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

Abstract

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 flow-cytometric 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.

Introduction

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 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 well-differentiated 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.