Bufo melanostictus, Schneider

Toxicon 58 (2011) 85–92

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

Anticancer activity of a low immunogenic protein toxin (BMP1) from Indian toad (Bufo melanostictus, Schneider) skin extract Antony Gomes a, *, Biplab Giri a,1, Aftab Alam a, Sanghamitra Mukherjee a, Pushpak Bhattacharjee a, Aparna Gomes b a

Laboratory of Toxinology and Experimental Pharmacodynamics, University of Calcutta, Department of Physiology, 92 Acharya Prafulla Chandra Road, Kolkata 700 009, India b Drug Development Division, Indian Institute of Chemical Biology, CSIR, 4 Raja S.C. Mallick Road, Kolkata 700 032, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 March 2011 Received in revised form 14 May 2011 Accepted 17 May 2011 Available online 26 May 2011

Earlier, a protein (BMP1, MW-79kDa) had been isolated from Indian toad (Bufo melanostictus) skin aqueous extract possessed anticancer activity against EAC bearing mice (Bhattacharjee et al., 2011). In the present study, the anti-proliferative and apoptogenic activities of BMP1 have been evaluated in leukemic (U937 and K562) and hepatoma (HepG2) cells. BMP1 dose dependently inhibited U937 and K562 cell growth having IC50 values of 49 mg/ml and 30 mg/ml respectively. The anti-proliferative activity of BMP1 was observed in MTT assay, proliferating cell nuclear antigen (PCNA) expression and cell cycle arrest study. Flow-cytometric data revealed that BMP1 arrested cell cycle in U937 and K562 cells at Sub-G1 and G1 phases. The BMP1-induced dose dependent expressions of CDKIs (p21cip1 and p27kip1) and inhibition of CDK2 and PCNA expression in HepG2 cells support the inhibition of cell proliferation due to G1 arrest. BMP1-induced apoptosis analyzed by annexin-V binding study and the DNA fragmentation by comet assay were correlated with the sub-G1 arrest. The parallel induction of bax and p53 expression in HepG2 cells and the up-regulation of caspase 3 and caspase 9 due to BMP1 treatment indicated the involvement of p53-dependent intrinsic pathway of apoptosis. BMP1 was found to be low immunogenic in nature. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Common Indian toad Bufo melanostictus BMP1 Anti-proliferative Apoptogenic CDK Low immunogenic protein

1. Introduction Toad’s granular gland secretions contain biogenic amines, steroids, peptides and proteins (Clarke, 1997), not Abbreviations: AO, acridine orange; BMP1, Bufo melanostictus protein 1; CDKI, cyclin-dependent kinase inhibitor; DAPI, 40 ,6-diamidino-2phenylindole; EAC, Ehrlich ascites carcinoma; EtBr, ethidium bromide; 5FU, fluorouracil; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PCNA, proliferating cell nuclear antigen; 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 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2011.05.008

only contain toxic defense molecules but also possess potent therapeutic activities against microbial infection, diabetes, cardiovascular disorder and cancer (Gomes et al., 2007a). 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; Qi et al., 2010). The anticancer activity of aurein peptides

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A. Gomes et al. / Toxicon 58 (2011) 85–92

was 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). Maximins, isolated from skin secretions of Bombina maxima, showed cytotoxicity to tumor cells, but at the same time it was toxic to mice (Lai et al., 2002). Earlier from this laboratory, it was found that the skin extract of common Indian toad (Bufo 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 compound, BM-ANF1 had been isolated from Bufo melanostictus that possesses anticancer properties (Gomes et al., 2007b). Very recently from this laboratory, a protein toxin (BMP1) had been isolated which was demonstrated to be anti-proliferative and apoptogenic against mouse Ehrlich ascites carcinoma with limited toxicity (Bhattacharjee et al., 2011). In the present communication, further detail studies have been carried out on the anticancer activities of BMP1 against leukemic (U937 and K562) and hepatoma (HepG2) cancer cells.

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. 2.4. Anti-proliferation study 2.4.1. U937 and K562 cell count and determination of IC50 One hundred microliters of U937 and K562 cell suspension containing 105 cells/ml of sterile RPMI 1640 was added to each well in a sterile 96-well plate. BMP1 was dissolved in sterile RPMI medium and cancer cells were cultured in the presence and absence of BMP1 (10–200 mg/ ml). Ara-C (100 mg/ml) and Imatinib mesylate (100 mg/ml) were used as standard drugs. The number of viable cells was counted after 24 h and 48 h by trypan blue dye exclusion method using a phase contrast microscope (Olympus, CK40), and the percentage inhibition of cell growth was calculated as compared to control (Agrawal et al., 1989) (n ¼ 6). The IC50 of BMP1, defined as the concentration of compound that causes 50% reduction in viable cell count in 24 h, was calculated (Reed and Muench, 1938).

2. Materials & methods 2.1. Materials U937, K562 and HepG2 cells were procured from National Center for Cell Sciences (Pune, India), 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), RPMI 1640 (HiMedia, India), Triton X-100 (SRL, India) and Trypan blue (SRL, India) were used. All antibodies are from Santa Cruz Biotechnologies Inc., USA and Cell Signaling Tech., 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 lethality study and New Zealand strain male rabbits (1.6 0.1 kg) were used for antiserum development. 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. Collection and preparation of toad skin aqueous extract (TSAE) Adult toads (Bufo 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

2.4.2. MTT assay MTT assay of control and BMP1-treated (50 mg/ml) cells was done to confirm the cytotoxic effect (Kawada et al., 2002). Control and treated cells were cultured in sterile 96-well plates incubated at 37 C in 5% CO2 incubator for 24 h (n ¼ 6). 20 ml MTT (5 mg/ml) was then added in each well and allowed to incubate further in same condition for 3 h. 100 ml of DMSO was added to each well to dissolve the formazan crystal formed. The OD was recorded at 570 nm with microplate reader (Merck-MIOS Mini, Model No. 309). Percentage growth inhibition was equal to [1-(OD of treated/OD of control)] 100. 2.4.3. Flow-cytometric analysis of cell cycle arrest To assay the stage of cell cycle arrest in a flowcytometer, control and BMP1 (IC50 dose, 24 h) treated U937 and K562 cells were fixed in ethanol overnight, washed and treated with DNase free RNase A (10 mg/ml) at 37 C for 30 min and stained with 200 ml propidium iodide (50 mg/ml) 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 and analyzed by Modfit software (Becton Dickinson) (Giri et al., 2009). 2.4.4. Western immunoblotting The effect of BMP1 on the expression of cell cyclerelated proteins (PCNA/p21/p27/p53) and pro-apoptotic bax was evaluated through Western blotting. HepG2 cells were treated with BMP1 (15, 30, 60 mg/ml) for 48 h and were seeded onto 60-mm plates, washed with PBS at termination and lysed in lysis buffer containing protease inhibitor mixture (Roche Applied Science, Indianapolis, IN), 1 mM phenylmethylsulfonyluoride, 10 mM NaF, 1 mM Na3VO4, and 50 mM b-glycerophosphate and phosphatase


inhibitor cocktail (Sigma). Protein concentration was quantitated using a modified Bradford assay (Bio-Rad protein assay, Bio-Rad Laboratories Hercules, CA), and 100 mg of total protein, solubilized in SDS-sample buffer and electrophoresed in 10% polyacrylamide (PAGE)-SDS gel system, was electrotransblotted onto a PVDF membrane and blocked with reconstituted non-fat dried milk. Following serial exposure of membranes to primary antibodies, proteins were detected by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (1:5000; Amersham Biosciences, Piscataway, NJ), followed by enhanced chemi-luminescence (ECL from Amersham Biosciences). 2.5. Apoptosis study 2.5.1. Fluorescence microscopy Fluorescence microscope (Motic, Germany) was used to study the nuclear integrity and membrane permeability of the leukemic cells. Both the BMP1 (IC50, 48 h) treated and untreated control cells were collected separately and centrifuged at 1000 rpm for 5 min. The pellet was rinsed twice and re-suspended in PBS. It was then treated with ethidium bromide and acridine orange solution (100 mg/ml of PBS) and observed under a fluorescence microscope for the qualitative determination of apoptotic cells. 2.5.2. Comet assay Comet assay of U937 and K562 cells was performed under alkaline condition following method of Giri et al. (2006). Both the BMP1 (IC50, 48 h) treated and untreated control cells were collected separately and 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 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. The slides were stained with EtBr (10 mg/ml), covered with a cover glass, and analyzed within 1 h at 100 magnification using a fluorescence 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. 2.5.3. Annexin-V binding Flow-cytometric analysis was done to assess the apoptotic activity induced by the BMP1 (Vermes et al., 1995). In brief, 2 106 cultured U937 cells were treated with BMP1 at IC50 doses for 24 h and centrifuged at 4 C. The cells were suspended in annexin–hepes buffer and centrifuged twice. The pellets were re-suspended in the

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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). Flowcytometric 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. 2.5.4. Caspase assay U937 cells were cultured in presence or absence of BMP1 (1/2 IC50, 48 h). Treated and non-treated cells were lysed and their caspase 3 activities were determined colorimetrically using Sigma Caspase 3 Assay Kit according to the manufacturer’s instructions. Caspase 9 was determined using LEHD-pNA as a substrate, as per the manufacturer’s instructions (colorimetric assay kit, US Biochemical). 2.6. Effect on normal peripheral blood mononuclear cell Peripheral blood mononuclear cells (PBMNC) toxicity was determined as reported previously (Giri et al., 2006). Briefly, after informed consent peripheral blood was collected from volunteers and separated using histopaque from the plasma interface and washed twice with normal saline. The cell pellet was re-suspended in culture medium with density of 1 106 cells/ml and cultured in a CO2 incubator with 95% air and 5% CO2 at 37 C for 24 h in presence and absence of BMP1 (100 mg/ml) and standard anticancer drugs imatinib mesylate (100 mg/ml). PBMNCs were stimulated with 2.5 mg/ml of phytohemoagglutinin (Kondala et al., 2005). Cell viability was determined using trypan blue (0.4%) dye exclusion procedure and viable cells were counted in a phase contrast microscope (Olympus CK40) and percentage (%) inhibition of viable cells was calculated as compared with control. 2.7. Immunogenicity study 2.7.1. Immunization of rabbits Antiserum was raised in New Zealand strain male rabbits (1.6 0.1 kg). BMP1 was mixed (1:1, v/v) with Freunds’ complete adjuvant (FCA) and injected subcutaneously at first, second, and third weeks (one injection per week). After a week gap, booster injection without FCA was injected intravenously for another three weeks. In the next week, blood was drawn from the marginal ear vein. Serum was separated by centrifugation at 2000 rpm for 20 min. Complement inactivation was done by heating the serum at 56 C. The serum was stored at 4 C until further use. 2.7.2. Immunogel diffusion 1.5% agarose was prepared in distilled water having 0.01% sodium azide as an anti-fungal agent. The mixture was heated just to boil and then cooled and poured on a clear slide (4 ml/slide) and allowed to solidify. After solidification holes were made with borer. In the central hole the protein antigen BMP1 was given and the peripheral holes were loaded with BMP1-antiserum and kept for 48 h at 4 C in moist condition for diffusion and antigen–antibody reaction.

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After 48 h the gel was repeatedly washed with 0.9% saline for removal of excess antigen and antiserum (Ouchterlony, 1948). 2.7.3. Indirect hemagglutination assay This was performed according to the method of Stavitsky (1954). Serial 2 fold dilutions of serum sample were made in 25 ml of phosphate buffer saline in U-bottomed micro-titration plates. In the experimental plate serum sample was mixed with 25 ml of antigen coated tannic RBC (2.5% goat RBC suspension). In the control plates uncoated tannic RBC was used. After mixing, the RBC was allowed to stand at 37 C until control well showed an unequivocally negative (a small button) pattern. The value of highest dilution carrying visible hemagglutination was taken as the antibody titre. 2.7.4. Lethality neutralization of BMP1 by BMP1-antiserum Various doses of BMP1 were mixed with a fixed amount of BMP1-antiserum, incubated at 37 1 C for 60 min and centrifuged at 2000 rpm for 10 min. The supernatant was injected (i.p.) into male albino mice (18–20 gm). Lethality was assayed in terms of LD50 of the BMP1 (Litchfield and Wilcoxon, 1949). 2.8. 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. 2.9. Statistical analysis All the results were expressed in terms of mean SE, n ¼ 6 at each dose level unless otherwise mentioned. The level of significance was determined through Student’s t-test or one way ANOVA, p < 0.05 was considered significant.

3.1.3. Cell cycle analysis The flow-cytometric data of cell cycle revealed that after 48 h of BMP1 (IC50 dose) treatment in U937 cells, there was significant increase in DNA content as observed in the SubG1 and G1/G0 phases as 14.4 0.7% and 61.7 1.4% respectively. Where as the untreated control cells showed 4.9 0.4% in Sub-G1 and 48.7 1.5% in G1/G0 stages. In K562 cells with the treatment of BMP1 at IC50 dose, DNA contents were significantly increased in Sub-G1 and G1/G0 phases and found to be 15.1 0.8% in Sub-G1 and 60.4 1.1% in G1/G0 phases respectively as compared with respective controls as 5.1 0.6% and 47.8 1.4% (Fig. 1B). 3.1.4. Western immunoblotting of cell cycle proteins (PCNA, p21, p27, CDK2) To evaluate the effect of BMP1 on the expression of CDKIs, marker proteins like p21 and p27 and CDK2 level were analyzed through Western blotting techniques using HepG2 cells as a model of study. BMP1 (15–60 mg/ml) dose dependently increased the level of p21 and p27 expression at 48 h of treatment in HepG2 cells (Fig.1C). The CDK2 and proliferating cell nuclear antigen (PCNA) expression levels were significantly inhibited with same treatment situation indicating the anti-proliferative nature of BMP1 (Fig. 1C). To evaluate the p53-dependent induction of CDKIs, same blot was stripped and re-probed with p53 antibody which was represented in Fig. 3B. Dose dependent stimulation of p53 expression was found to be parallel with the p21 (in Fig. 1C) supports the dependency of p53 in cell proliferation control (Fig. 3B). 3.2. Apoptosis study 3.2.1. Fluorescence microscopy Fluorescence microscopic observations of the BMP1treated (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. 2).

3. Results 3.1. Anti-proliferation study 3.1.1. In vitro U937 & K562 cell growth inhibition Dose dependent studies of U937 and K562 cell count were done to evaluate the inhibition concentration 50% (IC50) value of the respective cell groups. BMP1 (10–200 mg/ ml 24 h, n ¼ 6) significantly inhibited cell count in U937 cells (27.4–61%) and K562 cells (21.3–78.1%) as compared with control untreated cells at a dose dependent manner. From the dose response curve, the IC50 values were found to be 49 mg/ml (0.62 mM) in U937 cells and 30 mg/ml (0.38 mM) in K562 cells. 3.1.2. MTT assay of U937 and K562 cell BMP1 (50 mg/ml) showed significant percentage of growth inhibition as 50.8%, 62.2% and 52.3% respectively in U937, K562 and HepG2 cells as compared with respective control (Fig. 1A).

3.2.2. Comet assay The mean length-to-width ratio (L:W) of the DNA mass observed in BMP1-treated (IC50 dose, 48 h) U937 cells was significantly greater (p < 0.001) compared with the normal control cells. Similarly, the mean frequency of tailed cell percentage (TC%) was 84.5 0.28% in BMP1-treated U937 cells, which was significantly different (p < 0.001) from the normal control (8.7 0.14%). The mean L:W of the DNA mass in BMP1-treated (IC50 dose, 48 h) K562 cells was significantly greater (p < 0.001) compared with the normal control cells. Similarly, the mean frequency of TC% was 87.2 0.21% in BMP1-treated K562 cells, which was significantly different (p < 0.001) from the normal control (9.1 0.10%) (Table 1). 3.2.3. Annexin-V binding study After BMP1 (IC50, 48 h) treatment of U937 cells, flowcytometric data revealed that BMP1 significantly increased the early (annexin-Vþ/PI) and late (annexin-Vþ/PIþ)


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Fig. 1. Effect of BMP1 on U937, K562 and HepG2 cell proliferation. Panel A: MTT assay; BMP1 at 50 mg/ml reduced the MTT values in U937, K562 and HepG2 cells as 50.8%, 62.2% and 52.3% respectively (values are mean SE, *p < 0.001, significant). Panel B: Cell cycle study; BMP1 could arrest the cell cycle at G1 and prevent proliferation of U937, K562 and HepG2 cells and simultaneously arrested at sub-G1 phase which support its apoptogenic nature (values are mean SE, *p < 0.001, significant). Panel C: BMP1 suppressed the PCNA and CDK2 expression dose dependently in HepG2 cells. At the same time BMP1 induced the expression of CDKIs (p21 and p27 proteins) dose dependently explain the anti-proliferating activity of BMP1.

apoptotic cells as 32.37 0.19% and 19.21 0.18% respectively compared to the respective controls as 2.18 0.16% early and 0.43 0.09% late apoptotic cells (Table 2). 3.2.4. Caspase assay BMP1-treated (1/2 IC50, 48 h) U937 cells showed 2 fold increase in the caspase 3 activity as compared to the control cells. BMP1-treated (1/2 IC50) U937 cells showed 3.15 fold increase in the caspase 9 activity as compared to the control cells (Fig. 3A). 3.2.5. Western immunoblotting of p53 and bax Apoptosis induction due to BMP1 treatment may be correlated with the cell cycle arrest and p53 induction. The comet assay experiments and the induction of caspases due to BMP1 treatment was a clue of DNA damage which may be linked with the p53-dependent induction of apoptosis. To test this, the same blot was immune-blotted with p53 antibody and a pro-apoptotic protein, bax antibody. It was demonstrated that BMP1 (15–60 mg/ml) dose dependently

increased the level of p53 expression in parallel with the bax expression in HepG2 cells (Fig. 3B). 3.3. Normal peripheral blood mononuclear cell (PBMNC) toxicity The viable human peripheral blood mononuclear cells (PBMNC) were counted after the treatment of BMP1 (100 mg/ml, about twice-IC50 dose of U937 cells and thriceIC50 dose of K562 cells) and imatinib mesylate (100 mg/ml). The count in BMP1 and imatinib mesylate-treated cells decreased significantly by 26% and 23% (p < 0.05) as compared to the control which were interestingly comparable in terms of normal cell toxicity. 3.4. Immunological study 3.4.1. Immunogel diffusion with BMP1-antiserum Rabbits exposed to BMP1 (1.4 mg) did not change significantly in body weight, food intake and other

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A. Gomes et al. / Toxicon 58 (2011) 85–92 Table 1 Effect of BMP1 on the alkaline comet assay of U937 and K562 cells. Experimental group

Tailed cells (%)

U937 Control U937 (BMP1-treated, IC50) K562 Control K562 (BMP1-treated, IC50)

8.7 84.5 9.1 87.2

Length–width ratio of DNA mass

0.14 0.28* 0.10 0.21*

1.06 1.79 1.08 1.83

0.03 0.11* 0.07 0.09*

Values as mean SE (n ¼ 4). *p < 0.001, significant.

behavior during and after completion of schedule. The antiserum produced faint precipitation bands against BMP1 in immunogel diffusion (Fig. 4). 3.4.2. Indirect hemagglutination study with BMP1-antiserum Antiserum titre was monitored by indirect hemagglutination test and expressed in terms of number of wells in control and experimental groups. The antibody titre of BMP1-antiserum was found to be very low (1:4).

Fig. 2. Fluorescence photomicrograph of U937 and K562 cells treated with BMP1. Upper Panel: A ¼ U937 control cells, B ¼ BMP1-treated U937 cells (a ¼ membrane blebbing, b ¼ nuclear fragmentation). Lower Panel: C ¼ K562 control cells, D ¼ BMP1-treated K562 cells (a ¼ membrane blebbing, b ¼ nuclear fragmentation). Magnification 400.

3.4.3. Lethality neutralization of BMP1 by BMP1-antiserum The LD50 dose of BMP1 was found to be 12.2 mg/kg (i.p.) in Swiss male albino mice. BMP1-antiserum was mixed with BMP1 (1:1 v/v i.e., 0.1 ml antiserum: 0.1 ml BMP1 containing LD50 dose). The mixture was incubated for half an hour at 37 C. After the mixture was centrifuged, supernatant was injected in male Swiss albino mice intraperitoneally. BMP1-antiserum failed to antagonize the lethal action of BMP1 indicating the low immunogenicity.

4. Discussion In the present study, dose dependent reduction of viable cell count and the MTT values were observed due to BMP1 treatment in U937 and K562 cells in vitro. The reduction OD value during MTT assay has a direct correlation with the growth inhibitory rate and inverse relation with proliferative rate (Andrew et al., 2002). Flow-cytometric analysis of cell cycle using BMP1 on U937 and K562 cancer cells revealed that it arrested the cell cycle at Sub-G1 and G1/G0 phases indicating its anti-proliferating and apoptogenic tendency. Mammalian cell cycle is known to be controlled by anti-mitogenic signals where it involves p21cip1 and p27kip1 that bind to and inhibit cyclin-dependent kinases (CDKs) required for the initiation of S phase. The protein p21cip1 binds to the DNA polymerase d processivity factor, proliferating cell nuclear antigen (PCNA) and inhibits PCNA-dependent DNA replication (Luo et al., 1995). BMP1 induced the expression of cyclin-dependent kinase inhibitors (CDKIs) p21cip1 and p27kip1 in HepG2 cells and reduced the CDK2 level dose dependently. The progression of Table 2 Effect of BMP1 on the annexin-V/PI binding of U937 cells. Fig. 3. Apoptogenic effect of BMP1 on U937 and HepG2 cells. Panel A: BMP1 showed 3.15 fold increase in caspase 9 activity and 2 fold increase in caspase 3 activity in U937 cells, which may be cause of DNA damage during apoptosis induction. Panel B: BMP1 induced p53 expression dose dependently which may be linked with p53-dependent DNA damage and apoptosis induction supported by the BMP1-induced dose dependent parallel expression of a pro-apoptotic protein bax.

Cell stages (binding status)

U937 Control (%)

Normal (annexin-V/PI) Early apoptotic (annexin-Vþ/PI) Late apoptotic (annexin-Vþ/PIþ) Necrotic (annexin-V/PIþ)

97.27 2.18 0.43 0.12

0.28 0.16 0.09 0.02

Values as mean SE (n ¼ 4). *p < 0.001, significant.

BMP1-treated U937 (%) 47.16 32.37 19.21 1.26

0.24 0.19* 0.18* 0.07


Fig. 4. Immunogel diffusion of BMP1 and BMP1-antiserum. BMP1 protein was loaded in the middle well (B) and the BMP1 antisera were loaded in two wells (A) in two sides of B. The BMP1-antiserum produced faint immunoprecipitation bands ([) against BMP1 proteins.

mammalian cells through different cell cycle phases is a highly regulated process which is largely controlled by cyclins, the regulatory units of CDKs (Ko et al., 1997). BMP1 induced cell cycle arrest at G1, down-regulation of PCNA and CDK2 level and simultaneous up-regulation of p21cip1 may explain its anti-proliferative activity. BMP1 induced p53 protein expression was dose dependent, which was corresponding with the expression of p21cip1. It is well known that p53-dependent induction of p21cip1 prevents entry of cells into S phase (David-Pfeuty et al., 2001). Among the transcriptional targets of p53, p21cip1 plays a key role in mediating G1 arrest (Waldman et al., 1995) following DNA damage (el-Deiry et al., 1994). BMP1induced moderate expression of p27kip1 was also observed in HepG2 cells. p27kip1 could mediate cell growth arrest and is thought to play a critical role in negative regulation of cell division (Sherr, 1996; Fero et al., 1996). The result of CDKIs induction in most cells is inhibition of cell proliferation, differentiation or apoptosis. Flow-cytometric analysis of annexin-V/PI binding is a very good tool for detecting early and late apoptotic events in intact cells. At 48 h, BMP1 showed significant annexin-V binding in the lower right and upper right quadrant indicating early and late apoptotic pattern of U937 cells. Annexin-V binding is based on the transposition of phosphatidyl serine from the inner to the outer face of cell membrane during the early stages of apoptosis (Vermes et al., 1995). Apoptosis leads to cytoskeletal disruption, cell shrinkage, membrane blebing, nuclear fragmentation and disruption of DNA into fragments (Fairbairn et al., 1996). BMP1-induced DNA fragmentation was demonstrated in single-cell gel electrophoresis (alkaline comet assay) experiment where BMP1 produced increase in comet length: width (L:W) ratio of DNA mass in U937 and K562 cells. There is a direct relation of increase in L:W of DNA mass with the extent of DNA fragmentation (Singh et al., 1988). Fragmentation of DNA due to apoptogenic trigger requires activation of nucleases, e.g., caspases. Caspase 3 share both caspase 9- (intrinsic) and caspase 8 (extrinsic)- mediated pathway of apoptogenic signaling (Ghobrial et al., 2005). It was found that BMP1 significantly increased caspase 3 and caspase 9 level in U937 cells which might be the cause for DNA damage indicating the contribution of intrinsic pathway of apoptosis in agreement with our recent observation in EAC cells (Bhattacharya et al., 2011). Similar caspase 3 mediated apoptosis in A549 cells induced by cinobufocini, a toad skin preparation had been reported (Wang et al., 2009). Bax, a pro-apoptotic protein is

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considered to be an important marker for the apotosis pathway. The early and late apoptosis induced by BMP1 as analyzed by annexin-V binding study was correlated with the G1 and sub-G1 cell cycle arrest. The DNA fragmentation was found to be prominent in comet assay when U937 cells were treated with BMP1. The induction of bax and p53 expression in HepG2 cells due to BMP1 treatment may be indicated by the p53-dependent apoptosis induction which was supported by the parallel induction of p21cip1, which may be cause for p53-dependent G1 arrest (el-Deiry et al., 1994). Interestingly, BMP1 was less toxic toward normal peripheral blood mononuclear cells than the cancer cells. But the toxicity testing was not complete, which requires further study in different normal cell lines. The low immunogenic property of BMP1 found in the present study revealed that the molecules may not react with the body proteins of the host, thus favoring less immunogenic effects. It is also true that detail cross reactivity of the BMP1 needs to be worked out before one may design further experiments for drug development against cancer. It may be concluded that, Indian toad (Bufo melanostictus) skin aqueous extract (TSAE) posses various bioactive compounds, one of them, the BMP1 showed antiproliferative and apoptogenic activity on leukemic (U937, K562) and hepatoma (HepG2) cancer cells with minimum toxicity toward normal cell indicating its specificity. Being a protein, BMP1 was interestingly low immunogenic in nature which is encouraging for further research for possible drug clues against cancer. However, further detail anticancer mechanism and functional studies are warranted. Acknowledgment 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 Agrawal, S., Ikeuchi, T., Sun, D., Sarin, P.S., Konopka, A., Maizel, J., Zamecnik, P.C., 1989. Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues. Proc. Natl. Acad. Sci. 86, 7790–7794. Andrew, K.J., Liu, H., Suzui, M., Vural, M.E., Xiao, D., Weinstein, I.B., 2002. Resveratrol induces growth inhibition, S-phase arrest, apoptosis, and changes in biomarker expression in several human cancer cell lines. Clin. Cancer Res. 8, 893–903. Bhattacharjee, P., Giri, B., Gomes, A., 2011. Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus, Schneider) skin. Toxicon 57, 225–236. 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. Cunha-Filho, G.A., Resck, I.S., Cavalcanti, B.C., Pessoa, C.O., Moraes, M.O., Ferreira, J.R., Rodrigues, F.A., Dos, Santos, M.L., 2010. Cytotoxic profile of natural and some modified bufadienolides from toad Rhinella schneideri parotoid gland secretion. Toxicon 56 (3), 339–348.

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