茶鹼衍生物抑制人類支氣管上皮癌化細胞株增生及生長之藥理研究

本研究先前所合成的黃嘌呤 (xanthine) 衍生物---KMUP-1，已被證實具備刺激內皮與平滑肌系統中一氧化氮合成酶 (NOS)/溶解型鳥糞嘌呤環化酶 (sGC)/環化鳥糞嘌呤單磷酸 (cGMP) 路徑的作用； KMUP-1也能在離體的天竺鼠氣管實驗中，引起部分上皮相依性的平滑肌鬆弛效果，所以本實驗欲證明KMUP-1是否也具活化上皮細胞內的eNOS作用，而且在低氧下是否參與調控eNOS之活化、血管之增生，及對抗腫瘤生長的作用。本研究以西方點墨法 (Western blot) 證實KMUP-1在低濃度時 (10-8M) 即可促使人類肺支氣管上皮細胞株NCI-H441的eNOS表現增加，這項作用於18小時達最高峰並隨濃度的提高而增加，且蛋白質的表現會因前處理eNOS抑制劑L-NAME (100μM)而降低。而KMUP-1對於癌細胞生長情形及癌化因子的影響，則利用流式細胞儀分析來觀察細胞週期的變化，另外合併西方點墨法觀看HIF-1α與VEGF的表現。結果發現KMUP-1在較高濃度 (10-6~10-4M) 能使H441細胞週期產生較明顯的抑制作用，其效果與劑量是呈正相關性的 (dose-dependent) ，且必須延長藥物作用的時間達48小時，至72小時為最顯著。此外，已知VEGF基因受到HIF-1之調控，尤其在缺氧的狀態下，因此在正常氧壓下觀察不到被誘發的HIF-1α及VEGF的蛋白表現。於低氧模式下則可發現，在未用藥物處理的細胞會大量減少G0/G1時期的比例，顯著增加S期，而在KMUP-1的作用下，則明顯的抑制內皮細胞DNA的合成，並將細胞週期停滯在G0/G1期。利用西方點墨法偵測到KMUP-1可使與血管新生 (angiogenesis) 有關的蛋白HIF-1α與VEGF之表現比控制組有明顯的減少；而eNOS的表現則不受到誘發增加。總而言之，由上述的實驗結果顯示KMUP-1可於正常生理狀態下 (normoxia) 調控一氧化氮合成酶的表現，卻不會誘導HIF-1α及VEGF的表現；也能造成細胞週期的停滯。而於缺氧情況下能顯著抑制腫瘤細胞的生長週期進行，並有輕微促進細胞凋亡的作用；減少HIF-1α與VEGF的蛋白表現，卻不會造成eNOS的表現增加，有利於抑制腫瘤的增生。

關鍵字

茶鹼衍生物；癌化細胞株；抑制增生及生長；流氏細胞儀；西方點墨法

並列摘要

KMUP-1, a xanthine derivate, has been demonstrated to stimulate the NOS/sGC/cGMP pathway in endothelial and smooth muscle cells. Additionally, it caused partially epithelium-dependent relaxations in isolated guinea-pig trachea previously published in this laboratory. This study was designed to identify whether KMUP-1 has the ability on the expression of endothelial nitric oxide synthase (eNOS) in lung adenocarcinoma epithelial cell H441. Besides, the endothelial-derived NO is able to induce pro-angiogenic and pro-tumor growth. The aim of this study was to investigate the effects of KMUP-1 on cancinogen cell under the hypoxic situation. Western blotting analysis demonstrated that KMUP-1 initiated an increase of the eNOS expression at the concentration of 10-8M. The maximal eNOS expression of KMUP-1 was achieved at 18 hr and an increase of eNOS protein was dose-dependent. The expression of eNOS was attenuated by a NOS inhibitor L-NAME. Using flow cytometry techniques to analyze cell cycles, KMUP-1 produced both time and concentration dependent inhibitions on H441 cell at S phase, arrested the cell cycle at G0/G1 phase. Notably, the effects were dramatically changed after 72 hr exposure to KMUP-1. Hypoxia-inducible factor-1 (HIF-1) is a hypoxia-activated transcription factor that can regulate vascular endothelial growth factor (VEGF) gene, especially under hypoxic condition. Indeed, the increases of protein expression of HIF-1α and VEGF were not observed under normoxia. In the hypoxic model, the data showed that KMUP-1-untreated cells decreased the percentage of G0/G1 phase and increased S phase significantly. After treatment with KMUP-1, the DNA synthesis was reduced and the cell cycle was arrested in G0/G1 phase. From the results of Western blotting showed that KMUP-1 caused a significant decrease of the protein expression of HIF-1α and VEGF, but did not show any change in the eNOS protein. The findings indicated that KMUP-1 increase the expression of eNOS and causes the cell cycle arrested at G0/G1 phase in bronchial epithelial cell, but did not induce the expression of HIF-1α and VEGF in normoxic condition. Under hypoxia, KMUP-1 inhibits the cell cycle, promotes apoptosis, and decreases the expression of HIF-1α and VEGF, but without affecting the expression of eNOS.

並列關鍵字

Xanthine derivate ； tumor cell line ； flow cytometry ； western blotting ； inhibition of proliferation and growth

參考文獻

Blagosklonny MV. (2001) Hypoxia-inducible factor: Achilles’ heel of antiangiogenic cancer therapy. Int J Oncol 132:257-62

Google Scholar

Boussat S, Eddahibi S, Coste A, Fataccioli V, Gouge M, Housset B, Adnot S, Maitre B. (2000) Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 279:L371-L38