5-플루오로우라실에 의한 사람 구강암세포주의 단백질 발현 변화에 대한 면역침전 고성능 액체크로마토그래피 분석 Protein Expression Changes Induced by 5-Fluorouracil in an Oral Cancer Cell Line as Determined by Immunoprecipitation High Performance Liquid Chromatography
pp.251-261
5-fluorouracil (5-FU) is a pyrimidine analog which can work as antineoplastic antimetabolite by blocking thymidylate synthetase conversion of deoxyuridylic acid to thymidylic acid in DNA synthesis. This study is aimed to know the anticancer effect of 5-FU on the expressions of important signaling proteins in KB cells through immunoprecipitation high performance liquid chromatography (IP-HPLC). KB cells were treated with 5 μM 5-FU and cultured for 12, 24, 48, 72, and 96 hours, and followed by IP-HPLC analysis using 32 antisera. 5-FU suppressed the proliferation of KB cells by decreases in the expressions of proliferation-related proteins, Ki-67, PCNA, CDK4, and MPM2 to 82.6%, 92.4%, 95.2%, and 95.9%, respectively, but increases of antiproliferation-related proteins, p16 and p21 to 106.7% and 125.5%, respectively, during 96 hours of experiment. This proliferation reduction was also negatively regulated by cMyc/MAX/MAD network signaling. The cellular protection and survival were consistently arrested by 5-FU treatment in KB cells. The expressions of NFkB, MDR, p-mTOR, and TNFα were decreased to 95.1%, 92.8%, 93.4%, and 90.3% in 48-72 hours, respectively, while cellular stress was increased by upregulation of p38 to 111.3% in 48 hours. And the expressions of pAKT1/2/3, hTERT, and AMPK were also decreased to 93.3%, 97.4%, and 89.3% in 24-48 hours, respectively, while the cellular transformation might be undergone by upregulation of TGF-β1 to 117% until 96 hours. Particularly, 5-FU treatment greatly induced the cellular apoptosis in KB cells by increased expressions of PARP, cPARP, caspase 9, c-caspase 9, caspase 8, and caspase 3 in the lack of p53/BAX and FASL/FAS signaling. The expressions of PARP and c-PARP were increased maximum to 119.2% in 24 hours, and followed by increases of caspase 9, c-caspase 9, caspase 8, and caspase 3 to 111.2%, 125.9%, 108.6%, and 116.3% in 72-96 hours. Therefore, it is presumed that 5-FU induced cellular apoptosis in KB cells may be derived from the overexpression of PARP due to the increased DNA defect caused by 5-FU, which can lead to ATP depletion and subsequent cellular apoptosis.
Ⅱ. 실험재료 및 방법
1. KB 세포 배양 및 단백질 추출
2. 면역침전 고수행 액체크로마토그라피 분석
Ⅲ. 연구결과
1. 5-FU 투여에 의한 KB 세포의 세포증식-관련 단백질 발현변화
2. 5-FU 투여에 의한 KB 세포의 cMyc/MAX/MAD 네트워크-관련 단백질 발현변화
3. 5-FU 투여에 의한 KB 세포의 세포방어 및 생존-관련 단백질 발현변화
4. 5-FU 투여에 의한 KB 세포의 p53-중재 세포자멸사-관련 단백질 발현변화
5. 5-FU 투여에 의한 KB 세포의 FAS-중재 세포자멸사-관련 단백질 발현변화
Ⅳ. 총괄 및 고찰
ACKNOWLEDGEMENTS
REFERENCES