Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Virginia B
Nida Ali Alsaffar - Howard University
Co-Author(s): Yayin Fang; Eric Walters
Despite their predominant role in detoxification, glutathione S-transferases (GSTs) contribute to other eukaryotic cell proliferation, motility, and homeostasis. The eukaryotic model, Dictyostelium discoideum, when deprived of nutrients, a program of cellular chemotaxis multicellular aggregation, and developmental morphogenesis that is regulated by synthesis and secretion of cAMP. D. discoideum expresses five DdGST isozymes (DdGSTα1-α5 class). However, is little known about their functions in development and morphogenesis of this organism. Pharmacologic treatment can serve to elucidate both enzymatic and non-enzymatic role(s) of DdGST enzymes in eukaryotic cells. Previous studies from our laboratory reported that the polyphenol curcumin inhibits DdGST enzyme activity, which correlates with reduced proliferation and altered development. In this report, we examined the effect of thymoquinone (TQ), a bioactive polyphenol constituent of black seed of Nigella Sativa extract, on DdGST activity and proliferation, growth, and development in D. discoideum. Methods: The D. discoideum AX4 strain was used in this study, and all experiments were performed with 0, 2.5, 5 and 7.5μM TQ. For proliferation, log-phase cells were treated and counted after 0,24, 48, and 72h exposure to TQ. To induce development, cells were starved and incubated in potassium buffer (1XKK2 buffer) containing TQ and plated onto non-nutrient agar to monitor development and aggregation. In vegetative amoeba, conjugation of GSH (glutathione) to 1-chloro-2,4-dinitrobenzene (CDNB) was used to determined DdGST activity at 72h after TQ treatment, and in starved cells at 0,4, and 6h of TQ exposure. Results: Cell proliferation was significantly reduced by 66% and 80% in the presence of 5μM, and 7.5μM, respectively, after 72h incubation. TQ delayed chemotaxis, and development of the organism, resulting in abnormal multicellular development within 24h. DdGST activity in proliferating cells was significantly reduced by 90% and 99% at 5μM and 7.5μM, respectively, after 72h TQ incubation. In starved cells, DdGST activity was reduced by 95% in the presence of 7.5μM at 4h. CDNB assays conducted with the purified DdGSTA2 isozyme in the presence of TQ showed a 91% reduction in activity. Conclusions, further research: Collectively, these observations suggest that TQ influences D. dicoideum proliferation and development through a direct interaction with DdGST isozymes. These findings warrant additional investigation regarding the specific binding site(s) between DdGSTs and TQ, to further elucidate the role of DdGSTs in the regulation of YakA-mediated cAMP signaling in D. dicoideum.
Funder Acknowledgement(s): The department of biochemistry and molecular biology
Faculty Advisor: Eric Walters, firstname.lastname@example.org
Role: conducted all the studies involving cell proliferation, starvation, and GST activity assay.