Extracts from Cladiella australis, Clavularia viridis and Klyxum simplex (Soft Corals) are Capable of Inhibiting the Growth of Human Oral Squamous Cell Carcinoma Cells

Many biomedical products have already been obtained from marine organisms. In order to search more therapeutic drugs against cancer, this study demonstrates the cytotoxicity effects of Cladiella australis, Clavularia viridis and Klyxum simplex extracts on human oral squamous cell carcinoma (SCC4, SCC9 and SCC25) cells using cell adhesion and cell viability assay. The morphological alterations in SCCs cells after treatment with three extracts, such as typical nuclear condensation, nuclear fragmentation and apoptotic bodies of cells were demonstrated by Hoechst stain. Flow cytometry indicated that three extracts sensitized SCC25 cells in the G0/G1 and S-G2/M phases with a concomitant significantly increased sub-G1 fraction, indicating cell death by apoptosis. This apoptosis process was accompanied by activation of caspase-3 expression after SCC25 cells were treated with three extracts. Thereby, it is possible that extracts of C. australis, C. viridis and K. simplex cause apoptosis of SCCs and warrant further research investigating the possible anti-oral cancer compounds in these soft corals.


Introduction
Marine organisms are a major reservoir of bioactive natural products with potential biomedical application; several marine natural products are regarded as potential therapeutic agents for the treatment of multiple disease categories [1]. Many bioactive marine natural products and their derivatives are produced by invertebrates, such as sponges, soft corals, tunicates, mollusks or bryozoans, and are evaluated advancedly in preclinical and even clinical trials [1,2]. Moreover, from 2005 to 2007, two of 13 natural products and natural products-derived drugs approved marketing worldwide are found from marine organisms [3]. These facts attract us to pay more efforts on the research of bioactive natural products from other marine invertebrates.
Though seemingly defenseless, soft corals produce various chemical substances to discourage predation, instead of a protective calcium carbonate skeleton of hard corals as a defense mechanism. These chemicals are toxic to either predators or some neighboring hard corals, and some of these substances may have properties that can be beneficial to humans too. Because the geographic setting of Taiwan, between the West Pacific Ocean and East China Sea and at the crossroads of the Philippine-Japan I. Arc, has produced reefs of special biogeographical interest, three kinds of Formosan soft corals, including Cladiella australis (Alcyoniidae), Clavularia viridis (Clavulariidae) and Klyxum simplex (Alcyoniidae), were collected to be evaluated for biomedical research.
Oral cancer is a major global public health problem, causing high morbidity and mortality that have not improved in decades [4]. Squamous cell carcinomas (SCCs) are the most common type of oral cancer. Although new surgery techniques and adjuvant measures, such as chemotherapy and radiotherapy, have progressed, patients with advanced oral SCCs still have a poor prognosis, with a 5-year survival rate of 65% [5]. To develop new methods and improve existing protocols for diagnosis and treatment of SCCs become mandatory.
In the last decade, the manipulation of apoptosis (programmed cell death) has received considerable attention as a novel and promising strategy for cancer chemoprevention and therapy [6,7]. Cell apoptosis is characterized and involved by a series of typical morphological events, for example membrane blebbing, shrinkage of the cell, chromatin condensation, nuclear fragmentation, fragmentation into membrane-bound apoptotic bodies translocation of membrane phosphatidylserine and sub-G 1 fraction, and rapid phagocytosis by neighboring cells [8]. Effective cancer therapeutic strategies often rely on preferential and efficient induction of apoptosis in tumor cells. The objective of this research was to evaluate the capability of ethyl acetate extracts of C. australis, C. viridis and K. simplex to inhibit the growth and induce cell apoptosis on human oral SCCs cells.

Tissue extraction and sample preparation
The soft corals of C. australis, C. viridis and K. simplex were collected 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, Kaohsiung, Taiwan. The tissues were freeze-dried and then exhaustively extracted with ethyl acetate. The ethyl acetate extracts were then filtered and concentrated under vacuum to provide a brownish semisolid crude extract. Organic extracts were dissolved freshly in dimethylsulfoxide (DMSO) and the final concentration of DMSO did not exceed 0.1% (v/v) throughout the experiments.

Cell culture and treatment
Human oral SCCs cells (moderate differentiation of SCC4, poor differentiation of SCC9 and well differentiation of SCC25) were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium supplemented with 0.4 μg/ml hydrocortisone. All culture cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in medium supplemented with 10% fetal bovine serum (Hazelton Product, Denver, PA, USA) and 1% penicillin-streptomycin at 37°C in 5% CO 2 humidified atmosphere. At various concentrations after the treatment, the cells were processed for the analyses of cell adhesion, cytotoxicity, morphology changes, cell cycle, and apoptosis.

Measurement of cell adhesion
Cells (1.5 × 10 5 cells/well) were subcultured into 24-well plates and incubated. After 24 h of incubation, the medium was changed by adding medium containing 1% bovine serum albumin (BSA) and with or without serial concentrations of extracts for 18 h. Attached cell number was estimated using a DNA carmine-based colorimetric method [9]. 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, 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 above [9]. A relationship between the cell number and resultant absorbance after the colorant extraction, for each cell density, was accomplished and cell-density titration-curve construction, which measured cell adhesion.

Assessment of cell morphological changes
Cells (1.5 × 10 5 cells/well) were plated in 24-well plates then treated with IC 50 concentrations of extracts for 18 h. After incubation, the medium was removed and cells were fixed in 4% paraformaldehyde and permeabilized in saponin (0.1% v/v in PBS-BSA). To assess specific apoptosis, Hoechst 33342 (1 μg/ml) (Promega, USA) was added to each well and further incubated at 37°C for 30 min 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
Cells (1.5 × 10 5 cells/well) were seeded in 24-well plates and incubated with or without IC 50 concentration of extracts for 18 h. Cells were then fixed in 70% ethanol/PBS, pelleted and resuspended in buffer containing 200 μg/ml RNase A and 0.01 mg/ml propidium iodide (PI). The cells were incubated in the dark for 15 min at room temperature and then analyzed by FACScan flow cytometer (Becton Dickinson, San Jose, CA). The cell distribution in each phase (sub-G 1 , G 0 /G 1 , S and G 2 /M phases) of the cell cycle was determined using Windows Multiple Document Interface software (WinMDI), including subG 1 -peak of apoptotic cells.

Determination of the activation of caspase-3 expressions
Cells were treated with IC 80 concentration of extracts for 18 h and the expressions of caspase-3 were studied. Cells were stained with mouse anti-cleaved caspasae-3 monoclonal antibodies (1:300) (Santa Cruz, CA) in 1× PBS containing 0.5% BSA (PBS-BSA) and 0.1% sodium azide (Sigma-Aldrich) for 45 min at 4°C. Cells were then washed twice with cold PBS and incubated with FITC-conjugated anti-mouse IgG (1:500) (Santa Cruz, CA) at 4°C for 30 min. Cells were washed with cold PBS and fixed in 4% paraformaldehyde. The cell nuclei were stained with 0.1 μg/ml of Hoechst 33342 (Promega, USA) and inspected using a fluorescent microscope (Nikon, TE2000-U, Japan).

Quantitative analysis of the activation of caspase-3 expressions
Cells were treated with IC 50 and IC 80 concentration of extracts for 18 h and the expressions of caspase-3 were studied. Cells were stained with mouse anti-cleaved caspasae-3 monoclonal antibodies (1:300) (Santa Cruz, CA) in 1× PBS containing 0.5% BSA (PBS-BSA) and 0.1% sodium azide (Sigma-Aldrich) for 45 min at 4°C. Cells were then washed twice with cold PBS and incubated with FITC-conjugated anti-mouse IgG (1:500) (Santa Cruz, CA) at 4°C for 30 min. Cells were washed with cold PBS and fixed in 4% paraformaldehyde. The cell nuclei were stained with 0.1 μg/ml of Hoechst 33342 (Promega, USA). For percentage of fluorescent staining analysis, caspase-3 expressions (Ex, 495 nm; Em, 525 nm) and the cell nuclei (Ex, 346 nm; Em, 460 nm) were measured from three independent experiments by Multi-Detection Microplate Reader (Synergy TM 2, BioTek Instruments, Inc., USA) and calculated using Gene5 TM software.

Statistical analysis
To evaluate the statistical significance of the difference of all the values, statistical analysis was performed on the means of the triplicates of at least three independent experiments using a two-tailed Student's t-test. P values less than 0.05 was considered significant for all tests.

Effect of soft corals extracts on cell adhesion
To investigate the influence of extracts (C. australis, C. viridis and K. simplex) on cell density, constant amounts of extracts (0-100 μg/ml) were treated with SCC4, SCC9 and SCC25 cells for 18 h and then the cell adhesion was analyzed. Carmine, a natural stain is widely used for chromosome staining in cytological studies, and chromosome-specific stain method is highly accurate in a broad spectrum of cell types and cell densities [9]. As shown in Figure 1, the addition of different concentrations (0−100 μg/ml) of extracts to cancer cells for 18 h inhibited cell attachment from the culture dish surface. Although the inhibition of three cell lines generally increases with concentration, the increase is not a linear function of concentration. The C. viridis extract was the most potent inhibitor of both cells adhesion, which is better than those of C. australi and K. simplex. These experimental results indicate that these extracts may influence cell connection on collagen fibers, thus probably raising cell cytotoxicity. To determine further the effects of extracts on cell viability, an enzymatic tetrazolium combined with high-sensitivity test, such as a MTS assay, is necessary.

Effect of soft corals extracts on cells growth
In order to examine cytotoxic effects of soft corals extracts, the MTS assay were carried out using various doses of extracts on SCC4, SCC9 and SCC25 cells. As shown in Table 1

Effect of soft corals extracts on cell morphological changes
To investigate if the viability decrease was due to a specific death type, cellular shape and nuclear morphology of exposed and C. australis, C. viridis and K. simplex extracts-treated SCC4, SCC9 and SCC25 cells were analyzed. As depicted in Figure 2, cells treated with soft corals extracts (IC 50 ) presented morphological characteristics of apoptosis, including chromatin condensation and nuclear fragmentation, were observed under fluorescence inverted microscope. Thereby, the results could imply that three extracts may cause apoptosis of human SCCs cells.

Effect of soft corals extracts on cell cycle distribution and apoptosis assay
To confirm that C. australis, C. viridis and K. simplex extracts mediate SCC25 cell apoptosis, the cell cycle distribution and specific DNA content in the sub-G 1 peak were further investigated with flow cytometric analysis. Treatment of cells with IC 50 concentration of the C. australis, C. viridis and K. simplex extracts suggests that the main characteristic of apoptosis is the cleavage of nuclear DNA into multiple fragments and causing increase in the sub-G 1 phase ( Figure 3A). Incubation with the three extracts showed a typical pattern of DNA content that reflected in the percentage of G 0 /G 1 and S-G 2 /M phases of the cell cycle together with a marked apoptotic sub-G 1 phase in SCC25 cells as listed in Figure 3B.

Effect of soft corals extracts on the expression of caspase-3
To determine whether caspase-3 activity are involved in SCC25 cells apoptosis induced by C. australis, C. viridis and K. simplex extracts, immunofluorescence analysis was performed. Up-regulation of caspase-3 expression upon exposure to IC 80 concentration of extracts in SCC25 was obtained in Figure 4A. A significant decrease in the percentage of DNA content in SCC25 cells with concentration of IC 50 concentration of extracts and was confirmed by cell cytotoxicity. The percentage of caspase-3 expression levels in C. australis, C. viridis and K. simplex extracts (IC 50 and IC 80 )-treated SCC25 cells were around 1.6-2.7, 1.6-3.0 and 1.6-3.9 times, compared with untreated control cells ( Figure 4B). Hence, these experimental results suggest that up-regulation of caspase-3 expressions by the extracts may partially account for the cell apoptosis phenomenon in SCC25 cells. Figure 4. Expression of caspase-3 in SCC25 cells after treatment with C. australis, C. viridis and K. simplex extracts. (A) An IC 80 concentration of extracts was added to SCC25 cells and the expression of caspase-3 were determined by immunofluorescent analysis as described in the Methods Section. (B) The IC 50 and IC 80 concentrations of extracts were added to SCC25 cells and the percentage of fluorescent stained caspase-3 expression levels were analyzed by Synergy TM 2 and calculated by Gene5 TM software. Each value represents the mean ± S.D. from three independent experiments. * Indicates P < 0.05, ** indicates P < 0.01, paired Student's t-test.

Discussion
Coral reef is a basic and important biomass in ocean. The ecological factors in coral reef, such as species competition and limiting resources in space and light, lead soft corals to develop a delicate chemical balance for self protection, range from chemical defense against predation to intra-specific cues for larval settlement. Meanwhile, some of these secondary metabolites have also shown potential as new medicines for the treatment of a variety of human diseases. During the past decades, intense attention has been focused on the anti-tumor property of marine natural products and their derivatives, such as pachymatismin from the marine sponge (Pachymatisma johnstonii), didemnin B from a Caribbean tunicate (Trididemnum solidum) and bryostatins from the bryozoan (Bugula neritina) [10]. Soft corals (coelenterata, octocorallia, alcyonaceae) are a rich source of steroids and terpenoids [2,11], in which most isolated diterpenes are cembranolides [12]. In addition, marine eicosanoids, such as clavulones, halogenated clavulones and prostanoids, have attracted much interest because of their novel structures and significant antitumor, antileukemic effect and antiviral activities [13]. These prostanoids have been found in the different species of marine organism such as C. viridis, Telesto riisei, and Gracilaria sp. [14]. Additionally, the C. viridis is rich in bioactive prostanoids with different structural types such as preclavulone lactones, clavirins, tricycloclavulone, and clavubicyclone [14,15].
The prostanoids have been suggested to display anti-tumor activities in several types of human tumors [14][15][16]. Recently, several new marine prostanoids isolated from C. viridis among these prostanoids, bromovulone III showed inducing of Fas clustering [17], enhancing of endoplasmic reticulum stress [10] and promising anti-tumor activity and apoptosis in human hormone-resistant prostate cancers and human hepatocellular carcinoma cells [10,17]. Previous reports have shown that eunicellin-based diterpenoids, cladiellane diterpenes and australins A-D from the ethanol extract of C. australis has cytotoxic activity against human breast cell lines (MCF-7, MDA-MB-231) and liver cell lines (HepaG2/DMEM-12) [18]. However, none thorough cytotoxicity research has been performed on K. simplex and the mechanisms underlying the anti-oral cancer efficiency of C. australis, C. viridis and K. simplex extracts and their secondary metabolites are poorly understood. Additional research is required to confirm the inhibitory effects and action mechanisms of C. australis, C. viridis and K. simplex extracts on different cancer cells and possible clinical use. This study shows the action mechanism of C. australis, C. viridis and K. simplex organic extracts in the human oral SCCs cells.
Cell density and cell proliferation is usually evaluated during in vitro studies to pharmacological effects of specific compounds [9]. In this study, cell adhesion and MTS assay revealed that C. australis, C. viridis and K. simplex extracts impeded SCC4, SCC9 and SCC25 cells growth in a concentration-dependent manner. The C. viridis was the most potent inhibitor of cells adhesion and exhibited the activity in inhibiting cell growth; that is, better than those of C. australis and K. simplex. Nevertheless, the intrinsic structure and properties of these three 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.
Although 5-fluorouracil, 5% imiquimod cream, and 3% diclofenac gel are available as agents for topical skin cancer field therapies. Nevertheless, previous reports have shown that a successful treatment of topical 5-fluorouracil cream is inevitably accompanied by pain, pruritus, burning, erythema, erosion, and scar, not only in diseased skin but also occurred in peripheral normal skin [19]. In addition, previous reports have demonstrated that clinical drugs such as 5-fluorouracil and imiquimod incubated with cancer cell lines exhibited obvious cytotoxicity after treatment extension to 72 h. The concentrations of 5-fluorouracil that induced cell death by 50% (IC 50 ) were approximately 46.3 μg/ml for SCC25 cells (data not shown). Therefore, no significantly cytotoxic difference between these extracts and 5-fluorouracil could be found at the same and short interaction time for 18 h. Maybe the more deeply research is necessary in the near future to realize the true effect of these compounds on oral cancer cells.
Cancer is characterized by deregulated cell proliferation combined with suppressed apoptosis [20]. Most chemotherapeutic drugs exert their cytotoxic action by inhibiting cancer cell growth and inducing apoptosis [21]. Previous studies have demonstrated that Sinularia sp. extracts exhibited 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 demonstrate that C. australis, C. viridis and K. simplex extracts cause apoptosis of SCCs cells.
Cells contain various pathways designed for protection from the genomic instability or toxicity that can result when DNA is damaged. Checkpoint proteins that control normal passage of cells through the cell cycle play a pivotal role of this response. During tumorigenesis, tumor cells frequently loose checkpoint controls, which facilitate tumor development. Therefore, one important approach for cancer chemotherapy is to regulate cell-cycle progression. Previous reports have shown that clavulone and the halogenated analogues are anti-tumor prostanoids isolated from the C. viridis. It has been reported that clavulones show the antitumor activities against human leukemia HL-60 cells through the arrest of the cell cycle progression from G 1 to S phase [25]. In this study, the cell cycle distribution indicates that C. australis, C. viridis and K. simplex extracts sensitized cells in the G 0 /G 1 and S-G 2 /M phases with a concomitant significant increase in the sub-G 1 phase. Additionally, the caspases family can be divided into major subgroups based on their substrate-specificity, sequence homology and biochemical functions; particularly, caspase-3 plays a pivotal role in the terminal phase of apoptosis [26]. This study has revealed that C. australis, C. viridis and K. simplex extracts may accelerate caspase-3 activation, thus resulting in apoptosis of SCC25 cells. This study first presented evidence that human oral SCCs cells are sensitive to ethyl acetate extracts from C. australis, C. viridis and K. simplex. In conclusion, the present evidence suggests that C. australis, C. viridis and K. simplex extracts exerts its cytotoxicity effect through inhibition of the cell cycle and induction of apoptosis underwent activation of caspase-3 in SCCs cells. But the exact pathway such as what is the receptor involved in the caspase-3 activation is still unclear in these studies. Apparently, further investigations are needed for future studies.