Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 292873, 9 pages
CI431, an AqueousCompoundfromCionaintestinalisL.,
InducesApoptosis through a Mitochondria-Mediated Pathway in
1Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2Graduate University, Chinese Academy of Sciences, Beijing 100049, China
3College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
Correspondence should be addressed to Xiukun Lin, email@example.com
Received 21 January 2011; Accepted 21 May 2011
Copyright © 2011 Linyou Cheng et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In the present studies, a novel compound with potent anti-tumor activity from Ciona intestinalis L. was purified by acetone
fractionation, ultrafiltration, gel chromatography and High Performance Liquid Chromatography. The molecular weight of the
highly purified compound, designated CI431, was 431Da as determined by HPLC-MS analysis. CI431 exhibited significant
cytotoxicity to several cancer cell types. However, only a slight inhibitory effect was found when treating the benign human
liver cell line BEL-7702 with the compound. To explore its mechanism against hepatocellular carcinoma, BEL-7402 cells were
treated with CI431 in vitro. We found that CI431 induced apoptotic death in BEL-7402 cells in a dose- and time-dependent
manner. Cell cycle analysis demonstrated that CI431 caused cell cycle arrest at the G2/M phase, and a sub-G1 peak appeared after
24h. The mitochondrial-mediated pathway was implicated in this CI431-induced apoptosis as evidenced by the disruption of
mitochondrial membrane potential. The results suggest that the CI431 induces apoptosis in BEL-7402 human hepatoma cells by
intrinsic mitochondrial pathway.
It is now clear that the oceans are not only home to a tre-
mendous diversity of species but that their inhabitants pro-
duce also a wealth of natural products . Since the 1950s,
many structurally diverse natural products with astounding
bioactivities have been discovered from marine organisms
. These compounds are mainly isolated from sessile an-
imals, such as sponges, tunicates, corals, mollusks, and bry-
ozoans [3, 4].
Among sessile animals, tunicates have received the most
attention. More commonly known as Ascidiacea, members
of the class Ascidiacea (Ascidians) are the most highly in-
vestigated tunicates, since they present a benthonic stage in
their life, making their collection easier. The chemistry of
ascidians has become one of the most active fields of marine
natural products; it has been amply demonstrated that these
significant bioactivities. Most of these products fall within
the area of cancer therapy , and a significant number of
ascidian-derived compounds have entered into preclinical
and clinical trials as antitumor agents [3, 6].
Didemnin B, is perhaps the most studied marine natural
product. This cyclic peptide was isolated from the Caribbean
tunicate Trididemnum solidum . Early investigation into
the bioactivity of this compound revealed its strong antipro-
liferative effects in vitro against a variety of human tumor
cell lines. It was developed by NCI and went through phase
II clinical trials but was withdrawn because it proved to be
too toxic. Although didemnin B was never carried into Phase
III trials, activity focused on developing the compound as
a potential cancer treatment helped pave the way for the
rest of the marine-derived products following it into the de-
amide , and ET-743 , all of them were compounds
with efficient antitumor activity isolated from Ascidians.
Hepatocellular carcinoma (HCC) is the fifth most com-
mon cancer , with a 5-year survival rate of less than 5%
and is the fourth leading cause of cancer death worldwide
2Evidence-Based Complementary and Alternative Medicine
00:00 01:1002:20 03:30
05101520 2530 35
MWD E: signal = 280, 16, reference = 360, 100
MWD D: signal = 214, 16, reference = 360, 100
MWD B: signal = 230, 16, reference = 360, 100
Quaternary pump: %B
Figure 1: Elution patterns on column chromatography. The fraction of acetone precipitation was applied to Sephadex G25 column (a).
The active fraction from Sephadex G25 chromatography was applied to P2column (b). The active fraction from P2was further purified by
high-performance liquid chromatography (c). The arrow indicates the active component during the purification.
[13–15]. Its incidence has been increasing over the past
few decades in some areas such as Europe, USA, and East
Asia [16, 17]. Despite the high mortality and frequency
of this cancer, surgical resection is an available option for
only a small proportion of patients because metastases are
often present when the cancer is discovered. In addition,
because of the inherent chemotherapy-resistant nature of
effective at improving patient survival . Thus, novel
strategies and agents, which have greater targeting on HCC
of enormous potential.
Previous studies have shown that several agents derived
from Ascidiae can induce the apoptosis of many cancer cells
. However, Ciona intestinalis L., a selected species in the
present study, has not been studied for its anticancer effects.
Therefore, we attempted to investigate the growth-inhib-
itory and apoptotic effects of components from Ciona
intestinalis L. against human liver cancer cells. A bioguid-
ed isolation was performed to purify the active components
from the species. We found that a component, CI431, was a
potent inhibitor against human hepatoma Bel-7402 cells and
may be developed as a novel class of anticancer agents.
2.1. Materials. Ciona intestinalis L. was obtained from the
Xunshan Fishery Company of Rongcheng, China. The an-
imals were identified by professor Fuhua-li at Institute of
Oceanology, Chinese Academy of Sciences. The human he-
patocellular cancer BEL-7402, human colorectal cancer
HCT116, human cervical cancer Hela cells as well as human
lung adenocarcinoma A549, breast cancer MCF-7, and
human benign liver cell BEL-7702 cells were obtained from
American Type Culture Collection.
2.2. Extraction and Purification of the Compound from Ciona
and incubated at 70◦C for 30min and were then centrifuged
at 8,000rpm for 30min. Next, the supernatants were added
to 3 volumes of acetone at 4◦C for 12h and were cen-
trifuged at 10,000rpm for 30min once more. The acetone
supernatants were collected and then were disposed through
a 5kDa ultrafiltration membrane (Millipore, USA). The
residue with size <5kDa was lyophilized and dissolved in
a small amount of distilled water. The prepared extraction
Evidence-Based Complementary and Alternative Medicine3
5 10 2040
Relative inhibition rate (%)
Figure 2: CI431 inhibited the growth of several types of cancer
cells. Human hepatoma BEL-7402, human colon cancer HCT116,
human breast cancer MCF-7, human cervical cancer Hela cells, and
human lung adenocarcinoma A549 as well as human benign liver
cell BEL-7702 cells were incubated in the absence or presence of
certain concentrations of CI431 for 48h at 37◦C. MTT assay was
performed to determine the growth inhibition of different cancer
cells and benign BEL-7702 cells by CI431. The experiments were
performed more than three times.
solution was applied to a Sephadex-G25 column and eluted
with distilled water.
All of the fractions were collected and analyzed for cyto-
toxicity by MTT assay. The active fraction was lyophilized
and loaded to a P2 Gel (Bio-Rad Laboratories, Inc) column.
The column was eluted with distilled water. The active frac-
tion was pooled and subjected to reverse-phase liquid chro-
matography on a C18 column (Agilent C18 4.6 × 250mm)
using an Agilent apparatus. The chromatography was per-
formed at a flow rate of 0.5mL/min, using 0.1% TFA as
solvent A and 99.9% acetonitrile containing 0.1% TFA as
solvent B. The gradient was 10–60% of solvent B for 50min.
The elution profile was monitored by online measurement
of the absorbance at 214 and 280nm. The fractions were
pooled and freeze dried. All of the fractions were collected
and analyzed by MTT assay.
2.3. Cell Culture. The carcinoma cells BEL-7402, Hela,
HCT116, and benign liver cell BEL-7702 were cultured in
RPMI-1640 medium. The MCF-7 cell lines were cultured in
DMEM medium. The A549 cell lines were cultured in F12
medium. All cells were incubated in media supplemented
with 10% fetal bovine serum in a humidified atmosphere
containing 5% CO2 at 37◦C. The media and sera were
purchased from Sigma Chemical.
2.4. Assessment of Cytotoxicity. The inhibitory effects of the
antitumor agents on the growth of cancer cells as well as
benign cells BEL-7702 were assessed in vitro by MTT assay
. Four thousand cells per well were seeded into a 96-well
microplate. Cells were cultured in 180μL media of RPMI-
1640, 12K, or DMEM for 24h. The purified compound with
different concentrations was added to the medium. After
48h, MTT solution (50μL, 0.5mg/mL) was added into each
well, and the cells were incubated for another 4h. After
adding 150μL DMSO to each aspirated well, the plate was
gently agitated until the color reaction was uniform. The
OD590 was determined by a microplate reader (Bio-Tek
Instruments, USA) with subtraction of background absorb-
2.5. CI431-Induced Morphological Changes of BEL-7402 Cells.
Morphological alterations of BEL-7402 cells after CI431
treatment were investigated using phase contrast microscopy
and SEM. The cells were seeded and cultured in 96-well
plates, as described above. After incubation with 50μg/mL
CI431, the morphology of cells was observed under the
CKX41 phase contrast microscopy (Olympus, Japan) and
photographed at 0, 12, and 24h, respectively. In the SEM
experiment, BEL-7402 cells were grown onto poly-L-lysine-
coated coverslips in 6-well plates for 24h to allow firm
attachment. Then, they were treated with 50μg/mL CI431
and incubated for 0, 12, and 24h. The medium containing
CI431 was removed, and subsequently the cells were fixed in
glutaraldehyde. After fixation overnight at 4◦C, the coverslip
was dehydrated in ethanol and dried in a critical point dryer.
Cells on coverslip were coated with gold and analyzed by the
S-3400N SEM (Hitachi, Japan).
2.6. DAPI (4?-6-diamidino-2-phenylindole) Staining. The
cells BEL-7402 were grown onto poly-L-lysine-coated cov-
erslips in 6-well plates and treated with CI431 as described
above for the SEM assay. Then the medium containing
CI431 was removed, and subsequently the cells were fixed
in paraformaldehyde. After fixation overnight at 4◦C, the
coverslips were stained with DAPI solution followed by
observation with a fluorescence microscope .
2.7. Flow Cytometric Analysis. The cells BEL-7402 were
incubated with 20, 40, and 80μg/mL of CI431 for 24h. The
cells were harvested and fixed in ice-cold 70% (v/v) ethanol
for 24h at 4◦C. After centrifugation, the cell pellet was
resuspended in PBS; the cells were subjected to propidium
iodide (PI) staining and then analyzed by a flow cytometry
(FACSCalibur; BD Biosciences, USA) .
2.8. Mitochondrial-Membrane Potential (Δψm). The JC-1
Mitochondrial Apoptosis Detection Kit was used to detect
Δψm disruption. JC-1 is selectively accumulated within
intact mitochondria to form multimer J-aggregates emitting
fluorescence light at 590nm (red) at a higher membrane
at a low membrane potential. Thus, the fluorescence color of
JC-1 represents mitochondrial-membrane potential, which
can be analyzed by FACS system . According to the
manufacturer’s protocols, cells were seeded in 12-well plates
at a density of 3 × 105cells/mL and treated with CI431 at
doses of 20, 40, and 80μg/mL for 24h. After treatment with
4Evidence-Based Complementary and Alternative Medicine
KYKY-2800B SEM SN:1526
KYKY-2800B SEM SN:1530
KYKY-2800B SEM SN:1532
Figure 3: Morphological analysis of BEL-7402 cells induced by CI431. BEL-7402 cells were grown on poly-L-lysine-coated coverslips for
24h to allow firm attachment and treated with 50μg/mL CI431 for certain time intervals. Cells were fixed on coverslips coated with gold and
analyzed by using the KYKY-2800B SEM. The cells were untreated (a) or treated with CI431 for 12 (b), 18 (c), and 24h (d). The untreated
BEL-7402 cells showed a normal smooth surface. In contrast, the cells treated with CI431 became rounded, and the surface of the cell
membrane was markedly disrupted (Scale bar = 10μm).
CI431 200μL, prewarmed incubation buffer containing
0.2μL MitoCapture was added to each well and plates were
incubated for 15min at 37◦C in a 5% CO2incubator. Then,
cells were analyzed by a fluorescence spectrophotometer (F-
2.9. Statistical Analysis. All experiments were done three
times in triplicate (n = 9), and the results were expressed
as means ± SD (standard deviation). A one-way analysis
of variance (ANOVA) and the Duncan test were used for
multiple comparisons (SPSS program, ver 10.0).
3.1. Extraction and Purification of the Compound from Ciona
intestinalis L. In order to isolate novel antitumor agents
from Ciona intestinalis L., we developed a purification
protocol involving heat-inactivation, acetone precipitation,
chromatography HPCL. Briefly, acetone precipitation, and
ultrafiltration concentration were carried out to obtain
a pool of antitumor agents as previously described. The frac-
tion obtained after acetone precipitation was concentrated
through a 5kDa ultrafiltration membrane. The fraction of
acetone precipitation solution, which contained several ma-
jor compounds below 5kDa, showed strong inhibitory ac-
tivity against several tumor cell lines.
The fraction of acetone precipitation compounds was
applied to a Sephadex-G25 column and eluted with distilled
water (Figure 1(a)). The active fractions were further puri-
fied by P2 column (Figure 1(b)). After gel chromatography,
matography using a C18 column, and purified to homo-
geneity (Figure 1(c)). The purified agent, designated CI431,
was revealed as a single MW by MS.
3.2. Assessment of Cytotoxicity. MTT assays were performed
to investigate the effects of CI431 on the proliferation of
sixcell lines. As shown in Figure 2, CI431 (over 50μg/mL)
had a significant growth-inhibiting effect on the five cancer
cell lines and a slight growth-inhibiting effect on the benign
liver BEL-7702 cell line. Results indicated that CI431 had
Evidence-Based Complementary and Alternative Medicine5
Figure 4: DAPI staining assay. The cells BEL-7402 were grown on poly-L-lysine-coated coverslips in 6-well plates and treated with CI431.
After incubation for 24, the cells were stained with DAPI and then observed under the fluorescence microscopy. BEL-7401 cells in the
control medium were stained homogeneously with DAPI (a), whereas treatment with CI431 led to marked chromatin condensation and
nuclear fragmentation together with the appearance of small structures like apoptotic bodies. These observations indicated that the cells
treated with CI431 entered apoptosis (b, c).
a significant grow-inhibiting effect on the five cell lines in
a time-dependent manner (data not shown) and in a dose-
dependent manner (Figure 2). Among these cell lines, the
BEL-7402 cells were much more sensitive than the other cell
lines. From this result, BEL-7402 cells were chosen for the
3.3. CI431-Induced Morphological Changes of BEL-7402 Cells.
The morphologic changes of the cell membrane were
clearly visualized by SEM. Remarkable alterations of the
cell membrane of BEL-7402 cells were observed after CI431
treatment. The architecture of untreated BEL-7402 cells
morphology of the cells started to change after incubation
with CI431. The cells detached from the substratum, became
spindle shape (Figure 3(b)), and separated from each other
after exposure to CI431 for 12h. Membrane bulge and de-
tachment from cytoplasmic inclusion were observed in 18h
and 24h after CI431 treatment (Figures 3(c) and 3(d)). The
untreated BEL-7402 cells showed a normal smooth surface.
In contrast, the cells treated with CI431 became rounded,
and the surface of the cell membrane was markedly dis-
3.4. DAPI (4?-6-diamidino-2-phenylindole) Staining. We
characterized the changes in the nuclear morphology by
staining with DAPI. BEL-7401 cells in the control medium
were stained homogeneously with DAPI, whereas treatment
with CI431 led to marked chromatin condensation and
nuclear fragmentation together with the appearance of small
structures like apoptotic bodies, a biochemical hallmark of
apoptosis. The sizes of CI431-treated BEL-7402 cells in-
creased as compared with those of untreated cells. Double
nucleated cells or giant multinucleated cells can be easily
found. These observations indicated that the cells treated
with CI431 entered apoptosis (Figure 4).
3.5. CI431 Induces Apoptosis and G2/S Phase Arrest in
BEL-7402 Cells. To gain an insight into the antiprolif-
eration mechanism of CI431, the cell cycle distribution
of CI431-treated cells was determined by flow cytometry
analysis. The results showed that CI431 significantly induced
6Evidence-Based Complementary and Alternative Medicine
Figure 5: Effect of CI431 on cell cycle distribution. BEL-7402 cells were seeded at 3 x 104/cm2in 10-cm dishes and treated with CI431 at 20
while the RNAs were removed by digestion with RNase A. The DNA contents of the cells were determined with the FACSCalibur cytometer.
The results showed that CI431 significantly induced a G2/S phase arrest with a decrease in G0/G1phase population at 20, 40, and 80μg/mL,
while a dramatic increase of sub-G1 phase (hypodiploid cells) was observed at doses higher than 80μg/mL.
a G2/S phase arrest with a decrease in G0/G1 phase popu-
lation at 20, 40, and 80μg/mL, while a dramatic increase
of sub-G1 phase (hypodiploid cells) was observed at doses
higher than 80μg/mL (Figure 5).
3.6. Mitochondrial-Membrane Potential (Δψm). To investi-
gate the involvement of the mitochondrial pathway, depolar-
ization of the mitochondrion was analyzed by loading with
JC-1. BEL-7402 were exposed to CI431 at doses of 20, 40,
and 80μg/mL for 24h, and the ratio of green fluorescence
intensity to red fluorescence intensity was used for quantita-
tive analysis of the disruption of Δψm. As shown in Figure 6,
after treatment with CI431, Δψm began to decrease, and the
ratio is 0.61 ± 0.07 (blank), 1.92 ± 0.19 (20μg/mL), 5.08 ±
0.45 (40μg/mL), and 8.28 ± 0.61 (80μg/mL), respectively,
indicating disruption of mitochondrial function.
Over 17,000 biologically active compounds have been iden-
tified from marine sources, mainly isolated from sessile an-
imals, such as sponges, tunicates, corals, mollusks, and bry-
ozoans [3, 4]. Many of these substances are potent cytotoxins
that are of great interest for anticancer drug development.
The discovery of new marine drug candidates is a highly
efficient process due to the availability of sophisticated
Evidence-Based Complementary and Alternative Medicine7
500 550 600
Figure 6: Cytofluorometric analysis of Δψm BEL-7402 cells were seeded in 12-well plates at a density of 3 × 105cells/mL and treated
with CI431 at doses of 20, 40, and 80μg/mL for 24h. After treatment with CI431 200μL pre-warmed incubation buffer containing 0.2μL
MitoCapture was added to each well and plates were incubated for 15min at 37◦C in a 5% CO2incubator. Then, cells were analyzed by a
Fluorescence Spectrophotometer (F-4500, HITACHI, Japan). After treatment with CI431, Δψm began to decrease, and the ratio of green
fluorescence intensity to red fluorescence intensity was 0.61 ± 0.07 (blank, a), 1.92 ± 0.19 (20μg/mL, b), 5.08 ± 0.45 (40μLgmL, c) and
8.28 ± 0.61 (80μg/mL, d), respectively, indicating disruption of mitochondrial function.
screening, dereplication, and characterization techniques. In
this study, we describe a novel protocol which allows ef-
ficient and rapid purification of low-molecular weight
marine drug candidates with antitumor activity from Ciona
intestinalis L. We have utilized for their isolation a combina-
tion ofvspace.5ptextraction/purification procedures, includ-
ing heating, acetone fractionation, gel chromatography and
high performance liquid chromatography. The proposed
protocol resulted in obtaining homogeneous preparations
of one compound from Ciona intestinalis L. with MW of
431Da, which exhibited strong antitumor activity. The MW
identity of the purified compound was determined by mass
We used the MTT assay to test the cytotoxic effects of
CI431 and found that CI431 exhibited potent cytotoxicity
against various types of cancer cell lines in a dose- and
time-dependent manner but inhibited the viability of Bel-
7702, human benign liver cells, very slightly. That CI431 has
opposite effects on tumor cells and normal cells is intriguing.
cells. When the cells were stained with different concentra-
tions of CI431 for 48h, marked morphological changes were
clearly observed, including chromatin condensation, nuclear
and cytoplasmic fragmentation, and apoptotic body appear-
ance. Further studies using PI staining and flow cytometry
analysis showed that CI431 significantly induced a G2/S
8Evidence-Based Complementary and Alternative Medicine
phase arrest with a decrease in G0/G1 phase population, and
17.3% hypodiploid cells were observed when the dose up to
60μg/mL. Considering figures of apoptosis bodies obtained
from staining with DAPI and 17.3% hypodiploid BEL-
7402 cells reserved from flow cytometry, we concluded that
CI431 could induce the apoptosis of BEL-7402 cell lines. In
addition, marked mitochondrial-membrane-potential chan-
ges were clearly observed especially after treatment with
0.1mg/mL of CI431 for 24h. Loss of mitochondrial-mem-
brane potential (ΔΨm) is an early event in apoptosis. It
indicated that maybe CI431 induced BEL-7402 cell apoptosis
through a mitochondria-mediated apoptosis pathway.
This study describes for the first time an efficient method
for the purification of a kind of antitumor compound from
Ciona intestinalis L. It could effectively induce apoptosis in
HCC cell lines, mediated through a disruption of mitochon-
drial membrane potential. Our results suggest that CI431
is a promising drug candidate for the treatment of HCC.
However, the specific mechanism of the apoptosis-inducing
effect of CI431 has not yet been elucidated. The effective and
further characterized. Because some secondary metabolites
are generated in organisms just in a very short period of their
how to obtain enough chemicals for structure analyzing and
preclinical testing has become a crucial problem in marine
medicine research. In this study, the yield rate of CI431 from
Ciona intestinalis L. was extremely low. In order to elucidate
its structural formula, more samples should be collected,
isolated, and purified.
This study was supported by 863 High Technology Project
(no. 2007AA091403 and 2007AA09Z408) from the Chinese
Ministry of Science and Innovative Drug Development
Projects of China (2009ZX09103-661 and 2009ZX09102).
The authors are grateful to Dr. Chunguang Wang at the
College of Life Science and Technology, Tongji University for
her carefulreview of the study, and also to all members of the
laboratory for their continuous technical advice and helpful
discussion. They would also like to thank Dr. Fred Bogott at
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