腎上腺素對H460肺癌細胞之AMPK路徑的影響

Abstract

運動有益於癌症的效果已被許多研究證實，但其中的機轉卻一直沒有被建立。AMPK (AMP-activated protein kinase) 為能量恆定的重要調節者，由於其抑制腫瘤生長的效果已應用於抗癌藥物的發展，因此運動活化AMPK的作用被認為是運動有益於癌症的機轉。在本論文中，我們先建立葡萄糖與血清的刺激和AMPK活性反應之間的關係，結果發現AMPK活性的反應會出現完全不同的變化。因此我們進一步使用運動時負責調節能量代謝作用的荷爾蒙代表運動的刺激，從細胞模式建立荷爾蒙、AMPK與肺癌細胞之間的關係，進而了解運動透過AMPK影響癌症的可能性。本論文的目的為探討荷爾蒙在細胞模式中對於H460肺癌細胞內AMPK活性與細胞生長情況的影響。研究的方法以腎上腺素、生長激素、胰島素和昇糖素作為刺激原，將H460肺癌細胞培養於含10 % 血清的DMEM培養基，以固定濃度的荷爾蒙作用3、6、12、24與48小時後，進行各時間點的細胞存活分析，並測量細胞內AMPK的蛋白質表現量與活性。結果顯示荷爾蒙的作用與細胞生長情況之間沒有時間依存的關係，腎上腺素在第3小時、生長激素在第24小時皆可顯著增加約7 %的細胞 (p< .05），胰島素的作用在第3、12、24與48小時顯著增加約7 ~ 11 % 的細胞 (p < .05），而昇糖素分別在第3與12小時增加約8 % 與5 % 的細胞 (p < .05）。此外，荷爾蒙的作用與AMPK活性的變化之間也沒有時間上的依存關係，但荷爾蒙皆可在第3小時抑制AMPK活性約26 ~ 50 %，在第48小時則引起AMPK活性增加約57 ~ 148 %。上述的結果證明腎上腺素的作用在第3小時會顯著增加H460肺癌細胞的生長，同時抑制細胞內AMPK的活性，推測高濃度腎上腺素在3小時內的作用可能會因抑制AMPK活性而造成腫瘤生長。For improving the cancer disease, exercise has been widely accepted with its benefit, but the mechanism of action between exercise and cancer has not been elucidated yet. AMPK (AMP-activated protein kinase) is an important regulator in energy homeostasis. According to the published literatures, activated AMPK suppresses the growth of tumor, which is potential to develop as a drug target for developing anticancer drug. Therefore, an increased AMPK activity by exercise is speculated as key role between exercise and cancer. In this thesis, we first established the AMPK responses with different stimulation of glucose and serum. Results appeared that there is different patent on AMPK activity following serum (containing with growth factors) stimulation compared with glucose stimulation. Furthermore, we used different hormones, which are generated during exercise, as stimulator in cell culture to establish the relationship among hormone, AMPK and lung cancer cell, and then investigate whether exercise influences cancer development through alteration of AMPK activity. The purpose in this thesis is to investigate the effects of hormone on AMPK activity and cell growth in H460 lung cancer cells. H460 lung cancer cells were incubated with DMEM medium including 10 % serum, and then epinephrine, growth hormone, insulin and glucagon were added to H460 cell culture, respectively, for analyzing the cell viability, AMPK activity and AMPK protein amount at 3, 6, 12, 24, 48 h. We found that there was no time-dependant relationship between the effect of hormone and cell growth. Epinephrine and growth hormone significantly increased cell viability about 7 % at 3 h and 24 h, respectively (p< .05). Insulin significantly increased cell viability from 7 to 11 % at 3, 12, 24 and 48 h (p < .05). Glucagon significantly increased cell viability 8 % and 5% at 3 and 12 h separately (p < .05). Furthermore, there was no time-dependant relationship between the effect of hormone and AMPK activity. We found all hormones suppress AMPK activity from 26 to 50 % at 3 h, but enhance AMPK activity from 57 to 148 % at 48 h. In conclusion, epinephrine significantly increases cell viability, which was coincident with a decreased AMPK activity. These data suggest that high level of epinephrine could cause the tumor growth mediated by a decreased AMPK activity within 3 hours.