poor prognosis, with a 5-year survival rate of 65% [13]. Thus, new anticancer drugs are required to enhance current protocols for diagnosis and treatment of SCCs.
Apoptosis is an important phenomenon in exerting antitumor response to cancer therapy and is also a valuable marker for predicting tumor response following anticancer treatment. Cell death can be apoptotic, or result from morphological changes such as membrane blebbing, cell shrinkage, chromatin condensation, and nuclear fragmentation with formation of apoptotic bodies. Translocation of membrane phosphatidylserine and sub-G 1 fraction is a form of programmed cell death that occurs naturally in cells and can be beneficial to cancer therapy [14]. Ability to manipulate the machinery of cell death is an obvious goal of medical research, and effect on regulation of apoptosis might lead to new possibilities for oral cancer treatment [15]. Hence, this study evaluated the induction of cell apoptosis of the genus Sinularia extracts on human SCC25 cells and premalignant keratinocytes (HaCaT).

Material.
The six soft corals of the genus Sinularia including S. grandilobata, S. parva, S. triangula, S. scabra, S. nanolobata and S. gibberosa were collected by hand via scuba along the coast of Southern Taiwan, at a depth of 10-15 m and were stored in a freezer until extraction. A voucher specimen was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Taiwan.

Preparation of Extracts.
The tissues of six soft corals of the genus Sinularia were freeze-dried and then exhaustively extracted with ethyl acetate (two times). The ethyl acetate extracts were then filtered and concentrated under vacuum to provide a brownish semisolid crude extract. Organic extracts were dissolved at a concentration of 10 mg/mL in 100% dimethyl sulfoxide (DMSO) at stock solution. Stock solution was diluted to the desired final concentrations with growth medium just before use. The final DMSO concentration did not exceed 0.1%.

Cell Adhesion
Assay. Cells (1.5 × 10 5 cells/well) were subcultured into 24-well plates and incubated. After 24 hours of incubation, the medium was changed by adding DMEM/F12 or DMEM containing 1% bovine serum albumin (BSA) and with or without serial concentrations ofextracts for 18 hours. Attached cell number was estimated by means of a DNA carmine-based colorimetric method [16]. Briefly, cells were fixed with 100% methanol, dried and stained with alcoholic/HCl carmine. Colorant was extracted with 0.01 N NaOH, and absorbance was determined at 540 nm. The cell number was estimated using a titration curve of cell density (SCC25: y = 5 × 10 −8 x + 0.0143; R 2 = 0.9849; HaCaT: y = 3 × 10 −8 x + 0.0019; R 2 = 0.9904), and results were given as a percentage of the cell number with respect to control cells. For the titration curve, cells were plated at densities ranging form 1 × 10 3 to 7 × 10 5 cells/well in 24-well plates using serial dilutions of concentrated cell suspensions. After adhesion, some wells of each density were harvested with trypsin and cells were counted in a hemacytometer; meanwhile, parallel cultures were fixed and stained as described before [16]. A relationship between the cell number and resultant absorbance after the colorant extraction, for each cell density, was accomplished and celldensity titration-curve construction, which measured cell adhesion.

Growth-Inhibition
Assay. Cells (1.5 × 10 4 cells/well) were seeded in each 100 μL of 96-well multidishes for at least 24 hours prior to use. The cells were treated with serial concentrations of extracts for 18 hours. After replacing new medium, the effects on cell growth were determined by a colorimetric tetrazolium MTS [3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carbox-ymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt] assay according to the manufacturer's procedure (CellTiter 96 AQ, Promega, Madison, Wis, USA). The absor-bance at 490 nm was measured by a spectrophotometer (Dydatech, Alexandria, Va, USA). Values are expressed as the percentage of mean cell viability is relative to the untreated cultures. The IC 50 and IC 80 were calculated from the drug concentration that induced 50% and 80% of cell survival rate. All determinations were performed in triplicate and statistically analyzed by Student's t-test.

Determination of Morphological Changes of Cells.
Cells (1.5 × 10 5 cells/well) were plated in 24-well plates then treated with IC 50 concentrations of extracts for 18 hours. After incubation, the medium was removed and cells were fixed in 4% paraformaldehyde and permeabilized in saponin (0.1% v/v in PBS-BSA). Morphological analysis was performed using phase contrast inverted light microscope (Nikon, TE2000-U, Japan) at 200× magnification. To assess specific apoptosis, Hoechst (1 μg/mL) (Sigma, USA) was added to each well and further incubated at 37 • C for 30 minutes in the dark. Living and apoptotic cells were visualized through blue filter of fluorescence inverted microscope (Nikon, TE2000-U, Japan) at 200× magnification.

Assessment of Cell-Cycle Distribution and Apoptotic Cells by Flow
Cytometry. Cells (1.5 × 10 5 ) were seeded in 24well plates and incubated with or without IC 50 and IC 80  An enzymatic test such as MTS assay is required to further evaluate the effect of extracts on cell cytotoxicity.

Growth-Inhibition Assay Effect of the Genus Sinularia
Extracts. MTS assay was conducted to examine the relationship between concentrations of the genus Sinularia extracts and the cytotoxicity of SCC25 and HaCaT cells. Cells were treated with extracts at increasing concentrations of 0-100 μg/mL for 18 hours, and the percentage of cell viability was analyzed. Organism extracts were dissolved in DMSO, and a parallel experiment demonstrated that the final concentration of DMSO in the medium (0.1%) did not produce any impact on SCC25 and HaCaT cell cytotoxicity (data not shown). As revealed in Figure 1, all of the extracts inhibited SCC25 and HaCaT cell growth in a dose-dependent manner. The concentrations of extracts causing 50% and 80% cell growth inhibition (IC 50 and IC 80 ) were determined and are presented in   was undertaken to obtain additional information about the cytotoxicity of soft corals of the genus Sinularia extracts. Rounding was observed following incubation with extract under concentration of IC 50 for 18 hours to observe the morphological alterations in the cells. Some sensitive cells were then detached from the surface, and membrane blebbing was shown by using a phase-contrast-inverted microscope. The typical nuclear condensation, nuclear fragmentation, nuclear shrinking, and apoptotic bodies of the cells were then demonstrated by Hoechst staining (see Figure 2). Results of these experiments indicate that the genus Sinularia extracts cause apoptosis of human SCC25 and HaCaT cells.

Influence of the Genus Sinularia Extracts on Cell-Cycle
Distribution and Apoptosis. The cell cycle distribution of SCC25 and HaCaT cells was analyzed with flow cytometry after exposure to the genus Sinularia extracts (see Figure 3). Results of treatment of cells with IC 50 and IC 80 concentrations of extracts reveal that the main character of apoptosis is the cleavage of nuclear DNA into multiple fragments and reflected G 0 /G 1 and S-G 2 /M phase together with a dose-dependent increase in sub-G 1 phase (corresponding to apoptotic cells). As shown in Table 3, the percentage of G 0 /G 1 , S, and G 2 /M phases in SCC25 cells incubated with extracts (IC 80 ) for 18 hours was, respectively, 23.7-77.2%, 18.2-64.6%, and 11.4-37.9% less than those in control cells. The sub-G 1 fraction of apoptotic SCC25 cells following incubation with (IC 80 )-treated cells was around 36.8-79.6 times that of control cells. The percentage of sub-G 1 phases in (IC 80 )-treated HaCaT cells was approximately 65.3-91.1 times that in untreated control cells. The G 0 /G 1 and G 2 /M phase fractions were, respectively, about 9.2-25.7% and 46.0-76.4% less in the (IC 80 )-treated cells than in the control cells, and not much change in the S phase populations. The apoptotic cell death induced by treatment with Sinularia extracts by flow cytometry was thus very similar to that seen with Hoechst staining (see Figure 2). These results show that the genus Sinularia extracts-mediated inhibition of SCC25 and HaCaT cells viability might predominantly induce cells from the G 0 /G 1 and G 2 /M phases to apoptosis.

Discussion
Oral cancer, which is one of the most disfiguring cancers, may lead to facial distortion. It is also known to exhibit field cancerization, resulting in development of second primary tumors [12]. Consequently, the development of new antioral cancer drugs, and study of their medicinal value, has become highly significant. The marine environment is a major reservoir of bioactive natural products with potential biomedical application; several marine natural products are seen as potential sources of therapeutic agents for the treatment of multiple disease categories. The majority of marine natural products and their derivatives are formed from invertebrates including soft corals, sponges, tunicates, mollusks, or bryozoans and are currently in advanced preclinical evaluation [1]. However, relatively few attempts have been made to explore resources of structurally unique chemistry for cytotoxic mechanism. This study presents the action mechanism of soft corals of the genus   The previously characterized genus Sinularia of secondary metabolites is mainly chemicals that are structurally related to terpenoids. Previous studies have indicated that sinugrandisterols A-D, trihydroxysteroids, and oxygenated terpenoids from the S. grandilobata impede the proliferation of different cancer cell lines, such as human liver carcinoma (HepG2 and Hepa59T/VGH), human breast cancer cells (MCF-7 and MDA-MB-231), human oral epidermoid carcinoma (KB), and human lung cancer cells (A549) [16]. Three norcembrane-base diterpenoids, leptocladolide A, 1epi-leptocladolide A, 7E-leptocladolide A and ineleganoid were isolated from Taiwanese soft coral S. parva, and these compounds have been revealed to exhibit significant cytotoxic activity against KB and Hepa59T/VGH cancer cell line [17]. Four amphilectane-type diterpenoids, sinulobatins A-D [18], two norsesquiterpenoids, nanonorcaryophyllenes A-B, two diterpenoids, nanolobatins A-B, nordoterpenoids nanolobatin C, (+)-5-hydroxymethyl-5-methylfuran-2-one, and (+)-5-acetoxymethyl-5-methylfuran-2-one were isolated from the S. nanolobata. Sinulobatins A-D and nanolobatins A-B exhibited moderate cytotoxicity against KB cancer cells. Nanonorcaryophyllenes A-B and nanolobatin C demonstrated no significant cytotoxicity against the tested cell lines, such as KB cells [19]. Additionally, previous reports have shown that polyoxygenated sterols from the formosan soft coral Sinularia gibberosa significantly inhibit the upregulation of the proinflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins of LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells and cytotoxic activity against HepG2 (human liver carcinoma), MCF-7, MDA-MB-23 (human breast carcinoma), and A549 (human lung carcinoma) cells [20]. Extracts of soft corals Sinularia compressa have been adopted to explore the antibacterial potential of Bacillus pumilus and Pseudomonas vesicularis [21]. In this work, cell adhesion and cytotoxicity assay indicated that soft corals of the genus Sinularia extracts prevented SCC25 and HaCaT cell growth in a concentrationdependent manner. Moreover, the extracts of S. parva and S. nanolobata were found to be more effective inhibitors of cell viability than S. grandilobata, S. triangula, S. scabra, and S. gibberosa, suggesting the S. parva and S. nanolobata extracts could be investigated in the further to forage for a potential antioral cancer compounds. In the previous research, it was found that cembranoids extracted from S. parva and S. nanolobata showed cytotoxicity in some cell lines [17][18][19]. Nevertheless, the intrinsic structure and properties of these six soft corals are still not to be clarified. Additionally, the relation yield of compounds purified form soft corals is too few to carry out apoptosis experiments. This study is a preliminary test for cytotoxic activity of soft corals, and very few correlated researches could be found. At least, these results could provide the useful information to determine whether it is worthy to further isolate the natural product or not.
As previously reported, acylspermidines from the soft coral, Sinularia Sp. showed potent cytotoxicity against A431 cells [22] and NAKATA cells [23], and induced apoptotic DNA fragmentation and condensation of chromatin in A431 cells obtained from SCC [24]. In this study, morphologic alterations, nuclear chromatin condensation, and formation of apoptotic bodies indicate that extracts of soft corals of the genus Sinularia are cytotoxic. The cell cycle distribution demonstrates that extracts sensitized the cells in the G 0 /G 1 and G 2 /M phases with a concomitant significant increase in the sub-G 1 fraction. Experimental results of this work indicate that extracts from soft corals of the genus Sinularia kill not only SCC25, but also HaCaT cells through apoptosis. In summary, these studies demonstrate that the soft corals of the genus Sinularia extracts could be a warrant further research investigating the possible antioral cancer compounds in these medicinal marine organisms of soft corals.