Toxicology in Vitro xxx (2011) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of
mitochondria and reduces S-phase population in human breast cancer cells (MCF-7)
Jamil Zargan a, Sadiq Umar a, Mir Sajad a, M. Naime b, Shakir Ali b, Haider A. Khan a,⇑
a
b
Clinical Toxicology Laboratory, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
Department of Biochemistry, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
a r t i c l e
i n f o
Article history:
Received 9 December 2010
Accepted 5 September 2011
Available online xxxx
Keywords:
Scorpion venom
Odontobuthus doriae
Apoptosis
Proliferation
MCF-7
Caspase-3
Mitochondrial membrane potential
BrdU
a b s t r a c t
Venom of some species of scorpions induces apoptosis and arrests proliferation in cancer cells. This is an
important property that can be harnessed and can lead to isolation of compounds of therapeutic importance in cancer research. Cytotoxicity was investigated using MTT reduction and confirmed with lactate
dehydrogenase release following venom exposure. Apoptosis was evaluated with determination of mitochondrial membrane potential, reactive nitrogen species assay, measurement of Caspase-3 activity and
DNA fragmentation analysis. To confirm that venom can inhibit DNA synthesis in proliferating breast cancer cells, immunocytochemical detection of BrdU incorporation was done. Our results demonstrated that
venom of Odontobuthus doriae not only induced apoptosis but lead to the inhibition of DNA synthesis in
human breast cancer cells (MCF-7). Cell viability decreased with parallel increment of LDH release in dose
dependent manner after treatment with varying concentrations of venom. Moreover, venom depleted
cellular antioxidants evidenced by depression of GSH and Catalases and concomitantly increased reactive
nitrogen intermediates (RNI). These events were related to the depolarization of mitochondria and associated Caspase-3 activation following venom treatment in a concentration dependent manner. Finally,
fragmentation of nuclear DNA following venom treatment confirmed the apoptotic property of the said
venom. These results suggest that venom of O. doriae can be potential source for the isolation of effective
anti-proliferative and apoptotic molecules.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Recent investigations in vitro as well as in vivo have demonstrated that scorpion venom has ability to induce apoptosis and
inhibition of DNA synthesis in variety of cells (Soroceanu et al.,
1998; Omran, 2003; Wang and Ji, 2005; Das Gupta et al., 2007)
with unclear mechanism. Venom of Heterometrus bengalensis induced apoptosis and antiproliferative effect in human leukemic
cell lines (Das Gupta et al., 2007) and Buthus martensii Karsch
(Wang and Ji, 2005) and Leirus quinquestriatus (Omran, 2003) venoms inhibit growth of primary brain and glioma tumors, respectively. Results of these findings strongly indicate the presence of
compounds in scorpion’s venom that can bring about a breakthrough in pursuit of proapoptotic components. It is clear that
the induction of apoptosis and inhibition of proliferation in tumor
cells are widely recognized as common factors in the biological response of cancer and key mechanisms to a variety of therapeutic
manoeuvres (Dowsett et al., 1999). Several studies have been attempted to find the anti apoptotic and antiproliferative properties
of scorpion’s venoms (Petricevich, 2010).
⇑ Corresponding author. Tel.: +91 9910940516; fax: +91 1126059663.
E-mail address: [email protected] (H.A. Khan).
Previous studies have demonstrated that scorpion venom
contains a rich source of polypeptides and enzymes with a variety
of biological functions (Calıskan et al., 2009). Different species of
scorpions have been reported to have around 70 peptides (Possani
et al., 1999). It was reported that biological effects of scorpion venom are mainly due to the presence of low-molecular-weight proteins that exert powerful effects on excitable cells (Jalali et al.,
2005) through interaction with different cellular ion channels such
as Na+ (Ji et al., 2002), K+ (Ji et al., 2003) and Cl (De Bin et al., 1993)
without mechanistic characterization. Advanced methods of fractionation, chromatography and peptide sequencing have made it
possible to characterize the components of scorpion venoms (Petricevich, in press).
Odontobuthus doriae (Thorell, 1876) is one of the medically
important Buthide scorpions of Middle East (Mirakabadi et al.,
2006). Envenomation after scorpion sting can cause various
effects ranging from local pain, inflammation and necrosis to
muscle paralysis, and it can be deadly for children (Jalali et al.,
2007).
Two of the protein toxins including OD1 (Jalali et al., 2005) and
OdK1 (Abdel-Mottaleb et al., 2006) have been identified from
venom of O. doriae that target voltage-gated Na+ and K+ ion channels, respectively.
0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2011.09.002
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
2
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
In this study we highlight the cytotoxic effects of O. doriae
venom and a possible mechanism of apoptosis and antiproliferation on the human breast cancer cells (MCF-7).
2. Materials and methods
2.1. Antibodies and reagents
DMEM (Sigma–Aldrich, USA), Fetal bovine serum (Gibco, USA),
Trypsin–EDTA (Sigma–Aldrich, USA), Trypan blue (Sigma–Aldrich,
USA), PBS (Gibco, USA), Penicillin–streptomycin solution (Sigma–
Aldrich, USA), Antibiotic–antimycotic (Invitrogen, USA), Phenol red
(Sigma–Aldrich, USA), In vitro Toxicology Assay kit: Lactic dehydrogenase based (Sigma–Aldrich, USA), Griess reagent (Sigma–Aldrich,
USA), JC-1[5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide] (Sigma–Aldrich, USA), DMSO [Dimethylsulfoxide] (Sigma–Aldrich, USA), Triton X-100 (Sigma–Aldrich, USA),
Agarose (Sigma–Aldrich, USA), Fixative (Sigma–Aldrich, USA), BrdU
[5-Bromo-20 -deoxyuridine] (Sigma–Aldrich, USA), DAB [Diaminobenzidine] (Sigma–Aldrich, USA), Primary monoclonal anti
bromodeoxyuridine (BrdU) antibody (Sigma–Aldrich, USA), NGS
[Normal goat serum] (Gibco, USA), Anti-mouse IgG (Fab specific)peroxidase antibody produced in goat (Sigma–Aldrich, USA), U Bottom 12-wells plate (Sigma–Aldrich, USA), U Bottom 96-wells plate
(Sigma–Aldrich, USA). All the other chemicals and reagents were
of analytical grade and purchased locally.
2.2. Scorpion venom
Scorpions were collected from southern regions of Tehran province in Iran. Crude venom was obtained monthly by mild electrical
simulation and was solubilized in sterile double distilled water.
After centrifugation at 8000 g for 15 min at 4 °C, supernatant
was immediately freeze dried and stored at 20 °C until further
use (Borges et al., 2006). Venom was reconstituted in serum free
DMEM without phenol red and protein content was estimated by
Bradford method (Bradford, 1976). Venom was disinfected with
1.5% antibiotic–antimycotic solution.
2.3. Cell culture
MCF-7 cells were procured from national facility for animal tissue and cell culture (NCCS), Pune, India. Cells were grown in 75 ml
plastic flasks in DMEM medium supplemented with 10% heat inactivated fetal bovine serum, 10 ll/ml penicillin–streptomycin solution or 1% antibiotic–antimycotic and incubated in CO2 incubator
(37 °C, 5% CO2 and humidified atmosphere). The medium was replaced three times a week. For enumeration, 100 ll of cells concentration were stained with trypan blue (0.2%) and cells were
counted using a haemocytometer.
2.4. Cell treatments
For treatment with venom, cells were seeded in sterile 12 or 96well plate with care taken to keep the cell number almost equal in
all the wells. Following overnight incubation (37 °C and 5% CO2),
medium was aspirated and medium with different concentration
of venom was added. The seeding density was different in different
tests as indicated therein.
2.5. Determination of cell viability (MTT reduction)
Cytotoxicity of scorpion venom was carried out by MTT reduction assay (Mosmann, 1983). Cells were seeded in 96-well plate at
a density of 2 104 cells per well and incubated overnight. Media
was removed and cells exposed to medium containing varying concentrations of venom (10, 25, 50, 100 and 200 lg/ml) for 24 h 20 ll
of MTT stock solution (5 mg/ml) was added to each well. This was
followed by incubation for 1 h. When purple colored precipitates
were visible under the microscope, media was carefully discarded.
For solubilization of formazan crystals (MTT formazan), 200 ll of
DMSO (dimethylsulfoxide) was added to each well and cells were
incubated in dark at room temperature for 2 h. The color (purple)
developed was measured at 570 nm by a microplate reader (BioRad, USA).
2.6. Lactate dehydrogenase assay (LDH)
Release of lactate dehydrogenase (LDH) was analyzed as a marker for cell membrane integrity to determine cell viability (Decker
and Lohmann-Matthes, 1988) after exposure with venom. Cells
were seeded in 96-well plate at a density of 2 104 cells/well in
culture medium. After overnight incubation, medium was replaced
and cells were exposed to varying concentrations of venom (10, 20,
50 and 100 lg/ml). Cells were incubated for 24 h and LDH activity
was measured in the cell lysate and supernatants using in vitro toxicology assay kit (Sigma) in accordance with the manufacturer’s
instructions. The absorbance was determined at 490 nm using
plate reader.
2.7. Reactive nitrogen species assay
Nitrite production was determined in the supernatants of cultured cells (Ding et al., 1988). The cells were seeded in 96-well
plate at density of 2 104 cells/well. Cells were incubated overnight. Thereafter media was discarded and cells exposed to medium containing venom (50, 100 lg/ml). After 24 h media from
each well was transferred to fresh tube. After centrifugation at
500 g for 5 min at 4 °C, 100 ll of the supernatant were transferred to fresh 96-well plate, mixed with an equal volume of Griess
reagent (0.04 g/ml in PBS, pH 7.4) and incubated at room temperature for 10 min. The absorbance was measured within 30 min at
540 nm by a microplate reader (Bio-Rad, USA). Nitrite concentration in control and treated cells were calculated using sodium nitrite standard reference curve and expressed as lM/ml.
2.8. Reduced glutathione (GSH)
Total reduced glutathione (GSH) was measured following the
method of Sedlak and Linsay (1968). Cells were seeded in 12-well
plate at a density of 5 105 cells/well in culture medium. After
overnight incubation, culture medium was replaced with medium
containing 50 and 100 lg/ml of venom for 24 h. After harvesting,
the cells were washed with PBS (pH 7.4) and pelleted by centrifugation at 5000 g for 5 min at 4 °C. The cells were freeze fractured
by incubation in 20 °C for 30 min and were resuspended in 200 ll
chilled cell lysis buffer containing 20 mM Tris–HCl (pH 7.5),
150 mM NaCl, 1 mM EDTA and 1% Triton X-100 for 30 min at room
temperature. Thereafter, cell lysates were sonicated for 10–15 min
and centrifuged at 2000 g for 10 min and supernatant was collected. After estimation of protein by Bradford assay (Bradford,
1976), an aliquot of supernatant was deproteinized by adding an
equal volume of 10% TCA and was allowed to stand at 4 °C for
2 h. The contents were centrifuged at 500 g for 15 min and
supernatant was collected. 20 ll sample was mixed with 75 ll
lysis buffer, 55 ll Tris buffer (pH 8.5) containing 0.02 M EDTA
and 25 ll DTNB [5,50 dithio bis (2-N benzoic acid)]. Absorbance
of the chromogen (yellow) was read at 412 nm by a microplate
reader (Sajad et al., 2010). The result was expressed as lgm GSH/
mg protein using molar extinction coefficient of 13600.
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
3
2.9. Catalase activity
2.12. DNA fragmentation analysis
Catalase activity in the cell lysates was assayed as per Sinha
(1972). Cells were seeded in 12-well plate at a density of 5 105
cells/well in culture medium. After overnight incubation, medium
was replaced with another medium containing 50 and 100 lg/ml
of venom for 24 h. After harvesting, the cells were pelleted by centrifugation at 5000 g for 5 min at 4 °C. Thereafter, cells were
freeze fractured by incubation in 20 °C for 30 min following incubation with 200 ll lysis buffer [20 mM Tris–HCl (pH 7.5), 150 mM
NaCl, 1 mM EDTA and 1% Triton X-100] for 30 min at room temperature. Lysates were sonicated for 10–15 min, centrifuged at
2000 g for 10 min and supernatant was collected. After estimation of protein by Bradford assay (Bradford, 1976), in an eppendroff tube 5 ll of sample/well was mixed with 50 ll of lysis buffer,
20 ll of distilled water and 25 ll of hydrogen peroxide (15%). After
shaking, the samples were incubated for 2 min at 37 °C and mixed
with 100 ll of dichromate acid reagent (0.1 M potassium dichromate in glacial acetic acid) following heating in a water bath at
100 °C for 10–15 min until the color of the sample was changed
to greenish/faint greenish. 200 ll/well of sample was transferred
into flat bottomed 96-well plates and absorbance was read at
570 nm in a plate reader (Sajad et al., 2010). The results were converted into activity using molar extinction of Catalases as 43.6 and
express as micro moles of hydrogen peroxide consumed/min/mg
protein.
Cells were seeded at a density of 5 105 cells/well in 12-well
plate and incubated overnight. Thereafter media was carefully aspirated and cells were exposed to 500 ll medium containing 50 and
100 lg/ml of venom and incubated for 24 h. The cells were harvested and centrifuged at 500 g for 5 min at 4 °C. Pellet was then
washed twice with 0.5 ml TE buffer (Tris–EDTA). Cells were lysed in
50 ll of chilled DNA lysis buffer (0.5% Triton X-100, 25 mM EDTA
and 25 mM Tris–HCl, pH 7.4) for 30 min on ice. Extraction of DNA
was carried out by adding 50 ll of 0.1 mg/ml proteinase K,
150 mM NaCl and 0.2% (w/v) SDS and incubated at 50 °C for 3 h. Nucleic acid was extracted by adding equal volume of solution containing phenol/chloroform/Isoamyl alcohol (25:24:1) (Kweon
et al., 2004). After centrifugation at 1000 g for 5 min at 10 °C,
supernatant was transferred to a fresh tube. DNA was precipitated
by two volumes of cold absolute ethanol and pelleted by centrifugation (20000 g for 30 min at 4 °C). Supernatant was carefully discarded by rapidly inverting tubes and DNA was washed twice by
ice-cold 70% ethanol. It was then air dried for 5–10 min and mixed
in 50 ll TE buffer containing RNase (0.2 mg/ml). After incubation at
55oC for 1 h, DNA was stored at 20 °C until use. The extracted DNA
was analyzed by loading 10–20 lg into 1.5% agarose gel containing
ethidium bromide (1 lg/ml). The gel was visualized with ethidium
bromide by UV transilluminator (Nugen Scientific, India with UV
Photo MW version 11.01 for windows).
2.10. Mitochondrial membrane potential (wm) determination
Mitochondrial membrane potential was measured with specific
cationic dye JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) (Smiley et al., 1991). Briefly, cells were
seeded in 12-well plate at a density of 1 106 cells/well and incubated overnight. Medium was replaced and cells were exposed to
50 and 100 lg/ml of venom and incubated for 24 h. The harvested
cells were incubated in 0.5 ml JC-1 (10 lM) for 8 min at room temperature in dark. After centrifugation for 5 min at 500 g and
washing twice by PBS (pH 7.4) to remove unincorporated dye, pellets were resuspended in 2 ml PBS (pH 7.4). Red fluorescence (excitation 570 nm, emission 610 nm) and green fluorescence
(excitation 490 nm, emission 535 nm) were measured using a
spectrofluorimeter (spectrometer LS50B, Perkin Elmer). The mitochondrial membrane potential was estimated as the ratio of green
to red fluorescence.
2.11. Measurement of Caspase-3 activity
Caspase-3 activity was evaluated using a commercially available Caspase-3/CPP32 Colorimetric assay kit (Biovision) according to the manufacturer’s instructions. Briefly, cells were
seeded in 12-well plate at a density of 1 106 cells/well overnight. Medium was discarded and cells were exposed to different
concentrations of venom (50 and 100 lg/ml) for 24 h. After harvesting, the cells were washed with PBS (pH 7.4) and pelleted by
centrifugation at 5000 g for 5 min at 4 °C. The cells were
resuspended in 100 ll chilled cell lysis buffer [10 mM Tris,
1 mM EDTA, and 1% Triton X-100; pH 7.4] and incubated on
ice for 20 min. Aliquots were analyzed for protein (Bradford,
1976), and lysate volume equivalent to 50 lg of protein was
brought to 100 ll with lysis buffer in 96-well plate and mixed
with reaction buffer (containing 10 mM DTT). All the samples
were incubated for 1 h at 37 °C after adding 5 ll of 4 mM
DEVD-pNA (200 lM). The absorbance was read at 405 nm in
microplate reader (Bio-Rad, USA).
2.13. Immunocytochemical study (BrdU incorporation)
Cells were seeded in 96-well plate at density 2 104 cells/well
and incubated overnight. Cells were exposed to 50 and 100 lg/ml
of venom and incubated for 22 h and pulsed with 10 lM (Dover
and Patel, 1994) 5-bromo-20 -deoxyuridine for 2 h. Medium was
carefully discarded and the cells were subsequently washed twice
with PBS (pH 7.4) and dehydrated using ethanol. Optimal fixation
of cells was achieved by 4% paraformaldehyde for 30 min and after
washing twice with PBS (pH 7.4) containing 1 N HCl on ice for
10 min and followed by 2 N HCl for 10 min at room temperature.
After brief rinsing in PBS (pH 6.0), cells were incubated in methanol
containing 0.3% H2O2 for 15 min and followed by incubation in
blocking buffer (1.5% NGS, 0.5% BSA and 0.1% Triton X-100) for
30 min. Finally cells were incubated with monoclonal anti-BrdU
antibody (1:100) in 10% NGS at 4 °C overnight. Cells were washed
twice with PBS (pH 7.4) and immediately incubated with 1:200
anti-mouse secondary antibody for 1 h with constant shaking at
room temperature. Cells were washed two times with PBS (pH
7.4) and peroxide complex was visualized with DAB (3,30 -diaminobenzidine tetrahydrochloride). Cells were briefly counterstained using Basic Fuschin (0.1%) in ethanol. After dehydration
in ethanol (70%, 80%, 90% and 95%), brown positive stained cells
were photographed with an inverted microscope (Nikon, Japan).
For analysis of BrdU-immunoreactive cells, a person blind to the
experimental protocol was employed for counting. Cells were
counted using Image J software with cell counting jar (National
Institutes of Health, USA). The numbers of BrdU-labeled cells were
averaged from seven different photomicrographs in three replicate
wells. For data analysis, numbers were expressed as percentage of
BrdU (+) cells per photograph.
2.14. Statistical analysis
All the data presented are mean ± SEM .Analysis between the
groups was carried out with one way ANOVA (analysis of variance)
with post hoc analysis by Tukey Kramer multiple comparison method. Any variation with p < 0.05 was considered to be significant.
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
4
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
3. Results
3.1. Analysis of cell morphology
Changes in the cell morphology were monitored for 24 h after
exposure with 50 and 100 lg/ml of venom. The cells changed from
round shape to polygonal with slightly granulated contents and
distinct edges after overnight incubation at 37 °C and 5% CO2.
Under similar conditions, exposure with 50 lg/ml of venom
exhibited increasing cellular contents, shrinkage, swelling, rupture
of membrane (black arrows) and release of cytosolic contents after
24 h. Venom (100 lg/ml) of presented similar changes but with
marked acute necrosis (Fig. 1). In other experiments, the round
shaped cells without overnight incubation were exposed to similar
concentration of venom. In this case, cells did not switch to polygonal form after overnight incubation. These cells, 24 h after exposure with 50 lg/ml of venom, exhibited aggregation, swelling,
rupture of membrane and release of cytosolic contents. A
100 lg/ml dose resulted in loss of cells with acute necrosis with increased cellular adhesiveness (Fig. 1).
3.2. Cell viability (MTT assay)
Venom decreased cell viability in a dose dependant manner
(Fig. 2). At a dose of 10, 20, 50, 100 and 200 lg/ml of venom; cell viability decreased to 86.8 ± 2.38, 80.6 ± 0.37, 67.7 ± 3.3, 62.2 ± 0.76
and 39.9 ± 1.2%, respectively. Venom (10 lg/ml) affected cell viability significantly (p < 0.01), but a dose of 20 lg or more, decreased the
cell viability to a greater significant level (p < 0.001).
3.3. Effect on lactate dehydrogenase (LDH)
Lactate dehydrogenase release was evaluated to confirm results
of MTT assay. Release of LDH increased in dose dependent manner
(Fig. 3). At a dose of 10, 20, 50 and 100 lg/ml of venom; release of
LDH increased to 19.73 ± 0.66 (p < 0.05), 21.51 ± 0.74 (p < 0.001),
Fig. 2. MTT assay in MCF-7 cells after 24 h exposure with varying concentrations of
venom. Cell viability was expressed as the proportion of absorbance values
normalized to the control group after subtracting the blank absorbance from
samples and the control. The data are expressed as the mean ± SEM of three
independent experiments. Significances indicated are shown in comparison to
control. ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
29.89 ± 0.51 (p < 0.001) and 35.08 ± 0.98% (p < 0.001), respectively.
No significant increase in LDH was observed at 20 lg/ml as compared to 10 lg/ml but at two higher doses (50 and 100 lg/ml),
LDH increased significantly (p < 0.01). Therefore these two doses
were considered as optimal dose to carry out all other analysis.
3.4. Reactive nitrogen intermediates (RNI)
NO production increased in the cells after exposure with venom
in a concentration dependant manner. The selected optimal doses
of venom increased concentration of nitrite (NO production) in the
supernatant significantly as compared to control (Fig. 4). Dose
100 lg/ml of venom increased the nitrite content from
6.34 ± 0.14 lM/ml in controls to 9.38 ± 0.46 lM/ml (p < 0.001)
Fig. 1. Microscopic graphs (X 200) of cultured breast cancer cell line (MCF-7) after 24 h of incubation with varying doses (50 and 100 lg/ml) of O. doriae venom. (A) The round
shaped cells after seeding in 96 wells plate. (B) Incubated round cells with 50 lg/ml of venom show loss of neurite out growth, aggregation (black arrows) and swelling. (C)
Incubated round cells with 100 lg/ml of venom exhibit aggregation, swelling, rupture of membrane and release of cytosolic contents. (D) The round cells present polygonal
form with granulated contents, homogeneous and distinct edges after overnight incubation at 37 °C and 5% CO2. (E) Polygonal cells after incubation with 50 lg/ml of venom
exhibit increasing cellular contents, shrinkage, swelling (white arrows), rupture of cell membrane (black arrows) and release of cellular contents. (F) Polygonal cells incubated
with 100 lg/ml of venom present similar acute changes and necrosis.
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
Fig. 3. LDH release in MCF-7 cells after 24 h exposure with varying concentrations
of O. doriae venom (10, 20, 50,100 lg/ml). The % LDH release from the cells was
determined by dividing absorbance of the culture supernatants to absorbance of
supernatant plus cells lysate. The data were expressed as the mean ± SEM of three
independent experiments carried out in triplicate. Significances indicated are
shown in comparison to control. N.S (non significant), ⁄p < 0.05, ⁄⁄⁄p < 0.001.
5
Fig. 5. Glutathione (GSH) was estimated in cell lysate of control and treated cells
with 50 and 100 lg/ml of scorpion venom and expressed as lgm GSH/mg protein
using molar extinction coefficient of 13600. The data were expressed as the
mean ± SEM of three independent experiments carried out in triplicate. Significances indicated are shown in comparison to control. ⁄⁄⁄(p < 0.001).
3.7. Change in mitochondrial membrane potential (wm)
where as at 50 lg/ml, 8.32 ± 0.07 lM/ml (p < 0.01) concentration
was observed.
3.5. Reduced glutathione (GSH)
Treatment of cells with the two selected doses of venom
resulted in decrease in glutathione level in a dose dependent
manner (Fig. 5). GSH in control cells was 0.245 ± 0.01 lgm GSH/
mg protein whereas in treated cell with 50 and 100 lg/ml,
decrement was 0.035 ± 0.005 (p < 0.001) and 0.031 ± 0.007 (p <
0.001) lgm GSH/mg protein, respectively (Fig. 5). Depletion in glutathione level was not significant between the two doses as compared to each other.
3.6. Catalase activity
The two concentration of venom viz. 50 lg/ml and 100 lg/ml
significantly decreased the catalase activity as compared to control. At 50 and 100 lg/ml of venom, the activity reduced to
2.75 ± 0.22 (p < 0.001) and 2.44 ± 0.40 (p < 0.001), respectively, in
comparison to 16.82 ± 0.52 lmol of hydrogen peroxide consumed/min/mg protein in control (Fig. 6). Again the decrement in
catalase activity between the two doses was not significant.
The ratio of green/red fluorescence intensity of JC-1 in control
cells was 0.17 ± 0.006 and in treated cell with 50 and 100 lg/ml,
increment was 0.21 ± 0.03 and 0.62 ± 0.014, respectively (Fig. 7).
Therefore mitochondrial membrane potential as a function of
green/red fluorescence decreased significantly at the two concentrations of venom viz. 50 lg/ml (p < 0.05) and 100 lg/ml (p < 0.001).
3.8. Caspase-3 activity
The elevation of Caspase-3 following venom treatment is indicative of cellular apoptosis either by breakage of DNA or lysis of
cytoskeletal protein. Upregulation of Caspase-3 expression was
checked by colorimetric method dependent on release of pNA
(p-nitroanilide) in cell lysates of MCF-7 cells. Caspase-3 activity increased in a dose dependent manner (Fig. 8). At 50 and 100 lg/ml
of venom, the activity increased to 0.033 ± 0.001 (p < 0.05) and
0.0587 ± 0.005 (p < 0.001) lM pNA liberated/h/ml, respectively, in
comparison to 0.021 ± 0.001 lM pNA liberated/h/ml in control.
Also the increment in caspase activity was in close agreement with
change in mitochondrial potential.
3.9. DNA fragmentation analysis
DNA was extracted from the cells after venom treatment and
resolved on 1.5% agarose. Both the concentrations viz. 50 lg/ml
Fig. 4. Nitric oxide production was determined by measuring nitrite in culture
medium of control and treated cells with 50 and 100 lg/ml of scorpion venom.
Concentration of NO was determined by comparison of absorbance value to the
nitrite standard curve and expressed as lM/ml. The data were expressed as the
mean ± SEM of three independent experiments carried out in triplicate. Significances indicated are shown in comparison to control. ⁄⁄(p < 0.01), ⁄⁄⁄(p < 0.001).
Fig. 6. Catalase activity was estimated in cell lysate of control and treated cells with
50 and 100 lg/ml of scorpion venom and expressed as micro moles of hydrogen
peroxide consumed/min/mg protein. The data were expressed as the mean ± SEM of
three independent experiments carried out in triplicate. Significances indicated are
shown in comparison to control. ⁄⁄⁄(p < 0.001).
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
6
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
Fig. 7. Mitochondrial membrane potential was estimated by using JC-1(10 lM)
probe in control and sample cells (treated with 50 and 100 lg/ml for 24 h). Red
fluorescence (excitation 570 nm, emission 610 nm) and green fluorescence (excitation 490 nm, emission 535 nm) were measured. The ratio of green/red fluorescence as a measure of mitochondrial membrane potential was calculated. The data
were expressed as the mean ± SEM of three independent experiments carried out in
triplicate. Significances indicated are in comparison to control. ⁄(p < 0.05),
⁄⁄⁄
(p < 0.001).
and 100 lg/ml induced fragmentation of DNA in 200–400 bp
regions as compared to the control cells (Fig. 9).
3.10. BrdU incorporation
Immunocytochemistry was carried out to check whether O. doriae venom can affect the synthesis of DNA in cells using a thymine
analog BrdU which gets incorporated during DNA synthesis. The
scorpion venom decreased the number of nuclei undergoing DNA
synthesis (Fig. 10). At 50 lg/ml, numbers of cells incorporating
BrdU was reduced to 12.51 ± 0.96% (P < 0.01), while 100 lg/ml of
venom inhibited incorporation to 5.39 ± 1.05% (P < 0.001) as
compared to control (24.3 ± 1.6).
4. Discussion
Scorpion venom can induce apoptosis in different types of cells
including cancer cells (Gomes et al., 2010). This is an important
property which can lead to isolation of compounds of therapeutic
importance in cancer research. In this study, we analyzed apoptosis and antiproliferative effects of scorpion venom (O. doriae) to
Fig. 8. Caspase-3 activity was determined by measuring DEVD-pNA hydrolysis and
was performed in lysates of control and samples (treated with 50 and 100 lg/ml for
24 h). The absorbance of cell lysates and buffers were subtracted to calculate the
caspace-3 activity. Comparison of the absorbance of pNA of a treated sample with
an untreated control allows determination of the fold increase in Caspase-3 activity.
Data represent mean ± SEM in triplicate. Significances indicated are shown in
comparison to control. ⁄(p < 0.05), ⁄⁄⁄(p < 0.001).
Fig. 9. Genomic DNA fragmentation analysis following, treatment of scorpion
venom (O. doriae) in MCF-cells. DNA was extracted, and resolved on a 1.5% agarose
gel and visualized with ethedium bromide. C, control cells. M, DNA Marker. 50,
treated cells with 50 lg/ml of venom. 100, treated cells with 100 lg/ml of venom.
elucidate this probable mechanism of action in breast cancer cells.
Scorpion venoms contain enzymes, such as acetylcholinesterase,
phospholipase A2, alkaline phosphatase and proteolytic enzymes
with gelatinolytic activity (Incesu et al., 2005). Cells after venom
treatment exhibited increasing necrosis in a dose dependent manner. It suggests that proteolytic enzymes are responsible for necrotic activity (Incesu et al., 2005).
Morphological findings suggested reduction in cell viability. Our
investigation showed reduced MTT and increased LDH level in the
treated cells. These parameters are used as markers for cell membrane integrity and index for cytotoxicity (Decker and LohmannMatthes, 1988). These results lend support to the finding where
cell viability of MCF-7 decreased after treatment with scorpion
venom in a dose dependent manner.
Since venom is a complex mixture of several organic and inorganic compounds, we investigated venom induced oxidative
stress on the cells. Venom was found to reduce the cellular antioxidant level which may be due to induction of reactive oxygen
species.
Nitric oxide has been previously demonstrated to affect mitochondrial functioning by degrading the endomembrane system
(Finocchietto et al., 2009) and altering the structure of several
ion channels (Leonelli et al., 2009). More over, nitrite is a potent
reactive intermediate and in presence of superoxide, can form
peroxynitrite (Liaudet et al., 2009). All these probable mechanisms can lead to deleterious consequences on the cellular health.
The primary product of nitric oxide metabolism, nitrite was assayed in MCF-7 cells following venom treatment. Scorpion venom
of O. doriae elevated NO production which was clearly observed
after 24 h of treatment. Earlier studies demonstrated that induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-a-induced apoptosis in MCF-7 (Binder et al.,
1999; Mooney et al., 2002). Moreover other studies have also suggested role of nitric oxide in induction of apoptosis in mammalian
cells like human neuroblastoma cells (Zargan et al., 2011) and rat
hippocampal neuronal cells (Sajad et al., 2009). Elevated nitrite
can damage the biomembranes including mitochondrial membranes and can aid in the formation of permeability transition
pore (Vieira and Kroemer, 2003) and leakage of pro-apoptotic fac-
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
7
Fig. 10. Effect of scorpion venom on the synthesis of DNA in MCF-7 cells visualized by immunocytochmeical analysis of thymidine analog BrdU (pulsed for 1 h) using
monoclonal antibody. (A) Fixed cells without the BrdU incorporation. (B) Normal cells pulsed with BrdU (arrow). (C) inhibition of the DNA synthesis following treatment with
50 lg/ml of venom (arrow). (D) 100 lg/ml of venom shows inhibition of DNA synthesis to a greater extent as compared to the lower dose. (E) Frequency of BrdU positive cells.
50, treated cells with 50 lg/ml of venom. 100, treated cells with 100 lg/ml of venom. Data represent mean ± SEM and significances indicated are shown in comparison to
control. ⁄⁄(p < 0.01), ⁄⁄⁄(p < 0.001).
tors including cytochrome-c in the cytoplasm (Caroppi et al.,
2009).
In order to evaluate the mitochondrial involvement in the venom induced rise in nitrite level, a cationic dye JC-1 with dual fluorescence properties was used. Scorpion venom was found to induce
depolarization in mitochondria in breast cancer cells.
Caspase-3 is a key protease required for the execution of
apoptosis (Wimmer et al., 2004). We investigated its activity to
find out whether scorpion venom can lead to apoptosis by elevation of Caspase-3 activity. It was observed that Caspase-3 activities increased in the cell lyastes of MCF-7 cells following
treatment with venom of O. doriae. These results were in close
agreement with enhanced depolarization of mitochondrial membrane and production of nitric oxide in MCF-7. Recent investigations in spider and O. doriae venom have demonstrated their
ability to induce apoptosis in HeLa and SH-SY5Y cells, respec-
tively, resulting from an increase in Caspase-3 activity (Li et al.,
2005; Zargan et al., 2011).
Caspace-3 has been demonstrated to activate Caspase-8 and
induce the cleavage of DNA repair polypeptide, PARP making it
inactive thus enhancing the chances of DNA damage. In order to
understand whether Caspase-3 leads to the fragmentation of
DNA in MCF-7 cells after venom treatment, we resolved DNA on
an agarose gel. Our result showed fragments of broken DNA,
confirming apoptosis. Finally to confirm that venom can inhibit
DNA synthesis in proliferating breast cancer cells, BrdU incorporation was checked with immunocytochemistry. Venom decreased
the DNA synthesis in a concentration dependent manner. These results are in conformity with findings where scorpion venom of
H. bengalensis and O. doriae inhibited proliferation in cells by cell
cycle arrest and DNA fragmentation (Das Gupta et al., 2007; Zargan
et al., 2011).
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
8
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
Fig. 11. Venom of O. doriae induces reactive nitrogen intermediates (RNI) and
reactive oxygen species(ROS) in MCF-7 cells. Nitric oxide depolarizes mitochondria.
Furthermore nitric oxide can lead to formation of permeability transition pore and
leakage of pro-apoptotic factors leading the elevation of caspases in the cytoplasm.
Together ROS and RNI can lead to the fragmentation of nuclear DNA and trigger
apoptosis thereof.
These studies highlight the apoptogenic and growth arresting
properties of venom of O. doriae and probable mechanism of cell
death in MCF-7 (Fig. 11). The most practical importance of this
study lies in the investigation of anti-proliferative and apoptosis-inducing properties of O. doriae venom fraction constituents
for developing new drugs for cancer and other incurable
diseases.
5. Conflict of interest statement
None declared.
Acknowledgments
Authors acknowledge and thank NCCS, Pune for timely shipment of the cell lines. Biochemistry Department of Hamdard University also acknowledges the support of DST to establish
infrastructure for animal tissue culture facility under FIST program.
References
Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L.,
Tytgat, J., 2006. The first potassium channel toxin from the venom of the Iranian
scorpion Odontobuthus doriae. FEBS Letters 580, 6254–6258.
Binder, C., Schulz, M., Hiddemnn, W., Oellerich, M., 1999. Induction of inducible
nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced
apoptosis in MCF-7 and other epithelial tumor cells. Laboratory Investigation
79, 1703–1712.
Borges, A., Silva, S., Op den Camp, H.J., Velasco, E., Alvarez, M., Alfonzo, M.J.,
Jorquera, A., De Sousa, L., Delgado, O., 2006. In vitro leishmanicidal activity of
Tityus discrepans scorpion venom. Parasitology Research 99, 167–173.
Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical
Biochemistry 72, 248–254.
Calıskan, F., Hulya, S., Yalcın, S., 2009. A preliminary study for the detection of
gelatinolytic proteases from the scorpion Androctonus crassicauda (Turkish
black scorpion) venom. Turkish Journal of Biochemistry 34, 148–153.
Caroppi, P., Sinibaldi, F., Fiorucci, L., Santucci, R., 2009. Apoptosis and human
diseases: mitochondrion damage and lethal role of released cytochrome c as
proapoptotic protein. Current Medicinal Chemistry 16, 4058–4065.
Das Gupta, S., Debnath, A., Saha, A., Giri, B., Tripathi, G., Vedasiromoni, J., Gomes, An.,
Gomes, Ap., 2007. Indian black scorpion (Heterometrus bengalensis Koch) venom
induced antiproliferative and apoptogenic activity against human leukemic cell
lines U937 and K562. Leukemia Research 31, 817–825.
De Bin, J., Maggio, J., Strichartz, G., 1993. Purification and characterization of
chlorotoxin, a chloride channel ligand from the venom of the scorpion.
American Journal of Physiology 264, 361–369.
Decker, T., Lohmann-Matthes, M., 1988. A quick and simple method for the
quantitation of lactatedehydrogenase release in measurements of cellular
cytotoxicity and tumor necrosis factor (TNF) activity. The Journal of
Immunological Methods 15, 61–69.
Ding, A.H., Nathan, C.F., Stuehr, D.J., 1988. Release of reactive nitrogen intermediates
and reactive oxygen intermediates from mouse peritoneal macrophages:
comparison of activating cytokines and evidence for independent production.
The Journal of Immunology 141, 2407–2412.
Dover, R., Patel, K., 1994. Improved methodology for detecting bromodeoxyuridine
in cultured cells and tissue sections by immunocytochemistry. Histochemistry
102, 383–387.
Dowsett, M., Archer, C., Assersohn1, L., Gregory1, R.K., Ellis, P.A., Salter, J., Chang, J.,
Mainwaring, P., Boeddinghaus, I., Johnston, S.R.D., Powles, 1.T.J., Smith, I.E.,
1999. Clinical studies of apoptosis and proliferation in breast cancer. EndocrineRelated Cancer 6, 25–28.
Finocchietto, P., Franco, M., Holod, S., Gonzalez, A., Converso, D., Arciuch, V., Serra,
M., Poderoso, J., Carreras, M., 2009. Mitochondrial nitric oxide synthase: a
masterpiece of metabolic adaptation, cell growth, transformation, and death.
Experimental Biology and Medicine 234, 1020–1028.
Gomes, A., Bhattacharjee, P., Mishra, R., Biswas, A.K., Dasgupta, S.C., Giri, B., 2010.
Anticancer potential of animal venoms and toxins. Indian Journal of
Experimental Biology 48, 93–103.
Incesu, Z., Calıskan, F., Zeytinoglu, H., 2005. Cytotoxic and gelatinolytic
activities of Mesobuthus Gibbosus (Brullé, 1832) venom. Revista Cenic Ciencias
Biológicas 36.
Jalali, A., Bosmans, F., Amininasab, M., Clynen, E., Cuypers, E.,
Zaremirakabadi, A., Sarbolouki, M.N., Schoofs, L., Vatanpour, H., Tytgat,
J., 2005. OD1, the first toxin isolated from the venom of the scorpion
Odontobuthus doriae active on voltage-gated Na+ channels. FEBS Letters
579, 4181–4186.
Jalali, A., Vatanpour, H., Hosseininasab, Z., Rowan, E.G., Harvey, A.L., 2007. The effect
of the venom of the yellow Iranian scorpion Odontobuthus doriae on skeletal
muscle preparations in vitro. Toxicon 50, 1019–1026.
Ji, Y., Wang, W., Wang, Q., Huang, Y., 2002. The binding of BmK abT, a unique
neurotoxin, to mammal brain and insect Na+ channels using biosensor.
European Journal of Pharmacology 454, 25–30.
Ji, Y., Wang, W., Ye, J., He, L., Li, Y., Yan, Y., Zhou, Z., 2003. Martentoxin, a novel K+
channel-blocking peptide: purification, cDNA and genomic cloning, and
electrophysiological and pharmacological characterization. Journal of
Neurochemistry 84, 325–335.
Kweon, S.-M., Lee, Z.-W., Yi, S.-J., Kim, Y.-M., Han, J.-A., Paik, S.-G., Ha, K.-S., 2004.
Protective role of tissue transglutaminase in the cell death induced by TNF-a in
SH-SY5Y neuroblastoma cells. Journal of Biochemistry and Molecular Biology
37, 185–191.
Leonelli, M., Torrao, A.s., Britto, L.R.G., 2009. Unconventional neurotransmitters,
neurodegeneration and neuroprotection. Brazilian Journal of Medical and
Biological Research 42, 68–75.
Li, G., Shan, B.-e., Chen, J., Liu, J.-h., Song, D.-x., Zhu, B.-c., 2005. Effects of spider
Macrothele raven venom on cell proliferation and cytotoxicity in HeLa cells.
Acta Pharmacologica Sinica 26, 369–376.
Liaudet, L., Vassalli, G., Pacher, P., 2009. Role of peroxynitrite in the redox
regulation of cell signal transduction pathways. Frontiers in Bioscience 1,
4809–4814.
Mirakabadi, A.Z., Jalali, A., Jahromi, A.E., Vatanpur, H., Akbary, A., 2006. Biochemical
changes and manifestations of envenomation produced by odentobuthus doriae
venom in rabbits. Journal of Venomous Animals and Toxins including Tropical
Diseases 12, 67–77.
Mooney, L.M., Al-Sakkaf, K.A., Brown, B.L., Dobson, P.R.M., 2002. Apoptotic
mechanisms in T47D and MCF-7 human breast cancer cells. British Journal of
Cancer 87, 909–917.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. Journal of Immunological
Methods 65, 55–63.
Omran, M.A.A., 2003. In vitro anticancer effect of scorpion Leiurus quinquestriatus
and Egyptian cobra venom on human breast and prostate cancer cell lines.
Journal of Medical Science 3, 66–68.
Petricevich, V.L., 2010. Scorpion venom and the inflammatory response. Mediators
of Inflammation, in press. ID903295.
Possani, L.D., Becerril, B., Delepierre, M., Tytgat, J., 1999. Scorpion toxins specific for
Na+ channels. European Journal of Biochemistry 264, 287–300.
Sajad, M., Asif, M., Umar, S., Zargan, J., Rizwan, M., Ansari, S. H., Ahmad, M., Khan,
H.A., 2010. Amelioration of inflammation induced oxidative stress and tissue
damage by aqueous methanolic extract of nigella sativa linn. in arthritic rats.
Journal of Complementary and Integrative Medicine 7.
Sajad, M., Zargan, J., Chawla, R., Umar, S., Sadaqat, M., Khan, H.A., 2009. Hippocampal neurodegeneration in experimental autoimmune encephalomyelitis
(EAE): potential role of inflammation activated myeloperoxidase. Molecular and
Cellular Biochemistry 328, 183–188.
Sedlak, J., Linsay, R.H., 1968. Estimation of total protein bound and nonprotein
bound sulphydryl group in tissue with Ellman’s reagents. Analytical
Biochemistry 25, 192–205.
Sinha, A.K., 1972. Colorimetric assay of catalase. Analytical Biochemistry 47, 389–
394.
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002
J. Zargan et al. / Toxicology in Vitro xxx (2011) xxx–xxx
Smiley, S.T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T.W., Steele,
G.D., Chen, L.B., 1991. Intracellular heterogeneity in mitochondrial membrane
potentials revealed by a J-aggregate forming lipophilic cation JC-1. PNAS 88,
3671–3675.
Soroceanu, L., Gillespie, Y., Khazaeli, M., Sontheimer, H., 1998. Use of chlorotoxin for
targeting of primary brain tumors. Cancer Research 58, 4871–4891.
Thorell, T., 1876. Etudes scorpiologiques. Atti della Societa Italiana di Scienze
Naturali 19, 75–272.
Vieira, H., Kroemer, G., 2003. Mitochondria as targets of apoptosis regulation by
nitric oxide. IUBMB Life 55, 613–616.
9
Wang, W., Ji, Y., 2005. Scorpion venom induces glioma cell apoptosis in vitro and
inhibits glioma tumor growth in vivo. Journal of Neuro-Oncology 73, 1–7.
Wimmer, T., Cohen, G., Saemann, M.D., Horl, W.H., 2004. Effects of Tamm-Horsfall
protein on polymorphonuclear leukocyte function. Nephrology, Dialysis,
Transplantation 19, 2192–2197.
Zargan, J., Sajad, M., Umar, S., Naime, M., Ali, S., Khan, H.A., 2011. Scorpion
(Odontobuthus doriae) venom induces apoptosis and inhibits DNA synthesis in
human neuroblastoma cells. Molecular and Cellular Biochemistry 384, 173–181.
Please cite this article in press as: Zargan, J., et al. Scorpion venom (Odontobuthus doriae) induces apoptosis by depolarization of mitochondria and reduces
S-phase population in human breast cancer cells (MCF-7). Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.09.002