Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin

Toxicon 57 (2011) 225–236

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Toxicon journal homepage: www.elsevier.com/locate/toxicon

Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin Pushpak Bhattacharjee, Biplab Giri 1, Antony Gomes* Laboratory of Toxinology and Experimental Pharmacodynamics, University of Calcutta, Department of Physiology, 92, Acharya Prafulla Chandra Road, Kolkata 700009, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 August 2010 Received in revised form 21 November 2010 Accepted 24 November 2010 Available online 7 December 2010

A protein (BMP1) was purified from common Indian toad (Bufo melanostictus, Schneider) skin through DEAE cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the BMP1 was found to be 79 kDa. BMP1 (0.5 and 1 mg/kg/day, i.p.) significantly decreased the number of viable Ehrlich ascites carcinoma (EAC) cells, thereby increased the lifespan of EAC bearing mice (p < 0.001). MTT values reduced significantly with the treatment of BMP1 (0.5 and 1.0 mg/kg/day, i.p. for 3 days) on EAC cells indicated its antiproliferative activity. This was also supported by flowcytometric data on the cell cycle arrest at G1 in EAC cells. BMP1 (1 mg/kg) reduced the solid tumor weight and volume of about three times further support the antiproliferative nature. Fluorescence and confocal microscopic study on EAC cells after BMP1 (0.5 mg/kg/ day, i.p. for 3 days) treatment indicated certain features of apoptosis, like nuclear fragmentation, membrane blebbing, and vacuolization of cells. DNA fragmentation was clearly observed in alkaline comet assay. Apoptosis induced by BMP1 was further confirmed through flow-cytometric analysis of annexin-V binding study, sub-G1 arrest in the cell cycle and found to be mediated through caspase 3 dependent pathway. LD50 of BMP1 was found to be 12.2 mg/kg, i.p. in male Swiss albino mice. BMP1 treatment at 0.5 mg/kg and 1.0 mg/kg for 10 days did not alter any hematological and biochemical parameters in mice, but after 30 days of treatment produce significant rise in total leucocyte count, neutrophil percentage, serum urea, creatinine, GOT, LDH and decrease in lymphocyte percentage as compared to respective control. In conclusion, BMP1, a protein molecule isolated from Indian toad (B. melanostictus, Schneider) skin, showed antiproliferative and apoptogenic activity on EAC cancer cell with limited toxicity. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Common Indian toad Bufo melanostictus BMP1 Antiproliferative Apoptogenic Caspase 3 Toxicity study

1. Introduction Abbreviations: AO, acridine orange; BMP1, Bufo melanostictus Protein 1; DAPI, 40 ,6-diamidino-2-phenylindole; EAC, Ehrlich ascites carcinoma; EtBr, ethidium bromide; 5-FU, fluorouracil; GOT, glutamate oxaloacetate transaminase; HPLC, high performance liquid chromatography; i.p., intraperitoneal; LD50, lethal dose 50%; LDH, lactate dehydrogenase; TSAE, toad skin aqueous extract. * Corresponding author. Tel.: þ91 33 23508386/6387/6396/1397x229; fax: þ91 33 2351 9755/2241 3288. E-mail addresses: [email protected], [email protected] (A. Gomes). 1 Present address: West Bengal State University, Department of Physiology, Barasat, North 24 Parganas, Kolkata 700126, India. 0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2010.11.015

Amphibian skins are a treasure house of bioactive peptides and proteins (e.g. neuropeptide, substance-P like peptides, bombasin, maximines, bombinin, etc.) that are not only toxic defense molecules but also possess potent therapeutic activities against various pathophysiological conditions like microbial infection, diabetes, cardiovascular disorder and cancer (Gomes et al., 2007a). Among the amphibian, toad, particularly genus Bufo, is found to be a convenient and useful source of granular gland secretions, which commonly

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contains biogenic amines, steroids, peptides and proteins (Clarke, 1997). Use of toad skin has already been mentioned in traditional folk and medicine like, Chan Su, Senso. Chan Su, the traditional Chinese medicine preparation from the dried white secretion of the auricular and skin glands of toad (Bufo bufo gargarizans) induced apoptosis in T24, human bladder carcinoma cell line (Ko et al., 2005). Bufalin and other steroid molecules isolated from Chan Su, showed anticancer property against leukemia, carcinoma, melanoma and other cancer cells (Zhang et al.,1992; Kamano et al.,1998; Cunha-Filho et al., 2010). Cinobufocini injection, a preparation containing certain components of Chan Su, showed anticancer effects in clinical and experimental studies (Wang et al., 2005; Cao and Luo, 2007; Qi et al., 2010). Magainin 2, a 23-residue alpha-helical defense antibiotic peptide molecule isolated from Xenopus, and its two synthetic analogues could rapidly and irreversibly lyse hematopoietic tumor and solid target cells through the concentrations that were relatively non-toxic to welldifferentiated cells (Ohsaki et al., 1992). The anticancer activity of aurein peptides were established by the National Cancer Institute, USA (Rozek et al., 2000). Citropin 1.1, and its other analogous synthetic peptide, A4K14-citropin 1.1, showed anticancer activity on 60 different human cell lines as tested by US National Cancer Institute (Doyle et al., 2003). Practice of natural toxins for therapeutic management may lead to frequent toxicities. A case study revealed that a 43-year-old Korean male patient who had a history of having eaten raw flesh of frog in Keoje Island for the purpose of treatment of arthritis by the oriental custom developed sparganosis within 7 years. It is probable that human sparganosis is acquired by ingestion of raw flesh of frog in that country (Seo et al., 1964). Two patients developed severe illness after eating the toad skin probably Bufo melanostictus Schneider, in southeastern Laos. One boy died, and one developed a digoxin toxicity-like syndrome with bradycardia and heart failure but survived (Keomany et al., 2007). In experimental study it was found that Maximins, isolated from skin secretions of Bombina maxima, was cytotoxic to tumor cells, but at the same time it was also toxic to mice (Lai et al., 2002). Thus it was revealed that drug development from natural toxins may lead to the occurrence of toxicities, which need to be evaluated along with its therapeutic potential. Earlier from this laboratory, it was found that the skin extract of common Indian toad (B. melanostictus, Schneider) possessed significant antineoplastic activity on EAC cells and human leukemic cell lines (Das et al., 1998; Giri et al., 2006). Later a non-protein crystalline molecule, BM-ANF1 had been isolated from the common Indian toad, B. melanostictus that possess anticancer property (Gomes et al., 2007b). In the present communication a protein molecule has been identified that showed antiproliferative and apoptogenic activity in experimental cancer models. 2. Materials and methods 2.1. Materials Acridine orange (Sigma, USA), annexin-V (Sigma, USA), DMSO (SRL, India), Bradford reagent (Sigma, USA), EDTA

(Sigma, USA), ethidium bromide (Sigma, USA), 5-fluorouracil (Sigma, USA), heparin (Sigma, USA), low melting point agarose (Promega, USA), methanol (Spectrochem, India), normal melting point agarose (Promega, USA), propidium iodide (Sigma, USA), PVDF (Pall, USA), RPMI1640 (HiMedia, India), Triton X-100 (SRL, India), trypan blue (SRL, India) were used. All antibodies are from Santa Cruz Biotechnologies Inc., USA. The other chemicals were purchased locally and were analytical grade unless otherwise mentioned. 2.2. Animals Male Swiss albino mice (20  2 g) were used for in vivo experiments. They were given synthetic pellet diet and clean tap water ad libitum and maintained at 24  1  C with 55  5% relative humidity and day and night cycles of 12 h each. All animal experiments were approved by the University Animal Experimental Ethics Committee, and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA), Government of India. 2.3. Ascites tumor Ehrlich ascites carcinoma (EAC) cells were maintained intraperitoneally in Swiss albino mice in the ascitic form. Tumor cells were collected by aspiration with a syringe aseptically, centrifuged for 5 min at 1000 rpm, washed and diluted with 0.9% saline and tumor cell number was adjusted to 1 106 cells/ml by counting the number with a haemocytometer using a phase contrast microscope (Olympus, CK40). Cell viability was evaluated by the trypan blue dye exclusion assay and only cell suspensions that presented more than 95% viability were used. Aseptic condition was maintained throughout the transplantation procedure. 2.4. Collection and preparation of toad skin aqueous extract (TSAE) Adult toads (B. melanostictus, Schneider) of both sexes (70  10 g) were collected commercially. After pithing the toad, its skin was taken out leaving the head region including parotid gland intact. The skin was taken into a ceramic glass mortar and grinded with sea sand and distilled water. The extract was filtered, centrifuged. The concentration of the freshly prepared toad skin aqueous extract (TSAE) was expressed in terms of its protein content. 2.5. DEAE cellulose ion exchange chromatography Crude TSAE was subjected to ion exchange chromatography (IEC) using DEAE cellulose column (20  80 mm) and phosphate buffer (0.01 M, pH 7.5). The flow rate was adjusted at 25 ml/h. Phosphate buffer (0.02 M, pH 7.2) containing increasing molarities of NaCl (0.02, 0.05, 0.1, 0.2, 0.5 and 1.0 M) was used to elute the proteins from the column. Fractions (5 ml) were collected at room temperature (24  1  C) and protein levels were estimated (Lowry

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et al., 1951). The active fraction was tested through the EAC cell count using EAC mice.

570 nm was estimated by measuring with an ELISA reader (Giri et al., 2006).

2.6. High performance liquid chromatography

2.8.3. Flow-cytometric analysis of cell cycle arrest To assay the stage of cell cycle arrest in a flow cytometer (Giri et al., 2009), control and BMP1 (1 mg/kg/day for 3 days) treated EAC cells were fixed in ethanol overnight, washed, treated with DNase free RNase A (10 mg/ml) at 37  C for 30 min and stained with propidium iodide (200 ml from 50 mg/ml stock) and kept at dark for 15 min. Intracellular DNA 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). Data analysis was performed with Cell Quest program.

DEAE cellulose purified TSAE was further purified through HPLC (WATERS 600, USA). Protein-Pak 300 SW column (7.5  300 mm) and 2487 g Absorbance detector, using 10 mM sodium phosphate buffer containing 0.1 M sodium chloride (pH 7.5) with a flow rate 1 ml/min. Detection of protein peak was done at 280 nm and the retention time was calculated. The pure isolated protein was designated as Bufo melanostictus protein 1 (BMP1). 2.7. Molecular weight determination Molecular weight of the BMP1 was determined by SDSPAGE (10% acrylamide slab gel containing 1% SDS), using standard molecular weight marker proteins (Sigma, USA). The migration of BMP1 and molecular weight marker proteins were recorded and calibration curve was prepared by plotting the relative mobility of different marker proteins against logarithmic molecular weight after the method of Laemmli (1970). 2.8. EAC cell proliferation study 2.8.1. EAC cell count The 1 105 Ehrlich ascites carcinoma cells were inoculated in mice by the intraperitoneal route (i.p.) and treated with 0.1 ml of either 0.9% saline, BMP1 (0.5 and 1 mg/kg/ day, i.p.) and 5-FU (10 mg/kg/day, i.p.) for 10 days in control, treated and standard groups of animals, respectively (n ¼ 6). Treatment with BMP1 and 5-FU was started 24 h after tumor implantation and continued for 10 days. On 11th day, mice from each group were sacrificed and tumor cells were collected by repeated intraperitoneal wash with phosphate buffer saline PBS (pH 7.4). Viable cells were counted with trypan blue dye exclusion assay using phase contrast microscope (Olympus, CK40) (Sur and Ganguly, 1994). 2.8.2. MTT assay The 1 105 Ehrlich ascites carcinoma cells were inoculated in mice by the intraperitoneal route (i.p.) and treated with 0.1 ml of either 0.9% saline, BMP1 (0.5 and 1 mg/kg/ day, i.p.) and 5-FU (10 mg/kg/day, i.p.) for 3 days in control, treated and standard groups of animals, respectively (n ¼ 8). Treatment with BMP1 and 5-FU was started 24 h after tumor implantation and continued for 3 days. On 4h day, mice from each group were sacrificed and tumor cells were collected by repeated intraperitoneal wash with phosphate buffer saline PBS (pH 7.4) and resuspended in 3 ml of RPMI-1640 medium each of the above groups. 100 ml of suspended EAC cells were placed in each well of 96 wells plate in duplicate for each animal of each group. 50 ml of MTT solution (5 mg/ml of RPMI-1640 stock) was added and incubated for 3 h in a humidified CO2 incubator (5% CO2 and 95% air). The formazan granules formed by viable cells were dissolved in DMSO and the absorbance at

2.8.4. EAC induced solid tumor in mice EAC induced solid tumor developed by the method of Badr El-Din et al. (2008). Briefly, the mice were divided into three groups (n ¼ 6). EAC cells (1 105 cells/mouse) were injected into the right hind limb of all the animals intramuscularly. Mice of group I were tumor control. Groups II and III received BMP1 (1 mg/kg) and 5-FU (5 mg/kg) intraperitoneally, respectively, for 20 consecutive days. After 20 day of tumor cell implantation, all animals were sacrificed and their tumor weight and volume were noted. 2.9. Bioassay of EAC cell viability Mice were inoculated with 1 105 EAC cells (i.p.). On day 3, mice received 0.1 ml of either 0.9% saline or BMP1 (1 mg/kg, i.p. for 3 day), or 5-FU (10 mg/kg, i.p. for 3 day), respectively, in control, treated and standard groups (n ¼ 8). On 4th day tumor cells from each group of mice were counted and harvested in cold saline, pooled, centrifuged and reinoculated (1 105 cells/mouse) i.p. into three groups of 8 mice each. On day 7 after reinoculation, all animals from each group were sacrificed and EAC cell count/mouse was estimated (Sur and Ganguly, 1994). 2.10. Survivability of EAC bearing mice EAC bearing mice were treated with 0.1 ml of either 0.9% saline (i.p.), BMP1 (1 mg/kg/day, i.p.) and 5-FU (5 mg/kg/day, i.p.) for 20 days in control, treated and standard groups of animals, respectively (n ¼ 8). Then the mice from each group were kept in the laboratory for survival analysis. The (T/C%) value defined as the ratio of the mean survival time (in days) of treated group divided by that of the control group and expressed as percent and was considered as a criterion for the antitumour activity (Sur and Ganguly, 1994). 2.11. Morphological study 2.11.1. Fluorescence microscopy Fluorescent microscope (Motic, Germany) was used to study the cellular characterization, nuclear integrity and membrane permeability of the EAC cells. Both the BMP1 treated (0.5 mg/kg/day, i.p. for 3 days) and untreated control cells were collected separately and centrifuged at

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1000 rpm for 5 min. The pellet was rinsed twice and resuspended in PBS. It was then treated with ethidium bromide (EtBr) and acridine orange solution (AO) (100 mg/ ml of PBS) and observed under a fluorescence microscope for the qualitative determination of apoptotic cells. 2.11.2. Confocal microscopy Confocal laser scanning microscope (Leica TCS-SP2 system, Leica microsystem, Germany) was used to study cellular integrity in the EAC cells. Briefly, control and BMP1 treated (0.5 mg/kg/day, i.p. for 3 days) EAC cells were washed in ice-cold phosphate buffer saline (PBS) and were stained with EtBr (100 mg/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. 2.12. Single-cell gel electrophoresis (alkaline comet assay) Comet assay of EAC cells was performed under alkaline condition following the method of Giri et al. (2006) with minor modifications. EAC cells bearing mice were treated with BMP1 (1 mg/kg/day) for 10 days. On 11th day the cells were collected then washed with cold PBS by centrifuging at 1000 rpm for 5 min in cold centrifuge at 4  C. Slides were initially coated with a layer of normal melting point agarose (0.75% in PBS). After solidification 85 ml of cell–agarose (LMP) suspensions containing 104 cells were placed. After solidification, third layer of low melting point agarose (100 ml) 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 1 h 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. The slides were stained with EtBr (10 mg/ml), covered with a cover glass, and analyzed within 1 h at 100 magnification using a fluorescent microscope (Motic BA400, Germany) with green filter. The photograph was taken through the attached digital camera. The comet length–width ratio (L:W), tailed cell % (TC%) was recorded by randomly counting about 100 cells per slide, considering no overlap of counting, using Motic Images Plus 2.0 software. 2.13. Flow-cytometric analysis of apoptosis Flow-cytometric analysis was done to assess the apoptotic activity induced by the BMP1 (Vermes et al., 1995). BMP1 (1 mg/kg/day) treated, 5-FU treated (10 mg/ kg/day) and untreated EAC cell were collected. The cells were suspended in annexin–HEPES buffer and centrifuged twice. The pellets were resuspended in the same buffer (100 ml) 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 program.

2.14. Caspase 3 and 9 analysis EAC cells were collected after BMP1 treatment (1 mg/ kg/day) for 10 days. BMP1 treated and non-treated cells were lysed and their caspase 3 activities were determined using Sigma Caspase 3 colorimetric assay kit according to the manufacturer’s instructions. Caspase 9 was determined using LEHD-pNA as a substrate, as per the manufacturer’s (US Biochemical) instructions. 2.15. Toxicity study 2.15.1. LD50 determination LD50 value of BMP1 was determined in male Swiss albino mice (20  2 g) through i.p. route according to the method of Litchfield and Wilcoxon (1949). Swiss albino mice at different dose of BMP1 (5, 10, 15, 20, 25 mg/kg, i.p.) and mortality were recorded up to 24 h of observation. Eight mice were taken in each dose level. 2.15.2. Biochemical and hematological study Swiss albino male mice were treated with BMP1 (0.5 and 1 mg/kg, i.p.) for 10 days and 30 days intraperitoneally. In the control animals, 0.9% saline in equal volume was injected. The behavioural changes, food water intake, body weight and body temperature were noted with respect to the control animals. After 10 days and 30 days, urinary parameters were analyzed by multistix, Bayer Diagnostics, India. Hematological parameters and serum biochemical parameters were analyzed using standard protocol. 2.16. Animal ethical committee permission All animal experiments were approved by University animal ethics committee, and were in accordance with the guideline of the Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA), Government of India. 2.17. Statistical analysis All the results were expressed in terms of mean  SE, n ¼ 8 at each dose level unless otherwise mentioned. The level of significance was determined through one way ANOVA, p < 0.05 was considered significant. 3. Results 3.1. Purification of TSAE by ion exchange chromatography Crude TSAE (500 mg), applied on a DEAE cellulose column (20  80 mm), eluted with phosphate buffer (0.2 M, pH 7.2) and stepwise NaCl (dissolved in 0.2 M phosphate buffer, pH 7.2) gradient, produced five protein peaks (P1, P2, P3, P4, and P5). P3 (tube no. 55) was eluted with 0.02 M NaCl, possess antiproliferative activity on EAC cell. Yield of P3 was found to be 3.0  0.7%, protein loss was 5.6  0.2% and 91.4  2.4% protein was recovered by this process (Fig. 1A).

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3.2. High performance liquid chromatography HPLC elution pattern of ion exchange eluted P3 fraction showed one sharp peak at the retention time 7.34 min and one hump (Fig. 1B). For the sake of convenience, HPLC eluted peak at 7.34 min possess antiproliferative activity on EAC cells was provisionally designated as BMP1 (B. melanostictus Protein 1). 3.3. Molecular weight of BMP1 On SDS-PAGE the molecular weight of BMP1 was found to be 79 kDa (Fig. 1C). 3.4. EAC cell proliferation study 3.4.1. EAC cell count EAC cell count on 11th day was inhibited after 10 days treatment of BMP1 (0.5 and 1 mg/kg/day, i.p.) and 5-FU (10 mg/kg/day, i.p.) on EAC bearing mice. BMP1 treated group produced 15.1% and 63.6% inhibition of EAC cell,

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respectively, with compared to control, whereas 5-FU treated standard group produced 55% inhibition of EAC cell. 3.4.2. MTT assay EAC cells treated with 0.1 ml of either 0.9% saline, BMP1 (0.5 and 1 mg/kg/day, i.p.) and 5-FU (10 mg/kg/day, i.p.) for 3 days in control, treated and standard groups of animals, respectively. Treatment with 0.5 mg BMP1, 1 mg BMP1 and 5-FU showed 74.2  1.54%, 25.9  1.41% and 35.4  1.16% MTT values, respectively, considering the control as 100%. Alternatively it can be explained that BMP1 at the above two doses reduced the MTT values 25.8% and 74.1%, respectively, in EAC cells whereas 5-FU reduced the MTT values about 64.6% in comparison to the control group (Fig. 2, Panel A). 3.4.3. Flow-cytometric analysis of cell cycle After BMP1 (1 mg/kg/day for 3 days) treatment of EAC cells flow-cytometric data of cell cycle revealed that there was significant increase in DNA content in the G1 phase (58.4  1.7%) as compared to untreated control cells (46.6  1.4%) whereas, DNA content decreased in S phase (16.5  0.7%) and G2/M phase (7.5  0.5%) in treated EAC cell

Fig. 1. Purification steps of BMP1 from toad skin extract. (A) DEAE cellulose ion exchange chromatography profile of toad skin extract; Peak P3 (tube no. 55) was eluted with 0.2 M NaCl, possess anticancer activity on EAC cell. (B) HPLC profile of fraction P3 (inset figure). P3 fraction on HPLC showed retention time of 7.34 min showed similar anticancer activity and provisionally designated as BMP1; (C) SDS-PAGE profile of BMP1 (lane b) as compared to standard molecular weight marker (lane a). Relative mobility of BMP1 was found to be 1.9–7 kDa (log MW).

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Fig. 2. Effect of BMP1 on EAC cell proliferation in MTT assay, cell cycle and EAC induced solid tumor. Panel A: MTT assay; BMP1 at the 0.5 mg/kg and 1 mg/kg doses reduced the MTT values 25.8% and 74.1%, respectively, in EAC cells whereas 5-FU reduced the MTT values about 64.6% in comparison to the control group (values are mean  SE, *p < 0.01, **p < 0.001, significant). Panel B: cell cycle study; present data provided the information that BMP1 could arrest the cell cycle at G1 and prevent proliferation of EAC cells and simultaneously arrested at sub-G1 phase which is indicative in support of apoptogenic nature of BMP1(values are mean  SE, *p < 0.001). Panel C: EAC induced solid tumor; EAC cell injection in right hind leg muscle of male albino mice caused development of the solid tumor, control tumor weight 3.2 g (a), BMP1 (1 mg/kg/day for 20 days) and 5-FU (5 mg/kg/day for 20 days) decreased the tumor weight significantly as 0.89 g (b) and 0.1 g (c), respectively, as compared with control.

as compared to untreated control cell (28.7  1.2%) and (19.9  1.0%), respectively. These data provided the information that BMP1 could arrest the cell cycle at G1 and prevent proliferation of EAC cells indicating its antiproliferative action. Again the sub-G1 phase of BMP1 treated EAC cells showed significant increase in DNA content (17.6  0.8%) as compared to the control (4.8  0.3) indicating the apoptogenic nature of BMP1 (Fig. 2, Panel B; Table 1). 3.4.4. EAC induced solid tumor EAC cell injection in right hind leg muscle of male albino mice caused development of the solid tumor, weight

3.2  0.46 g. BMP1 (1 mg/kg/day for 20 days) and 5-FU (5 mg/kg/day for 20 days) decreased the tumor weight significantly as 0.89  0.23 g and 0.1  0.04 g, respectively, as compared with control (Fig. 2, Panel C; Table 2). 3.5. Bioassay of EAC cell viability BMP1 significantly decreased the EAC cell viability. BMP1 (1 mg/kg, i.p. for 3 day) and 5-FU (20 mg/kg, i.p. for 3

Table 2 Effect of BMP1 and 5-FU on tumor weight and diameter of EAC induced solid tumor in Swiss albino mice. Table 1 Effect of BMP1 on cell cycle distribution in EAC cells. Group

Sub-G1 (%)

G1 (%)

S (%)

G2-M (%)

Control BMP1 (1 mg/kg)

4.8  0.3 17.6  0.8*

46.4  1.4 58.4  1.7*

28.7  1.1 16.5  0.7

19.9  1.0 7.5  0.5

Values shown are mean  SE (n ¼ 4), *p < 0.001.

Group of animals

Tumor weight (g)

Tumor diameter (mm)

Control BMP1 (1 mg/kg  20 days) 5-FU (5 mg/kg  20 days)

3.2  0.4 0.89  0.2* 0.1  0.04*

9.25  1.2 3.77  0.4* 0.6  0.14*

Values shown are mean  SE (n ¼ 6), *p < 0.001.

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day) produced 73  1.5% and 83.1 1.6% reduction in viability of EAC cells, respectively, as compared to untreated control group. After reinoculation EAC cell viability decreased by 88.6  1.1% and 82.4  3.8% for BMP1 and 5-FU treated group, respectively. 3.6. Survivability of EAC mice Treatment with BMP1 increased the mean survival time of EAC bearing mice, which was found to be 92 days (T/C % ¼ 209.1) as compared to EAC bearing untreated control

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mice (44 days) 5-FU, the standard anticancer agent increased the mean survival time by 81 days (T/C% ¼ 184.1). 3.7. Morphological study 3.7.1. Fluorescence microscopy Fluorescence microscopic study of EAC cells after BMP1 treatment revealed the presence of apoptotic cells. BMP1 treatment caused increase in early and late apoptotic cells accompanied with nuclear fragmentation and membrane blebbing as compared to untreated EAC cell (Fig. 3, Panel A).

Fig. 3. Fluorescence photomicrograph of EAC cells. Panel A: left panel – control cells, right panel – BMP1 treated cells; [a ¼ Nuclear fragmentation, [b ¼ Membrane blebbing found in BMP1 treated cells. Confocal photomicrograph of EAC cells (Panels B and C): Panel B: left – control cells, right – BMP1 treated cells (ethidium bromide staining); Panel C: left – control cells, right – BMP1 treated cells (DAPI staining); [a ¼ vacuolization, [b ¼ marginalization of fragmented nuclei observed in BMP1 treated cells as compared with the respective control.

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Fig. 4. Effect of BMP1 on alkaline comet assay of EAC cells. (A) Control cells, and (B) BMP1 treated cells. Comet like DNA mass is indicated by arrow ([). The length–width ratio of EAC cell DNA mass of BMP1 treated cells (2.9  0.17) was significantly (p < 0.001) different from the control cells (1.3  0.05).

3.7.2. Confocal microscopy The apoptotic and cytotoxic activity of BMP1 was observed by confocal microscopic study. BMP1 treated EAC cells were characterized with nuclear fragmentation and marginization, vacuolization as compared to untreated EAC cell (Fig. 3, Panels B and C).

2.2 and 1.7 fold increase in the activity of caspase 3, respectively, as compared to the control cells. The activity of caspase 9 in EAC cells treated with BMP1 and 5-FU showed 1.6 and 1.8 fold increase as compared to the EAC control cells (Fig. 5, Panel B). 3.11. Toxicity study

3.8. Single-cell gel electrophoresis BMP1 (1 mg/kg, i.p. for 10 days) treatment produced comet like DNA mass as compared to the untreated control cells in the alkaline comet assay. Mean tailed cells (TC) (%) and mean length:width ratios (L:W) was analyzed in control and BMP1 treated EAC cells at 11th day of inoculation. The mean length to width ratio of the DNA mass observed in BMP1 treated EAC cells was significantly greater compared with the normal control cells. The length–width ratio of control cells was 1.3  0.05, whereas in BMP1 treated EAC cells, the ratio was 2.9  0.17. Similarly, the mean frequency of tailed cells was (90.5  0.3) in BMP1 treated EAC cells, which was significantly different from the normal control (6.5  0.25) (Fig. 4). 3.9. Flow-cytometric analysis of apoptosis The four different quadrants of flow-cytometric data represent four different states of cells. The lower left (LL), annexin/PI represent normal healthy cells. The lower right (LR), annexinþ/PI and upper right (UR), annexinþ/ PIþ quadrant represent early and late apoptotic cells, respectively. And the upper left quadrant (UL), annexin/ PIþ represent only necrotic cells. BMP1or 5-FU treatment of EAC cell produced 56.4  0.9% and 57.8  0.8% cells, respectively, in LR quadrant and 34.5  0.5% and 9.3  0.4% cells, respectively, in UR quadrant as compared to 21.9  0.1% cells in LR quadrant and 11.9  0.04% cells in UR quadrant of untreated control cells (Fig. 5, Panel A). 3.10. Caspase 3 and 9 analysis The activity of caspase 3 and 9 in EAC cells was studied after BMP1 treatment. BMP1 and 5-FU treated cells showed

3.11.1. LD50 LD50 value of BMP1 was found to be 12.2 mg/kg, i.p. in male Swiss albino mice. 3.11.2. Biochemical and hematological study BMP1 treatment (0.5 and 1 mg/kg, i.p., n ¼ 8) for 10 days and 30 days did not produce any significant change in food, water intake, body weight and body temperature during the period of investigation. BMP1 treatment did not produce any significant change in urinary parameter of male Swiss albino mice as compared to control. BMP1 (0.5 and 1 mg/kg, i.p.  10 days, n ¼ 8) did not cause any significant change in the blood hematological and serum biochemical markers as compared with control mice. BMP1 treatment after 30 days of treatment produce significant rise in total leukocyte count (control: 8.13  0.27  103 cells/dl, BMP1 treated (0.5 mg/kg): 10.4  0.2  103 cells/dl, BMP1 treated (1 mg/kg): 9.8  0.2  103 cells/dl, p < 0.05) and neutrophil percentage (control: 61.5  1.0%, BMP1 treated (0.5 mg/kg): 71.0  1.5%, BMP1 treated (1 mg/ kg): 76.5  0.8%, p < 0.05) and decreased lymphocyte percentage (control: 30.3  0.9%, BMP1 treated (0.5 mg/kg): 20.5  1.2%, BMP1 treated (1 mg/kg): 14.6  0.7%, p < 0.05) (Table 3). BMP1 (0.5 mg/kg and 1 mg/kg, i.p.  30 days, n ¼ 8) significantly increased the serum biochemical markers as compared with control mice urea (control: 35.7  1.6 mg/dl, BMP1 treated (0.5 mg/kg): 43.5  1.3 mg/dl, BMP1 treated (1 mg/kg): 43.4  1.4 mg/dl, p < 0.05); creatinine (control: 0.83  0.1 mg/dl, BMP1 treated (0.5 mg/kg): 0.94  0.03 mg/ dl, BMP1 treated (1 mg/kg): 0.98  0.03 mg/dl, p < 0.05); GOT (control: 40.1  0.7 IU/l, BMP1 treated (0.5 mg/kg): 57.9  1.3 IU/l, BMP1 treated (1 mg/kg): 58.4  3.4 IU/l, p < 0.05) and LDH (control: 204.5  8.9 IU/l, BMP1 treated

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Fig. 5. Effect of BMP1 on Annexin-V/PI binding of EAC cells (Panel A). Annexin-V binding was analyzed through flow cytometry in control (A), BMP1 (B) treated and C-5-FU treated (C) EAC cells. The lower left (LL), annexin/PI represent normal healthy cells. The lower right (LR), annexinþ/PI and upper right (UR), annexinþ/PIþ quadrant represent early and late apoptotic cells, respectively. And the upper left quadrant (UL), annexin/PIþ represent only necrotic cells. BMP1or 5-FU treatment of EAC cell produced significant numbers of early and late apoptotic cells as compared with untreated control cells. Effect of BMP1 on caspase 3 and 9 activity of EAC cells (Panel B). BMP1 and 5-FU treated cells showed 2.2 and 1.7 fold increase in the activity of caspase 3, respectively, and 1.6 and 1.8 fold increase in caspase 9 activity as compared with the EAC control cells. Values are shown as fold increase in comparison to the respective control.

(0.5 mg/kg): 263.0  8.9 IU/l, BMP1 treated (1 mg/kg): 267.5  8.0 IU/l, p < 0.05) (Table 4). 4. Discussion BMP1, a high molecular weight protein (MW – 79 kDa) was isolated from the Indian toad (B. melanostictus) skin aqueous extract (TSAE) by ion exchange chromatography followed by HPLC. BMP1 treatment after EAC induction in mice may cause direct interaction of BMP1 with EAC cell. Decrease in viable tumor cell count in BMP1 treated EAC mice as compared to untreated EAC mice, showed the

antiproliferative nature of BMP1. The decreased MTT values due to BMP1 treatment in EAC cells and the cell cycle arrest at G1 is an indication of the antiproliferative action of BMP1. EAC induced solid tumor in mice and BMP1 reduces rather hold the growth or proliferation of solid tumor in the present study also supported its antiproliferative action. It is assumed from the present study that BMP1 is more effective in EAC cells when it is exposed directly within the peritoneal cavity than the 5-FU but in solid tumor 5-FU seems to be more effective than BMP1 when the MTT data was compared with the solid tumor data considering the respective dose. BMP1 was also tested in different human

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Table 3 Effect of BMP1 on hematological parameters of male Swiss albino mice. Group of animals

Control (0.9% NaCl)

BMP1 (0.5 mg/kg)

BMP1 (1 mg/kg)

Hemoglobin (g%) Reticulocyte (%) Total RBC count  105/ml Total WBC count  103/ml Neutrophil (%) Lymphocyte (%) Eosinophil (%) Monocyte (%)

10.33  0.22 0.77  0.12 3.40  0.05

10.45  0.29 0.50  0.09 3.46  0.11

10.23  0.30 0.74  0.08 3.19  0.11

8.13  0.27

10.43  0.19*

9.80  0.25*

61.5  1.05 30.38  0.96 5.25  0.49 2.88  0.29

71.0  1.55* 20.50  1.2* 5.38  0.46 3.13  0.48

76.5  0.82* 14.63  0.7* 5.13  0.48 3.75  0.41

Values are shown as mean  SE of respective groups (n ¼ 8), *p < 0.001.

cancer cell lines (U937, K562, HepG2 and C6) which showed reliable and promising results (data not shown). A reliable criterion for assessing the potential of anticancer agent is the prolongation of lifespan of the animals (Clarkson and Burchenal, 1965). Reduction of this tumor burden caused increase in lifespan of EAC bearing mice after BMP1 treatment as compared to EAC bearing untreated mice. Reduction of this tumor burden by BMP1 was may be due to its direct interaction with EAC cell in mouse intraperitoneal cavity. This interaction leads to the morphological changes of EAC cells. The morphological changes incurred during apoptosis are unique and should be a deciding factor concerning the mechanism of cell death. Cell death by apoptosis is characterized by cell chromatin condensation, together with visible nuclear fragmentation formation of apoptotic bodies and disruption of DNA into fragments. These changes may be used as markers for apoptosis (Arends et al., 1990). The effect of non-protein compound that has been isolated from the methanolic skin extract of B. melanostictus, on U937 and K562 cells has been explored by Gomes et al. (2007b) and it was found that the viable cells excluded ethidium bromide and were

Table 4 Effect of BMP1 on serum biochemical parameters of male Swiss albino mice. Group of animals

Control (0.9% NaCl)

BMP1 (0.5 mg/kg)

BMP1 (1 mg/kg)

Glucose (mg/dl) Urea (mg/dl) Creatinine (mg/dl) Uric acid (mg/dl) Cholesterol (mg/dl) Triglygerides (mg/dl) HDL (mg/dl) LDL (mg/dl) Total protein (g/dl) Albumin (g/dl) Globulin (g/dl) ALP (IU/l) GOT (IU/l) GPT (IU/l) Total bilirubin Creatinine kinase (IU/l) LDH (IU/l) Iron (mM/dl)

92.8  3.9 35.7  1.6 0.83  0.1 5.83  0.3 183.2  10.2 87.8  1.5 40.5  0.6 125.2  10.1 8.4  0.3 4.06  0.07 4.3  0.32 207.1  8.9 40.1  0.7 64.7  3.4 0.95  0.08 81.0  2.7 204.5  8.9 91.2  2.0

91.1  3.9 43.5  1.3* 0.94  0.03 5.80  0.3 193.2  9.5 86.3  1.2 40.5  0.5 135.5  9.9 7.9  0.2 4.0  0.08 3.9  0.25 198.5  6.4 57.9  1.3* 65.0  3.9 0.94  0.07 84.5  2.9 263.0  8.9* 58.9  1.7*

92.4  3.3 43.4  1.4* 0.98  0.03* 6.09  0.2 176.8  13.7 85.5  1.8 42.6  0.5 117.1  13.7 8.4  0.2 4.08  0.06 4.36  0.22 204.5  8.1 58.4  3.4* 61.5  4.4 0.9  0.06 81.1  2.6 267.5  8.0* 79.7  1.8*

Values are shown as mean  SE of respective groups (n ¼ 8), *p < 0.001.

permeable to acridine orange, which reacts with DNA to yield a green nuclear fluorescence. Nonviable cells show red/orange fluorescence because on entry of ethidium bromide, which reacts with DNA. In the present study it was observed that the BMP1 treatment increased the number of brightly stained apoptotic cells. Both fluorescence and confocal microscopic studies revealed that BMP1 treatment induced apoptotic characteristic in EAC cell. Apoptosis mediated by BMP1 was measured by fluorescence activated cell sorter (FACS) technique. During apoptosis, the PS residues are translocated in the membrane and are externalised. BMP1 affected the phospholipid distribution over the plasma membrane of EAC cell as observed by annexin-V/PI binding study. By dual staining with annexin-V FITC and propidium iodide it was possible to identify live cell, early apoptotic cells and late apoptotic cells (Darzynkiewicz et al., 2001; Wising et al., 2005). The increased number of early apoptotic cell and late apoptotic EAC cells after BMP1 treatment confirmed that BMP1 inhibited cancer cell growth by inducing apoptosis. In the cell cycle study, BMP1 induced sub-G1 arrest in EAC cells further indicates its apoptogenic action. BMP1 mediated DNA fragmentation due to apoptosis in intact EAC cell was also detected by alkaline comet assay. BMP1 treated EAC cell after lysis when subjected to electrophoresis at alkaline medium shows comet formation as compared to control EAC cell. There is a direct relation between increase in length–width ratio of DNA mass with the extent of DNA fragmentation (Singh et al., 1988). Similar increase in length–width ratio was found after BMP1 treatment in EAC cell as compared to control. Also, DNA extracted from EAC cell when subjected to agarose gel electrophoresis showed ladder formation in BMP1 treated EAC cell as compared to untreated control (data not shown). Fragmentation of DNA due to apoptogenic trigger requires activation of nucleases, e.g., caspases. Caspase 3 share both caspase 9- and caspase 8-mediated pathway of apoptogenic signaling. Caspases also affect cytoskeletal structure, cell cycle regulation, and signaling pathways, ultimately leading to the morphologic manifestations of apoptosis, such as DNA condensation and fragmentation, and membrane blebbing (Thornberry and Lazebnik, 1998). Therefore, caspase 3 expression level was studied in EAC cells after BMP1 treatment. It was found that caspase 3 activities of EAC cells were increased. Similar caspase 3 mediated apoptosis in A 549 cells induced by cinobufocini, a toad skin preparation has been established by Wang et al. (2009). The intrinsic and extrinsic apoptotic pathways converge to caspase 3, which cleaves the inhibitor of the caspase-activated deoxyribonuclease leading to nuclear fragmentation and apoptosis. The intrinsic and extrinsic caspases (upstream) are caspase 9 and caspase 8, respectively, that converge to caspase 3 (Ghobrial et al., 2005). In the present study caspase 9 activity in BMP1 treated EAC cells was also found to be increased which suggested the contribution of intrinsic pathway in BMP1 mediated apoptosis. In a clinical case report study it was previously found that consumption of B. melanostictus skin preparation in southeastern Laos developed severe illness. One boy died,

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and one developed a digoxin toxicity-like syndrome with bradycardia and heart failure but survived (Keomany et al., 2007). Though the present compound was a purified protein but as it was derived from B. melanostictus skin extract, its toxicity profile was examined. LD50 value of Bufalin, one of the potent anticancer molecules isolated from toad skin was found to be 0.14 mg/kg (Bagrov et al., 1995). At the same time LD50 value of BMP1 was found to be 12.2 mg/kg which is much higher as compared to the other molecules isolated from skin extract. BMP1 treatment for 10 days showed antiproliferative activity in EAC bearing mice without any significant toxicity sign. But like bufalin, an anticancer ingredient of toad skin (Nasu et al., 2005), chronic BMP1 exposures may cause certain impairment in renal function. Because, BMP1 treatment for 30 days showed significant increase in serum urea and creatinine levels although no significant change was found in urinary parameters. On the other hand after 10 days of treatment BMP1 showed no significant changes of any serum or urinary parameters. It may be concluded that, Indian toad (B. melanostictus) skin aqueous extract (TSAE) possess various bioactive compounds, one of them, the BMP1, a protein molecule isolated from Indian toad (B. melanostictus, Schneider) skin, showed antiproliferative and apoptogenic activity on EAC cancer cell with limited toxicity. However, further works on the detail anticancer mechanism and toxicity of BMP1 are warranted. Acknowledgement This work was partly funded by a research grant from University Grant Commission, Govt. of India, New Delhi, India (Ref no. F. No. 32-530/2006(SR) dated 08.03.2007). Conflict of interest We declare that there is no conflict of interest among the authors. References Arends, M.J., Morris, R.G., Wyllie, A.H., 1990. Apoptosis. The role of the endonuclease. Am. J. Pathol. 136 (3), 593–608. Badr El-Din, N.K., Noaman, E., Ghoneum, M., 2008. In vivo tumor inhibitory effects of nutritional rice bran supplement MGN-3/ biobran on Ehrlich carcinoma-bearing mice. Nutr. Cancer 60 (2), 235–244. Bagrov, A.Y., Roukoyatkina, N.I., Pinaev, A.G., Dmitrieva, R.I., Fedrova, O.V., 1995. Effects of two endogenous Naþ, K(þ)-ATPase inhibitors, marinobufagenin and ouabain, on isolated rat aorta. Eur. J. Pharmacol. 274, 151–158. Cao, Y.H., Luo, H.S., 2007. Clinical observation of cinobufacini injection used to treat advanced primary liver cancer. Zhong Guo Yi Xue Wen Zhai Lao Nian Yi Xue 1, 8–10. Clarke, B.T., 1997. The natural history of amphibian skin secretions, their normal functioning and potential medicinal applications. Biol. Rev. Camb. Philos. Soc. 72, 365–379. Clarkson, B.D., Burchenal, J.H., 1965. Preliminary screening of antineoplastic drugs. Prog. Clin. Cancer 1, 625–629. Cunha-Filho, G.A., Resck, I.S., Cavalcanti, B.C., Pessoa, C.O., Moraes, M.O., Ferreira, J.R., Rodrigues, F.A., DosSantos, M.L., 2010. Cytotoxic profile of natural and some modified bufadienolides from toad Rhinella schneideri parotoid gland secretion. Toxicon 56 (3), 339–348. Darzynkiewicz, Z., Bedner, E., Smolewski, P., 2001. Flow cytometry in analysis of cell cycle and apoptosis. Semin. Hematol. 38, 179–193.

235

Das, M., Dasgupta, S.C., Gomes, A., 1998. Immunomodulatory and antineoplastic activity of common Indian toad (Bufo melanostictus, Schneider) skin extract. Indian J. Pharmacol. 30, 311–317. Doyle, J., Brinkworth, C.S., Wegener, K.L., Carver, J.A., Llewellyn, L.E., Olver, I.N., Bowie, J.H., Wabnitz, P.A., Tyler, M.J., 2003. nNOS inhibition, antimicrobial and anticancer activity of the amphibian skin peptide, citropin 1.1 and synthetic modifications. Eur. J. Biochem. 270, 1141–1153. Ghobrial, I.M., Witzig, T.E., Adjei, A., 2005. Targeting apoptosis pathways in cancer therapy. CA. Cancer. J. Clin. 55, 178–194. Giri, B., Gomes, A., Debnath, A., Saha, A., Biswas, A.K., Dasgupta, S.C., Gomes, A., 2006. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract on U937 and K562 cells. Toxicon 48, 388–400. Giri, B., Gomes, A., Sengupta, R., Banerjee, S., Nautiyal, J., Sarkar, F.H., Majumdar, A.P., 2009. Curcumin synergizes the growth inhibitory properties of Indian toad (Bufo melanostictus Schneider) skin-derived factor (BM-ANF1) in HCT-116 colon cancer cells. Anticancer Res. 29 (1), 395–401. Gomes, A., Giri, B., Saha, A., Mishra, R., Dasgupta, S.C., Debnath, A., Gomes, A., 2007a. Bioactive molecules from amphibian skin: their biological activities with reference to therapeutic potentials for possible drug development. Indian J. Exp. Biol. 45, 579–593. Gomes, A., Giri, B., Kole, L., Saha, A., Debnath, A., Gomes, A., 2007b. A crystalline compound (BM-ANF1) from the Indian toad (Bufo melanostictus, Schneider) skin extract, induced antiproliferation and apoptosis in leukemic and hepatoma cell line involving cell cycle proteins. Toxicon 50, 835–849. Kamano, Y., Kotake, A., Hashima, H., Inoue, M., Morita, H., Takeya, K., Itokawa, H., Nandachi, N., Segawa, T., Yukita, A., Saitou, K., Katsuyama, M., Pettit, G.R., 1998. Structure-cytotoxic activity relationship for the toad poison bufadienolides. Bioorg. Med. Chem. 6, 1103–1115. Keomany, S., Mayxay, M., Souvannasing, P., Vilayhong, C., Stuart, B.L., Srour, L.M., Newton, P.N., 2007. Toad poisoning in Laos. Am. J. Trop. Med. Hyg. 77 (5), 850–853. Ko, W.S., Park, T.Y., Park, C., Kim, Y.H., Yoon, H.J., Lee, S.Y., Hong, S.H., Choi, B.T., Lee, Y.T., Choi, Y.H., 2005. Induction of apoptosis by Chan Su, a traditional Chinese medicine, in human bladder carcinoma T24 cells. Oncol. Rep. 14, 475–480. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680–685. Lai, R., Zheng, Y.T., Shen, J.H., Liu, G.J., Liu, H., Lee, W.H., Tang, S.Z., Zhang, Y., 2002. Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima. Peptides 23, 427–435. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose effect experiments. J. Pharmacol. Exp. Ther. 96, 99–102. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Nasu, K., Nishida, M., Ueda, T., Takai, N., Bing, S., Narahara, H., Miyakawa, I., 2005. Bufalin induces apoptosis and the G0/G1 cell cycle arrest of endometriotic stromal cells: a promising agent for the treatment of endometriosis. Mol. Hum. Reprod. 11, 817–823. Ohsaki, Y., Gazdar, A.F., Chen, H.C., Johnson, B.E., 1992. Antitumor activity of magainin analogues against human lung cancer cell lines. Cancer Res. 52, 3534–3538. Qi, F., Li, A., Zhao, L., Xu, H., Inagaki, Y., Wang, D., Cui, X., Gao, B., Kokudo, N., Nakata, M., Tang, W., 2010. Cinobufacini, an aqueous extract from Bufo bufo gargarizans Cantor, induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells. J. Ethnopharmacol. 128 (3), 654–661. Rozek, T., Wegener, K.L., Bowien, J.H., Olver, I.N., Carver, J.A., Wallace, J.C., Tyler, M.J., 2000. The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis. Eur. J. Biochem. 267, 5330–5341. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Seo, B.S., Rim, H.J., Yoon, J.J., Lee, D.J., 1964. A case report of sparganosis. Korean J. Parasitol. 2 (3), 179–182. Sur, P., Ganguly, D.K., 1994. Tea plant root extract (TRE) as antineoplastic agent. Planta. Med. 60 (2), 106–109. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 281, 1312–1316. Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C., 1995. A novel assay for apoptosis, flow cytometric detection of phosphatidylserin expression on early apoptotic cells using fluorescein labeled annexin-V. J. Immunol. Methods 184, 39–51.

236

P. Bhattacharjee et al. / Toxicon 57 (2011) 225–236

Wang, J., Jin, Y., Xu, Z., Zheng, Z., Wan, S., 2009. Involvement of caspase-3 activity and survivin downregulation in cinobufocini-induced apoptosis in A 549 cells. Exp. Biol. Med. (Maywood) 234 (5), 566–572. Wang, Y., Li, J.M., Yang, C.M., Zhang, L., Shen, Z.X., 2005. Study on apoptosis of NB4 cells induced by cinobufagin and its mechanism. Tumor 6, 534–537.

Wising, C., Azem, J., Zetterberg, M., Svensson, L.A., Ahlman, K., Lagergard, T., 2005. Induction of apoptosis/necrosis in various human cell lineage by Haemophilus ducreyi cytolethal distending toxin. Toxicon 45 (6), 767–776. Zhang, L., Yoshida, T., Kuroiwa, Y., 1992. Stimulation of melanin synthesis of B16-F10 mouse melanoma cells by bufalin. Life Sci. 51, 17–24.