ELUCIDATING THE EFFECTS OF MORINGA OLEIFERA EXTRACT ON REGULATING APOPTOSIS IN HUMAN BREAST CANCER CELL LINES

Abstract

Moringa oleifera L (Family: Moringacea) is a type of perennial angiosperm tree that consists of a few plant species. Moringa is a common vegetable used by inhabitants of tropical and sub-tropical countries. At present, M. oleifera is noted to possess a wide range of biological roles including anti-cancer, neuroprotective, anti-inflammatory and hepato-protective activities. In addition, there are numerous studies that have reported many other therapeutic effects of M. oleifera, which include anti-rheumatoid arthritis, anti-infertility, diuretic, thyroid regulation, anti-depression, anti-diabetes, pain relief and atherosclerosis. Over the last decade, the bioactivity of M. oleifera has achieved vast attention due to these bio-activities. The anti-cancer activities could be attributed to the presence of several bioactive compounds such as β-sitosterol-3-O-β-D-glucopyranoside, 4-(α-L-rhamnosyloxy) benzyl isothiocyanate and niazimicin in the Moringa oleifera leaves extracts. It has been noted that Moringa oleifera leaf extracts have anticancer effects, which is reported to take place through induction of apoptosis. To date, the ability of extracts from M. oleifera leaf to induce apoptosis in breast cancer cells has not been fully elucidated. This study involves evaluating the ability of extracts of Moringa oleifera, from various solvents, to modulate apoptosis. Cell based assays using two types of human breast cancer cell lines (MCF-7 and MDA MB-231) as well as a normal breast epithelial cell line (MCF-10A) were used in the experiments. Expression of several genes (human Caspase9, Bcl2, Ccnd1 and Brca2 genes) in the cells when treated with the extracts were also investigated. A commercial source of dried Moringa oleifera leaf powder was subjected to sequential extraction using n-hexane, followed by ethyl acetate, methanol and water-based extraction. The effect of these extracts (0, 10, 20, 40, 60, 80, 100 μg/mL) on cell proliferation was evaluated using the MTT cell proliferation assay. Data analysis was carried out on the MTT assay results. The half maximal inhibitory concentration (IC50) of M. oleifera extracts following 72 hours of exposure was found to be: (a) MCF-7 [IC50 for ethyl acetate (hex-EA): 95 g/mL; water: 100 g/mL and quercetin: 90 g/mL] and MCF-10A [IC50 for ethyl acetate (hex-EA): 52 g/mL]. The IC50 values for MDA MB-231 cells could not be attained in the studied concentrations range. Difficulty in obtaining the IC50 values for MDA MB-231 cells might be due to as this cell line is a triple-negative breast cancer [lack ER, PR and human epidermal growth factor receptor 2 (HER2) amplification)], which means it is an aggressive kind of breast cancer with limited therapeutic approach. The RNA from MCF-7 and MCF-10A cells treated with various extracts for 72 hrs of exposure was extracted for gene expression studies. The genes studied were [human Caspase9, Bcl2, Ccnd1 and Brca2 genes]; all of which are reported to be involved in the regulation of apoptosis. Following this, the purified RNA was analysed for gene expression using the real-time PCR approach. Gene expression studies showed that Caspase9, Bcl2, Ccnd1 and Brca2 genes were upregulated in MCF-7 cells treated with water extract of Moringa oleifera leaf powder. In the MCF-10A cells, the expression of the Bcl2 gene was downregulated in cells treated with hexane extract whilst the expression of the Ccnd1 gene was downregulated in cells treated with ethyl acetate (hex-EA) and water extracts. The findings from this study suggest that Moringa oleifera extracts can differentially regulate genes related to apoptosis in MCF-7 cells. More studies need to be conducted to elucidate the molecular signalling pathways.