摘要:細胞代謝是維持細胞生長極重要且必需的複雜經過,有許多酵素與營養素皆參與其中。癌細胞是一種生長快速的細胞,癌組織的細胞代謝旺盛,尤其是蛋白質合成大大增強,甚至奪取正常組織的蛋白質分解產物以合成癌組織所需要的蛋白質。在這過程中,麩醯胺酸(glutamine),一個體內最豐富的氨基酸,是癌細胞成長與代謝所需最重要的物質之一。已有許多研究發現阻斷麩醯胺酸的運輸可以降低癌細胞的生長和擴張。細胞中麩醯胺酸是合成許多抗氧化酵素的原料,而細胞內抗氧化酵素的合成是靠轉錄因子NRF2(nuclear factor E2 related factor 2)來調節,NRF2除了增進細胞抗氧化酵素的合成,是細胞抗氧化能力的決定者外,近年來許多研究也發現NRF2能夠促進癌細胞增生與惡化,更成為決定癌細胞抗藥性的重要指標。綜合以上所述,NRF2表現量高的癌細胞中,會產生較多量的抗氧化酵素,使細胞消耗更多的麩醯胺酸,由此我們推測細胞內NRF2的表現量應與細胞對於麩醯胺酸的需求量有關聯。因此我們針對在全球死亡率最高的兩種癌症,乳癌和肺癌,探討NRF2高表現細胞內麩醯胺酸代謝的變化,並且運用阻斷麩醯胺酸代謝的藥物,尋求對抗高抗藥性(高NRF2表現)癌細胞之治療方法。另外,我們更針對NRF2所引致的抗氧化酵素大量表達尋找標靶治療的藥物,期望可以降低乳癌和肺癌癌腫復發與轉移的機率,得到更全面的治療效果。在未來三年的研究計畫中,我們的研究重點如下列:第一年計畫目標:乳癌細胞中,NRF2表現量與麩醯胺酸缺乏(glutamine deprivation)之關係:(1)探討在乳癌細胞中,NRF2的表現量與細胞的抗氧化壓力能力有無直接相關,並且了解NRF2與細胞內多種抗氧化壓力酵素表現量的關聯,最後利用藥物或基因轉殖方式,增加或是減少NRF2的表現來研究NRF2與細胞外麩醯胺酸供給量之關係。(2)利用不同的siRNA干擾NRF2所控制的下游基因表達,尋找在NRF2控制的抗氧化酵素中,那一個酵素是細胞對於外界麩醯胺酸缺乏反應中最重要的控制因子。(3)在NRF2高表達的乳癌細胞中,能否藉由麩醯胺酸缺乏毒殺細胞,達到對抗癌症的治療。第二年計畫目標:在肺癌中,NRF2與肺癌細胞對於麩醯胺酸缺乏的敏感度之關聯性:(1)利用肺癌細胞microarray的資料尋找NRF2及其相關基因與肺癌細胞抗藥性的關聯。(2) 並且檢驗高表達NRF2肺癌細胞與麩醯胺酸缺乏敏感度之關係。(3) 最後檢測抑制麩醯胺酸代謝路徑是否能夠毒殺NRF2高表達之肺癌細胞或增加肺癌細胞對於化療藥物的敏感性。第三年計畫目標:尋找對於高抗藥性乳癌或肺癌更好的治療藥物並利用動物實驗驗證體外細胞實驗中的結果:(1)高表現量的NRF2,會開啟下游抗氧化酵素的製作,包括有GCLC、GCLM、NQO1和GSH等,提高癌細胞對抗外來刺激的能力,使細胞產生抗藥性。因此我們希望利用針對這些抗氧化酵素產生毒性的藥物來治療高抗藥性乳癌或肺癌,例如可透過NQO1對細胞產生毒殺的β-lapachone。利用麩醯胺酸代謝路徑的抑制劑與β-lapachone共同處理細胞,期望能以這樣的複合療法更全面的治療高抗藥性之乳癌及肺癌細胞。(2) 最後利用動物實驗來驗證所有在體外細胞實驗上看到的結果,並且以麩醯胺酸代謝路徑的抑制劑對動物體內之NRF2高表達的乳癌和肺癌做治療,期望可將阻斷麩醯胺酸代謝應用於實際的癌症治療。
Abstract: Cell metabolism is an important and complicated process maintaining cell growth. Cancercells not only grow faster, but also have greater protein synthesis ability than normal cells, and they take most nutrients from normal tissue to maintain their rapid metabolism.Glutamine, the most abundant amino acid in human body, is one of the most important materials for tumor cell growth. Many researchers have found that blocking the transportation of glutamine into cancer cells can decrease the tumor growth and expansion.Glutamine is also an essential component for making anti-oxidant enzyme production, including GCLC, GCLM, NQO1, and GSH. The expressions of those enzymes are regulated by a transcription factor, nuclear factor E2 related factor 2 (NRF2). NRF2 expression leads to the production of cellular anti-oxidant enzymes and contributes to the antioxidant ability of cancer cells, NRF2 is also found to increase the growth and metastasis of cancer cells, as well as an important maker for drug resistance of tumor. Combining all the clues above, higher NRF2 expression tumors produce more anti-oxidant enzymes, decrease the harmful effect of drugs, and lead the cells become high drug resistant tumor. Therefore, we hypothesize that the expression level of NRF2 is associated with glutamine requirement in tumor cells.In this project, we focus on the breast and lung tumor, the most lethal tumors throughout the world. Our goal is to profile the glutamine metabolism change in high NRF2 expression tumor cells, and to find a more efficient cancer therapeutic strategy through inhibiting the glutamine metabolism in tumors with a higher NRF2 level. Moreover, we aim to discover the therapeutics targeting the anti-oxidant enzymes that are regulated by NRF2. With this method, we will able to kill those high drug-resistant tumors more efficiently without tumor recovery and metastasis.In the next three years, our goal is to find out the correlation between NRF2 and glutamine deprivation response in tumor cells, the aims for each year are listed below:First year: The relationship between NRF2 expression and the sensitivity to glutamine deprivation in breast tumor.(1) To investigate the relationship between NRF2 expression level and ①the production ofanti-oxidant enzymes by NRF2; ②anti-oxidant ability; and ③glutamine deprivation response by manipulating the NRF2 expression.(2) To find out the key components of glutamine deprivation response amoung thoseNRF2-regulated anti-oxidant enzymes with siRNA technique.(3) To investigate whether glutamine deprivation could be used as an anti-cancer therapy against tumor cells with high NRF2 expression.Second year: The linkage between NRF2 level and glutamine deprivation sensitivity in lung tumor.(1) To find the relationship between NRF2-related genes and the drug-resistance in lung cancer cells with microarray data analysis.(2) To investigate the correlation between NRF2 expression level and glutamine deprivation sensitivity.(3) To exam whether the toxic effect of glutamine metabolism pathway blockage in tumor cells can facilitate the anti-tumor effect of some drugs used in clinical chemotherapy.Third year: Finding new drugs for targeting high NRF2-expression cancer and using an animal model to verify the hypothesis that higher NRF2 expressing cells are more sensitive to glutamine deprivation.(1) High level of NRF2 facilitates the production of anti-oxidant enzymes, including GCLC, GCLM, NQO1 and GSH, to maintain the intracellular oxidative stress balance, which leads cancer become more drug-resistant. We want to find more efficient drugs to kill those cancer cells with high drug-resistant by targeting those anti-oxidant enzymes, such as using β-lapachone to kill those cells targeting with high NQO1 level. We also want to combine drugs targeting to glutamine metabolism pathway and β-lapachone to get betterchemotherapy effect in drug-resistant breast and lung cancers.To use animal model to verify the anti-tumor effect of glutamine metabolism inhibitors or combine therapy (glutamine metabolism inhibitors+β-lapachone) on drug-resistant breast and lung tumors.