探討花生四烯酸在TNF-α誘發NF-κB傳訊下促進乳癌增生之調控機制

Abstract

花生四烯酸(Arachidonic acid ,AA ; C20:4 n-6)為多元不飽和脂肪酸,其多 種代謝產物在發炎反應中扮演調控角色。慢性發炎與癌化間關係於近年來多受關 注,在發炎反應中扮演整合中樞的轉錄因子NF-κB於乳癌中也發現過度活化。在 本實驗室先前研究中發現大鼠乳腺腫瘤之重量與AA含量成正比,顯現該脂肪酸 對於乳癌增生可能具調控作用。故本實驗以添加不同濃度AA的細胞模型模擬不 同重量腫瘤環境,觀察在TNF-α活化NF-κB傳訊過程中AA是否會透過影響Akt路 徑改變NF-κB相關蛋白表現量,並另以SD大鼠的乳腺腫瘤進行分析驗證。 本研究細胞實驗部份利用雌激素受體α陽性(ER+)人類乳癌細胞株MCF-7,培 養在含有0.1%胎牛血清的DMEM培養液中,以不同濃度(0、10、50、100 μM)的 AA處理,而後施以10 ng/mL TNF-α(視觀察項目更改刺激時間)。結果顯示p-Akt 活化程度會隨著AA濃度上升而增加,在10、50、100 μM時分別為控制組(0 μM) 的0.97、1.33、1.52倍。在免疫螢光染色方面,施以100 μM AA時也觀察到NF-κB 單體p65進入細胞核之現象。給予100 μM AA時,Akt磷酸化程度以90分鐘為分界 點先升後降,活化程度為控制組(0 分鐘)的4.83倍。至於細胞質內的NF-κB抑制 分子IκBα則會隨施加時間增加而逐漸分解,於20分鐘分解比例即佔79%,且該效 應在120分鐘內均維持在80%左右,直至180~240分鐘分解狀況方趨緩。在核質分 離樣本中觀察到,構成NF-κB常見二聚體中的p65蛋白質,隨AA濃度增加進入細 胞核比例亦漸增,在10、50、100 μM下分別是控制組的1.23、1.45以及1.59倍。 若添加Akt抑制劑阻斷傳訊時,可觀察到IκBα蛋白表現量比僅添加AA 100 μM 高,自控制組的46.3%回復至57.7%,顯現兩者傳訊途徑具上下游關係。然而, NF-κB下游的蛋白cyclin-D1隨AA濃度上升(0、10、50、100 μM)表現量卻漸減, 分別為控制組(0 μM)的45.4%、28.6%至28.1%。 動物組織實驗方面則印證了細胞實驗的結果,將乳腺腫瘤依照重量分類,進 行蛋白質萃取、電泳檢定,觀察目標蛋白p-Akt、p65、IκBα和cyclin-D1之表現與 腫瘤重量間是否具相關性。結果顯示,乳腺重量與Akt磷酸化、IκBα分解量之間 具正比關係,R值分別為0.6993、0.5319,且前者具統計顯著性(p<0.001)。而NF-κB 單體p65和NF-κB下游蛋白cyclin-D1表現,前者隨重量增加而上升(R值為0.6282, p=0.0287),後者則隨重量增加表現漸減(R值為0.7652,p<0.0001)。結果均與細胞 實驗觀察達一致。

本實驗發現,以TNF-α促使乳腺癌細胞株NF-κB活化時,添加AA會以活化 Akt的方式增強該傳訊途徑下游蛋白表現,且於動物模式中亦得到相同結論。故 推論AA在乳腺腫瘤中是以協助性角色影響NF-κB傳訊途徑,促進腫瘤增生、惡 化。應用於臨床上或許可透過降低目標器官中AA含量使乳癌惡化程度降低,進 而達到治療與延緩轉移之目的。

Our Lab previous found that arachidonic acid (AA, 20:4n-6) levels are 10 times higher in mammary tumor tissue than in the normal mammary gland, and are positively correlated with the tumor weight. However, the mechanism is not clear. We then proposed AA may enhance nuclear factor kappaB (NF-κB) activation studied in human breast cancer cell line MCF-7 and rat mammary tumors. We hypothesized that AA enhanced tumor necrosis factor-α (TNF-α)-induced NF-κB signaling for the growth of the breast cancer cells. We also studied the correlation between NF-κB signaling and tumor weight in rat mammary tumor. MCF-7 cells were pretreated with 0, 10, 50 and 100 μM AA for 48 hours, and then were stimulated with TNF-α. Western blot analysis was performed on whole cell lysates, cytosol and nuclear fractions for the NF-κB signaling protein expression. AA supplementation resulted in increasing pAkt /Akt levels, IκBα degradation, nuclear p65 expression and reduced cyclin D1 expression in MCF-7. In study of rat mammary tumors, the tumor weight were positively correlated with pAkt/Akt ratio (r=0.6993, p=0.0009), were inversely correlated with IκBα expression (r=0.5319, p= 0.0751), were positively correlated with nuclear p65 expression (r=0.6282, p=0.0287) and were inversely correlated with cyclin D1 expression (r=0.7652, p <0.0001) in the mammary tumor. It was concluded that AA enhanced Akt signaling resulted in increasing IκBα degradation and nuclear p65 expression for the growth of breast cancer.