Acacetin, which is present in damiana (Turnera diffusa ) and black locust (Robinia pseudoacacia ), has several pharmacologic activities such as antioxidant, anti-inflammatory, and anti-proliferative effects on cancer cells. However, the effect of acacetin on head and neck cancers has not been clearly established. This study aimed to examine the effects of acacetin on cell growth and apoptosis induction in FaDu human pharyngeal carcinoma cells. These were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, Live/Dead cell assay, 4′,6-diamidino-2-phenylindole dihydrochloride staining, caspase-3 and caspase-7 activation assay, and immunoblotting in FaDu cells. Acacetin induced FaDu cell death in a dose-dependent manner, with an estimated IC50 value of 41.9 µM, without affecting the viability of L-929 mouse fibroblasts as normal cells. Acacetin treatment resulted in nuclear condensation in the FaDu cells. It promoted the proteolytic cleavage of procaspase-3, -7, -8, and -9 with increasing amounts of the cleaved caspase isoforms in FaDu cells. Acacetin-induced apoptosis in FaDu cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting showed downregulation of the anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-xL, but upregulation of the mitochondria-dependent pro-apoptotic proteins Bax and Badin FaDu cells after acacetin treatment. These findings indicate that acacetin inhibits cell proliferation and induces apoptotic cell death in FaDu human pharyngeal carcinoma cells via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondria-mediated intrinsic apoptotic pathway.

Introduction
Materials and Methods
    1. Materials
    2. Cell line and cell cultures
    3. Cell viability test (MTT assay)
    4. Live/Dead cell assay
    5. DAPI staining
    6. Determination of caspase-3/-7 activation
    7. Immunoblotting
    8. Data analysis
Results
    1. Cytotoxic effect of acacetin in L-929 cells and FaDucells
    2. Induction of apoptosis by acacetin in FaDu cells
    3. Extrinsic death receptor-dependent and intrinsicmitochondria-dependent apoptotic signalingpathways induced by acacetin in FaDu cells
Discussion
References

• Kyeong-Rok Kang(The Institute of Dental Science, Chosun University)
• Jae-Sung Kim(The Institute of Dental Science, Chosun University)
• Tae-Hyeon Kim(The Institute of Dental Science, Chosun University)
• Jeong-Yeon Seo(The Institute of Dental Science, Chosun University)
• Jong-Hyun Park(The Institute of Dental Science, Chosun University)
• Jin Woong Lim(The Institute of Dental Science, Chosun University)
• Sun-Kyoung Yu(The Institute of Dental Science, Chosun University)
• Heung-Joong Kim(The Institute of Dental Science, Chosun University)
• Sang Hun Shin(The Institute of Dental Science, Chosun University)
• Bo-Ram Park(Department of Dental Hygiene, College of Health and Welfare, Kyungwoon University)
• Chun Sung Kim(The Institute of Dental Science, Chosun University)
• Do Kyung Kim(The Institute of Dental Science, Chosun University) Correspondence to