Evaluation of ricinus communis semi-purified extracts' potential as anti-metastatic agents using metastatic breast cancer (MCF-7) cancer cells

Abstract

The malignancy of cancer cells is responsible for the high death rate in patients diagnosed with metastatic cancers. Medicinal plants represent a reservoir of bioactive compounds that can be useful in the management of cancer. In this study, semi-purified extracts of Ricinus communis leaves were evaluated for their potential to serve as an anti-metastatic agent by using in vitro assays that tested their effects on a number of processes related to metastasis. The exhaustive extraction procedure was employed to generate the crude acetone extracts of R. communis leaves. The crude extracts were then subjected to solvent-solvent fractionation to yield six semi-purified extracts (n-butanol, Chloroform, Ethyl-acetate, n-hexane, Methanol + H2O and H2O). Thin layer chromatography (TLC) was done to determine the phytochemical composition of the semi-purified extracts as well as their antioxidant potential. Non-polar fractions showed to have a diverse mixture of phytochemicals with, however, very limited antioxidant activity. On the other hand, polar fractions showed to have phytochemical compounds with strong antioxidant potential. TLC guided the selection of n-hexane and n-butanol as fractions of great phytochemical diversity and antioxidant activity, respectively. The selected fractions were then assessed for their effect on the viability of normal fibroblasts (BUD-8) and breast (MCF-7) cancer cells using the MTT assay. The n-butanol fraction was shown to significantly decrease the viability of BUD-8 at concentrations above 200 µg/ml. The n-hexane fraction, however, showed to significantly affect the viability of the cells even at lower concentrations. On the positive side, the reduced viability of BUD-8 cells after exposure to both fractions was followed by an increase in cell proliferation after 24 hours suggesting that the extracts exhibited cytostatic rather than cytotoxic effects. Treatment of MCF-7 cells with different concentrations (100-500 µg/ml) of the fractions showed a dose- and time-dependant decrease in cell viability. Hoechst stain also confirmed the non-toxicity of the fractions to MCF-7 cells at 100 and 200 µg/ml. The fractions also showed to possess free radical scavenging activities by reducing the amount of intracellular ROS as demonstrated by the DCFH-DA fluorescent assay. Fluorescence intensity was strongly reduced in cells treated with the fractions and elevated in H2O2-treated and untreated MCF-7 cells. The effect of the fractions on metastasis was assessed by determining their effects on MCF-7 cell migration, attachment and invasiveness using wound healing assay, adhesion assay and Boyden chamber invasion assay, respectively. The wound healing assay showed the fractions to have strong inhibitory activities on the migration of MCF-7 cells. The xii

ability of the cells to attach to cell culture treated plates was also greatly reduced in cells treated with the fractions. The n-butanol fraction was demonstrated to exhibit a time- and dose-dependent inhibition on MCF-7 cell invasion by reducing the cells’ capability to penetrate through the matrigel matrix to the bottom of the porous membrane. Gelatin-zymography was done to assess the effect of the n-butanol fraction on activity of MMP-2 and MMP-9. The fraction showed to completely inhibit the gelatinolytic activity of MMP-2 and no band corresponding to the molecular weight of MMP-9 was observed, suggesting that MCF-7 cells produce undetectable levels of MMP-9. The n-butanol fraction further showed to down-regulate the expression of a range of proteins such as MMP-9, uPA, VEGF, TGF-β1 implicated in metastasis and angiogenesis determined using the human angiogenesis antibody array kit. This study demonstrated that the fractions of R. communis extracts have the ability to inhibit major processes of the metastatic cascade by down-regulating the expression of proteins relevant to metastasis. Thus, the fractions can be considered as potential anti-metastatic agents functional in the regulation and/or treatment of malignant of cancers.