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.
As the human mixed saliva plays important roles for the protection, regeneration, immunity, and molecular transfer/ signaling in the oral and gastro-intestinal mucosa, the salivary contents have great implications for the general health of human body. Nevertheless, the analysis method of human saliva has not been well developed up to date, because the proteins of mixed saliva are rapidly interacted with each other and easily degraded by proteolytic enzymes and microorganisms. This study aims to develop an immunoprecipitation-based high performance liquid chromatography (IP-HPLC) for the analysis of human mixed saliva. The representative IP-HPLC analyses were performed to compare among different subjects in variable general conditions. Compared to the normal control the subjects suffered from bacterial infections of gastro-intestinal enteritis, chronic periodontitis, and acute necrotizing gingivo-stomatitis showed dramatic increase of LL-37 level depending on the severity of diseases, while the subject suffered from Herpes stomatitis, a viral infection showed great increase of β-defensin 2. These data indicate that LL-37 in human mixed saliva is more responsible to the bacterial infections of gastro-intestinal enteritis, chronic periodontitis, and acute necrotizing gingivo-stomatitis, while β-defensin 2 is more responsible to the viral infection of Herpes stomatitis. This study also suggeststhat the IP-HPLC be easily applicable to the wide range of biological samples for the quantitative analysis of an objective protein.
본 논문에서는 식품 중 퀴놀론계(QNs) 합성항균제(marbofloxacin, norfloxacin, danofloxacin, ciprofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid 그리고 flumequine) 9종을 분석하기 위하여, 액-액 추출 과정을 거쳐서 형광검출기가 장착된 액체크로마토그래피를 이용하여 QNs를 효율적으로 동시분석하는 방법을 확립하였다. 컬럼은 Zorbax Eclipse XDB-C8(150 mm×4.6 mm, 5 μm), 이동상 용매는 200 mM ammonium acetate buffer (pH 4.5)와 ACN로 기울기 용리를 사용하였으며, 유속은 1.5 ml/min, 그리고, 주입량은 10 μl로 설정하여 분석하였다. 확립 된 분석조건으로, 표준검정 곡선은 10-500 μg/kg의 농도범위에서 상관계수가 0.9989 이상의 양호한 직선성을 나타내었으며, 회수율은 50, 100 그리고 500 μg/kg의 농도에서 89.6-106.5 %로, 향상된 추출효율을 나타내었다. 검출한계는 1-16 μg/kg이었고, 정량한계는 3-47 μg/kg이었으며, 일내(intra-day)와 일간(inter-day) 정밀도(CV%)는 0.2-15.0 %, 0.5-11.7 %이었다. 따라서, 확립된 분석방법은 광어 및 계란 중의 QNs을 효과적으로 분석하는데 이용될 수 있을 것으로 사료된다.