비임상시험관리기준에서 독성시험 전 분석법 밸리데이션은 농도 설정 및 시료 제조 측면에서 중요하다. 시험기관에서는 의뢰받은 시험물질 2종에 대한 밸리데이션을 고성능액체크로마토그래피를 이용 하여 수행한 결과 특이성, 시스템 적합성, 직선성, 일내 재현성, 균질성, 안정성, 농도분석, 품질관리를 판정 기준에 만족하는 분석방법을 확립 및 검증하였다. 하지만 의뢰기관의 시험성적서상 표준물질 함량은 시험 기관의 결과보다 1.34배, 1.17배 높은 결과로 나타나 비임상시험관리기준에 적합한 분석법 밸리데이션을 통한 결과 도출이 신뢰성과 안정성 확보 측면에서 중요함을 확인하였다.

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.