Search for Molecular Targets by Proteome Analysis and Its Use to Taylored-Made Therapy against Oral Cancer

通过双向聚丙烯酰胺凝胶电泳和MALDI-TOFMS分析，我们发现维生素A在口腔癌细胞系中特异性诱导了一种主要组织相容性复合物I类链相关分子A（云母）蛋白，它是大多数NK细胞、LAK细胞和细胞毒性T细胞上的活化受体NKG 2D的配体。RT-PCR和流式细胞仪分析表明，维生素A衍生物如全反式维甲酸（ATRA）分别诱导口腔癌细胞系云母的mRNA和蛋白表达，我们分析了100例口腔鳞状细胞癌（OSCC）和103例随机选择的无关对照的云母多态性（GCT三联重复序列跨膜区多态性）。结果发现口腔癌患者中云母5.1表型的比例为30%，而对照组为15%（X ~ 2 = 16.203，P值=0.00006）。5.1等位基因患者NK活性低于其他等位基因患者。流式细胞仪分析显示，所有细胞系的细胞表面均表达云母蛋白。据报道，肿瘤有办法逃避身体的免疫系统。作为这些系统之一，在血清中存在可溶性形式的云母（sMICA）。我们用ELISA法检测了45例口腔鳞癌患者血清中的sMICA。我们发现口腔鳞癌患者sMICA水平显著高于对照组。这些结果提示，口腔鳞癌的易感性与云母微卫星多态性密切相关，5.1等位基因可能与口腔鳞癌的发生有关，血清sMICA水平可能成为口腔鳞癌诊断、治疗和预后的一个新的分子靶点/生物标志物。

By two-dimensional PAGE and MALDI-TOFMS analysis, we have found that a major histocompatibility complex class I chain-related molecule A (MICA) protein, the ligand of NKG2D which is activation receptor on the most NK cells, LAK cells, and cytotoxic T cells, was specifically induced by vitamin A in oral cancer cell lines. RT-PCR and FACS analysis revealed that vitamin A derivative such as ATRA induced mRNA and protein expression of MICA in oral cancer cell lines, respectively.We analysed MICA polymorphism (GCT triplet repeat polymorphism in the transmembrane domain) in 100 cases with Oral Squamous Cell Carcinoma(OSCC), and 103 randomly selected unrelated controls by direct sequencing and fragment analysis. Five distinct MICA polymorphisms were studied.As a result, we have found that the percentage of the patient who has MICA 5.1 phenotype among total oral cancer patients is 30%, but that of control is 15% (X^2=16.203,P value=0.00006). NK activity of the patient of 5.1 allele is lower than that of the others of other alleles. By flow-cytometric analysis, MICA protein on the cell surface was detected in all cell lines. It has been reported that tumors have ways of evading the body's immune system. As one of the system of these, there are soluble forms of MICA (sMICA) in the serum. We have examined sMICA in the serum of 45 OSCC patients by ELISA. We have found the sMICA levels in OSCC patients were significantly higher than those in controls. On the other hand, sMICA serum concentration strongly correlated with the clinical state of OSCC patients and might serve as a novel biomarker of OSCC.These results suggest that the susceptibility to OSCC is closely linked to the MICA microsatellite polymorphism and 5.1 allele might confer the risk of OSCC, and that serum levels of sMICA may serve as a novel molecular target/biomarker of OSCC for its diagnosis, therapy, and prognosis.