# Cytotoxic Effect of the GenusSinularia Extracts on  Human SCC25 and HaCaT Cells

Authors: Guey-Horng Wang; Tzung-Han Chou; Rong-Jyh Lin; Jyh-Horng Sheu; Shih-Hao Wang; Chia-Hua Liang
Journal: Journal of Toxicology
 (2009)

---

## Abstract

Soft corals of the
genusSinularia 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.


---

## Body

## 1. 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 [5–8]. Many of these
metabolites have been found to be cytotoxic, or to possess other biological
activities [9–11]. 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 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-G1 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).


## 2. Materials and Methods

### 2.1. Material

The six soft corals of the genusSinularia 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.


### 2.2. Preparation of Extracts

The tissues of six soft corals of the
genusSinularia 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%.


### 2.3. Cell Lines and Cell Culture

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).


### 2.4. Cell Adhesion Assay

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.


### 2.5. Growth-Inhibition Assay

Cells (1.5 × 104 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-di-methyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-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.


### 2.6. Determination of Morphological  Changes of Cells

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 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.


### 2.7. Assessment of Cell-Cycle Distribution and Apoptotic Cells
by Flow Cytometry

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 μg/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.


### 2.8. 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’st-test. P values less than .05 were considered significant for all tests.


## 2.1. Material

The six soft corals of the genusSinularia 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.


## 2.2. Preparation of Extracts

The tissues of six soft corals of the
genusSinularia 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%.


## 2.3. Cell Lines and Cell Culture

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).


## 2.4. Cell Adhesion Assay

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.


## 2.5. Growth-Inhibition Assay

Cells (1.5 × 104 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-di-methyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-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.


## 2.6. Determination of Morphological  Changes of Cells

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 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.


## 2.7. Assessment of Cell-Cycle Distribution and Apoptotic Cells
by Flow Cytometry

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 μg/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.


## 2.8. 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’st-test. P values less than .05 were considered significant for all tests.


## 3. Results

### 3.1. Influence of the GenusSinularia Extracts on Cells Adhesion

To investigate six soft corals of the
genusSinularia 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 μg/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 μg/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 μg/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.Table 1
Percentage of SCC25 and HaCaT cells adhesion by different concentrations of the
genusSinularia extracts.
Cell linesTreatment (μg/mL)No. 123456SCC250100.0±0.9100.0±1.0100.0±0.6100.0±3.4100.0±2.2100.0±1.0174.7±1.0118.5±0.9105.1±8.794.1±5.8110.9±7.7101.7±6.7570.4±1.971.3±4.879.7±2.994.0±1.970.4±6.794.9±2.51070.4±1.970.4±3.860.3±3.881.4±2.970.4±1.988.2±8.62071.3±2.969.6±6.759.5±6.799.1±3.871.3±8.688.1±8.54070.4±3.866.2±2.960.3±3.881.4±2.971.3±0.981.6±2.96053.5±7.338.3±0.151.0±2.951.0±6.766.2±2.963.7±1.910041.7±1.921.5±0.344.3±0.953.5±1.555.2±3.849.3±8.7HaCaT0100.0±1.8100.0±1.6100.0±3.4100.0±0.9100.0±6.2100.0±5.4178.4±1.889.9±2.395.7±2.599.3±3.797.4±4.5100.0±2.7580.4±0.991.9±3.797.5±3.495.9±0.988.9±7.284.3±5.31079.1±0.983.1±7.693.8±0.188.6±1.886.3±9.874.5±1.82080.4±8.283.8±8.680.9±0.879.9±2.882.4±1.869.3±1.84077.7±2.981.1±1.874.7±2.575.2±3.779.8±1.869.3±5.46075.0±4.644.7±7.463.6±0.975.9±2.073.9±1.770.6±0.510064.2±8.843.3±9.360.5±0.261.8±2.945.8±4.451.7±4.5(i) Results are the average of three independent experiments.
(ii)S. grandilobata, 1; S. parva,
2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5; S. gibberosa, 6.


### 3.2. Growth-Inhibition Assay Effect of  the GenusSinularia Extracts

MTS
assay was conducted to examine the relationship between concentrations of the
genusSinularia 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
(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 μg/mL for SCC25, and 33.6, 30.3, 49.1, 26.8, 22.6, and 32.9 μg/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 μg/mL for SCC25, and 64.7, 54.2, 80.6, 70.5, 62.5, and 74.7 μg/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.Table 2
Cell viability of the genusSinularia extracts in SCC25 and HaCaT cells.
No.Cell linesSCC25HaCaTIC50 (μg/mL)IC80 (μg/mL)IC50 (μg/mL)IC80 (μg/mL)136.7±5.675.9±3.333.6±3.164.7±1.8234.0±2.571.3±0.930.3±4.254.2±2.9332.2±2.968.7±1.849.1±2.380.6±3.7438.9±1.593.0±4.226.8±5.070.5±3.6531.4±6.870.7±3.122.6±2.862.5±4.8639.1±1.9127.1±8.732.9±3.774.7±1.5(i) Results are the average of three independent experiments.
(ii)S. grandilobata, 1; S. parva,
2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5; S. gibberosa, 6.Dose-dependency effects of the genusSinularia extracts on the cell growth inhibition of SCC25 and HaCaT cells. Percentage of viable in cells
treated with 0–100 μg/mL concentrations of Sinularia extracts for 18 hours and determined by MTS assay. Data are means ± S.D. from three independent
experiments. (•) S. grandilobata, 1; (∘) S. parva, 2; (▾) S. triangula, 3; (▿) S. scabra, 4; (▪) S. nanolobata, 5; and (□) S. gibberosa, 6.
(a)(b)


### 3.3. Impact of the GenusSinularia Extracts on
Cell Morphology Changes

A morphological study of SCC25 and HaCaT cells was undertaken to obtain
additional information about the cytotoxicity of soft corals of the genusSinularia 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.Figure 2
Morphological changes in SCC25 and HaCaT cells after the genusSinularia extracts treatment. A constant concentration of extracts (IC50) was
added to the cells for 18 hours. The change was inspected by a
phase-contrast-inverted microscopy. The cells were then fixed in 4%
paraformaldehyde and DNA
stained with Hoechst. The nuclei of the cells were visualized using a
fluorescent microscope (200×). S. grandilobata, 1; S. parva, 2; S. triangula,
3; S. scabra, 4; S. nanolobata, 5; and S. gibberosa, 6.


### 3.4. Influence of the GenusSinularia 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 genusSinularia 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.Table 3
The changes of cell
cycle distribution of the genusSinularia extracts in
SCC25 and HaCaT cells.
Cell linesNo.Treatment (μg/mL)Sub-G1 phaseG0/G1 phaseS phaseG2/M phaseMean%Mean%Mean%Mean%SCC25Control01.0100.052.3100.019.8100.027.2100.01IC5032.53250.037.772.116.583.313.850.7IC8064.16410.021.941.97.336.96.825.02IC5018.71870.046.889.415.779.319.872.8IC8058.95890.024.146.18.341.98.832.43IC5023.62360.054.3103.812.261.610.538.6IC8069.86980.018.635.66.834.44.817.64IC5033.43340.037.772.114.472.714.854.4IC8080.18010.013.425.63.618.23.111.45IC5024.32430.051.197.712.563.112.546.0IC8036.83680.040.477.212.864.610.337.96IC5033.73370.032.862.715.578.318.367.3IC8079.67960.012.423.74.422.23.613.2HaCaTControl00.8100.059.5100.016.210023.7100.01IC5035.34412.534.357.613.180.916.770.5IC8069.48675.06.711.37.948.816.167.92IC5019.42425.042.871.917.8109.920.385.7IC8052.36537.515.325.715.193.217.473.43IC5025.13137.545.276.016.4101.213.958.6IC8056.17012.513.222.216.110015.163.74IC5039.14887.538.264.213.180.99.640.5IC8071.38912.58.113.69.961.110.946.05IC5035.84475.035.459.515.495.113.858.2IC8058.97362.511.920.014.388.315.163.76IC5057.47175.010.217.112.879.019.783.1IC8072.99112.55.59.28.351.213.356.1(i)S. grandilobata, 1; S. parva, 2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5; S. gibberosa, 6.Figure 3
Effect of the genusSinularia extracts on SCC25 and HaCaT cells
apoptosis. Flow cytometric analysis of the cell cycle distribution of cells
after treatment with extracts (IC50 and IC80) for 18 hours
as described in Materials and Methods. S. grandilobata, 1; S. parva, 2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5 and S. gibberosa, 6.


## 3.1. Influence of the GenusSinularia Extracts on Cells Adhesion

To investigate six soft corals of the
genusSinularia 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 μg/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 μg/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 μg/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.Table 1
Percentage of SCC25 and HaCaT cells adhesion by different concentrations of the
genusSinularia extracts.
Cell linesTreatment (μg/mL)No. 123456SCC250100.0±0.9100.0±1.0100.0±0.6100.0±3.4100.0±2.2100.0±1.0174.7±1.0118.5±0.9105.1±8.794.1±5.8110.9±7.7101.7±6.7570.4±1.971.3±4.879.7±2.994.0±1.970.4±6.794.9±2.51070.4±1.970.4±3.860.3±3.881.4±2.970.4±1.988.2±8.62071.3±2.969.6±6.759.5±6.799.1±3.871.3±8.688.1±8.54070.4±3.866.2±2.960.3±3.881.4±2.971.3±0.981.6±2.96053.5±7.338.3±0.151.0±2.951.0±6.766.2±2.963.7±1.910041.7±1.921.5±0.344.3±0.953.5±1.555.2±3.849.3±8.7HaCaT0100.0±1.8100.0±1.6100.0±3.4100.0±0.9100.0±6.2100.0±5.4178.4±1.889.9±2.395.7±2.599.3±3.797.4±4.5100.0±2.7580.4±0.991.9±3.797.5±3.495.9±0.988.9±7.284.3±5.31079.1±0.983.1±7.693.8±0.188.6±1.886.3±9.874.5±1.82080.4±8.283.8±8.680.9±0.879.9±2.882.4±1.869.3±1.84077.7±2.981.1±1.874.7±2.575.2±3.779.8±1.869.3±5.46075.0±4.644.7±7.463.6±0.975.9±2.073.9±1.770.6±0.510064.2±8.843.3±9.360.5±0.261.8±2.945.8±4.451.7±4.5(i) Results are the average of three independent experiments.
(ii)S. grandilobata, 1; S. parva,
2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5; S. gibberosa, 6.


## 3.2. Growth-Inhibition Assay Effect of  the GenusSinularia Extracts

MTS
assay was conducted to examine the relationship between concentrations of the
genusSinularia 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
(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 μg/mL for SCC25, and 33.6, 30.3, 49.1, 26.8, 22.6, and 32.9 μg/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 μg/mL for SCC25, and 64.7, 54.2, 80.6, 70.5, 62.5, and 74.7 μg/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.Table 2
Cell viability of the genusSinularia extracts in SCC25 and HaCaT cells.
No.Cell linesSCC25HaCaTIC50 (μg/mL)IC80 (μg/mL)IC50 (μg/mL)IC80 (μg/mL)136.7±5.675.9±3.333.6±3.164.7±1.8234.0±2.571.3±0.930.3±4.254.2±2.9332.2±2.968.7±1.849.1±2.380.6±3.7438.9±1.593.0±4.226.8±5.070.5±3.6531.4±6.870.7±3.122.6±2.862.5±4.8639.1±1.9127.1±8.732.9±3.774.7±1.5(i) Results are the average of three independent experiments.
(ii)S. grandilobata, 1; S. parva,
2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5; S. gibberosa, 6.Dose-dependency effects of the genusSinularia extracts on the cell growth inhibition of SCC25 and HaCaT cells. Percentage of viable in cells
treated with 0–100 μg/mL concentrations of Sinularia extracts for 18 hours and determined by MTS assay. Data are means ± S.D. from three independent
experiments. (•) S. grandilobata, 1; (∘) S. parva, 2; (▾) S. triangula, 3; (▿) S. scabra, 4; (▪) S. nanolobata, 5; and (□) S. gibberosa, 6.
(a)(b)


## 3.3. Impact of the GenusSinularia Extracts on
Cell Morphology Changes

A morphological study of SCC25 and HaCaT cells was undertaken to obtain
additional information about the cytotoxicity of soft corals of the genusSinularia 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.Figure 2
Morphological changes in SCC25 and HaCaT cells after the genusSinularia extracts treatment. A constant concentration of extracts (IC50) was
added to the cells for 18 hours. The change was inspected by a
phase-contrast-inverted microscopy. The cells were then fixed in 4%
paraformaldehyde and DNA
stained with Hoechst. The nuclei of the cells were visualized using a
fluorescent microscope (200×). S. grandilobata, 1; S. parva, 2; S. triangula,
3; S. scabra, 4; S. nanolobata, 5; and S. gibberosa, 6.


## 3.4. Influence of the GenusSinularia 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 genusSinularia 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.Table 3
The changes of cell
cycle distribution of the genusSinularia extracts in
SCC25 and HaCaT cells.
Cell linesNo.Treatment (μg/mL)Sub-G1 phaseG0/G1 phaseS phaseG2/M phaseMean%Mean%Mean%Mean%SCC25Control01.0100.052.3100.019.8100.027.2100.01IC5032.53250.037.772.116.583.313.850.7IC8064.16410.021.941.97.336.96.825.02IC5018.71870.046.889.415.779.319.872.8IC8058.95890.024.146.18.341.98.832.43IC5023.62360.054.3103.812.261.610.538.6IC8069.86980.018.635.66.834.44.817.64IC5033.43340.037.772.114.472.714.854.4IC8080.18010.013.425.63.618.23.111.45IC5024.32430.051.197.712.563.112.546.0IC8036.83680.040.477.212.864.610.337.96IC5033.73370.032.862.715.578.318.367.3IC8079.67960.012.423.74.422.23.613.2HaCaTControl00.8100.059.5100.016.210023.7100.01IC5035.34412.534.357.613.180.916.770.5IC8069.48675.06.711.37.948.816.167.92IC5019.42425.042.871.917.8109.920.385.7IC8052.36537.515.325.715.193.217.473.43IC5025.13137.545.276.016.4101.213.958.6IC8056.17012.513.222.216.110015.163.74IC5039.14887.538.264.213.180.99.640.5IC8071.38912.58.113.69.961.110.946.05IC5035.84475.035.459.515.495.113.858.2IC8058.97362.511.920.014.388.315.163.76IC5057.47175.010.217.112.879.019.783.1IC8072.99112.55.59.28.351.213.356.1(i)S. grandilobata, 1; S. parva, 2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5; S. gibberosa, 6.Figure 3
Effect of the genusSinularia extracts on SCC25 and HaCaT cells
apoptosis. Flow cytometric analysis of the cell cycle distribution of cells
after treatment with extracts (IC50 and IC80) for 18 hours
as described in Materials and Methods. S. grandilobata, 1; S. parva, 2; S. triangula, 3; S. scabra, 4; S. nanolobata, 5 and S. gibberosa, 6.


## 4. 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 Sinularia extracts (S. grandilobata, S. parva, S. triangula, S. scabra, S. nanolobata and S. gibberosa) in the most common type of
human oral squamous cell carcinomas SCC25 cells and human premalignant
keratinocytes HaCaT cells. The cell adhesion and cell viability assays demonstrate the
cytotoxicity effects of six extracts on both cells. Extracts induced
morphological changes of chromatin condensation, DNA fragmentation, and sub-G1 peak in a DNA histogram of SCC25 and HaCaT cells, indicating cell death
by apoptosis.The previously characterized genusSinularia 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, 1-epi-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 concentration-dependent 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 [17–19]. 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 G0/G1 and G2/M phases with a concomitant significant increase in the sub-G1fraction. 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.



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*Source: 634868-2008-10-13.xml*