Dasgupta 2007
Dasgupta 2007
Dasgupta 2007
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 [email protected] (A. Gomes).
0145-2126/$ see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.leukres.2006.06.004
818
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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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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.
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)
5.3 0.2
6.7 0.2*
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.
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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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
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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