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.

Immunoprecipitation-based high performance liquid chromatography (IP-HPLC) is a type of modified enzyme-linked immunosorbent assay (ELISA) that uses protein A/G (or antibody)-conjugated beads instead of the antibody-conjugated wells used in ELISA. In order to determine the fidelity of IP-HPLC, the author used 83 antisera to identify protein expression changes caused by cisplatin treatment in KB human oral cancer cells. KB cells were cultured for 12 or 24 hours with 10 ug/mL cisplatin. The results obtained by IP-HPLC were comparable with published cisplatin data, although ELISA was not conducted in the present study. Cisplatin dominantly reduced the levels of proteins associated with cell proliferation, transcription factors, growth factors, cytoskeletal proteins, and cellular differentiating factors, but on the other hand, apoptosis-related factors, oncogenes, and protective proteins were usually up-regulated, presumably to address cisplatin-induced DNA damage. In particular, cisplatin directly inactivated genomic DNA by down-regulating histone H1 and demethylase and by up-regulating deacetylase. Cisplatin also rapidly induced p53 overexpression and mitochondria-mediated endogenous apoptosis occurred after 12 hours of cisplatin treatment, although this was almost completely replaced by FASL/FAS-mediated exogenous apoptosis after 24 hours. This preliminary study was conducted to investigate the anticancer effect of cisplatin on the KB human oral cancer cells and to determine the fidelity of IP-HPLC data. It was concluded that IP-HPLC is useful for identifying profile changes of genome wide essential proteins and signaling changes of major molecular pathways.

The molecular mechanisms of the carcinogenesis of oral squamous cell carcinomas (OSCCs) are highly variable and result in different features of tumor progression, i.e., local tissue destruction and metastasis to regional lymph nodes. A case of OSCC arising from proliferative verrucous leukoplakia (PVL) was analyzed for its protein expression profile by immunoprecipitation (IP) – high performance liquid chromatography (IP-HPLC) by using 72 antisera and comparing results with those of KB cells. OSCC arising from PVL showed stronger expressions of proteins associated with cell proliferation (MPM2, PCNA, eiF5A, DHS, DOHH), cell survival (pAKT, MDM2, survivin), matrix proteolysis (elaffin), tumor suppression (p16, p21, PTCH1), the WNT/β-catenin pathway (SHH, WNT1, APC, β-catenin, snail), proinflammation (TNFα), angiogenesis (HIF, CMG2, vWF), and cellular protection (HSP-70, FAK, caveolin) and of oncoproteins (STAT3, 14-3-3, K-RAS, PUMA, PIM1) and growth factors (EGFR, bFGF) than KB cells. On the other hand, KB cells showed stronger expressions of proteins associated with apoptosis (caspase-3, caspase-8, caspase-9, PARP, FAS, FASL, TGase-1, BCL2, BAD, BID, BAK, FLIP), matrix proteolysis (MMP-2, MMP-9), transcription signaling (NFkB, p38, E2F-1, HO-1), and tumor suppression (p53, RB1, PTEN) and of oncoproteins (DMBT1, CEA) and growth factor (TGF-β1, c-erbB2, VEGF) than OSCC arising from PVL. These data indicate the cells of OSCC arising from PVL are more resistant and more robust than KB cells. Furthermore, they suggest the oncogenic signalings of OSCC arising from PVL play important roles in the aggressive growth and rapid tumor metastasis to regional lymph nodes

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.