尼古丁調控人類肺臟癌細胞（A549）細胞週期相關蛋白表現之分子機制研究

香菸和肺癌有著密不可分的關係，在香菸的成分中對生理影響最大的就是尼古丁。在本篇論文中我們在探討尼古丁調控細胞週期相關基因表現。首先使用吸菸者在血液中的常存在的濃度0.1-10mM，觀察到在10分鐘時ERK1/ERK2發現到磷酸化形式ERK1/2有明顯增加。在kinase assay中也觀察到ERK1/2的活性明顯上升，由於先前的文獻指出，ERK可以活化NF-kB，於是用Electrophoretic mobility shift assay (EMSA)來觀察轉位後的蛋白是否具有結合kB DNA結合位置的能力，結果顯示，在30分鐘時，NF-kB出現最大量的結合活性。利用transfections技術，證明尼古丁擁有活化NF-kB調控基因的能力。接著我們觀察長時間的細胞週期蛋白表現，發現cyclin D1及cyclin D3隨著時間而增加，p27先上升後下降，再分別利用PD98059及PDTC證明調控細胞週期的路徑是有經過MEK→ERK→NF-kB。最後我們用20mM天然的多酚類EGCG來抑制尼古丁的活性，觀察到尼古丁的效應被緩解。由以上的發現我們第一次發現到尼古丁的對細胞週期的調控有包含NF-kB的路徑，且此效應會被天然的抗氧化劑所緩解。

關鍵字

肺癌；尼古丁；細胞週期；多酚類；抗氧化劑

並列摘要

Cigarette has been demonstrated to be a strong association with lung cancer. Of the many components of tobacco, Nicotine has extensive effects in physiological roles. In this study, we investigated the molecular mechanisms of Nicotine on the cell cycle-regulatory protein expression in human pulmonary epithelial cells (A549). The concentrations of nicotine in an individual who smokes, on an average, ranges from 0.1 to 10 mM. First , 1mM of Nicotine was able to phosphorylate ERK1/2 at 10 mins after Nicotine treatment. Second, we found that Nicotine increased translocation of the nuclear factor kB subunits, p50/p60, and enhanced DNA kB site binding activity by western blot and electrophoresis mobility supershift assay. Transactivation of NF-kB were determined by transfection of a luciferase reporter construct containing several kB binding sites. The MEK1 inhibitor, PD98059, was able to inhibit kB binding activity induced by Nicotine. The expression of cell cycle regulatory protein, cyclin D1 and D3 was time-dependent manner after nicotine addition. The cyclin dependent kinase inhibitor protein, p27 increased in first 3 hrs and then decreased on Nicotine treated cells. The NF-kB inhibitor, PDTC, was able to inhibit the Nicotine-regulated the expressions of cell cycle proteins. These results suggested that Nicotine regulated cyclin D1, cyclin D3 and p27 throught NF-kB cascade pathway. Finally, the Nicotine effects was suppressed by the natural antioxidant compound, epigallocaechin gallate (EGCG). The present study first demonstrated that Nicotine-regulated cell cycle regulatory protein expression involve MEK→ERK→NF-kB pathway in human pulmonary epithelial cells (A549) and the effect were suppressed by natural antioxidant compound, epigallocaechin gallate(EGCG).

並列關鍵字

Cigarette ； lung cancer ； Nicotine ； ERK1/2 ； cell cycle

參考文獻

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尼古丁調控人類肺臟癌細胞（A549）細胞週期相關蛋白表現之分子機制研究

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