Anti-cancer and Anti-inflammatory Properties ofZiziphus NummulariaEthanolic Extract in Triple Negative Human Breast Cancer Cells MDA-MB-231

Abstract ID 91433 Poster Board 079 Breast cancer continues to be one of the most diagnosed cancers in women worldwide with triple-negative breast cancer (TNBC) that makes up approximately 15% to 20%. TNBC is challenging to treat because it does not respond to hormonal therapies and often develops resistance to chemotherapy. Natural products have long been utilized in traditional medicine as remedies to improve health and treat illnesses. Importantly, they have a key role in modern drug discovery. Recently, there has been an increasing interest in the search for bioactive agents from natural sources as alternative or complementary modalities to conventional treatments and synthetic medicines; especially for treatment of cancer which incidence and mortality rates have been on the rise worldwide. Ziziphus nummularia, a small bush belonging to the Rhamnaceae family, has been widely used in traditional medicine for the treatment of various diseases. Its traditional therapeutic uses may be attributed to its richness in bioactive compounds and the wealth of its pharmacological properties, including antioxidant, anti-inflammatory, anti-cancerous activities. However, its phytochemical composition or chemo-preventive effects against the aggressive TNBC are still poorly explored. In the present study, an ethanolic extract of leaves of Z. nummularia (ZNE) was prepared and chromatographically fractionated. ZNE reduced the viability of MDA-MB-231 cells, a TNBC cell line, with the ZNE Fraction 6 (F6) exhibiting the strongest activity. ZNE and F6 are rich in phytochemicals and HPLC-PDA-MS/MS analysis identified several compounds with F6 being particularly rich in Caffeoyl-benzene. Both ZNE and F6 showed potent antioxidant activities 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 at G1, and triggered apoptosis- and autophagy-mediated cell death. This was confirmed by the decrease in Ki67 and BCL-2 protein levels and the increase in P38, P21, P27, Rb, caspase 3, BAX and LC3B. ZNE and F6 inhibited metastasis-related cellular processes as well. Namely, ZNE- and F6- treated MDA-MB-231 cells had a reduced cell migration, invasion, and adhesion to collagen. This was supported by the decrease in MMP-9 and integrin β1 levels. In addition, ZNE and F6 also reduced the production of inducible nitric oxide synthase (iNOS) and inhibited in-ovo angiogenesis. It was also found that ZNE has potent anti-inflammatory properties by suppressing the LPS-stimulated inflammatory responses in RAW 264.7 macrophage cells. It significantly reduced the mRNA and protein expression of iNOS, cyclooxygenase-2 (COX-2) by targeting the NF-κB pathway. Taken together, our findings reveal that Z. nummularia is rich in phytochemicals that can attenuate the malignant phenotype of TNBC and may offer innovative avenues for the discovery of new drug leads for treatment of TNBC and other cancers.