- 學位論文
麩胱甘肽轉移酶抑制非小細胞肺癌轉移之研究
The Mechanisms of Glutathione S-Transferase M2 on Metastasis in Human Non-Small Cell Lung Cancer Cells
摘要
麩胱甘肽轉移酶(glutathione S-transferase, GST) 為參與解毒的phase II代謝酵素。過去本實驗室發現在肺癌細胞中，當GST-M2基因的啟動區高度甲基化將導致GST-M2表現量下降，同時也發現臨床上GST-M2基因的喪失將會影響肺癌患者的存活率，然而GST-M2和臨床特徵的相關性以及在肺癌細胞參與細胞移行的機制尚不清楚；另一方面有研究指出在肺癌病患的組織中發現有過多的重金屬鎳的堆積。因此，本研究擬探討環境中重金屬鎳是否會透過epigenetic modification造成GST-M2啟動區的甲基化以及肺癌的GST-M2喪失與腫瘤轉移及其預後是否有關。首先CL1-0細胞經氯化鎳暴露48小時後，GST-M2的表現量會下降。然而使用DNMT 抑制劑5-aza-dC之後，GST-M2的表現量有回復的現象。為釐清GST-M2和細胞的移行機制，在A549細胞建立pcDNA vector control及GST-M2過度表現的兩株細胞株。用boyden chamber及wound healing分析細胞的移行能力，發現GST-M2過度表現可以抑制細胞的移行能力。使用glutathione S-transferase 抑制劑8-Anilino-1-naphthalenesulfonic acid(ANS)可回復GST-M2所抑制的細胞移行能力，顯示GST-M2抑制細胞的移行是透過GST-M2的活性。接著利用細胞移行能力較弱的CL1-0細胞株，透過shRNA的方式建立GST-M2 knockdown的細胞株，分析其細胞的移行能力。從結果發現GST-M2 knockdown時，細胞的移行能力明顯的增加。GST-M2可透過啟動Connective tissue growth factor(CTGF)的啟動區增加CTGF的表現量。在silenced GST-M2的細胞外加CTGF可回復細胞移行能力，因此GST-M2是透過CTGF達到抑制細胞移行的能力。在動物模式中，以靜脈注射的方式植入pcDNA/A549及GST-M2/A549細胞，結果顯示GST-M2高度表現可抑制腫瘤的生長及轉移的能力。本研究證明GST-M2除了扮演解毒酵素角色外，還具有抑癌的新功能。
並列摘要
Glutathione S-transferase Mu2 (GST-M2) is a phase II detoxification enzyme. Low expression of GST-M2 in lung cancers occurs due to hypermethylation of its promoter. Lung cancer with the GST mu-null genotype is also associated with shorter survival. However, a correlation between GST-M2 and important clinical parameters, as well as the migration of GST-M2 defective cells in lung cancer, has not been established. On the other hand, recent reports have found that nickel accumulation in lung cancer patient’s tissue. In our study, we investigate whether the reduction of GST-M2 was due to the GST-M2 promoter hypermethylation by nickel exposure and the role of GST-M2 in cell migration and actin disassembly in lung cancer cells. The expression of GST-M2 was decreased under NiCl2 exposure to 48 hours. The GST-M2 mRNA expression was restored in the CL1-0 cells under 5-aza-dC treatment. To investigate the correlation between GST-M2 and the mechanism of cell migration, the migration ability of overexpressed or silenced GST-M2 cancer cells was performed on wound healing and Boyden chamber assays. It reveals that the cell migration ability was inhibited in GST-M2 overexpressed cell line which depends on its enzymatic activity relative to control cancer cells. GST-M2 can induce connective tissue growth factor (CTGF) expression by driving the CTGF proximal promoter. Moreover, the cell migration was inhibited in the presence of r-hCTGF on GST-M2 silenced cells. With or without GST-M2 expression on tumor growth and metastasis were investigated in xenograft tumor model. Overexpression of GST-M2 dramatically reduced tumor growth and metastasis in a xenograft mice model. These data highlight that GST-M2 acts as a novel tumor suppressor by inhibiting cancer cell migration in lung cancer and animal models.