Leukemia Research 31 (2007) 817825

Indian black scorpion (Heterometrus bengalensis Koch)

venom induced antiproliferative and apoptogenic activity against
human leukemic cell lines U937 and K562
Shubho Das Gupta a , Anindita Debnath a , Archita Saha b , Biplab Giri b , Gayatri Tripathi c ,
Joseph Rajan Vedasiromoni a , Antony Gomes b , Aparna Gomes a,
a

Drug Development Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India
b Laboratory of Toxinology and Experimental Pharmacodynamics, Department of Physiology,
University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India
Cellular Physiology Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India
Received 31 May 2006; received in revised form 31 May 2006; accepted 6 June 2006
Available online 28 July 2006

Abstract
Venoms are rich source of several bioactive compounds that possess therapeutic potentials. The different constituents of scorpion venom
can modulate cell proliferation, cell growth and cell cycle. In the present communication, the cytotoxic activity of Indian black scorpion
(Heterometrus bengalensis) venom was explored on human leukemic U937 and K562 cells. Scorpion venom induced U937 and K562 cell
growth inhibition and the IC50 value calculated to be 41.5 g/ml (U937) and 88.3 g/ml (K562). The scorpion venom showed characteristic
features of apoptosis such as membrane blebbing, chromatin condensation and DNA degradation in both the cells as evidenced by confocal,
fluorescence, scanning electron microscopy. Scorpion venom (IC50 dose, 48 h) induced DNA fragmentation as evidenced by comet formation.
Flow-cytometric assay revealed a significant amount of apoptotic cells (early and late) due to scorpion venom treatment. The venom induced
cell cycle arrest was observed with maximum cell accumulation at sub-G1 phase. Thus, the Indian scorpion (H. bengalensis) venom possessed
antiproliferative, cytotoxic and apoptogenic activity against human leukemic cells.
2006 Elsevier Ltd. All rights reserved.
Keywords: Scorpion venom; Leukemic cell; Cytotoxicity; Apoptosis

1. Introduction
From times immemorial venoms are being used as sources
of drugs to cure different ailments [1]. In Chinese traditional
medical practice, Buthus martensii Karsch venom is used to
treat various ailments for more than 2000 years [2]. It has been
used to treat epilepsy, acute and chronic convulsion, tetanus,
subcutaneous nodules, etc. as written in Herbarin Chinese
herb data. Scorpion venoms contain several small molecular
weight peptides having wide pharmacological activities such
as anti-epileptic [3], antimicrobial [4] and channel blocking
Corresponding author. Tel.: +91 33 24831980x108;
fax: +91 33 2473 5197/0284.
E-mail address: gomes aparna@yahoo.com (A. Gomes).

0145-2126/$ see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.leukres.2006.06.004

activities [58]. Membrane channel blockers are known to

control certain cellular behavior in the metastatic cascade [9]
and also play a key role in cellular mitogenesis [10]. This
hypothesis may arouse the curiosity to study the antiproliferative and cytotoxic potentiality of scorpion venom. Venom of
B. martensii Karsch induced glioma cell apoptosis in vivo and
thereby inhibited glioma tumour growth [11]. Leirus quinquestriatus venom also inhibited primary brain tumour [12].
Out of the 90 species of venomous scorpions of India [13],
Heterometrus bengalensis is highly prevalent in the state of
West Bengal of eastern India. Although the pharmacological
properties of H. bengalensis Koch venom have been partially
studied [1417], its cytotoxic profile has not been established.
The present investigation is an effort to assess the antiproliferative and apoptotic efficacy of H. bengalensis Koch

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venom, against two human leukemic cell lines U937 and

K562.

(PBS) and were stained with ethidium bromide (100 g/ml of

PBS). The cells were then mounted on slides. Finally it was
observed under Leica Confocal Microscope. Images were
acquired from argon/krypton laser using 590 nm filter.

2. Materials and methods

2.1. Chemicals
RPMI 1640 and fetal bovine serum were purchased
from Gibco, USA. Agarose, annexin V FITC, ethidium bromide, ethylene diamine tetraacetic acid (EDTA),
glutaraldehyde, MTT [3-(4,5-dimethylthiozol-2-il)-2,5-2,5dipheniltetrazoliumbromide], osmium tetraoxide, propidium
iodide, proteinase K, RNase A, Trypan blue were purchased
from SigmaAldrich, USA. All the other reagents were of
analytical grade and purchased locally.
2.2. Cell cultures
U937 and K562 cell lines were obtained from National
Facility for animal tissue and cell culture, Pune, India. The
cells were cultured in RPMI 1640, supplemented with 10%
heat inactivated fetal bovine serum, penicillin (100 units/ml),
streptomycin (100 g/ml), gentamycin (100 g/ml) and
incubated at 37 C in a humidified atmosphere containing
5% CO2 inside a CO2 incubator.
2.3. Scorpion venom
Live adult scorpions (H. bengalensis) were housed in wellventilated wooden cages with food and water, supplied ad
libitum. Venom was collected by electrical stimulation of
the telson (20 V, 500 mA) of live animals and was pooled,
lyophilized and stored in a vacuum desiccator at 4 C. The
lyophilized venom was weighed, dissolved in RPMI and was
centrifuged for 15 min at 10,000 rpm. The supernatant was
next passed through nitrocellulose filter (Millipore 0.45 m)
and kept at 4 C until further use.
2.4. Cell growth study in vitro
Both U937 and K562 cells were seeded at a concentration
of 105 cells/1 ml of RPMI 1640 in a 96-well sterile plate. The
cells were then treated with scorpion venom at different concentrations (10200 g/ml) and their effects on cell growth
were observed after 48 h by Trypan blue exclusion method
and MTT [18] assay. IC50 value was calculated [19].
2.5. Morphological studies of leukemic cells
2.5.1. Confocal microscopy
Confocal laser scanning microscope (Leica TCS-SP2 system, Leica microsystem, Germany) was used to study nuclear
integrity in the leukemic cells (U937 and K562) [20]. Briefly,
control and scorpion venom-treated cells (both U937 and
K562) were washed in ice cold phosphate buffer saline

2.5.2. Fluorescence microscopy

Fluorescent microscope (Motic, Germany) was used to
study the nuclear integrity and membrane permeability of the
leukemic cells. Both the scorpion venom-treated (IC50 , 48 h)
and untreated control cells were collected separately and centrifuged at 1000 rpm for 5 min. The pellet was rinsed twice
and resuspended in PBS. It was then treated with ethidium
bromide and acrydine orange solution (100 g/ml of PBS)
and observed under a fluorescence microscope for the qualitative determination of apoptotic cells.
2.5.3. Scanning electron microscopy
Changes of the cell membrane were visualized by scanning electron microscopy [21]. Both U937 and K562 cells
were incubated for 48 h with scorpion venom at IC50 concentration. The cells were then mounted on glass cover slip.
The cells were washed with cold PBS (pH 7.2) and fixed with
2% glutaraldehyde in PBS for 3 h. After washing, the cells
were again fixed with 1% osmium tetraoxide in cacodylate
buffer for 1 h. Dehydration was done with ascending concentrations of ethanol in deionised water. After absolute drying,
the cells were embedded in poly lysine coated thick cover
glasses. Finally the cells were observed under a scanning
electron microscope (Leica, Model S440) with 15 KV accelerating voltage.
2.6. Comet assay
DNA damage of U937 and K562 cells induced by scorpion venom was studied by single cell electrophoresis assay
or comet assay [22]. U937 and K562 cells were treated with
scorpion venom at IC50 for 48 h. The cells were washed twice
with cold PBS (pH 7.2) and centrifuged at 4 C. Slides were
initially coated with a layer of normal melting point agarose
(0.75% in PBS). After solidification 85 l of cellagarose
suspension containing 104 cells were placed. After solidification, third layer of low melting point agarose (100 l)
was applied. Slides were immersed in cold lysis buffer (10%
DMSO, 100 mM EDTA, 2.5 M NaCl, 10 mM Tris, 1% Triton X-100, pH 10) at 4 C for an hour in the dark and then
were placed in electrophoresis unit containing fresh buffer
(1 mM EDTA, pH 13.5, 300 mM NaOH) for 20 min. Electrophoresis was conducted at 18 V for 20 min. The slides
were placed in neutralization buffer (0.4 M TrisHCl, pH
7.5) for 5 min. Finally the slides were stained with ethidium
bromide (10 g/ml) and were observed under a fluorescence
microscope (Motic, Germany) with green filter at 100 magnification. The comet score was recorded by counting 100
cells/slide. Comet tail length and width were measured utilizing Motic images Plus 2.0 software.

S. Das Gupta et al. / Leukemia Research 31 (2007) 817825

Fig. 1. Effect of scorpion (Heterometrus bengalensis) venom on growth

inhibition of U937 () and K562 () cells. Data are mean S.E (n = 6)
taking control as 100%.

2.7. Flow-cytometric analysis of apoptosis

Flow-cytometric analysis was done to assess the apoptotic
activity induced by the scorpion venom [23]. In brief, 2 106
cultured cells (U937 and K562) were treated with venom at

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IC50 dose for 48 h and centrifuged at 4 C. The cells were suspended in annexinhepes buffer and centrifuged twice. The
pellets were resuspended in the same buffer (100 l) containing annexin V FITC and propidium iodide. After 15 min of
incubation in dark at room temperature analysis was done
by flow cytometer (Becton Dickinson FACS caliber single
laser cytometer). Flow-cytometric reading was taken using
488 nm excitation and band pass filters of 530/30 nm (for
FITC detection) and 585/42 nm (for PI detection). Data analysis was performed with Cell Quest (Macintosh platform)
program.
2.8. Flow-cytometric analysis of cell cycle arrest
To assay the stage of cell cycle arrest in a flow cytometer [24], control and scorpion venom (IC50 dose, 48 h) treated
U937 and K562 cells were fixed in ethanol overnight, washed,
treated with DNase free RNase A (10 g/ml) at 37 C for
30 min and stained with propidium iodide (200 l from
50 g/ml) and kept at dark for 15 min. Intracellular DNA

Fig. 2. Confocal microscopic images of control U937 (a), K562 (c) and scorpion venom-treated U937 (b), K562 (d) cells using propidium iodide. The control
cells showed intact nucleus and the venom-treated cells showed apoptotic bodies in both the cells indicated by arrows. Magnification (1000).

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content was measured by the amount of red fluorescence in a

flow cytometer (Becton Dickinson FACS caliber single laser
cytometer) using 488 nm argon laser light source and 623 nm
band pass filter using Cell Quest software (Becton Dickinson). Analysis of flow-cytometric data was performed using
ModFit software. A histogram of DNA content (x-axis, PIfluorescence) versus counts (y-axis) was displayed.
2.9. Statistical analysis
All the data are expressed in terms of percentage decrease
from the control values. Statistical analysis was done by Students t-test. P < 0.05 was considered as significant.

3. Results
3.1. Cell growth assay and MTT assay
Scorpion venom inhibited growth of U937 and K562 cells
at a concentration of 10200 g/ml (Fig. 1). The IC50 value
for U937 was calculated to be 41.5 g/ml and that for K562
was 88.3 g/ml. Reduction in the OD value in MTT assay also
confirmed the cytotoxic nature of scorpion venom. The IC50
concentration was used to detect the apoptogenic changes
during other experiments.

3.2. Morphological studies

3.2.1. Confocal microscopy
The cytotoxic and apoptotic activity of the scorpion
venom was observed by confocal microscopic studies.
Scorpion venom-treated (IC50 dose/48 h) U937 and K562
cells clearly exhibited chromatin condensation, margination and prominent nuclear disintegration, whereas confocal images of untreated control cells depicted intact nuclei
(Fig. 2).
3.2.2. Fluorescence microscopy
Fluorescence microscopic observations of the scorpion
venom-treated (IC50 dose/48 h) U937 and K562 cells stained
with ethidium bromide and acridine orange, revealed the presence of apoptotic cells (early and late) as compared to the
control cells. An array of nuclear changes were observed
including chromatin condensation and apoptotic body formation which are indicative of an apoptotic process comprising
of both early and late apoptotic stages (Fig. 3).
3.2.3. Scanning electron microscopy
Under scanning electron microscope it was observed that
the scorpion venom (IC50 dose/48 h) treated U937 and K562
cells showed a very high degree of membrane blebbing as
compared to the control cells. Venom-treated U937 and K562

Fig. 3. Fluorescence microscopic images of control U937 (a), K562 (c) and scorpion venom-treated U937 (b), K562 (d) cells using acridine orange and ethidium
bromide. Control U937 and K562 cells showed bright green fluorescence with no nuclear damage or membrane structure disruption. But the venom-treated
U937 and K562 cells showed chromatin disintegration as well as membrane blebbing. The cells clearly expressed signs of apoptosis where some are in early
apoptotic stage while others are in a late apoptotic condition as indicated by short and long arrowheads. Magnification (100). (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4. Scanning electron microscopic images of control U937 (a), K562 (c) and scorpion venom-treated U937 (b), K562 (d) cells. The control cells showed intact
plasma membrane, but the treated cells clearly show deep ridges and furrows as well as severe membrane blebbing as indicated by arrowhead. Magnification
(3000).

Fig. 5. Comet assay of control U937 (a), K562 (d) and scorpion venom-treated U937 (b) K562 (e) cells. a and d are the U937 and K562 control cells without
any comet shaped structures. b and e are the venom-treated U937 and K562 cells clearly showed comet formations indicating DNA breakage. c and f represent
single venom-treated U937 and K562 cells, respectively.

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Table 1
Comet tail length in scorpion venom-treated cells as compared to the control. Results shown are mean S.E.
Groups

Comet image
length (m)

Control U937 cells

Treated U937 cells

5.3 0.2
6.7 0.2*

Control K562 cells

Treated K562 cells

5.2 0.2
9.4 0.2*

Percentage increase
in image length (%)

Comet image
width (m)

Percentage increase
in image width (%)

Length/width
ratio

26.4

4.8 0.2
4.9 0.1

1.1
1.4

80.7

4.7 0.2
6.2 0.2*

31.9

1.1
1.5

P < 0.05.

cells also showed the deep crevices, ridges and perforations

on the cell surface (Fig. 4).
3.2.4. Comet assay
Under a fluorescence microscope, a significant number
of the scorpion venom (IC50 dose/48 h) treated U937 and
K562 cells with ethidium bromide staining showed distinct

comet formation indicating DNA fragmentation, while no

comet was seen in U937 and K562 control cells (Fig. 5). The
length width ratio and the percentage of comet formations in
both untreated and treated U937 and K562 cells are shown in
Table 1. The mean length to width ratio of treated U937 and
K562 cells were significantly greater than the untreated cells
(Table 1).

Fig. 6. Flow-cytometric analysis of U937 and K562 cells staining with annexin V FITC and PI. Leukemic cells were treated with scorpion venom at IC50
dose for 48 h. Control and treated cells were analyzed by FACS. Dual parameter dot plot of FITC-fluorescence (x-axis) vs. PI-fluorescence (y-axis) shows
logarithmic intensity. (a) Represents values for U937 control cells, (c) represents values for K562 control cells, (b) represents values for treated U937 cells and
(d) represents values for treated K562 cells. n = 3. The figure and values indicate results of one experiment.

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3.3. Flow-cytometric analysis of apoptosis

3.4. Flow-cytometric analysis of cell cycle arrest

Scorpion venom (IC50 dose/48 h) treated U937 and K562

cells were analyzed by flowcytometer to study the apoptotic status. Annexin V/PI population can be regarded
as normal cells, while annexin V+/PI cells are the early
apoptotic ones. Annexin V+/PI+ are the cells at late apoptosis or necrotic stage. Only the PI+ cells are considered to be the dead ones. After incubation of U937 and
K562 cells with the scorpion venom the percentage of both
the annexin V+/PI and annexin V+/PI+ cells increased
significantly as compared to their untreated counterparts.
The flow-cytometric data revealed that after 48 h of treatment 36.5% U937 and 27.2% K562 cells are in LR Quadrant (early apoptotic cell) and 11.4% U937 and 14.6%
K562 cells are in UR Quadrant (late apoptotic/necrotic cell)
(Fig. 6).

The cell cycle data revealed that after 48 h treatment

with scorpion venom at IC50 dose, apoptosis specific
increase in Sub-G1 peak was noticed in both the cell
lines. Hypoploid DNA content was increased 10-fold in
treated U937 cells (4.1% against 43%) and nine-fold in
treated K562 cells (5.4% against 53.3%) as compared with
untreated cell lines. However DNA content was found to
decrease in G0G1 phase in treated U937 cells (32.1%
against 22.8%) and treated K562 (27.5% against 7.5%)
cells, in S phase in treated U937 cells (10.3% against
7.8%) and treated K562 cells (15.5% against 3.1%) and
in G2/M phase in treated U937 cells (41.1% against
10.1%) and treated K562 cells (38.8% against 4.5%). These
results indicated that scorpion venom arrested the cell cycle
(Fig. 7).

Fig. 7. Flow-cytometric analyses of propidium iodide stained cell cycle phase distribution of control U937 cells (a) and K562 cells (c), along with scorpion
venom-treated U937 cells (b), and K562 cells (d). DNA histogram displaying DNA content (x-axis denotes PI fluorescence y-axis denotes count). n = 3. The
figure and values indicate results of one experiment.

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4. Discussion
Scorpion venoms are a natural treasure trove of various
bioactive molecules. Venoms contain many pharmacologically active substances having wide range of biological
actions from antimicrobial peptide to channel blocker. Lahiri
and Chaudhuri [14] have worked extensively on the pharmacological aspects of venom of Indian black scorpion, H.
bengalensis showing the presence of phospholipases [16],
kinin releasing substance [17] and a smooth muscle contractile material [15]. Literature survey revealed that venom of
B. martensii karsch inhibited glioma tumour growth [11] and
that venom of L. quinquestriatus also inhibited primary brain
tumour [25].
The present study was conducted to evaluate the antiproliferative and apoptogenic activity of H. bengalensis venom
on human leukemic cell lines U937 and K562. The antiproliferative and cytotoxic effect of H. bengalensis venom were
supported by cell count by Trypan blue exclusion method and
MTT assay. The hallmark of carcinogenesis is uncontrolled
cellular growth and proliferation. In normal cells, cell proliferation and DNA replication is monitored by cell cycle check
point and apoptosis. Apoptosis or programmed cell death
is beneficial in cancer therapy [26]. Apoptosis is characterized by several morphological changes such as membrane
blebbing, cell shrinkage, chromatin condensation, nuclear
fragmentation and formation of apoptotic bodies [27]. The
induction of apoptosis by venom was evidenced from the
morphological alteration as observed under confocal microscope. Nuclear fragmentation and margination were clearly
seen in treated U937 and K562 cells in comparison with the
control cells. Florescence microscopic observations clearly
indicated nuclear disintegration when stained by ethidium
bromide and acridine orange. Normal untreated cells (containing double strand DNA) show a clear bright green fluorescence. Apoptotic cells having denatured DNA showed
more intense red and reduced green fluorescence. On the contrary live cell (control and early apoptotic cell) with intact
membrane excludes ethidium bromide but dead cell (late
apoptosis and necrotic) cannot exclude the dye and stain
red [28]. Fluorescence microscopic images showed presence
of early and late apoptotic cells in both treated cell lines.
The crevices and ridges on the cell surface were seen under
the scanning electron microscope. Severe membrane blebbing and apoptotic bodies could be clearly visualized from
the photographs. DNA fragmentation is one of the features
of apoptosis. The comet tail results from the migration of
DNA fragments resulting from apoptosis, according to their
respective size through the agarose gel [22]. Scorpion venom
increased the length width ratio in both the cells and increased
the sizes of tail length revealing the fragmentation of DNA in
both the cells after treatment. In the early phase of apoptosis
translocation of phosphatidylserine occurs from inner to the
outer layer and this phosphatidylserine binds with annexin V.
In the late phase of apoptosis or secondary necrosis along
with translocation, cell membrane looses its integrity and

becomes leaky [23]. Thus, by dual staining with annexin V

FITC and propidium iodide it is possible to identify live cell,
early apoptotic cells and late apoptotic cells [29,30]. Venom
given at IC50 dose for 48 h affected the phospholipid distribution over the plasma membrane. The increased number
of early apoptotic cell and late apoptotic/necrotic cell were
observed in both cell lines pointing to the fact that venom
triggered apoptosis of leukemic cell lines. Cell cycle study
revealed that the venom induced a marked accumulation of
the treated cells in a sub-G1 phase and the DNA content
was decreased in other three phases (G0/G1, S and G2/M).
Thus, it was confirmed that the venom arrested cell cycle
and also produced apoptosis in the leukemic cells U937 and
K562.
All the evidence, from morphological changes, comet
assay and flow cytometry indicated that H. bengalensis
venom possesses antiproliferative and apoptogenic effect on
U937 and K562 leukemic cell lines. An attempt to isolate
the component(s) present in H. bengalensis venom, that are
actually responsible for the said action and the pathway by
which it is causing cytotoxicity is being undertaken.

Acknowledgement
The work has been financially supported partly by the
Department of Science and Technology, Government of India
(vide reference no. SR/SQ/AS-54/2002 dated 09.09. 2004).

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