探討花生四烯酸與缺氧對人類乳癌細胞株及大鼠乳腺腫瘤中血管新生相關蛋白之影響

Abstract

花生四烯酸 (Arachidonic acid，AA；C20：4 n-6) 為多元不飽和脂肪酸，其代謝過程中有許多產物會導致腫瘤生成 (tumorgenesis) 與惡化 (progression)。先前本實驗室發現大鼠乳腺腫瘤中AA含量占總脂肪酸之比例會隨著腫瘤重量增加而隨之遞增，顯示AA於乳癌進程中為重要的調控者，而缺氧是腫瘤轉移的重要因子。本實驗目的在於探討缺氧與AA協同下對乳癌血管新生 (angiogenesis) 之影響。利用人類乳腺癌細胞MCF-7添加0, 10, 50, 100 μM AA伴隨正常氧氣或缺氧來模擬腫瘤中環境，觀察AA在乳癌的血管新生過程中之可能作用機制。研究結果發現添加AA後會活化Akt路徑、提升HIF-1α在細胞內的表現、促進NF-κB (p65)蛋白與HIF-1α轉移並累積於細胞核內表現，進而提升血管新生指標蛋白VEGF的表現量；且在缺氧環境狀態下AA對Akt的磷酸化、轉錄因子NF-κB (p65)及HIF-1α蛋白質轉移至細胞核內及向上調控VEGF蛋白的表現量等促進的效果更為顯著。進一步利用人類臍靜脈內皮細胞 (HuVECs) 培養在經50 μM AA與缺氧依序處理之MCF-7條件培養基中，能提升HuVECs細胞的增生(proliferation)，且能誘導及驅使該細胞的移行。 在大鼠乳腺腫瘤組織中發現其腫瘤重量與p65、HIF-1α蛋白質在細胞核中所佔比例呈正相關性，R值分別為0.5198與0.5912 (p < 0.05)；而VEGF的表現量則隨著乳腺腫瘤重量的增加隨之遞增，其R值為0.8412 (p < 0.0001)。 總括而論，本研究指出AA能調控MCF-7在正常氧氣及加強缺氧逆境下Akt之磷酸化、NF-κB與HIF-1α的活化及VEGF表現量之提升，進而促進HuVEC細胞存活與增進其移行之能力。且在大鼠乳腺腫瘤中亦得到相映證之結論。故推論AA會調控及促進乳癌的惡性化程度—血管新生之現象。

We previous found that arachidonic acid (AA, 20:4n-6) levels are 10 times higher in rat mammary tumor than in the normal mammary gland and are positively correlated with tumor weight. However, the mechanism is not clear. Here, we examined the effect of AA and hypoxia on angiogenesis studied in human breast cancer cell line and rat mammary tumors. MCF-7 Cells were pretreated with 0, 10, 50 and 100 μM AA for 48 hrs and then were incubated in normoxia or hypoxia for 6 hrs. Western blot analysis was performed on whole cell lysates, cytosol and nuclear fraction for the NF-κB signaling, HIF-1α and VEGF expression. AA supplementation resulted in up-regulation of pAkt/Akt, nuclear p65, HIF-1α and VEGF expression. The AA treated MCF-7 medium increased the proliferation and migration of HuVEC cells. In addition, these effects were all enhanced in hypoxia with AA supplementation. In study of rat mammary tumors, the tumor weight were positive correlated with nuclear HIF-1α (r = 0.6091, p = 0.0073) and VEGF (r = 0.8412, p < 0.0001). We proposed that angiogenesis is enhanced by AA especially in the hypoxia through increasing NF-κB signaling, HIF-1α and VEGF protein expression.