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Indian black scorpion (Heterometrus bengalensis Koch) venom induced antiproliferative and apoptogenic activity against human leukemic cell lines U937 and K562
Leukemia Research 31 (2007) 817–825
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
c
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
activities [5–8]. 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 [14–17], 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 Sigma–Aldrich, 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 (10–200 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 cell–agarose 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 Tris–HCl, 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.
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Fig. 1. Effect of scorpion (Heterometrus bengalensis) venom on growth inhibition of U937 (䊉) 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 annexin–hepes 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 Student’s 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 10–200 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
2
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 G0–G1 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).
References [1] Pal SK, Gomes A, Dasgupta SC, Gomes A. Snake venom as therapeutic agents: from toxin to drug development. Indian J Exp Biol 2002;40:1353–8. [2] Wang R, Moreau P, Deschamps A, de Champlain J, Sauve R, Foucart S, et al. Cardiovascular effects of Buthus martensii (Karsch) scorpion venom. Toxicon 1994;32:191–200. [3] Wang CG, He XL, Shao F, Liu W, Ling MH, Wang DC, et al. Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch. Eur J Biochem 2001;268:2480–5. [4] Torres-Larios A, Gurrola GB, Zamudio FZ, Possani LD. Hadrurin a new antimicrobial peptide from the venom of scorpion Hadrurus aztecus. Eur J Biochem 2000;267:5023–31. [5] Garcia ML, Hanner M, Knaus HG, Slaughter R, Kaczorowski GJ. Scorpion toxins as tools for studying potassium channels. Meth Enzymol 1999;294:624–39. [6] Romi-Lebrun R, Martin-Eauclaire MF, Escoubas P, Wu FQ, Lebrun B, Hisada M, et al. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca2+ activated K+ channels. Eur J Biochem 1997;245:457–64. [7] Sabatier JM, Fremont V, Mabrouk K, Crest M, Darbon H, Rochat H, et al. Leiurotoxin I, a scorpion toxin specific for Ca (2+)-activated K+ channels. Structure–activity analysis using synthetic analogs. Int J Pept Protein Res 1994;43:486–95. [8] Strong PN, Clark GS, Arumugam A, De-Allie FA, Joseph JS, Yemul V, et al. Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus. Arch Biochem Biophys 2001;385:138–44. [9] Laniado ME, Fraser SP, Djamgoz MB. Voltage gated K(+ ) channel activity in human prostrate cancer cell lines of markedly different
S. Das Gupta et al. / Leukemia Research 31 (2007) 817–825
[10] [11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19] [20]
metastatic potential: distinguishing characteristics of PC-3 and LNCaP cells. Prostrate 2001;46:262–74. Gallagher JD, Fay MJ, North WG, McCann FV. Ionic signals in T47D human breast cancer cells. Cell Signal 1996;8:279–84. Wang WX, Ji YH. Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro. J Neurooncol 2005;73:1–7. Soroceanu L, Gillespie Y, Khazaeli MB, Sontheimer H. Use of Chlorotoxin for targeting of primary brain tumors. Cancer Res 1998;58:4871–9. Tikadar BK, Bastwade DB. The fauna of India. Scorpions, scorpionida: arachnida, vol. III. Calcutta: Zoological Survey of India; 1983. Lahiri SC, Chaudhuri AK. Action of the scorpion (Heterometrus bengalensis C.L. Koch) venom on smooth muscle. Indian J Exp Biol 1982;20:545–8. Kar PK, Sarangi B, Datta A, Gomes A, Lahiri SC. Separation and partial characterization of smooth muscle contractile material in the venom of the scorpion Heterometrus bengalensis. Toxicon 1983;21:353–61. Kar PK, Sarangi B, Datta A, Gomes A, Lahiri SC. Occurrence of phospholipase in the venom of the scorpion (Heterometrus bengalensis). Indian J Med Res 1986;83:332–7. Lahiri SC, Nag Chowdhury AK. Release of kinin by the scorpion (Heterometrus bengalensis C.L. Koch) venom. Indian J Exp Biol 1983;21:198–202. Cao Z, Li Y. Chemical induction of cellular antioxidants affords marked protection against oxidative injury in vascular smooth muscle cells. Biochem Biophys Res Commun 2002;292:50–7. Muench H, Reed LJ. A simple method of estimating 50 per cent end points. Am J Hyg 1938;27:493–7. Grujic M, Zavasnik-Bergant T, Pejler G, Renko M. Actinonin induces apoptosis in U937 leukemia cells. Cancer Lett 2005;223: 211–8.
825
[21] Nottola SA, Makabe S, Stallone T, Familiari G, Correr S, Macchiarelli G. Surface morphology of the zona pellucida surrounding human blastocysts obtained after in vitro fertilization. Arch Histol Cytol 2005;68:133–41. [22] Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184–91. [23] Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Meth 1995;184:39–51. [24] Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ. Resveratrol an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Cancer Lett 1999;140:1–10. [25] Soroceanu L, Manning Jr TJ, Sontheimer H. Modulation of glioma cell migration and invasion using Cl− and K+ ion channel blockers. J Neurosci 1999;19:5942–54. [26] Chinkwo KA. Sutherlandia frutescens extracts can induce apoptosis in cultured carcinoma cells. J Ethnopharmacol 2005;98:163–70. [27] Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 1997;27:1–20. [28] Belloc F, Dumain P, Boisseau MR, Jalloustre C, Reiffers J, Bernard P, et al. A flow cytometric method using Hoechst 33342 & propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells. Cytometry 1994;17:59–65. [29] Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol 2001;38:179–93. [30] Wising C, Azem J, Zetterberg M, Svensson LA, Ahlman K, Lagergard T. Induction of apoptosis/necrosis in various human cell lineage by Haemophilus ducreyi cytolethal distending toxin. Toxicon 2005;45:767–76.
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
c
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
activities [5–8]. 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 [14–17], 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 Sigma–Aldrich, 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 (10–200 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 cell–agarose 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 Tris–HCl, 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.
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Fig. 1. Effect of scorpion (Heterometrus bengalensis) venom on growth inhibition of U937 (䊉) 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 annexin–hepes 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 Student’s 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 10–200 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
2
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 G0–G1 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).
References [1] Pal SK, Gomes A, Dasgupta SC, Gomes A. Snake venom as therapeutic agents: from toxin to drug development. Indian J Exp Biol 2002;40:1353–8. [2] Wang R, Moreau P, Deschamps A, de Champlain J, Sauve R, Foucart S, et al. Cardiovascular effects of Buthus martensii (Karsch) scorpion venom. Toxicon 1994;32:191–200. [3] Wang CG, He XL, Shao F, Liu W, Ling MH, Wang DC, et al. Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch. Eur J Biochem 2001;268:2480–5. [4] Torres-Larios A, Gurrola GB, Zamudio FZ, Possani LD. Hadrurin a new antimicrobial peptide from the venom of scorpion Hadrurus aztecus. Eur J Biochem 2000;267:5023–31. [5] Garcia ML, Hanner M, Knaus HG, Slaughter R, Kaczorowski GJ. Scorpion toxins as tools for studying potassium channels. Meth Enzymol 1999;294:624–39. [6] Romi-Lebrun R, Martin-Eauclaire MF, Escoubas P, Wu FQ, Lebrun B, Hisada M, et al. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca2+ activated K+ channels. Eur J Biochem 1997;245:457–64. [7] Sabatier JM, Fremont V, Mabrouk K, Crest M, Darbon H, Rochat H, et al. Leiurotoxin I, a scorpion toxin specific for Ca (2+)-activated K+ channels. Structure–activity analysis using synthetic analogs. Int J Pept Protein Res 1994;43:486–95. [8] Strong PN, Clark GS, Arumugam A, De-Allie FA, Joseph JS, Yemul V, et al. Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus. Arch Biochem Biophys 2001;385:138–44. [9] Laniado ME, Fraser SP, Djamgoz MB. Voltage gated K(+ ) channel activity in human prostrate cancer cell lines of markedly different
S. Das Gupta et al. / Leukemia Research 31 (2007) 817–825
[10] [11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19] [20]
metastatic potential: distinguishing characteristics of PC-3 and LNCaP cells. Prostrate 2001;46:262–74. Gallagher JD, Fay MJ, North WG, McCann FV. Ionic signals in T47D human breast cancer cells. Cell Signal 1996;8:279–84. Wang WX, Ji YH. Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro. J Neurooncol 2005;73:1–7. Soroceanu L, Gillespie Y, Khazaeli MB, Sontheimer H. Use of Chlorotoxin for targeting of primary brain tumors. Cancer Res 1998;58:4871–9. Tikadar BK, Bastwade DB. The fauna of India. Scorpions, scorpionida: arachnida, vol. III. Calcutta: Zoological Survey of India; 1983. Lahiri SC, Chaudhuri AK. Action of the scorpion (Heterometrus bengalensis C.L. Koch) venom on smooth muscle. Indian J Exp Biol 1982;20:545–8. Kar PK, Sarangi B, Datta A, Gomes A, Lahiri SC. Separation and partial characterization of smooth muscle contractile material in the venom of the scorpion Heterometrus bengalensis. Toxicon 1983;21:353–61. Kar PK, Sarangi B, Datta A, Gomes A, Lahiri SC. Occurrence of phospholipase in the venom of the scorpion (Heterometrus bengalensis). Indian J Med Res 1986;83:332–7. Lahiri SC, Nag Chowdhury AK. Release of kinin by the scorpion (Heterometrus bengalensis C.L. Koch) venom. Indian J Exp Biol 1983;21:198–202. Cao Z, Li Y. Chemical induction of cellular antioxidants affords marked protection against oxidative injury in vascular smooth muscle cells. Biochem Biophys Res Commun 2002;292:50–7. Muench H, Reed LJ. A simple method of estimating 50 per cent end points. Am J Hyg 1938;27:493–7. Grujic M, Zavasnik-Bergant T, Pejler G, Renko M. Actinonin induces apoptosis in U937 leukemia cells. Cancer Lett 2005;223: 211–8.
825
[21] Nottola SA, Makabe S, Stallone T, Familiari G, Correr S, Macchiarelli G. Surface morphology of the zona pellucida surrounding human blastocysts obtained after in vitro fertilization. Arch Histol Cytol 2005;68:133–41. [22] Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184–91. [23] Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Meth 1995;184:39–51. [24] Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ. Resveratrol an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Cancer Lett 1999;140:1–10. [25] Soroceanu L, Manning Jr TJ, Sontheimer H. Modulation of glioma cell migration and invasion using Cl− and K+ ion channel blockers. J Neurosci 1999;19:5942–54. [26] Chinkwo KA. Sutherlandia frutescens extracts can induce apoptosis in cultured carcinoma cells. J Ethnopharmacol 2005;98:163–70. [27] Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 1997;27:1–20. [28] Belloc F, Dumain P, Boisseau MR, Jalloustre C, Reiffers J, Bernard P, et al. A flow cytometric method using Hoechst 33342 & propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells. Cytometry 1994;17:59–65. [29] Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol 2001;38:179–93. [30] Wising C, Azem J, Zetterberg M, Svensson LA, Ahlman K, Lagergard T. Induction of apoptosis/necrosis in various human cell lineage by Haemophilus ducreyi cytolethal distending toxin. Toxicon 2005;45:767–76.