Cytotoxicity of the crude extract and constituents of the bark of Fagara tessmannii towards multi-factorial drug resistant cancer cells

Abstract

Ethnopharmacological relevance: Fagara tessmannii Engl. is an African medicinal plant used in Cameroonian traditional medicine to treat various types of cancers.

Aim of the study: This work was designed to determine the cytotoxicity of the crude extract (FTB), fractions (FTBa-d) and compounds isolated from the bark of Fagara tessmannii, namely lupeol (1), fagaramide (2), zanthoxyline (3), hesperidin (4), nitidine chloride (5), fagaridine chloride (6), and β-sitosterol-3-O-β-_D-glucopyranoside (7). The study was extended to the mode of induction of apoptosis by FTB, compounds 5 and 6.

Materials and methods: The resazurin reduction assay was used to evaluate the cytotoxicity of samples. The cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were measured by flow cytometry. Column chromatography was used for the purification of FTB. Meanwhile, nuclear magnetic resonance (NMR) spectroscopic analysis was applied for structural elucidation.

Results: The crude extract, fractions FTBa, FTBc, FTBd as well as compounds 5 and 6 revealed cytotoxicity towards the 9 tested cancer cell lines. The IC₅₀ values ranged from 17.34 µg/mL (towards U87MG.ΔEGFR glioblastoma cells) to 40.68 µg/mL (against CCRF-CEM leukemia cells) for FTB, from 16.78 µg/mL (towards U87. MGΔEGFR cells) to 37.42 µg/mL (against CEM/ADR5000 leukemia cells) for FTBa, from 19.47 µg/mL (towards U87. MG glioblastoma cells) to 41.62 µg/mL (against CCRF-CEM cells) for FTBc, from 14.17 µg/mL (against HCT116p53^-/- colon adenocarcinoma cells) to 22.28 µg/mL (towards CEM-ADR5000 cells) for FTBd, from 1.75 µM (against CCRF-CEM cells) to 23.52 µM (against U87. MGΔEGFR cells) for compound 5, from 1.69 µM (against CCRF-CEM cells) to 22.06 µM (against HepG2 hepatocarcinoma cells) for compound 6 and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin. FTB induced apoptosis in CCRF-CEM cells mediated by enhanced ROS production. Compound 5 induced apoptosis through caspases activation and increase ROS production. Meanwhile, 6 induced apoptosis mediated by caspases activation, MMP alteration and enhanced ROS production.

Conclusion: Fagara tessmannii as well as its constituents 5 and 6 revealed considerable cytotoxicity and may be suitable candidates deserving to be further explored to develop new anticancer drugs to combat sensitive and resistant phenotypes.

Keywords: 2´,7´-dichlorodihydrofluorescein diacetate; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Alkaloids; Apoptosis; Cytotoxicity; Fagara tessmanniii; Multi-drug resistance; Traditional medicine; dimethyl sulfoxide; doxorubicin; fagaramide; fagaridine chloride; hesperidin; hydrogen peroxide; lupeol; nitidine chloride; valinomycin; zanthoxyline; β-sitosterol-3-O-β-(D)-glucopyranoside.