Trifolium pratense leaves (red clover) has been used in Oriental and European folk medicine for the treatment of whooping cough, asthma, and eczema, and is now being used to treat and alleviate the symptoms, such as hot flushes, cardiovascular health effects that occur in postmenopausal women. However, relatively little scientific data is available on the physiological activity of this plant. Therefore, in this study, we investigated the anti-cancer activity of T. pratense leaves using methanol extract of T. pratense leaves (MeTP) on human FaDu hypopharyngeal squamous carcinoma cells. MeTP inhibited the viability of FaDu cells by inducing apoptosis through the cleavage of procaspase- 3, -7, and -9 and poly (adenosine diphosphate ribose-ribose) polymerase (PARP), downregulation of Bcl- 2, and upregulation of Bax, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Live & dead assay, 4’6-diamidino-2-phenylindole stain, fluorescence-activated cell sorting analysis, and Western blot analysis. In addition, colony formation was slightly inhibited when FaDu cells were treated with a non-cytotoxic concentration (0.125 mg/mL) of MeTP and almost completely inhibited when cells were treated with 0.25 mg/mL MeTP. Collectively, these results indicate that MeTP induced cell apoptosis via caspase- and mitochondrial-dependent apoptotic pathways, and inhibited colony formation of cancer cells in FaDu human hypopharyngeal squamous carcinoma cells. These findings suggest MeTP should be considered for clinical development as a chemotherapeutic option in oral cancer.

Introduction
Materials and Methods
    1. 실험재료
    2. MeTP 추출
    3. 세포 배양
    4. 세포 성장률 분석
    5. Colony formation 측정
    6. Live & dead assay
    7. DAPI 염색
    8. 유세포 분석
    9. Western blot 분석
    10. 실험자료의 통계학적 검정
Results
    1. MeTP에 의한 FaDu 암세포의 증식 억제
    2. Live & dead 염색을 이용한 MeTP에 의한 FaDu 암세포의 세포사멸
    3. DAPI 염색을 이용한 MeTP에 의한 FaDu의 세포핵 형태 변화
    4. 유세포분석을 이용한 MeTP에 의한 FaDu의 세포사멸
    5. MeTP에 의한 세포사멸관련 단백질 발현 유도
Discussion
References

• Seul Ah Lee(Department of Oral Biochemistry, College of Dentistry, Chosun University)
• Bo-Ram Park(Department of Dental Hygiene, College of Health and Welfare, Kyungwoon University)
• Chun Sung Kim(Department of Oral Biochemistry, College of Dentistry, Chosun University) Correspondence to