ABSTRACT
The present study focuses on isolation and evaluation of the anti-cancer activity of compounds from the leaves of Abrus precatorius. The bioassay-directed strategy was adopted using chromatographic, gas chromatographic-mass spectrum analysis, nuclear magnetic resonance and X-ray crystallography techniques for purification and characterization of active cytotoxic compounds. Further, MDA-MB-231 breast cancer cell lines and 7,12-dimethylbenz (a) anthracene (DMBA) induced virgin female Sprague Dawley (SD) rats were used for in vitro and in vivo cytotoxicity evaluation. Stigmasterol hemihydrate and 9,12-Octadecadienoic acid (Z,Z)-2-hydroxy-1-(hydroxymethyl)ethyl ester or (ß-monolinolein) were the two main cytotoxic constituents of leaf extract of A. precatorius, with an IC50 value of 74.2 and 13.2 µg/ml, respectively, in MDA-MB-231 cells. Additionally, the treatment with the stigmasterol and ß-monolinolein as a combinatorial drug therapy in DMBA-induced female SD rats led to recovery of body weight, decreased tumor weight and volume, without any toxic side effects. Immunohistochemical examination showed extensive cell death and low proliferation in the treated tumor tissues that was confirmed by results from H and E staining, TUNEL assay and Ki-67 index as compared to control animal group. The reversion of glycoprotein, lysosomal and tumor marker enzyme levels back to near-normal levels after treatment with the plant compounds clearly demonstrated the reduction of tumor burden in these animals. This is the first report on isolation and characterization of the two active cytotoxic components from leaves of A. precatorius. Additionally, the profound cytotoxic and tumor-suppressive effect of these two compounds as a combinatorial therapy provide an alternative option for breast cancer treatment.
ABSTRACT
Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.
