去甲氧基薑黃素引起人類非小細胞肺癌NCI-H460 細胞凋亡，DNA損傷和基因改變之研究

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題名:	去甲氧基薑黃素引起人類非小細胞肺癌NCI-H460 細胞凋亡，DNA損傷和基因改變之研究 Demethoxycurcumin Induced Apoptosis,DNA Damage and Gene Alterations in Human Lung Cancer NCI-H460 Cells
作者:	柯仰謦;Yang-Ching Ko
貢獻者:	臨床醫學研究所博士班
關鍵詞:	去甲氧基薑黃素;非小細胞肺癌;細胞凋亡;粒腺體路徑;demethoxycurcumin;non-small cell lung cancer;apoptosis;mitochondrial pathway
日期:	2015-06-23
上傳時間:	2015-11-04 17:03:11 (UTC+8)
出版者:	中國醫藥大學
摘要:	肺癌總死亡數已經躍居國人癌症死亡原因之首位。因此，研究與發現新的治療方式是相當急迫與必要的。過去細胞與活體研究都顯示，薑黃素對多種腫瘤細胞具有抑制作用。而近年來，一些報告發現，去甲氧基薑黃素可能比薑黃素有更強的活性。所以本研究以去甲氧基薑黃素為研究主題，探討對非小細胞肺癌的細胞研究。首先，在引起細胞凋亡試驗中，去甲氧基薑黃素誘導人類非小細胞肺癌(NCI-H460)細胞發生細胞型態改變與導致存活數的減少。去甲氧基薑黃素也誘導細胞停滯在S期，且隨著濃度愈高細胞凋亡與存活數的減少也愈明顯。其次，去甲氧基薑黃素對NCI-H460細胞株引起活性氧化物質及鈣離子釋放和降低粒腺體膜電位。Caspase-3、-8和-9活性在經去甲氧基薑黃素處理後則有上升情形。西方墨點法分析顯示，去甲氧基薑黃素作用後，與細胞週期相關蛋白，p21和p27表現增加，而CDC25A、cyclin A、E 和CDK2則受到抑制；促細胞凋亡的蛋白質，不論是與粒腺體、細胞凋亡或內質網路徑相關，比如AIF、Endo G、Fas ligand、Fas、PARP、GRP78、GADD153、IRE1-β、ATF6-α、ATF6-β 和caspase-4的表現?全部增加。還有，與細胞凋亡路徑有關蛋白，calpain 1表現也受到促進。若以免疫染色及?射掃瞄共軛焦顯微鏡觀察發現，在癌細胞凋亡變化中，去甲氧基薑黃素明顯促進AIF、Endo G、ATF6-β、IRE1-α和p-PERK從粒腺體或內質網轉移至細胞核內。細胞凋亡過程，常引起 DNA 損傷反應以反制 DNA 損傷。本研究發現去甲氧基薑黃素?單可引起 NCI-H460 細胞的 DNA 損傷，也可干擾其DNA損傷反應以加大細胞毒?效果。以去甲氧基薑黃素處理NCI-H460細胞後，在DAPI染色中，隨著去甲氧基薑黃素濃度的增加，可以觀察到細胞核有凝集的現象；而彗星試驗中可以觀察到，隨著去甲氧基薑黃素濃度的增加，細胞拖尾的情形愈明顯。西方墨點法顯示，去甲氧基薑黃素作用後，與DNA修復的相關蛋白質，比如BRCA1、 14-3-3σ、MDC1、MGMT、p53，表現受到抑制，而磷酸化p53和H2A.X表現則有所增加。以免疫染色及?射掃瞄共軛焦顯微鏡觀察發現在DNA損害相關變化中，則明顯看到去甲氧基薑黃素促進p-p53和 p-H2A.X的轉移。以DNA微陣?分析去甲氧基薑黃素作用後NCI-H460細胞內互補 DNA 發現，與細胞凋亡、DNA損傷和修補、細胞週期調控相關的基因均會受到去甲氧基薑黃素的改變。當中共有144個基因表現增加而 179個基因表現則受到抑制。例如，與DNA損傷和修補有關的ERCC6L 增加2.85倍；與細胞凋亡有關的TIPARP增加5.44倍；而與細胞週期調控相關的CCNE2增加3.32倍。結論顯示，在細胞實驗中，去甲氧基薑黃素確實誘導NCI-H460產生細胞凋亡，並在DNA損傷後，破壞後續修復反應。而基因研究可以讓我們了解去甲氧基薑黃素的相關細胞毒殺機制並可能在未來當作診斷或治療肺癌的生物標記。 Lung cancer is the most common cause of cancer deaths in Taiwan. Therefore, it is important to explore new strategies for lung cancer. It has been reported that curcumin inhibited various types of cancer cells in vitro and in vivo. In recent years, numerous reports have demonstrated the stronger activity of demethoxycurcumin (DMC) compared with curcumin in many aspects. We evaluated DMC’s effects in human lung cancer NCI-H460 cells in vitro. First, we investigated the effect of DMC on the cell death via the induction of apoptosis in NCI-H460 cells. Results indicated that DMC significantly induced cell morphological changes and decreased the percentage of viable NCI-H460 cells. In addition, DMC also induced apoptosis based on the arrest of S phase. Moreover, DMC promoted ROS and Ca2+ productions and decreased the levels of mitochondrial membrane potential. We also found that DMC promoted activities of caspase-3, -8 and -9. Western blotting results showed that DMC significantly promoted the expression of p21 and p27 but reduced the expressions of CDC25A, cyclin A, E and CDK2. AIF, Endo G, PARP, Fas ligand and Fas were up-regulated. Moreover, DMC promoted the expression of calpain 1, that are associated with apoptosis pathways. DMC promoted ER stress associated protein expression such as GRP78, GADD153, IRE1-β, ATF-6α, ATF-6β and caspase-4. The results from confocal laser microscopy also indicated that DMC promoted the release of AIF, Endo G, ATF-6β, IRE1-α and p-PERK from the mitochondria or ER to the nuclei, respectively. Following apoptosis, DDR usually will be elicited to counteract the damage. We demonstrated DMC not only induced apoptosis and DNA damage, but also disrupted NCI-H460 cell’s DDR. DNA damage and condensation in NCI-H460 cells treated with DMC were displayed by comet assay and DAPI staining. Western blotting indicated that DMC suppressed the protein levels associated with DNA damage and repair, such as BRCA1, 14-3-3σ (an important checkpoint keeper of DDR), MDC1, MGMT and p53 (tumor suppressor protein). DMC could activate phosphorylated p53 and H2A.X in NCI-H460 cells. Furthermore, we also used confocal laser systems microscopy to examine the protein translocation and results show DMC promoted the levels of p-p53 and p-H2A.X (S140) in NCI-H460 cells. By cDNA microarray hybridization, genes associated with DNA damage and repair, cell cycle check point and apoptosis could be altered by DMC; in particular, 144 genes were up-regulated and 179 genes were down-regulated significantly in NCI-H460 cells after exposure to DMC. For example, the associated with DNA damage and repair ERCC6L gene was increased 2.85-fold, the associated with cell apoptosis TIPARP gene expression was increased 5.44-fold and the associated with cell cycle distribution CCNE2 gene was increased 3.32-fold. In conclusion, very low concentration of DMC can induce apoptosis of human lung cancer NCI-H460 cells in vitro. DMC also disrupt DDR following DNA damage. These results suggest DMC has a potential to develop into a clinically useful therapy for human non-small cell lung cancer. These DMC-altered genes may offer information to understand the cytotoxic mechanism of this agent at the genetic level and gene alterations may be proven useful biomarkers or targets for the diagnosis and treatment of human lung cancer in the future.
顯示於類別:	[臨床醫學研究所] 博碩士論文

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