Ziziphus nummularia crude and fractionated extracts attenuate the malignant phenotype of human triple-negative breast cancer cells

Abstract

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has limited therapeutic options. Effects of Ziziphus nummularia against TNBC have not been investigated yet. An ethanolic extract of Ziziphus nummularia (Burm.f.) Wight & Arn. (ZNE) was prepared and chromatographically fractionated. Phytochemical composition of ZNE and its chromatographically isolated fraction (F6) was identified both qualitatively by spectrophotometric assays and analytically by HPLC-PDA-MS/MS. Effects of ZNE and F6 on the viability of several cancerous cell lines were tested by MTT assay. The anti-cancerous potential of ZNE and F6 was tested in vitro in MDA-MB-231 cells, a TNBC cell line. ZNE and F6 radical scavenging capacity was tested using DPPH assay, and their effects on reactive oxygen species (ROS) generation in vitro in cells by DCFDA staining. Propidium iodide-based FACS analysis was used for cell cycle analysis. Scratch wound healing and trans-well migration chamber assays were used to assess MDA-MB-231 cell migration and invasion. Western blotting analysis was used to analyse changes in the levels of cell cycle, apoptosis and autophagy proteins. Findings showed that ZNE and F6 reduced the viability of several cancerous cell lines including MDA-MB-231 cells. F6 decreased MDAMB-231 viability more than crude ZNE or F6. ZNE and F6 are rich in phytochemicals and HPLC-PDA-MS/MS analysis identified several metabolites that were previously reported to have anti-cancerous effects. Both ZNE and F6 showed potent antioxidant capacity in the DPPH assay, but promoted reactive oxygen species (ROS) production in MDA-MB-231 cells; an effect which was blunted by the antioxidant N-acetyl cysteine (NAC). NAC also blunted ZNE- and F6-induced reduction in TNBC cell viability. We also demonstrated that ZNE and F6 induced an arrest of the cell cycle, and triggered apoptosis- and autophagy-mediated cell death. ZNE and F6 inhibited metastasis-related cellular processes by modifying cell migration, invasion, and adhesion. Collectively, our findings reveal that Z. nummularia is rich in metabolites that can attenuate the malignant phenotype of TNBC and may provide novel approaches for the discovery of new drug leads for treatment of TNBC and other cancers.