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標題: 研究新穎的DNA甲基轉移酶抑制劑對人類胰臟癌細胞抗癌效用及分子機轉
The study of anticancer effects and molecular mechanisms by a novel DNA methyltransferase inhibitor on human pancreatic cancer cells.
作者: 黃茂軒
Mao-Hsuan Huang
關鍵字: 胰臟癌
patched domain containing 4
Pancreatic ductal adenocarcinoma
DNA-methyltransferase 1
patched domain containing 4
Hedgehog pathway
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摘要: 胰臟癌在美國是因癌症死亡人數的第四位,患有胰臟癌的病人只有8% 可以活到五年以上,是一種極為致命的疾病。主要原因是目前臨床用來治療胰臟癌的化療藥物,其抗癌效果皆不佳,最多只延長三個月的壽命。異常的DNA甲基化在胰臟的癌化過程中被認為扮演重要的角色,同時將近50%的胰臟癌病人DNA甲基轉移酶1 (DNMT1) 都有過度表達的現象。因此我們以DNMT1的表現當作篩選平台,從中找到一個新的DNMTs抑制劑,正丁烯基苯酞 (n-butylidenephthalide, n-BP),n-BP可以劑量依賴性和時間依賴性方式抑制DNMT1的表現。同時我們也發現n-BP治療人類胰臟癌細胞後,可以有效的抑制胰臟癌細胞生長。以流式細胞儀分析它對細胞週期的影響,結果顯示n-BP不只會造成細胞週期停滯於G0/G1,也會引發細胞凋亡的現象。在用基因轉殖技術維持DNMT1的表現後,n-BP造成的細胞抑制生長現象就被大幅降低。此外我們也發現n-BP是透過影響蛋白質穩定性去抑制DNMT1而不是透過RNA的轉錄。為了更進一步了解n-BP降低DNMT1後影響了哪些基因,我們利用微陣列分析技術掃描發現,patched domain containing 4 (PTCHD4) 是DNMT1的潛在下游基因。在透過siRNA沉默PTCHD4表現後,n-BP便無法降低腫瘤的生長。而且PTCHD4 是一個腫瘤抑制基因參與在Hedgehog路徑上,我們也看到n-BP能夠誘導PTCHD4進而抑制Hedgehog路徑的活化。更進一步,我們利用兩種動物模式來評估n-BP在活體內的療效。透過局部緩慢釋放高分子聚合物來攜帶n-BP進行治療,實驗的結果不論是皮下腫瘤或原位瘤的動物模式,BP都能有效的抑制胰臟癌的生長,同時藉由免疫組織切片染色,我們觀察到與細胞實驗一致的結果。總合以上結果證實,n-BP可能是透過降低DNMT1並增加PTCHD4的表現來抑制人類胰臟癌細胞的生長,同時在體外和體內都有很好的抗癌效果,顯示n-BP有潛力做為未來治療胰臟癌的標靶藥物。
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with only eight percent of the patients surviving beyond five years. One of the major reasons is the low effectiveness of anticancer drugs that only extends survival by 3 months. Aberrant DNA methylation has been considered to play an important role during carcinogenesis in PDAC, with approximately 50% of tumor tissues overexpressing the DNA methyltransferase 1 (DNMT1) protein. In the present study, we used DNMTs as a screening platform to find a new DNMT inhibitor, n-butylidenephthalide (n-BP). n-BP could inhibit DNMT1 expression in both dose-dependent and time-dependent manner. It also displays an effect in suppressing growth of PDAC cells, inducing cell cycle arrest at G0/G1 phase, and apoptosis. Growth suppression can be restored by the overexpression of DNMT1 in PDAC cells. Furthermore, we found n-BP-mediated DNMT1 suppression influenced the protein stability rather than changing the RNA expression. Through microarray analysis, we found that the patched domain contained 4 (PTCHD4) is the potential downstream gene of DNMT1. Following silencing of PTCHD4 expression by siRNA, n-BP decreased tumor growth inhibition. PTCHD4 is also a tumor suppressor gene involved in the Hedgehog pathway. We observed that n-BP could induce PTCHD4 and inhibit the activation of the Hedgehog pathway. Finally, in vivo, two animal models were used to evaluate the efficacy and survival after n-BP treatment by interstitial control release polymer delivery. The results show that n-BP could effectively inhibit PDAC tumor volume growth and extend animal survival. In summary, n-BP may inhibit the growth of human PDAC cells though reducing DNMT1 and increasing the expression of PTCHD4 both in vitro and in vivo.
文章公開時間: 2021-01-11
Appears in Collections:生命科學系所



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