The effects of Tulbaghia violacea (wild garlic) leaf and bulb extracts on an oesophageal cancer cell line (SNO)

Abstract

Ethnopharmacological relevance: Indigenous plants such as Tulbaghia violacea(TV) and Allium sativum (garlic) are traditionally used as natural remedies to treat a variety of ailments, including cancer. This study investigated the effects of TV leaf and bulb extracts and garlic extract on a cancerous oesophageal cell line (SNO). Materials and methods: The methylthiazoltetrazolium (MTT) assay was used to determine the IC50 of TV leaf (TVL) (250μg/ml) and TV bulb extracts (TVB) (25μg/ml) and garlic (500μg/ml). Extracts were treated individually and in combination for a period of 24 hours. Oxidative damage and intracellular glutathione levels were assessed using the Thiobarbituric Acid Reactive Substances (TBARS) Assay and GSH-Glo™ Luminometry Assay, respectively. The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess ATP activity. Induction of apoptosis and mitochondrial membrane potential were determined via the Caspase-Glo® 3/7 Assay, Caspase-Glo® 8 Assay, Caspase-Glo® 9 Assay and JC-1 Mitoscreen Assay, respectively. Morphological apoptotic changes were determined using the Hoechst 33342 stain. Expressions of p53, PARP and NFKB activities were determined by western blotting. Results: Bulb and leaf extracts of TV increased lipid peroxidation compared to the control (p>0.05), whilst garlic and combination of TV leaf and bulb (TVB + TVL) extracts significantly decreased lipid peroxidation relative to the control (p< 0.05). Endogenous glutathione levels significantly decreased in all TV treatments compared to the control (p<0.05).However, garlic was accompanied by insignificantly increased intracellular glutathione levels compared to the control (p> 0.05). The percentages of depolarised mitochondria in all treated cells were significantly decreased compared to untreated cells (p< 0.05). ATP levels increased significantly in garlic and combination (TVB + TVL) treated cells as compared to the control (p< 0.05), yet no significant differences were noted in TVL and TVB treatments (p> 0.05). Caspase8 and caspase 9 activities significantly increased in garlic and combination treated cells relative to the control (p<0.05). A similar trend was noted for caspase 3/7 activity in garlic and combination treatments (p< 0.05). However, initiator and executioner activities in TVL (p> 0.05) and TVB (p> 0.05) treatments did not significantly differ from the control (p> 0.05). All treatments (including garlic) resulted in increased DNA fragmentation and condensation. All treatments decreased p53 expression (p< 0.05), PARP expression (p< 0.05) and NFK B expression (p>0.05) compared to the control. Conclusions: All TV extracts and garlic induces apoptosis in the oesophageal cancerous SNO cell line through changes in oxidative stress, antioxidant systems, and nuclear chromatin condensation, as well as through induction of nuclear genes and signalling pathways. Since inhibition of apoptosis is a principal alteration in cancer, induction of apoptosis would result in a decrease in cancer cell growth. Thus, TV could be exploited as a potential anti-cancer agent.