Cytotoxicity of the venom from the
nematocysts of jellyfish Cyanea nozakii
Kishinouye
Toxicology and Industrial Health
28(2) 186–192
ª The Author(s) 2012
Reprints and permission:
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DOI: 10.1177/0748233711410910
tih.sagepub.com
Li Cuiping1, Li Pengcheng2, Feng Jinhua2,
Li Rongfeng2 and Yu Huahua2
Abstract
In this article, the cytotoxicity of the venom from the nematocysts of jellyfish Cyanea nozakii Kishinouye on
human hepatoma cells (Bel-7402, SMMC-7721) and human colon cancer cells (H630) was investigated first.
Of the three kinds of cells, the venom had the strongest cytotoxicity on H630 cells with the 50% lethal
concentration (IC50) of 5.1 mg/ml. However, the IC50 on Bel-7402 and SMMC-7721 was approximate 17.9 and
24.3 mg/ml, respectively. The cytotoxicity of the venom was affected by pH, temperature and storage conditions. At the pH 4.5–8.5, the venom displayed obvious cytotoxicity and the percentage of survival was about
50%. When pre-incubated at temperatures over 60 C for as short as 10 min, the percentage of survival sharply
improved from 4.6% up to 80%. The venom was stored in a more stable condition at 80 C and in lyophilized
state compared to other storage conditions used in this study. Lactate dehydrogenase release assay performed
on H630 cells indicated that exposure to the venom could result in damage to the cell membrane.
Keywords
Cytotoxicity, jellyfish, nematocysts, venom, Cyanea nozakii Kishinouye
Introduction
The jellyfish, a cnidarian, is known to produce
venoms for food predation and self-defence against
predator. Jellyfish venoms have many bioactive compounds with enzymatic activity, cardiac toxicity,
netrotoxicity, haemolysis, hepatocyte toxicity and
myotoxicity (Burnett and Calton, 1974; Gusmani
et al., 1997; Nagai et al., 2000; Nevalainen et al.,
2004; Radwan et al., 2000; Ramasamy et al., 2005).
Severe stings of human beings by jellyfish are frequently reported, resulting in burning pain, local
oedema, tingle tight breath, blood pressure depression, and even death. Such effects arise from the complex mixture of biologically active molecules that
make up jellyfish venoms (Chung et al., 2001). Therefore, the species and its venom have become a focus
of research for medical purposes. The biochemistry,
pharmacology and toxicology of jellyfish venoms
have been studied since 1960s, being searched for
active components as a new source of medicine. It has
been reported that the venoms are promising in curing
cardiovascular diseases and widely used in nerve
molecular biology (Wang et al., 2002). On the other
hand, the investigation of the bioactivity of jellyfish
venoms could be useful to prevent or decrease syndromes from envenomation of jellyfish. So, the study
on jellyfish venoms will be beneficial for human
health.
Cyanea nozakii Kishinouye (C. nozakii), a cnidarian of the class Scyphomedusae, the order Semaeostomeae and the family Cyaneidae, is a large-sized
plankton. C. nozakii distributes widely in the East
Sea, the Yellow Sea and the Bohai Sea of China and
flourishes in late summer to early autumn. C. nozakii
can seriously damage the fisher environment as its
venom causes the death of halobios (Dong et al.,
1
Ocean University of China, Qingdao College, Qingdao, China
Institute of Oceanology, Chinese Academy of Sciences, Qingdao,
China
2
Corresponding author:
Yu Huahua, Institute of Oceanology, Chinese Academy of
Sciences, 7 Nanhai Road, Qingdao 266071, China
Email: [email protected]
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Cuiping et al.
187
2005; Zhong et al., 2004). And it has garnered
attention because of its stinging capacity and the
resulting public health hazard. Recently, the haemolytic activity and lethal activity of the venom of C. nozakii (Feng et al., 2010) have been studied, but its
cytotoxicity has never been reported.
In recent years, the cytotoxicity assays have been
used to assess the toxicity of jellyfish venoms on different cells. Carli et al. (1996) reported that Aequorea
aequorea venom greatly affected the growth rate of
V79 cells during long-term experiments. And Rhizostoma pulmi venom showed remarkable cytotoxicity
on V79 cells and killed all treated cells at the concentration of 15,000 nematocysts/ml within 2 and 3 h.
Radwan et al. (2000) reported that the fishing tentacle
plus lappets nematocyst venom of Chrysaora achlyos
showed minimal cytologic damage to human liver
cells (STCC CCL-13) after 30 s exposure to 10 mg
protein/ml. On the other hand, the syndrome, skin
necrosis, induced by envenomation of jellyfish venom
was probably in connection with the cytotoxicity. The
present work is the first to report on the cytotoxicity of
the venom from jellyfish C. nozakii and the effect of
some factors such as pH, temperature and storage
conditions on the cytotoxicity. The aim of this study
was to verify and characterize the in vitro cytotoxicity
of the venom from the nematocysts of C. nozakii. This
will provide references for finding new anti-tumour
bioactive components from marine animals.
Materials and methods
Nematocyst preparation
The jellyfish C. nozakii was collected at the First
Bathing Beach in Qingdao, Shandong Province,
China, in August 2007. Tentacles were excised immediately and stored at 20 C until use. The preparation
of nematocysts was according to the method of Bloom
et al. (1998). Briefly, the refrigerated tentacles were
dipped in distilled sea water (v/v, 1:1) for 1 to 5 days
at 4 C. Each day the tentacles were stirred softly and
the suspension was filtered through a fine kitchen
sieve. The filtered fluid was settled for 24 h at 4 C.
Then, the final sediments were washed with sea water
by centrifugation at 15,000g for 20 min.
Venom preparation
Nematocysts were resuspended in cold Tris-HCl buffer (pH 7.8, 10 mM) and sonicated on ice for 20-s
periods at 50 MHz. Then, the suspension was clarified
by centrifugation at 15,000g for 20 min at 4 C and
used as crude extract from nematocysts of jellyfish
C. nozakii (CNN). Sample concentrations were determined by the method of Bradford (1976), using
bovine serum albumin (BSA) as a standard.
Cytotoxicity assay
Bel-7402, SMMC-7721 and H630 cells were used to
evaluate the cytotoxicity of CNN in the present article. Cytotoxicity was carried out by the method of
Chong et al. (2000) with a slight modification. Cells
were cultured in RPMI 1640 supplemented with
10% foetal bovine serum, 2 mM L-glutamine, 100
U/ml penicillin, 100 mg/ml streptomycin at 37 C with
5% CO2 atmosphere. Assays on cytotoxic effects
were conducted in 96-well flat-bottomed microtitre
plates. Cultured cells (90 ml, 5000 cells) were added
into a set of wells. Blank wells contained 90 ml of supplement media instead of cultured cells. After incubation for 24 h, 10 ml of different concentrations of CNN
was added to each well except for the control wells.
Control wells contained 10 ml of Tris-HCl buffer
(pH 7.8, 10 mM) instead of CNN. Then, microtitre
plates were incubated at 37 C with 5% CO2 atmosphere for 12, 24 and 48 h.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Assessment of cell viability was according
to the method of Mosmann (1983). At the end of each
incubation period, 20 ml of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml
in 10 mM, pH 7.4 phosphate buffer saline) were added
into each well and the cultures were further incubated at
37 C with 5% CO2 atmosphere. After 4 h, 100 ml of
dimethyl sulfoxide (DMSO) was added into the wells
to solubilize the formazan crystal products. The microtitre plates were incubated at 37 C for 10 h to make the
formazan crystals solubilizable. The absorbance was
measured at 490 nm using a microtitre plate reader. The
percentage of survival was estimated as the percentage
of control absorbance of reduced dye at 490 nm.
% survival ¼ ½ðAS AB Þ=ðAC AB Þ 100
where AB is the absorbance of the blank (without cultured cells), AC is the absorbance of the control (without CNN) and AS is the absorbance of mixture
containing cultured cells and CNN.
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188
Toxicology and Industrial Health 28(2)
cooling at 4 C and incubation with CNN for 12 h, the
cytotoxicity was determined according to the method
mentioned above.
The venom was stored for various days (2, 4, 6, 9
days) in liquid state at 4, 20, 80 C and in the lyophilized state at 80 C. Then, the cytotoxicity was
determined after incubation with CNN for 12 h
according to the method mentioned above.
(a)
100
SMMC 7721-48 h
80
H630-48 h
Bel 7402-64 h
%survival
60
40
20
Lactate dehydrogenase assay
0
0
20
40
60
80
The concentration of CNN (µg/ml)
(b)
100
12 h
%survival
80
24 h
48 h
60
40
20
0
0
20
40
60
80
The concentration of CNN (µg/ml)
Figure 1. (a) Effects of CNN on the production of formazan in three cell lines after incubation with different concentrations of venom for 48 or 64 h. (b) Effects of CNN
on the production of formazan in H630 cell lines after incubation with different concentrations of venom for 12, 24
and 48 h. Data were presented as means + SD of three
parallel measurements.
Cell membrane damage was carried out by the
method of Chong et al. (2000). After incubation with
CNN for a period of time, 100 ml of the media overlaying the cells was removed to assay for lactate dehydrogenase (LDH) released in the medium. Then, 100 ml of
1% (v/v) Triton X-100 was added into these wells to
lyse the cells for 10 min. Contents in the wells were
suspended by repeated piping and removed from wells
and assayed for cellular LDH. To determine the
quantity of LDH in a specific sample, the sample was
centrifuged (at 4 C at 7000 g for 5 min) to remove
cells or debris. Into each well of a UV-transparent
microtitre plates, 180 ml of 0.1 M phosphate butter at
37 C, 30 ml of samples, 10 ml of NADH (nicotinamide
adenine dinucleotide-reduced) solution (2.5 mg/ml)
and 30 ml of pyruvic acid solution (1 mg/ml) were
added in sequence. The decrease in absorbance at
340 nm was measured for 3 min. The above procedures
were used to assay both released and cellular LDH. The
data were expressed as the percentage of LDH
released.
% LDH released ¼ released LDH=ðreleased LDH
þ cellular LDHÞ 100%
Assay of cytotoxicity affected by pH, temperature Results
and storage conditions
Cytotoxicity of CNN
Experiments were carried to investigate the effects of
pH, temperature and storage conditions on the cytotoxicity of CNN. The CNN was diluted 10 times with
different buffers (acetate 10 mM of pH 4.5 and 5.5,
phosphate 10 mM of pH 6.5, Tris-HCl 10 mM of
pH 7.8 and 8.5, and biocarbonate 10 mM of pH 9.5
and 10.5). After incubation with different buffers for
3 h at 4 C, the cytotoxicity was determined after incubation with CNN for 12 h according to the method
mentioned above.
CNN was pre-incubated at 20, 40, 60, 80 C for different time (10, 30, 50 min), respectively. After
Cytotoxicity of CNN was studied on three different
cell lines including Bel-7402, SMMC-7721 and
H630. There were no significant differences in the
percentage of survival of all cell lines at high concentrations (37–73 mg/ml). However, of the three kinds of
cells, H630 cells were most sensitive to CNN at low
concentrations (2.5–19 mg/ml; Figure 1a), with the
approximate IC50 of 5.1 mg/ml. However, the IC50
on Bel-7402 and SMMC-7721 was approximately
17.9 and 24.3 mg/ml, respectively. Therefore, H630
cells were selected to evaluate the cytotoxicity of
CNN.
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Cuiping et al.
189
Effects of pH and temperature on the cytotoxicity
of CNN
The effects of pH on the cytotoxicity of CNN were
studied. As shown in Figure 2a, CNN had obvious
cytotoxicity at the pH 4.5–8.5 and the percentage of
survival was approximately 50%. However, the cytotoxicity was significantly inhibited at pH 3.5 and the
percentage of survival was about 93.6%.
The effects of temperature on CNN are shown in
Figure 2b. In general, the cytotoxicity of CNN was
temperature-dependent and it decreased with the
increasing temperature and prolonging reaction time.
No significant change in the cytotoxicity of the
venom was observed when CNN was incubated at
20 C for 50 min; incubation at 40 C for 50 min, the
cytotoxicity of CNN significantly decreased and the
percentage of survival was from 17.7 to 43.6%; at
high temperatures (60 and 80 C), a great loss of cytotoxicity was observed. The cytotoxicity was entirely
lost when CNN was kept at 60 C for 50 min and at
80 C for 10 min.
(a)
100
%survival
80
60
40
20
0
3.5
4.5
5.5
6.5
7.5
8.5
9.5
10.5
pH
(b)
100
80
10 min
30 min
50 min
%survival
The responses of H630 cells incubated with
different concentrations (2.5, 5, 10, 19, 37, 73 mg/ml)
of CNN for various periods of time, namely 12, 24 and
48 h, are shown in Figure 1b. The cytotoxicity of CNN
was time and dose-dependent. The cytotoxicity
improved with the increase of incubated time. At the
concentrations below 37 mg/ml, the obvious differences
in percentage of survival in three treated groups were
found. At the concentration of 10 mg/ml, the percentage
of survival was 65.6%, 43.2% and 35.9% after incubation for 12, 24 and 48 h, respectively. However, from
37 to 73 mg/ml the percentage of survival was slightly
different in the three treated groups. The cytotoxicity
also improved with the increase of the concentration
of CNN. For the three incubated periods, at the concentrations from 2.5 to 19 mg/ml, the percentage of survival
was markedly decreased from about 70% to 30%. The
approximate IC50 of CNN on H630 cells was 15.9, 8.8
and 5.1 mg/ml after incubation for 12, 24 and 48 h,
respectively.
60
40
20
0
25
40
60
80
Temperature (°C)
Figure 2. Effects of pH (a) and temperature (b) on the
cytotoxicity of CNN. Data were presented as means +
SD of three parallel measurements.
20 C. As shown in Figure 3, the cytotoxicity
decreased with time. Clearly, storage at 80 C and
in lyophilized state the cytotoxicity reduced slightly
in 9 days; storage at 4 C for 6 days, the cytotoxicity
did not impair remarkably. Nevertheless, storage at
4 C for 9 days, the cytotoxicity decreased sharply and
the percentage of survival was enhanced to 96.9%;
storage at 20 C for 6 days, the cytotoxicity was
lower than that of the venom stored at 4 C. But for
9 days, the cytotoxicity was higher than that of the
venom stored at 4 C.
The cytotoxicity of CNN after storage on different
conditions
LDH assay
In order to assess the stability to storage of CNN, the
cytotoxicity of CNN was investigated just after
extraction and after storage for different days at 4,
20, 80 C and in the lyophilized state storage at
Cytotoxicity of CNN on H630 cells was further
assessed using the LDH release assay. The results are
shown in Figure 4. LDH assay showed marked LDH
leakage. The percentage of LDH released was also
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190
Toxicology and Industrial Health 28(2)
100
4 °C
−20 °C
−80 °C
liophylized
%survival
80
60
40
20
0
2
4
6
Storage time (day)
9
Figure 3. Effects of different storage conditions on the
cytotoxicity of CNN. The percentage of survival of the
venom just after extraction was 39.7%. Data were presented as means + SD of three parallel measurements.
% LDH released
100
80
60
12 h
40
24 h
20
48 h
0
0
10 20 30 40 50 60 70 80
The concentration of CNN (µg/ml)
Figure 4. Effects of CNN on the percentage of LDH
released in H630 cell lines after incubation with different
concentrations of toxin for 12, 24 and 48 h. Data were presented as means + SD of three parallel measurements.
dose- and time-dependent. The percentage of LDH
released improved with the increase in concentration
and incubation time. At the concentrations from 2.5 to
19 mg/ml, the percentage of LDH released significantly
increased at three incubation time, but it improved
slightly at the concentrations from 37 to 73 mg/ml.
Discussion
Cytotoxic tests are usually employed to evaluate toxicity of venoms, such as sea anemone, dinoflagellate
and alga (Carli et al., 1996; Li et al., 2007; Naves
et al., 2006). This method is easy, ethical and uses
small amounts of active venom. Jellyfish venoms
clearly vary in activity and in composition (Bailey
et al., 2005). The results presented in this study
showed that the nematocysts of C. nozakii contained
powerful toxins with strong cytotoxicity on H630
cells. The approximate IC50 of CNN on H630 cells
was 5.1 mg/ml after incubation for 48 h, while the
IC50 of the Chiropsalmus quadrigatus venom on
human malignant glioma cells (U251 cells) was about
80.3 mg/ml after incubation for 48 h (Sun et al., 2002).
pH had marked effects on the bioactivities of the
venom from cnidarians. In previous studies, Marino
et al. (2004) reported that the Aiptasia mutabilis
venom displayed strong cytotoxicity at a pH of around
7.5. However, at pH 4.5 and 9.5, the cytotoxicity was
nearly lost entirely. The cardiovascular activity of the
venom from Chironex fleckeri (C. fleckeri) was active
at a pH rang of 5–9 and lost at pH 3 (Winter et al.,
2007). A 50% loss of haemolytic activity of the
venom from Carybdea marsupialis was found in a
few minutes after extraction by sonication in Krebs
Ringer phosphate buffer (pH 7.4), but the stability
was increased by lowering the pH values of the
extract medium (Rottini et al., 1995). In this article,
CNN had high cytotoxicity at a pH rang of 4.5–8.5,
while at pH 3.5, the cytotoxicity decreased greatly.
One possible reason for this result might be that pH
mainly affected the charges of CNN, and the charges
could influence the venom binding to the target membrane which was the first step to cell damage
(Edwards et al., 2002). On the other hand, pH could
influence the spatial structure of bioactive proteins
with changes in the proteinaceous conformation.
The present study revealed that the cytotoxicity of
CNN was temperature-dependent. When preincubated at 60 C, the cytotoxicity sharply reduced.
Moreover, at 80 C, even for as short as 10 min, the
cytotoxicity was destroyed entirely. It was indicated
that the venom was most likely of a protein nature
as the cytotoxicity of CNN was inactivated by excessive heat. Loss of cytotoxicity of CNN might be
attributed to change in molecular structure, heat denaturation of the protein and presence of proteases in the
venom. Some bioactivities of jellyfish venoms were
significantly affected by temperature. The myotoxicity of the C. fleckeri venom was lost within 24–46
h at room temperature and inactivated by heating at
42 C (Endean et al., 1993). Heat treatment at 60 C for
1 h inactivated the cytotoxicity of Chrysaora venom
(Ordóez et al., 1990).
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Cuiping et al.
191
The cytotoxicity of CNN was affected by storage
conditions. Storage at 80 C and in lyophilized state
did not affect the cytotoxicity greatly. And they were
the best methods to store CNN for long time. As the
cytotoxicity was relatively stable after storage at
4 C for 4 days, CNN could be stored at 4 C for short
time. Storage at 20 C for 6 days, the cytotoxicity
decreased greatly, and it was lower than that of the
venom stored at 4 C. May be there were one or more
components that could degrade the protein with cytotoxicity in CNN, which were more active at 20 C,
so the cytotoxicity of CNN stored at 20 C was lower
than that of CNN stored at 4 C.
Cytolytic toxins were known to operate by either of
two general mechanisms: enzymatic and stoichiometric,
with the stoichiometric lysins including pore formers
(Hessinger and Lenhoff, 1988). LDH release assay performed on H630 cells indicated that exposure to CNN
could result in damage to the cell membrane (Chong
et al., 2000), but its detailed underlying mechanism was
still unclear. In other studies, Man-of-war venom produced pore-like structures in the membranes of target
cells, which lead to colloid osmotic swelling with subsequent release of intracellular proteins and cell lysis
(Edwards et al., 2002). Li et al. (2006) reported that carp
hepatocytes died in apoptosis at low concentrations of
microcystins, while at high concentrations, they died in
the form of necrosis. The activation of calpain
and Ca2þ/calmodulin-dependent protein kinase was
believed to be critical in the microcystin-induced apoptolic process (Ding and Ong, 2003). Houck et al. (1996)
reported that the sea nettle venom did not form large
membrane channels similar to complement, nor did calcium appear to play a major role in the mechanism of
toxicity in hepatocytes. Intracellular accumulation of
toxic oxidative products of sea nettle jellyfish venom
metabolism may play an important role in the cytotoxicity of the venom (Cao et al., 1998).
Conclusion
The cytotoxicity of CNN on Bel-7402, SMMC-7721
and H630 cells was investigated for the first time in
this article. CNN could result in damage to the cell
membrane because of the release of LDH, and it had
the strongest cytotoxicity on H630 than on Bel-7402
and SMMC-7721. The cytotoxicity was affected by
incubation time, temperature and pH. The cytotoxicity of CNN was stable when stored at 80 C and
in lyophilized state, and they could be used as the storage conditions of CNN.
Funding
This work was financially supported by the National Natural
Science Foundation of China (Grant No.41006095), Qingdao Science and Technology Project (08-1-3-51-JCH),
Guangdong Province and Chinese Academy of Sciences
overall strategy Cooperative Project (2010B080703027) and
the Knowledge Innovation Program of the Chinese Academy of Sciences.
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