背景:麩胱甘肽轉移酶 (Glutathione S-transferase, GST) 是一種解毒酵素。GST-M2的低表現在肺癌細胞可以被偵測到；然而，GST-M2在肺癌細胞中調控機制並不清楚。在本研究中，我們探討在肺癌細胞中的GST-M2的表觀調控機制(epigenetic mechanism)。方法:在肺癌細胞以DNA甲基轉移酵素 (DNA methyltransferase；DNMT)抑制劑5’-aza-2’-deoxycytidine (5’-Aza-dC) 處理後，我們評估GST-M2的啟動子甲基化；Reporter activity assay、Chromatin immunoprecipitation (ChIP)、electrophoretic mobility-shift assay (EMSA) 以及shRNA 試驗被使用來測試是否Sp1甲基化影響結合在GST-M2 啟動子上，並且調控GST-M2的轉譯。即時定量PCR (real-timePCR)也被運用來決定73名非小細胞肺癌(non-small cell lung cancer [NSCLC])組織中，GST-M2與DNMT-3b mRNA程度。在肺癌細胞中，藉由5’-Aza-dC的處理，GST-M2的基因表現被回復；在肺癌細胞及非小細胞肺癌組織中，GST-M2也表現出高度的啟動子甲基化程度。結果:在肺癌細胞中，CpG甲基化減弱了Sp1結合至GST-M2啟動子。在肺部正常細胞中，Sp1的剔除減少了GST-M2的表現；並且在肺癌細胞中，DNMT-3b的靜默則增加了GST-M2的表現。此外，在具有低GST-M2表現的肺部腫瘤中相較於具有高GST-M2的肺部腫瘤中，DNMT-3b表現是顯著較高的，特別是女性與第一期病患分組。結論:GST-M2的表觀靜默能夠被辨識出Sp1調控的GST-M2轉譯表現，並且代表著導致在肺癌細胞和第一期NSCLC病患中所觀察到的GST-M2減少表現之機制。
BACKGROUND:Glutathione S-transferases M2 (GST-M2) is a detoxifying enzyme. Low expression of GST-M2 in lung cancer cells was detected. However, little is known about the regulation of GST-M2 in lung cancer cells. In this study, we investigated the epigenetic regulatory mechanisms of GST-M2 in lung cancer cells. METHODS:We evaluated the promoter methylation of GST-M2 following the DNA methyltransferase (DNMT) inhibitor, 5’-aza-2’-deoxycytidine (5’-aza-dC) treatment in lung cancer cells. Reporter activity assay, Chromatin immunoprecipitation (ChIP), electrophoretic mobility-shift assay (EMSA) and small interfering RNA (siRNA) assays were used to detect whether methylation of Sp1 affects the binding to the GST-M2 promoter and regulates GST-M2 transcription. Real-time PCR was applied to determine GST-M2 and DNMT-3b mRNA levels in 73 non-small cell lung cancer (NSCLC) tissues. RESULTS:GST-M2 expression was restored by the treatment with 5’-aza-dC in lung cancer cells. GST-M2 also exhibited high frequency of promoter hypermethylation in lung cancer cells and NSCLC tumor tissues. CpG hypermethylation abated Sp1 binding to the GST-M2 promoter in lung cancer. Knockdown of Sp1 in lung normal cells reduced GST-M2 expression, and silencing of DNMT-3b increased GST-M2 expression in lung cancer cells. In addition, DNMT-3b expression was significantly higher in lung tumors with low GST-M2 expression than that in lung tumors with high GST-M2 expression, especially in female and stage I groups. CONCLUSIONS: Epigenetic silencing of GST-M2 could be distinguished Sp1 mediated GST-M2 transcriptional expressions and represent the mechanism leading to the decreased expression of GST-M2 observed in lung cancer cells and stage I/II NSCLC patients.
