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Antiproliferative and apoptotic activities of extracts from carob tree (Ceratonia siliqua L.) in MDA-MB-231 human breast cancer cells
Breast carcinoma is the commonest cancer among women and the second highest cause of cancer death . Thus, the development of new therapeutic approach to breast cancer remains one of the most challenging areas in cancer research. Numerous experimental and epidemiological studies have shown that polyphenols can inhibit carcinogenesis by affecting the molecular events in the initiation, promotion or progression states. Extracts from pulps and leaves from carob tree are rich in phenolics [2–7] and display antioxidant [3,8,9] and antiproliferative activity on HeLa cells . The aim of this work was the evaluation of the antiproliferative and apoptotic potential of methanolic extracts (ME) of carob leaves and pulps, in a human breast cancer (MDA-MB-231 cells). Cells were treated with ME from leaves (0.025–0.4mg.ml-1), and from pulps (2.5–40mg.ml-1), for 24, 48 and 72h. Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. The ME significantly inhibited cell proliferation in a dose-dependent manner, and the best periods of incubation were 48 and 72h. Leaf extracts exhibited a significant higher capacity to inhibit cell proliferation than pulp extracts. The cell-cycle distribution was analyzed by flow cytometry after exposure to ME. Treatment with leaf extracts (0.4mg.ml-1), resulted in an increase in cells at the sub-G1 stage (apoptotic cells). To verify whether extracts induced the activation of proteins evolved in apoptosis, cells were exposed to ME and analyzed by Western Blotting. After exposure to ME from leaves (0.1, 0.2 and 0.4mg.ml-1) or from pulps (20 and 40mg.ml-1) for 48h, it was observed the cleavage of procaspase-3 and the reduction of the levels of the antiapoptotic protein Bcl-2. The results suggest that leaves and pulps from carob tree could be a source of phenolic compounds with potential anticancer activity.
Acknowledgements: L. Custódio thanks Portuguese Foundation for Science and Technology (FCT) for a post-doctoral grant (grant SFRH/BPD/20736/2004).
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