缺氧系統在抗癌藥物開發之應用: Silibinin 在正常氧及缺氧系統下，
對 HIF 蛋白及血管新生具有不同的調控作用
The application of hypoxia system on anticancer drug development:
Different effect of silibinin on HIF protein and angiogenesis in
normoxia and hypoxia system
中文摘要 癌症在現今的社會已成為一項相當普遍的疾病，目前更成為二十六年來國人十大 死因之首。而針對各項癌症全世界莫不競相努力研發更有效的抗癌藥物。但長期 研究顯示出癌症細胞比一般體細胞具有更強大的環境適應能力及耐受性，所誘導 的訊息傳遞系統也與正常細胞有非常多的差異。同時癌症細胞也能將自我的內在 環境改變，促進缺氧誘導因子-1α (Hypoxia-inducible factor – 1α)的產生，並 引發一連串的訊息使得癌細胞週邊的血管新生(angiogenesis)以提供更多的營養 送達。基於這些研究結果再回來探討癌症藥物的開發，發現許多藥物實驗都是在 正常氧分壓(normoxia)環境下並外加生長因子來模擬癌細胞血管新生的情形。以 至許多研究藥物於體外實驗有很好的效果，進入臨床實驗時卻失敗，而造成時間 與金錢上難以估算的損失。 因此本實驗主要在於確立缺氧系統的正確性，以及探討在充氧環境下與缺氧環境 下細胞之間是經由何種訊息傳遞。我們選用人類臍帶靜脈內皮細胞(human umbilical vein endothelial cells)作為實驗對象。在離體細胞實驗中，發現缺氧的細 胞除了缺氧誘導因子-1α蛋白質增加外，同時也誘使 VEGF 蛋白質表現有相同的 趨勢，顯示缺氧會促使生長因子增加來促進細胞生長。而缺氧誘導因子-1α會經 由PI3K/Akt 以及 Ras/ERK 兩個路徑誘導出來，因此在實驗中也發現 Akt 磷酸化 蛋白質會隨時間有逐漸增加的趨勢，而ERK 磷酸化蛋白質則在的趨勢。接著我 們藉由已知之血管新生抑制劑silibinin 來比較癌細胞在正常氧與缺氧系統下的訊 息傳遞，發現在兩種不同氧氣分壓與silibinin 作用下缺氧誘導因子-1α、VEGF、 pAkt、pERK 蛋白質的確有不同程度的表現。顯示以往傳統的離體藥物開發模式 可能必須要重新審視，將癌細胞於缺氧環境的特性也納入考慮，以期更接近癌細 胞的原始表現型態，也可為將來的新藥研發提供一個不同的參考。 英文摘要Cancer has become an universal disease in the nowaday’s society. It occupied heads of top ten causes of death in Taiwan during the past 26 years. Therefore, cancer research and anti-cancer drugs development are competing to effort more effective over the world.
Research has demonstrated cancer cells have a more powerful environment adaptability and tolerance than normal cells since long ago. And the signaling
transduction pathway also have a lot of difference in cancer cells. In addition, cancer cells can change the entraenvironment for itself and promotes the hypoxia inducible factor-1α expression. And initiates series of messages to cause the cell angiogenesis beside on the cancer cells to provide the more nutrition delivery.
Based on these findings to come back to explore the development of cancer drugs, we discovered many anti-cancer drugs developments are conduct the research under the normoxia environment and add the growth factors, ex. VEGF(vascular endothelial growth factor) to demonstrate the condition of cancer cell angiogenesis. Many research medicines have the highly therapeutic effects at in vitro experiment, but fail at in vivo experiment. These results make the loss on the time and money to estimate difficulty.
This experiment is to establish the correctness of the hypoxia system, and figure out the pathways between normoxia and hypoxia. We select the human umbilical vein endothelial cells (HUVECs) to take the experimental subjet. In the in vitro cell test, we find HIF-1α and VEGF protein induction and mRNA expression are increased in HUVECs treated with hypoxia than normoxia. The results Show that hypoxia will lead to increased growth factors to promote cell proliferation. HIF-1α can be produced from the PI3K/Akt pathway and Ras/ERK pathway, and we also find ERK and Akt can phosphorylated and increased dependent with time. The phosphorylation of ERK usual related to cell proliferation response, and the phosphorylation of Akt is known correlated with many cancer diseases. In recent years, studies have also found that iNOS and COX-2 caused by hypoxia in retinal neovascularization are also involved in the role of regulation and control. We use the well known angiogenesis inhibitor silibinin to compare the signal transduction under normoxia and hypoxia in cancer cells, and we find silibinin really has the different performance under two kind of different oxygen densities. The results also showed that for the traditional model of drug development in vitro may have to re-examine, and taking into account the characteristics in the hypoxic environment of cancer cells.
Above the synthesis states and from the experimental result inference , cancer cells not only overexpression HIF-1α in hypoxia, but also promote VEGF expression and phosphorylate pERK and pAkt to progress cells angiogenesis. This hypoxia system is closer to the performance of the original pattern in cancer cells, and can provide a different way to new drugs for the future development.
