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Title: A DNA Methyltransferase I Inhibitor Mithramycin A in Cancer Cells: A Pilot Study
Other Titles: Mithramycin A抑制肺癌細胞中的甲基轉移酵素及其轉移能力
Authors: 林若凱
Issue Date: Dec-2007
Publisher: 國立臺灣師範大學生命科學學系
Department of Life Science, NTNU
Abstract: 在抑癌基因啟動子的CpG島群上若發生了不正常的過度甲基化時,往往導致人類癌症的發生及進程。負責將CpG島群甲基化的是DNA甲基轉移酵素(5'-cytosine-methyltransferase,簡稱DNMT)。抑制形成CpG島群的甲基化的藥物正可以作為恢復抑癌基因的表現及活化癌細胞中抑制癌症的重要路徑的新穎標的。而Mithramycin A(簡稱MMA)是一個會與富含GC及CG序列的DNA結合的藥物,因此本研究檢測癌細胞在經過MMA的處理之後,是否會抑制CpG島群甲基化的情形。我們發現當以低劑量(10 nM)的MMA處理癌細胞14天後,會減少SLIT2基因的CpG島群過度甲基化的情形,並進而使這個具有抑制癌細胞轉移的SLIT2重現基因表達。同時間藉由膜穿透(transwell)實驗我們也發現MMA可降低具有高轉移能力的癌細胞CL1-5的穿越及移動能力。為了暸解MMA的作用途徑,我們使用西方點漬法發現MMA會使DNMT1蛋白明顯下降,但是DNMT1的基因表現不受影響。總結研究結果發現,MMA具有去DNA甲基化及抑制癌細胞轉移的潛力。而抑制的機轉可能藉由降低癌細胞中DNMT1的蛋白表達量,進而導致抑制癌細胞轉移的基因啟動子去甲基化且重新恢復表達。
Abnormal CpG island hypermethylation of multiple tumor suppressor genes (TSGs) can lead to the initiation and progression of human cancer. The cytosine of the CpG island on promoter region is methylated by 5'-cytosine-methyltransferases (DNMTs). Pharmacologic inhibitors of CpG island methylation provide a rational approach to reactivate the TSGs in tumor cells and restoring of critical cellular pathways in cancer cells. Mithramycin A (MMA) is known to be a GC and CG-rich DNA binding agent. We sought to determine whether MMA could inhibit CpG island methylation and DNMT expression in lung cancer cells. We found that MMA reduced CpG island methylation of anti-metastasis TSGs, SLIT2, which associated with the prevention of metastasis. When highly metastatic CL1-5 lung cancer cells were treated with low does (10 nM) of MMA for 14 days, they re-expressed mRNA levels for SLIT2 genes. MMA also inhibited the invasion phenotypes of CL1-5 cells as indicated by its inhibition of cancer cell migration using transwell assays. Western blots showed that DNMT1 protein levels were depleted after MMA. These data support the idea that MMA has demethylation and anti-metastasis effects on lung cancer cells. This inhibitory mechanism of MMA in DNA methylation may be mediated by the depletion of DNMT1 protein.
Other Identifiers: 816A7561-02C2-DD5E-EB23-D4ED8B5811D4
Appears in Collections:生物學報

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