Mechanistic Studies Of The Anticancer Activity Of The Extracts Of Eupatorium odoratum L. And Euphorbia hirta L.
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Date
2012-02
Authors
Harun, Faizah
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Publisher
Universiti Sains Malaysia
Abstract
Eupatorium odoratum L. (EO) and Euphorbia hirta L. (EH) are two plants which are commonly used traditionally to treat wounds, asthma, warts and tumors. Even though the anti-cancer properties were described previously in the species, little was known on the activity of these plants specifically and on mechanism of cell death induced by these plant extracts. Hence, in this study, the ability of the plant extract to induce cell death was evaluated. Human breast carcinoma (MCF-7), human cervical carcinoma (Hela), human ovarian carcinoma (CaOV-3) and Vero cells were evaluated for response to treatment with EO and EH extracts. The cells were treated with nine different concentrations of respective extracts at three different time intervals (24, 48 and 72 hours) and assessed for the following parameters. (1) cell death using MTT assay and graph pad prism software to assess the cytotoxicity effects and IC50 values, (2) morphology of the cells using visible and fluorescent microscopy, (3) apoptosis using DNA ladder assay and expression of BCl-2 and Bax proteins as markers, (4) autophagy using BCl-2 and LC3-A protein expression as markers and (5) cell cycle effects using flow cytometry. Ethyl acetate extracts of E. odoratum (EOea) showed potent cytotoxicity against Hela, CaOV-3 and MCF-7 cells at IC50 values ranging from 40-95 μg/ml while EOace showed promising effects on MCF-7 cells only. Ethyl acetate extracts of E. hirta (EHea) showed effective cytotoxic effects on Hela and CaOV-3 cells with IC50 values in the range of 40-55 μg/ml. The extracts were non-toxic to Vero cells. Morphological observations on treatment with EOea and acetone extract (EOace), showed typical features of autophagic mechanism of cell death in MCF-7 cells. This was confirmed by upregulated expression of LC3-A protein. This pattern was also observed with CaOV-3 cells even though at a lower degree. Lower expression of LC3-A and no DNA fragmentation were observed casting doubts over autophagy or apoptosis as the cell death mechanism. Hela cells treated with EHea showed initial autophagic signs with moderate LC3-A expression. At 24 hours, shrunken cells and nuclear condensation at 48 hours and detachment of the cells were seen by 72 hours suggesting apoptotic cell death. This was also supported by DNA ladder formation at 48 and 72 hours, reduced LC3-A protein expression and increased Bax protein expression at this time. Results of cell cycle analysis showed increase in G0/G1 cell population in Hela cells treated with EHea,
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Keywords
The anticancer activity of the extracts of , Eupatorium odoratum L. And Euphorbia hirta L