Procyanidin-rich extract of natural cocoa powder causes ROS-mediated caspase-3 dependent apoptosis and reduction of pro-MMP-2 in epithelial ovarian carcinoma cell lines

Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000 μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease in invasive potential of the cells. Apoptosis and MMP2 downregulation appear to be linked to the increase in intracellular ROS levels, caused due to the prooxidant effect of cocoa procyanidin extract.

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