D-antroquinonol及Antroquinonol 對非小細胞肺癌之細胞具有抗癌作用

D-antroquinonol and Antroquinonol Induce Anticancer Effects on Non-Small Cell Lung Cancer Cell Lines

顏怡婷(YI-TING YEN)

指導教授 ： 林若凱

共同指導教授 ： 邱士娟(Shih-Jiuan Chiu)

臺北醫學大學/藥學院/藥學院生技製藥產業碩士專班/碩士(2013年)

摘要

肺癌為全世界癌症死亡人口數中比例最高的癌症。造成肺癌發生的其中一種機制為表遺傳學改變，如DNA甲基化。當DNA序列上發生不正常甲基化時可能致癌基因活化或腫瘤抑制基因靜默，進而導致癌症的發生。當CpG 島內的胞嘧啶 (cytosine)被DNA甲基轉移酵素甲基化時可能會抑制DNA轉錄功能。甲基轉移酵素在文獻中被指出有過度表現現象，進而使腫瘤抑制抑制基因過度甲基化。本次實驗以DNMT1及DNMT3B兩種酵素活性進行數十種天然物篩選，其中對DNMT1具有酵素抑制潛力的有D-antroquinonol。而在研究中所使用的D-antroquinonol及antroquinonol是由臺灣牛樟芝固態培養液中純化而得。文獻指出，Antroquinonol 在其他癌細胞中具有抗癌作用，D-antroquinonol為Antroquinonol類似物，目前尚未發表過，是為一全新的化合物。此研究目標主要為探討給予數種肺癌細胞兩種化合物後，對癌細胞的存活率及爬行能力的影響，進而與腫瘤抑制基因的DNA甲基化程度改變、RNA和蛋白質表現量之關聯性。
根據實驗結果，給予CL1-5肺癌細胞兩種化合物後，在細胞爬行能力測試中發現，具有抑制癌細胞爬行能力。在細胞毒性試驗，D-antroquinonol與現行DNA甲基轉移酵素抑制劑azacitidine相比在數種肺癌細胞中更具有專一性，且對正常肺細胞高濃度給予下不會影響。D-antroquinonol不管時間依賴或是劑量依賴方式在CL1-5肺癌細胞均可使cyclin D2 (CCND2)基因的mRNA表現量增加，並可使細胞週期停留在G0/G1期。除此之外，當我們在H1299細胞中將CCND2基因進行siRNA轉染作用，細胞爬行能力增加。
根據上述實驗結果，我們推測D-antroquinonol為一具有抗癌潛力之藥物，並可抑制癌細胞爬行能力及增加CCND2基因表現量。

關鍵字

肺癌 ； D-antroquinonol ； antroquinonol

並列摘要

Lung cancer is the leading cause of mortality in both men and women worldwide. One of mechanisms contributing to NSCLC through epigenetic changes is DNA methylation. Promoter methylation may cause the silencing of tumor suppressor genes, resulting in abnormal growth of cancer cells and tumorigenesis. Overexpression of DNA methyl- trasferases (DNMTs) protein has been reported in various cancerous tissues in many studies. Therefore, it is needed to develop novel DNMTs inhibitors for cancer therapy. D-antroquinonol and antroquinonol, were purified and identified from Antrodia camphorate. We screened several compound by DNMT enzyme activity assay, and we discovered D-antroquinonol had DNMT1 enzyme activity. The aim of this study was to investigate the DNA methylation status and mRNA and protein expression level of multiple tumor suppressor genes in NSCLC cell lines and to analyze the association with cell viability and migration ability after D-antroquinonol or antroquinonol treatment.
In this study, results from wound-healing assay and transwell assay demonstrated that D-antroquinonol and antroquinonol inhibited cell migration ability of CL1-5 cells. D-antroquinonol also exhibited higher cytotoxicity toward different lung cancer cell lines than azacitidine. In addition, D-antroquinonol showed low cytotoxicity to normal lung cells. Analytical results from RT real-time PCR showed that D-antroquinonol up-regulated cyclin D2 (CCND2) gene expression in CL1-5 cells in time-dependent and dose-dependent manners, and caused cell cycle arrest at G0/G1 phase from flow cytometry analysis. Moreover, knockdown of CCND2 gene in H1299 cells caused increasing cell numbers of invasion.
According to the results above, we suggested that novel antroquinonol analog D-antroquinonol might be a potential anticancer drug which may inhibit metastasis and recover the expression of CCND2 tumor suppressor gene.

並列關鍵字

lung cancer ； D-antroquinonol ； antroquinonol

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