Int. J. Pharm. Sci. Rev. Res., 20(1), May – Jun 2013; nᵒ 25, 146-152 ISSN 0976 – 044X International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net 146 Shila Elizabeth Besra 1* , Moumita Ray 1 , Sayantan Dey 1 , Subhadeep Roy 1 , Nilanjana Deb 2 1. Drug Development/ Diagnostic & Biotechnology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India. 2. Gupta College of Technological Sciences, Ashram More, G. T. Road P.O. Asansol, West Bengal, India. * Corresponding author’s E-mail: besrashila@yahoo.com Accepted on: 08-03-2013; Finalized on: 30-04-2013. ABSTRACT Secretion extract of Bellamya bengalensis f. annandalei have been traditionally used for many ailments. The anti-leukemic activity of secretion extract of Bellamya bengalensis f. annandalei (SEBB) has been investigated against three human leukemic cell lines U937, K562 and HL-60 where IC 50 values are calculated to be 22.21µg/ml, 10.3µg/ml & 12.79µg/ml respectively. Morphologically, externalization of phosphatidyleserine has been found on all the treated cells but not in control cells. Cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies are characteristic features of apoptosis. Gel electrophoresis study shows fragmented DNA in the form of ladder and Flow cytometric analysis showed appreciable number of cells in early & late apoptotic stages. The cells are getting arrested in the sub-G 1 & G 1 phases of cell cycle. The apoptosis is mediated through activation of caspase-9 & Caspase-3. Keywords: Apoptosis, Bellamya bengalensis, Cytotoxic, Leukaemia, Secretion. INTRODUCTION ancer is a complex multifactorial disease of the cell. Cancer development and progression are dependent on cellular accumulation of various genetic and epigenetic events. 1 Radiotherapy, chemotherapy and surgical measure are the key tools for cancer treatment. In the haematopoietic malignancies, chemotherapeutic approaches are widely applied in practice. Drug discovery against cancer is ventured throughout the world especially from the natural products. 2 Animals are therapeutic arsenals that have been playing significant roles in the healing processes, magic rituals, and religious practices of peoples from the five continents. In the folk-traditional medicine, it has been mentioned that snail flesh is used as medicines for the cure of a number of ailments such as conjunctivitis and gastro intestinal disorders. In the northern Bihar, the flesh of Bellamya bengalensis is used as a traditional medicine against arthritis. 3 But no work has been done so far in the field of cancer with this fresh water snail. The present communication is an approach to study the anti- proliferative, cytotoxic and apoptotic activity of secretion extract of Bellamya bengalensis f. annandalei (SEBB) family Viviparidae has been investigated against three myeloid cell lines. MATERIALSAND METHODS Chemicals The following chemicals were used: RPMI 1640 medium (Gibco, USA), Fetal bovine serum (FBS), HEPES, L- glutamine, Penicillin- Streptomycin (Bio-west, Germany), Gentamycin (Nicholas, India), Ara-C (Arabinofuranosyl Cytidine), MTT [3-(4,5-dimethylthiozol- 2-il)-2,5-2,5-dipheniltetrazoliumbromide], Ethidium bromide and Acridine orange, Annexin V- FITC apoptosis detection kit, RNase, Propidium iodide, Caspase-3 were purchased from Sigma (St. Louis, MO, USA), Caspase- 9 assay kit (U.S. Biological, USA),Trypan blue, Proteinase –K, Agarose medium EEO(SRL, India) and other chemicals and reagents were of analytical grade and purchased from local firms. Cell cultures U937 (human leukemic monocyte lymphoma cell line), K562 (human myelogenous leukemia cell line) and HL-60 (human promyelocytic leukaemia cell line) obtained from National Facility for Animal Tissue and Cell Culture, Pune, India. The cells were cultured and routinely maintained in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, penicillin (100 units/ ml), streptomycin (100μg/ ml), Gentamycin (100μg/ ml) and were incubated at 37°C in a humidified atmosphere containing 5% CO2 inside a CO2incubator. Extraction and Preparation of Test Sample Live adult Bellamya bengalensis f. annandalei, family Viviparidae was collected commercially from the Kolkata market and authenticated by Dr. R. Venkitesan Scientist- ‘C’ O/ C Mollusca Section, Zoological Survey of India- Kolkata 53. Authentication No: F.No.229-10/ 98-Mal (1). Secretion was collected from the snail in a cold room, and then it was centrifuged for 10 min (4°C) at 1600 g to precipitate the residual debris, then it was partially purified by Amicon ultra filtration unit. After filtration the sample was lyophilised to powder form. It was kept in a glass container, sealed with parafilm and stored at -20°C. Stock solution (1 mg/ml) of secretion extract of Bellamya bengalensis f. annandalei has been designated as SEBB Apoptogenic Activity of Secretion Extract of Bellamya Bengalensis f. Annandalei via Mitochondrial Mediated Caspase Cascade on Human Leukemic Cell Lines C Research Article