Antioxidant, cytotoxic, anti-migratory, and pro-apoptotic effects of Bolanthus turcicus extracts on head and neck cancer cells.

Abstract

Purpose: Investigation of various plant extracts using in-vitro/in-vivo assays has emerged as a promising avenue for identifying potential pharmacophores that can be developed into therapeutic drugs. This study aims to assess the bioactive compounds and antioxidant capacity of the Bolanthus turcicus (B. turcicus) and to investigate the effects on head and neck cancer (HNC) cell lines.

Methods: Methanol (MeOH), ethyl acetate (EA) and aqueous (Aq) extracts were prepared from B. turcicus, and the amount of total phenolic content (TPC) and total flavonoid content (TFC) in the extracts were analyzed by the Folin-Ciocalteu and Aluminum chloride method, respectively. In addition, the total antioxidant capacity and iron reducing potential of B. turcicus extracts were determined by the Phosphomolybdenum and Ferric ion reducing antioxidant power (FRAP) method. The effect of B. turcicus on HEp-2, SCC-90, SCC-9, FaDu HNC cell viability, motility, and cell-nuclear morphology was evaluated by MTT, scratch-wound healing assay, and Pllalloidin-DAPI staining, respectively. The effect of B. turcicus on the expression of CASP-3, BAX, and BCL-2 genes at the mRNA, protein, and intracellular level was evaluated by quantitative PCR (qPCR), western blot, and immunofluorescence staining. Moreover, Annexin V-FITC/PI, was used in flow cytometry to investigate the effect of B. turcicus on apoptosis.

Results: The MeOH extract exhibited the highest phenolic content, flavonoid content and antioxidant activity (p < 0.05 for all). HNC cells treated with extracts indicated delayed wound healing and decreased motility (p < 0.05 for all). Analysis of annexin V-PI staining indicated that the B. turcicus extracts induced apoptosis but not viability and necrosis in the HNC cell (p < 0.05 for all). Moreover, qPCR data regarding the apoptotic mechanism showed that the extracts could induce apoptosis by upregulation of pro-apoptotic CASP-3 and BAX genes and downregulation of anti-apoptotic BCL-2 gene (p < 0.05 for all). The expression of protein and intracellular levels of CASP-3 and BAX were increased, while the BCL-2 was decreased in cells treated with the extracts (p < 0.05 for all). In addition, diffuse pycnosis and DNA condensation in HNC cell nuclei, confirming apoptotic cell death (p < 0.05 for all).

Conclusion: This study data indicated that B. turcicus extracts have antioxidant, cytotoxic, anti-migratory and pro-apoptotic activity. In conclusion, it has been shown that B. turcicus can be used as a potential therapeutic agent against HNC.