| Abstract: | 近二十年來全世界的癌症死亡率正快速上升，其中以肺癌的死亡率增加最快。儘管現今醫療持續進步，標靶治療藥物陸續上市，但肺癌病患之預後依舊不佳，因此急需新的藥物及治療方針來改善肺癌的治療效果。於過去的研究當中，誘發癌細胞產生細胞凋亡一直是開發抗癌藥物的首要方向，但是癌細胞能透過啟動許多訊息路徑來逃避細胞凋亡機制的啟動。細胞自體吞噬是一種非凋亡型細胞死亡，近幾年已成為治療癌症的熱門研究方向。靈芝免疫調節蛋白已知具有多方面的抗癌功效。由本實驗室之前之研究發現，松杉靈芝免疫調節蛋白reFIP-gts 能抑制A549 肺癌細胞中端粒?的表現，分別透過抑制 C-myc 結合至hTERT 啟動子上而減少hTERT 的表現量，以及改變端粒?於細胞中的座落位置。reFIP-gts 能刺激內質網壓力產生，而內質網壓力已知是促進細胞自噬的因子之一。此外，reFIP-gts 誘發的未成熟衰老過程中，可見酸性囊狀胞器大量增加，而酸性囊狀胞器增加也是細胞自噬的指標之一。基於上述理由，本計畫的研究目標是觀察靈芝免疫調節蛋白reFIP-gts能否誘發肺癌細胞產生細胞自噬並抑制癌細胞生長。第一年計畫，首先以各種細胞自噬分析法證實reFIP-gts能於肺癌細胞中誘發細胞自噬，包含(1)以 Real-time PCR及西方點墨法分析細胞自噬基因表現，如LC3 的轉變，(2)以acridine orange分析酸性囊狀胞器表現及(3)使用GFP-LC3 觀察細胞自噬小體形成。並使用細胞自噬專一抑制劑3-methyladenine 及bafilomycin-A1 印證與分析reFIP-gts 誘發的細胞自噬及類型。進一步使用各種化學抑制劑(Akt：LY294002；mTOR：Rapamycin；PKC： Go6976，Rottlerin；ERK：PD98059)及VZV-G pseudotyped lentivirus-shRNA 系統來釐清細胞自噬的活化路徑。已知於細胞中，細胞自噬對細胞存活具有的保護及抑制的相反能力。因此，第二年的重點是分析細胞自噬現象在reFIP-gts的抑癌功能中扮演的角色。為了定義細胞自噬的影響，我們以VZV-G pseudotyped lentivirus-shRNA系統抑制肺癌細胞中Beclin-1、ATG5 與LC3的表現，建立自噬能力缺陷之細胞株來進行實驗。並且，我們欲進一步釐清細胞自噬與細胞週期停滯、細胞老化、端粒?活性抑制之間的關係。使用流式細胞儀分析reFIP-gts 透過細胞自噬抑制細胞週期進行。使用 Senescence-Associated β-Galactosidase 與C12FDG 染色分析reFIP-gts 透過細胞自噬促使細胞走向未成熟衰老。為觀察reFIP-gts 透過自噬型蛋白降解抑制端粒?，我們使用西方點墨法與免疫沉澱法分析端粒?組成分子hTERT 是否有泛素化現象及是否與p62 及 LC3有交互作用來證實。在第三年，我們將以口服餵食方式給予reFIP-gts，分析reFIP-gts 於生物體中的抑癌能力。並且觀察reFIP-gts搭配cisplatin共同處理下抑制腫瘤的效果。此外，我們將偵測reFIP-gts 是否能於腫瘤中誘發細胞自噬及抑制端粒?活性。本研究提供靈芝免疫調節蛋白抗癌的新機制，證實reFIP-gts 有潛力成為對抗肺癌的化學預防藥劑，並提供多方面的生物指標，希望reFIP-gts 能進一步開發成為標靶治療藥物，提供未來個人化醫療之應用。
Lung cancer is one of the most common malignancies in the world. It is a leading cause of cancer-related death in men and women in many countries. Despite advanced therapy, involving target therapy, lung cancer patients still face poor prognosis. New drugs and strategies are needed to improve the clinical responses and outcomes of lung cancer patients. Over the past several years, apoptosis induction has been the major consideration in anti-cancer drug development. However, cancer cells trigger multiple pathways to escape from apoptosis. More recently, autophagy, one form of nonapoptotic cell death, has been extensively studied as a cancer therapy. Immunomodulatory protein, one major bioactive component of Ganoderma, has anti-tumor activity. In our previous studies, FIP-gts inhibited lung cancer A549 cell proliferation and suppressed telomerase activity by regulating the hTERT expression and localization. FIP-gts stimulates endoplasmic reticulum (ER) stress which is a well-documented autophagy inducer. FIP-gts induced acidic vesicular organelle (AVO) formation serves as a senescence marker and AVO development has been investigated in autophagic cells. Based on the previous studies, the aim of this study is to investigate that reFIP-gts inhibits lung cancer cell proliferation through activating autophagy. In first year, we prove that reFIP-gts induces autophagy in lung cancer cells by several autophagy assays, involving (1) autophagy-related gene (ATG) expression by real-time PCR and western blot, (2) acidic vesicular organelles (AVOs) development and (3) GFP-LC3 autophagosome formation. Autophagy specific inhibitors, 3-methyladenine and bafilomycin-A1, will be used to confirm and analyze the type of autophagy. We use chemical inhibitors (Akt : LY294002; mTOR : Rapamycin; PKC : Go6976, Rottlerin; ERK : PD98059) and VZV-G pseudotyped lentivirus-shRNA system to verify the autophagy activating pathway. Opposing effects of autophagy on cell survival and death have been documented. Therefore, the second year proposal aims to study the role of autophagy in GMI-mediated lung cancer cell proliferation inhibition. We establish the autophagy-defective lung cancer cells by VZV-G pseudotyped lentivirus-shRNA system for Beclin-1, ATG5 and LC3 silencing. Furthermore, we clarify the relationship between autophagy and cell cycle arrest, senescence, telomerase activity inhibition. The effects of reFIP-gts induced autophagy on cell cycle arrest were analyzed by flow cytometry. The effects of reFIP-gts induced autophagy on immature senescence were investigated by senescence-Associated β-Galactosidase and C12FDG staining. The ubiquitinized hTERT and the interaction between hTERT and p62 or LC3 will analyzed with western blot and immunoprecipitation. In the third year study, we will establish animal model to investigate the anti-cancer ability of reFIP-gts by oral administration in vivo. The effects of reFIP-gts and cisplatin co-treatment will also be observed. Moreover, we detect that reFIP-gts induces autophagy and telomerase activity inhibition in xenograft tumor. This study may prove that reFIP-gts may be a potential chemopreventive agent against lung cancer. We hope that reFIP-gts can be developed as a target therapy drug for personal therapy. |
