Sphingosine 1-phosphate induces autophagy in human prostate cancer PC-3 cells

神經鞘氨醇 1-磷酸鹽(sphingosine 1-phosphate, S1P)是一種在血清中含量豐富並且具有生物活性的小型磷脂鹽類，S1P可以透過五種G型蛋白偶合受器，S1P1-5，在不同細胞中引發各種不同的反應，包括細胞增生、發炎反應、細胞移行、血管新生以及細胞存活等細胞反應。另外，細胞自噬作用(autophagy)亦是一種常見的細胞反應，可使細胞在處於飢餓或是外在環境壓力下，行使降解反應將一些多餘的或是可以暫時回收利用的蛋白質、胞器以及細胞質消化掉。其消化的方式，是藉由細胞內因應刺激而產生的游離細胞膜將目標物包覆，而後和溶體結合進而達成目標分子消化、回收、再利用的目的，是一種維持細胞內平衡狀態的重要機制。近年來愈來愈多研究指出，細胞自噬作用可能扮演一個能夠壓抑癌症細胞生長的角色，而關於兩者之間的關係至今尚未完全明瞭。本實驗的重點在於S1P和細胞自噬之間的關係，並以人類前列腺癌PC-3細胞株為實驗對象。關於細胞自噬反應是藉由偵測細胞自噬指標蛋白microtubule-associated protein light chain 3-II (LC3-II)的表現量，以及在穩定表現EGFP-LC3的PC-3細胞株中觀察含綠螢光之LC3-II集中到細胞自噬體(autophagosome)的現象做為判斷標準。結果顯示，LC3-II的表現量不論是濃度上或是時間上都和S1P有顯著的正相關，而含綠螢光之細胞自噬體的出現亦被證明和濃度梯度有關。此實驗更進一步深入探查反應下游之訊息傳導途徑，包括Rho鳥苷三磷酸水解酶(Rho GTPase)相關分子(Rho, Rac and cdc42)、ERK1/2蛋白激酶、環氧合酶-1 (cyclooxygenase-1)以及環氧合酶-2 (cyclooxygenase -2)等。本實驗結果是第一次發現S1P能夠促進前列腺癌PC-3細胞株產生細胞自噬。因為細胞自噬與癌症的關係密切，所以進一步深入研究其細節是絕對必要的。Sphingosine 1-phosphate (S1P), a bioactive sphingolipid is abundant in serum. S1P can induce various responses in many cell types, such as cell proliferation, inflammation, migration, angiogenesis and cell survival through a family of S1P-specific G-protein-couple receptors (S1P1-5). Autophagy, one of main cellular activities, is a cellular degradation pathway which serves an adaptive role to protect cells under starvation or stress situations. Damaged or superfluous cellular proteins and organelles can be engulfed by autophagosomes, then digested when fused with lysosomes, and recycled back into the cytosol to maintain cellular homeostasis. Recently, scientists have begun to consider autophagy as a tumor-suppressing pathway, but the regulatory mechanisms are still controversial. In addition, the relationship between S1P and autophagy remains unclear. Herein, autophagy was characterized by using Western blotting analysis to detect microtubule-associated protein light chain 3-II (LC3-II, an autophagy marker) formation, and to observe the LC3-II molecules re-localization from cytosol to autophagosomes in EGFP-LC3 stably expressing cells by using fluorescence microscopy. These results suggested that S1P induced autophagy in PC-3 cells, a human prostate cancer cell line, is both time- and concentration-dependent. Furthermore, by using chemical inhibitors, Rho family GTPase (Rho, Rac, and Cdc42)-, ERK1/2-, COX-1-, and COX-2-dependent pathways were found to be involved in the induction of autophagy. In conclusion, the results demonstrated the molecular mechanisms of S1P-induced autophagy in PC-3 cells during starvation.摘要 Ibstract IIntroduction 1phingosine 1-phosphate 1utophagy overview 3utophagosome formation 4C3 and autophagy 5utophagy and cancer 6eagents 9ell culture 9mmunoblotting 10mmunocytochemistry 11tatistical analyses 11esults 131P induces autophagy in dosage- and time-dependent manners in PC-3 cells 131P induces relocalization of EGFP-LC3 in stably transfected PC-3 cells in a concentration-dependent manner 141P-induced autophagy is mediated by Clostridium difficile toxin B-, SC-560-, NS-398-, and PD98059-sensitive pathways 15iscussion 18eferences 22igures 31igure 1 31igure 2 33igure 3 36igure 4 38igure 5 40igure 6 4