終端是合成染色體末端端粒DNA的特殊核酸蛋白質，終端的活性在正常人類體組織中是受到抑制的，但在85～90%的腫瘤組織會表現出活性，所以抑制終端的活性可做為治療癌症的一個方向。薑黃素在薑黃植物中為橘黃色結晶，目前已知薑黃素可以抑制癌症細胞的增生並且誘導腫瘤細胞進入凋亡。本篇研究主要探討薑黃素是否透過氧化性傷害來抑制A549細胞的終端活性之機轉。首先，我們以染色體終端重複增幅步驟法測定，觀察薑黃素確實有效的抑制A549端粒活性，並且以西方點墨法和RT-PCR方式觀察到h.TERT表現亮隨著薑黃素濃度上升而明顯的減少。我們以Reporter gene assaay分析暫時轉染帶有不同終端啟動子於pGL3載體到A549細胞中其活性，結果指出薑黃素抑制h.TERT轉錄是透過Sp1，並且以DNA 親和性沉澱試驗證實薑黃素會抑制Sp1與端粒啟動子結合的能力。另外，我們以流氏細胞儀測得薑黃素會誘發A549細胞的鈣離子的產生，但是以BAPTA-AM和Nifedipine鈣離子的抑制劑處理A549細胞，發現h.TERT的表現量並沒有很顯著的回覆，因此認定薑黃素並非透過鈣離子的產生而抑制了終端的活性。此外，我們以染色體終端重複增幅步驟法測定，當A549細胞終處理乙醯基半胱胺酸發現，原本被薑黃素所抑制的終端活性有回復的現象，同時也偵測h.TERT的表現在mRNA與蛋白的部分均有回復的現象，而Sp1的表現也有回復的現象，我們利用了Reporter gene assay分析終端啟動子的活性也均有回復的現象。我們的結果指出薑黃素會誘發癌症細胞產生氧化性傷害，降低了Sp1與h.TERT結合的能力，進而抑制癌症細胞終端之活性。 Telomerase is a special ribonucleoprotein for synthesizing the telomeric DNA at the ends of chromosomes. Telomerase activity is suppressed in normal human somatic tissues, but it is activated in 85~90% tumor tissues. So inhibiting telomerase activity is a useful therapeutic strategy. Curcumin, the yellow pigment in turmeric, is known to inhibit proliferation of cancer cells by arresting them at various phases of the cell cycle and to induce apoptosis in tumor cells. This study mainly explored that curcumin suppress telomerase activity throuh ROS production in A549 cells. First, curcumin inhibited A549 telomerase activity by telomerase repeat amplification protocol. Using Western blot and RT-PCR, curcumin reduced h.TERT expression in a dose dependent manner. The reporter assay show that curcumin suppress the h.TERT promoter expression through Sp1. DNA affinity precipitation assay was used to confirm that curcumin inhibited Sp1 binding on the h.TERT promoter. In addition, we analyzed that curcumin could induce calcium ion production by Flow cytometry in A549 cells. A549 cells were treated with calcium ion inhibitor of BAPTA-AM and Nifedipine, which did not recover the h.TERT supperession by curcumin. Therefore, our data suggested that suppression of telomerase activity is independent on calcium ion production. Furthermore, N-acetyl cysteine (NAC) restored the telomerase activity suppression by curcumin. The mRNA and protein expression of h.TERT decreased by curcumin was reversed by NAC. NAC also restored the expression of Sp1 reduction and h.TERT that were downregulation by curcumin. Our results strongey suggested that curcumin induced ROS production which resulted in inhibiting Sp1 binding activity and h.TERT downregulation.
