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.

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%.

Human oral squamous cell carcinoma (SCC25) cells was purchased from the American Type Culture Collection (Rockville, Md, USA). Human premalignant keratinocytic cells (HaCaT) were a kind gift from Hamm-Ming Sheu (National Cheng Kung University Medical College, Tainan, Taiwan). Cells were cultured in medium supplemented with 10% fetal bovine serum (Hazelton Product, Denver, Pa, USA) and 1% penicillin-streptomycin at 37?C in 5% CO₂; specifically SCC25 cells in Dulbecco's Modified Eagle's Medium/F12 medium and HaCaT cells in Dulbecco's Modified Eagle's Medium medium (GIBCO, Grand Island, NY, USA).

Cells (1.5 ? 10⁵ 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^?8x + 0.0143; R² = 0.9849; HaCaT: y = 3 ? 10^?8x + 0.0019; R² = 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 ? 10³ to 7 ? 10⁵ 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 cell-density titration-curve construction, which measured cell adhesion.

Cells (1.5 ? 10⁴ cells/well) were seeded in each 100??L 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-di-methyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay according to the manufacturer's procedure (CellTiter 96 AQ, Promega, Madison, Wis, USA). The absorbance 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 IC₅₀ and IC₈₀ 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.

Cells (1.5 ? 10⁵ cells/well) were plated in 24-well plates then treated with IC₅₀ 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??g/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.

Cells (1.5 ? 10⁵) were seeded in 24-well plates and incubated with or without IC₅₀ and IC₈₀ concentrations of extracts for 18 hours. Cells were then fixed in 70% ethanol/PBS, pelleted and resuspended in buffer containing 200??g/mL RNase A and 0.01?mg/mL propidium iodide (PI). The cells were incubated in the dark for 15 minutes at room temperature and then analyzed by FACScan Flow Cytometer (Becton Dickinson, San Jose, Calif, USA). The cell distribution in each phase of the cell cycle was determined using WinMDI software, including subG₁-peak of apoptotic cells.

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 .05 were considered significant for all tests.

To investigate six soft corals of the genus Sinularia extracts (S. grandilobata, S. parva, S. triangula, S. scabra, S. nanolobata and S. gibberosa) inhibited SCC25 and HaCaT cells adhesion, cells were treated with different concentrations (0, 1, 5, 10, 20, 40, 60, and 100??g/mL) of extracts for 18 hours, and the cell adhesion assay was performed. Cells remained firmly attached to the culture dish at low concentrations (<40??g/mL) of extract, and a cytotoxic effect was not observed until almost 70% as indicated in Table 1. The number of attached cells decreased with rising concentrations of extracts (60?100??g/mL). This reveals that high concentration of extracts may affect cell adhesion on collagen fibers, thus increasing cell cytotoxicity. The cell adhesion assay shows that the extract of S. parva was the most effective inhibitor of cell survival and adhesion. However, cell adhesion alone does not indicate that a cell is alive. An enzymatic test such as MTS assay is required to further evaluate the effect of extracts on cell cytotoxicity.

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??g/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 (IC₅₀ and IC₈₀) were determined and are presented in Table 2. The (IC₅₀)s of S. grandilobata, S. parva, S. triangula, S. scabra, S. nanolobata and S. gibberosa were approximately 36.7l, 34.0, 32.2, 38.9, 31.4, and 39.1??g/mL for SCC25, and 33.6, 30.3, 49.1, 26.8, 22.6, and 32.9??g/mL for HaCaT cells. The (IC₈₀)s of six extracts were about 75.9, 71.3, 68.7, 93.0, 70.7, and 127.1??g/mL for SCC25, and 64.7, 54.2, 80.6, 70.5, 62.5, and 74.7??g/mL for HaCaT cells. The cell cytotoxicity assay demonstrates that S. parva and S. nanolobata exhibited the highest potency in inhibiting cell growth, and the results are corresponded to observe with cell adhesion assay.

A morphological study of SCC25 and HaCaT cells 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 IC₅₀ 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.

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 IC₅₀ and IC₈₀ concentrations of extracts reveal that the main character of apoptosis is the cleavage of nuclear DNA into multiple fragments and reflected G₀/G₁ and S-G₂/M phase together with a dose-dependent increase in sub-G₁ phase (corresponding to apoptotic cells). As shown in Table 3, the percentage of G₀/G₁, S, and G₂/M phases in SCC25 cells incubated with extracts (IC₈₀) 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-G₁ fraction of apoptotic SCC25 cells following incubation with (IC₈₀)-treated cells was around 36.8?79.6 times that of control cells. The percentage of sub-G₁ phases in (IC₈₀)-treated HaCaT cells was approximately 65.3?91.1 times that in untreated control cells. The G₀/G₁ and G₂/M phase fractions were, respectively, about 9.2?25.7% and 46.0?76.4% less in the (IC₈₀)-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 G₀/G₁ and G₂/M phases to apoptosis.