The mechanism of acacetin-induced apoptosis on Oral squamous cell carcinoma cell line

Abstract

Acacetin (5, 7-dihydroxy-40-methoxyflavone) – present in safflower seeds, plants, flowers, and Cirisium rhinoceros Nakai – has been reported to exhibit anti-peroxidative, anti-inflammatory, anti-plasmodial, and anti-proliferative activities. The objective of this study is to investigate the mechanism of acacetin-induced apoptosis of the oral squamous cell carcinoma cell line. Acacetin caused 50% growth inhibition (IC50) in HSC-3 cells at 25μg/ml over 24 hours in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase in sub-G1 cells and involved the activation of caspase-3 and PARP (poly-ADP-ribose) polymerase. Maximum caspase-3 activity was observed with 100μg/ml of acacetin treated for 24 hours. Caspase-8 and -9 activation, which mediate the activation of caspase-3, were confirmed. Acacetin caused a reduction in Bcl-2 expression leading to an increase in the Bax: Bcl-2 ratio. In addition, it caused a loss of mitochondrial membrane potential that induced the release of cytochrome c into the cytoplasm. Pretreatment with the casapse-3 (Z-DEVD-FMK), -8 (Z-IETD-FMK), and -9 inhibitor (z-LEHD-fmk) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c. The mitogen-activated protein kinases (MAPKs) were activated by acacetin and pre-treating the cells with each of the MAPKs specific inhibitors apparently inhibited the acacetin-induced cytotoxicity of the HSC-3 cells. Moreover, when the cells were treated with the MAPKs inhibitor, the PARP cleavage, increase in sub G1 cells, increase in the Bax: Bcl-2 ratio, and release into cytoplasm of cytochrome c – which were induced by acacetin – were inhibited. In conclusion, acacetin induces the apoptosis of the oral squamous cell carcinoma cell line, which has a close relation to its ability to activate the MAPK-mediated signaling pathways with the subsequent induction of a mitochondria- and caspase-dependent mechanism. These results strongly suggest that acacetin might have an effect on HSC-3 cells growth inhibition and therapeutic potential in oral squamous cell carcinoma.