YC-1抑制A549人類肺腺癌細胞在缺氧環境下所誘導的組織因子表現

YC-1 inhibits hypoxia-induced tissue factor expression in A549 human lung adenocarcinoma epithelial cells

謝侃言(Kan-Yen Hsieh)

指導教授 ： 吳志中

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2012年)

摘要

缺氧是引起腫瘤細胞轉移的重要因素之一，在缺氧的環境之下，會引起組織因子 (Tissue factor ，TF) 的表現量增加。TF是一種穿膜蛋白 (transmembrane protein)，經常在人類的腫瘤細胞上過度表現，並且在腫瘤引起的凝血以及血小板凝集上扮演重要的角色。在之前的研究指出TF會參與腫瘤轉移、生長和血管新生，因此TF似乎可以做為癌症治療的合理目標。
YC-1 是soluble guanylate cyclase (sGC) 的活化劑，先前的研究顯示YC-1可以增加血小板中的cGMP，並抑制血小板的活化。另外有文獻指出YC-1具有抑制hypoxia-inducing factor (HIF) 的作用。 在本篇研究當中我們發現了YC-1可以減少缺氧所引發的TF表現量，並抑制癌細胞所引起的pro-coagulant的活性。YC-1能夠引起A549細胞內vasodilator-stimulated phosphoprotein (VASP)在ser 239的磷酸化，這確認了YC-1能夠引起cGMP/ protein kinase G (PKG)路徑的活化。但是，在加入非選擇性phosphodiesterase (PDE) 抑制劑IBMX或者sGC抑制劑ODQ去調節後，發現TF的表現量並沒有因此改變。我們使用了另一個sGC活化劑BAY412272做為對照，發現BAY412272也能夠抑制缺氧引起的TF表現，並且在合併IBMX以及ODQ後也能影響BAY412272的效果。這證實了YC-1抑制缺氧引起的TF表現主要是經由cGMP不相關的路徑。
我們觀察到YC-1可以抑制缺氧環境下NF-кB進入細胞核的量，並抑制缺氧引起的NF-кB以及IкB的磷酸化。由於使用NF-кB抑制劑RO 106-9920也可以抑制缺氧引起的TF表現量，因此，可以推論YC-1抑制缺氧引起的TF表現乃經由防止NF-кB活化而來。

關鍵字

組織因子 ； YC-1 ； 缺氧 ； A549

並列摘要

Hypoxia is an important factor to induce cancer metastasis. Under the hypoxic environment, the expression of tissue factor (TF) in cancer cells can be increased that leads to a series of action associated with cancer metastasis. TF, a kind of transmembrane protein, is often overexpressed in human cancer cells and plays an important role in cancer induced blood coagulation, platelet aggregation. In previous studies, TF has been reported to participate in tumor growth and angiogenesis, thus it seems to be a reasonable target for cancer therapy.
YC-1, a soluble guanylate cyclase (sGC) activator, can increase intracellular cGMP levels, and inhibits platelet activation. In addition, YC-1 can also act as a hypoxia-inducing factor (HIF) inhibitor. We found that YC-1 reduced the hypoxia-induced TF expression in human lung cancer A549 cells, and inhibited the pro-coagulant activity of cancer cells. YC-1 treatment induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation at ser 239, indicating the activation of cGMP/protein kinase G (PKG) pathway. However, pretreatment of the phosphodiesterase inhibitor IBMX or the sGC inhibitor ODQ did not affect the effect of YC-1 on hypoxia-induced TF expression. Another sGC activator BAY412272 also inhibited hypoxia-induced TF expression and, the effect was potentiated and reversed by IBMX and ODQ, respectively. These results suggest that YC-1 inhibits hypoxia-induced TF expression in A549 cells through a cGMP-independent manner.
On the other hand, YC-1 reduced hypoxia-induced translocation of NF-кB to the nucleus and inhibited the phosphorylation of NF-кB and IкB. Treatment of A549 cells with the NF-кB inhibitor RO-106 9920 led to inhibition of hypoxia-induced TF expression. Therefore, these results suggest that YC-1 inhibits hypoxia-induced TF expression in A549 cells through preventing NF-кB activation.

並列關鍵字

tissue factor ； YC-1 ； hypoxia ； A549

參考文獻

參考文獻

Brasier AR (2006). The NF-κB regulatory network. Cardiovasc Toxicol 6: 111-30.

Blum S, Issbruker K, Willuweit A, Hehlgans S, Lucerna M, Mechtcheriakova D, Walsh K, von der Ahe D, Hofer E, Clauss M (2001). An inhibitory role of the phosphatidylinositol 3-kinase signaling pathway in vascular endothelial growth factor induced tissue factor expression. J Biol Chem 276:33428-34.

Camerer E, Gjernes E, Wiiger M, Pringle S, Prydz H (2000). Binding of factor VIIa to tissue factor on keratinocytes induces gene expression. J Biol Chem 275:6580-5.

Chen C, Zhou H, Wei F, Jiang L, Liu X, Liu Z, Ma Q (2011). Increased levels of hypoxia-inducible factor-1alpha are associated with Bcl-xL expression, tumor apoptosis, and clinical outcome in chondrosarcoma.