The hexane fraction of the Moringa oleifera Lam seed extract induces apoptosis, causes cell cycle arrest, and modulates expression of HSP60, NPM, PGK1, RCN1, and PDIA1 in MCF7 cells

Abstract

Moringa oleifera Lam is a medicinal plant that had been shown to have anticancer activity. Previously, the hexane fraction of the seeds (HF-CEE) was found to inhibit breast cancer (MCF7) cell proliferation at IC50 of 130 μg/ml. However, the mechanism by which HF-CEE inhibits MCF7 cells remained unknown. To elucidate the mechanism, we investigated the effect of HF-CEE on apoptosis, cell cycle, and the cell proteome of MCF7 cells. The HF-CEE was prepared by fractionating the non-polar fraction of the ethanolic extract of M. oleifera seeds using the hexane solvent. The antiproliferative effect of HF-CEE on the growth of MDA-MB-231 cells (another breast cancer cells) was performed using Presto Blue assay. The morphologies of the untreated and treated cells (MCF7, MDA-MB-231 and MCF 10A cells) were observed using inverted light microscope. The apoptotic and cell cycle arrest effects of HF-CEE on MCF7 cells were determined using flow cytometry analyses. 2-DE proteomics analysis was employed to identify the protein biomarkers of MCF7 cells that have their expression levels altered by HF-CEE treatment. qRT-PCR method was used to validate the 2-DE results. The gene ontology analysis was carried out using PANTHER server to identify the mechanisms and biological processes that are dependent on the functions of the identified biomarkers.Our results showed that HF-CEE selectively inhibited the proliferation of the breast cancer cells (MCF7 and MDA-MB-231) but not the MCF 10A non-tumor cells. MCF7 and MDA-MB-231 cells treated with HF-CEE showed morphological properties of dead cells, but MCF 10A cells did not. HF-CEE also induced apoptosis and cell cycle arrest at the S and G2/M phases in MCF7 cells. 2-DE analysis revealed that HF-CEE altered the regulation of several cancer-related proteins in MCF7 cells, such as heat shock protein 60 (HSP60), nucleophosmin (NPM), protein disulphide isomerase (PDIA1), phosphoglycerate kinase 1 (PGK1), and reticulocalbin 1 (RCN1). In conclusion, HF-CEE induced cell death in MCF7 cells via apoptosis, cell cycle arrest, and other mechanisms such as the alteration of the expression of proteins that are involved in glycolysis, cancer invasiveness and metastasis among others. Results of this study provide further insights into the mechanisms that underlie the antiproliferative effect of HF-CEE on MCF7 cells.