INDUCTION OF CASPASE-3 DEPENDENT APOPTOSIS, CELL CYCLE ARREST AND CYTOTOXICITY IN BREAST CANCER CELLS BY ABRUS PRECATORIUS
Objective: To evaluate the leaf extract of Abrus precatorius as a potential therapy for breast cancer treatment.
Methods: Aqueous leaf extracts of A. precatorius was prepared by the process of maceration. The collected filtrate was further partitioned successively into five solvent fractions starting from water, hexane, chloroform, ethyl acetate and butanol by solvent fractionation method using separating funnel. Finally, all the five fractions were subjected to cytotoxic activity assay. The molecular mechanism underlying cytotoxicity was determined by using various approaches viz., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Fluorescence-activated cell sortingÂ (FACS) analysis, Western blot analysis, casp-glow assay and Semi-quantitative reverse transcriptionÂ polymerase chain reaction (RT-PCR) analysis.
Results: Among five fractions only ethyl acetate fraction of A. precatorius (EAF-AP) showed significant cytotoxic activity on MDA-MB-231 cells, with an IC50 value of 47.3 Âµg/ml. Apoptosis was confirmed by the appearance of Sub G0/G1 (apoptotic) peak by FACS analysis. Western blotting results clearly indicated cleavage of Caspase-3 and PARP. Casp-glow assay confirmed activation of caspase-3, an important mediator of apoptosis. Semi-quantitative RT-PCR analysis showed an up-regulation of pro-apoptotic genes (p21, p53 and Bax) and down-regulation of the anti-apoptotic Bcl-2 gene, which is an important hallmark of an apoptosis.
Conclusion: All these results indicate that EAF-AP, induced apoptosis in MDA-MB-231 cells, making A. precatorius a potentially good candidate for anticancer drug development.
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