Apoptotic effect of betulinic acid in FaDu human head and neck squamous cell carcinomas

Betulinic acid (3-beta-hydroxy-lup20[29]-en-28-oic acid) has attracted significant attention due to its diverse biological and pharmacological activities, including anti-inflammatory, antimicrobial, antiviral, antidiabetic, antimalarial, anti-human immunodeficiency virus, and antitumor effects.However, its effectiveness against oral cancer remains unknown.This study aimed to evaluate the effect of betulinic acid on the induction of apoptosis in FaDu human pharyngeal carcinoma cells by performing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, LIVE/DEAD stain, 4',6-diamidino-2-phenylindole (DAPI) stain and western blot.In the MTT assay, LIVE/DEAD stain, and DAPI stain analyses, betulinic acid increased FaDu cell apoptosis in a concentration-dependent manner.Apoptosis induced by betulinic acid in FaDu cells was mediated by the expression of Fas and the activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase.Western blotting revealed that B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large were downregulated, while Bcl-2-associated death promoter and Bcl-2-associated X protein were upregulated by betulinic acid in FaDu cells.These findings indicate that betulinic acid inhibits cell proliferation in FaDu human pharyngeal carcinoma cells and induces apoptosis through both apoptotic receptor-mediated exogenous apoptosis and mitochondrialmediated endogenous apoptosis pathways.