NBS/2DE/MS法による薬剤応答性乳癌タンパク質マーカの探索

摘要

疾患における薬剤応答性タンパク質マーカの発見は，その治療や予後にきわめて重要である。著者らは，乳rn癌細胞のモデルとしてエストロゲン受容体陽性（ER＋）のセルラインMCF-7を用い，これを乳癌治療薬タモrnキシフェン（TAM）で処理した時に発現が変動するタンパク質を島津製作所が開発した定量的プロテオーム解rn析試薬NBSClと2DE/MSを組み合わせる方法により解析し，有益なる知見を得た。TAM処理と非処理の比較rnにおいて88個のスポットが明らかな発現差を示した。このうち44個は質量差6DaのNBSラベル化ペアペプチrnドとして同定でき，23種の異なるタンパク質が含まれていた。そのうち16個（70%）は，TAM処理やER活性rnとの関連が明らかにされていない。これらのタンパク質のうち，GRP78,CK19,PA2G4について生物学的評rn価として免疫プロット法による確認，続いてPA2G4については免疫組織化学染色法による評価を行った。さらrnにこのPA2G4については，対応する遺伝子発現と乳癌再発との関係を調べるため，二つの独立した乳癌患者集rn団のmRNAおよび予後に関するデータを使用して検討した結果，新しい予後診断因子になりえる可能性を見rn出した。以上の結果，NBS試薬と2DE/MSを組み合わせた解析手法は非常に有効な方法であるといえる。%The identification of drug-responsive protein biomarkers for disease is crucial to its medical treatment and prognosis. The present paper describes a novel approach for identifying drug-induced protein alternations, for which NBSC1 tryptophan labeling reagent developed by Shimadzu and 2DE/MS were combined. As a sample model, the estrogen positive breast cancer cell line MCF-7 was used. For comparative study, half of the cultured cells were treated by Tamoxifen (TAM) and the other half were not treated as control group. Of 88 protein spots that showed significant difference in expression between TAM treatment and non-treatment, 44, including 23 distinct protein species, were identified as NBS-labeled pair peptide with a mass difference of 6Da. Of the 23 TAM-treated proteins, 16 (i.e., 70%) have not been clarified concerning relationship to TAM or ER activity. To validate the NBS/2DE/MS results, GRP78, CK19 and PA2G4 proteins were selected and confirmed using the immunoblotting method. Then, PA2G4 was validated by the immuno-tissue-microarray analysis method. Furthermore, the relationship between gene expression and breast cancer recurrence was studied for PA2G4, where database on mRNA expression and disease-free survival in two independent breast cancer patient cohorts was utilized. The study findings suggest that PA2G4 has a possibility of becoming a novel prognosis factor for disease-free survival. Considering all the results obtained in this search, we can conclude that the combination of NBS and 2DE/MS is a very useful tool for research of protein markers.