研究雪松萃取液抗惡性腦瘤生物活性之影響

膠原母細胞瘤(Glioblastoma, GBM)為腦瘤中惡性度最高的惡性腦瘤，其特徵為死亡率高及預後差，並且臨床用於治療GBM的化療藥物普遍效果都不佳，過去文獻證實GBM具有高度血管新生的能力，因而導致腫瘤在顱內增生快速，但又因顱腔為固定空間，因此易壓迫正常腦組織而導致病人死亡。因此積極開發一個較佳的治療藥物成為目前臨床迫切性的需求。本實驗室先前透過一系列篩藥平台，篩選出對GBM有明顯抑制效果之雪松萃取液(Cedrus atlantica, CAt extract)，過去文獻顯示雪松具有抗菌、抑黴、抗氧化及緩解胃潰瘍等作用。雪松醇(Cedrol)為CAt extract的主要活性成分之一，並證實能抑制A549肺癌細胞生長，然而在GBM與動物體內之抑癌作用尚未被探討，因此本實驗目的為研究CAt extract及其活性成分Cedrol對抗GBM之抑癌作用機轉及原位治療效果。結果顯示CAt extract與Cedrol皆能抑制p53正常及缺陷之GBM細胞生長，相較臨床藥物Temozolomide (TMZ)，Cedrol有較佳的GBM細胞毒性，接著證實CAt extract與Cedrol皆能提高細胞內ROS生成並導致DNA損傷，誘發細胞週期停滯於G0/G1期，並增加Sub-G1的細胞百分比進而誘導細胞凋亡。在動物實驗中顯示CAt extract與Cedrol皆能有效抑制皮下與原位腫瘤生長並延長動物的生存時間，同時證實CAt extract與Cedrol在生理及病理分析上並沒有顯著毒性。合併Cedrol與TMZ能協同抑制GBM生長，並且能減少抗藥性蛋白O6-methylguanine methyl transferase（MGMT)、CD133及Multi-drug resistance protein 1 (MDR1)的表現，進而降低GBM細胞產生對TMZ之抗藥性。最後透過生物資訊與細胞實驗證實Cedrol能結合其標靶蛋白Androgen receptor (AR)，降低DHT與AR結合，抑制AR活化進入細胞核並降低其下游基因開啟，進而減少GBM細胞增生。總結上述，CAt extract與Cedrol皆具有良好對抗GBM之作用，希望在未來能開發成用於治療GBM的新一代抗癌藥物，或作為佐劑與臨床藥物搭配使用。

Glioblastoma (GBM) is one of the most malignant in brain tumor, and the patients have low survival rate and poor prognosis. There are almost no effective drugs for GBM clinically. Recent study showed that GBM has a high angiogenesis effect, which can cause tumor to grow rapidly, thereby contricting the normal tissue in cranial cavity and causing death. Therefore, it is important to develop novel drugs for GBM therapy. In our laboratory, through a series of screening platforms, we have identified that the Cedrus atlantica extract (CAt extract) has a significant inhibitory effect in GBM. In tranditional use, CAt is used in inhibition of bacteria, anti-oxidation, and anti-ulcer activities. Cedrol is one of the major components in CAt extract. It has been proven to inhibit the growth of A549 lung cancer cells in the past, but it has not been studied in GBM and there is no evidence related to therapeutic effects in animal experiments. Therefore, the purpose of this study is to explore the anti-GBM mechanism and therapeutic effects in situ of CAt extract and its active component Cedrol. The results show that both CAt extract and Cedrol inhibited the growth of p53-wild and defective GBM cells, and show better cytotoxicity than the clinical drug Temozolomide (TMZ). It was confirmed that CAt extract and Cedrol promote intracellular ROS production, lead DNA damage, induce cell cycle arrest at the G0/G1 phase, increase percentage of Sub-G1 phase and induce cell apoptosis. In animal experiments, both CAt extract and Cedrol effectively inhibit the growth of subcutaneous and orthotopic GBM and prolong the survival time of animals. Besides, it is show no significant toxicity in CAt extract and Cedrol-treated animals by physiological and pathological analysis. Further, the combination of Cedrol and TMZ synergistically inhibit the growth of GBM, and reduce the expression of drug resistance proteins, such as O6-methylguanine methyl transferase (MGMT), CD133 and Multi-drug resistance protein 1 (MDR1), thereby reducing the production of TMZ resistance in GBM cells. Finally, using bioinformatics and cell experiments to confirm that Cedrol binds to its target protein Androgen receptor (AR), reduces the binding of DHT and AR, inhibits the translocation of AR to the nucleus and decreases AR downstream gene expression, resulted in reducing GBM cell proliferation. Therefore, this study showed the CAt extract and Cedrol have great potential to develope into an anti-tumor agent for GBM therapy or be used as adjuvant for combination with clinical drug in the future.