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標題: 自噬作用對非小細胞肺癌抗藥性之調控
Autophagy-mediated drug resistance of non-small cell Lung cancer
作者: 盧奕瑄
Yi-Hsuan Lu
關鍵字: 自噬作用
Drug resistance
Graptopetalum paraguayense
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摘要: 非小細胞肺癌在化療時常衍生出多重抗藥性,造成臨床治療上之困境,因此極需克服。Rosiglitazone (ROSI)是一種PPAR-γ活化劑,被應用於糖尿病及癌症的治療上,Cisplatin則是目前應用於癌症化療上的第一線用藥。本實驗室近期的研究發現非小細胞肺癌細胞(H460)可透過不同作用機轉產生對抗抑癌藥物ROSI與Cisplatin的高抗藥性。自噬作用是細胞利用溶酶體將有缺失或過多之蛋白或胞器進行降解的一種過程,是細胞自我的保護機制之一,亦被認為是癌細胞產生抗藥性的原因之一。雖然自噬作用可提升癌細胞之抗藥性已在多種癌細胞中被報導,但其對H460細胞多重抗藥性的調控尚未被探討過。因此本論文主要在探討具有細胞保護功能之'自噬作用'是否參調控H460細胞對ROSI及Cisplatin的抗藥性,及其在肺癌治療上之應用。研究的子題目的包括:釐清ROSI與Cisplatin是否會透過提升非小細胞肺癌細胞(H460)之自噬作用來保護細胞之存活並提高細胞之抗藥性;確認ROSI是否會透過活化PPAR-γ啟動PKCδ/JNK-1/BcL-2/Beclin-1訊號路徑來提升自噬作用;並評估具有抑癌功能之石蓮花水萃物(WGP)是否可透過抑制自噬作用的生成來破壞H460細胞對ROSI及Cisplatin之抗藥性;另外,更利用Rapamycin(自噬作用促進劑)加劇ROSI對H460細胞自噬作用的提升,進一步評估過度自噬作用之生成是否可誘發細胞凋亡之發生。實驗中使用的分析方法主要包括,MTT分析法分析細胞存活率,以西方墨點法分析蛋白表現,以Annexin V/PI染色與DAPI染色分析細胞凋亡之發生,以DiOC6染色分析粒線體膜通透性之變化,以及以EMSA分析PPAR-γ之活性。研究結果顯示,ROSI能透過活化PPAR-γ/JNK-1/BcL-2/Beclin-1訊號路徑進而提升自噬作用之生成來促進H460細胞之生長並導致抗藥性的生成。Cisplatin亦能透過提升自噬作用來抑制H460細胞之凋亡,進而產生抗藥性。而自噬作用之抑制劑(3-MA)、PPAR-γ之抑制劑(GW9662)與WGP,均能透過抑制自噬作用來破壞H460細胞之抗藥性,具有成為抗癌藥物之功效。此外,若以Rapamycin加劇ROSI所提升之自噬作用,的確能誘導H460細胞邁向凋亡,破壞細胞對ROSI之抗藥性。整體而言,本論文之重要性在於首次證實自噬作用可以促進H460細胞多重抗藥性的生成;GW9662及WGP可有效抑制被ROSI活化的PPAR-γ/JNK-1/BcL-2/Beclin-1訊號路徑,是兩種新的自噬作用抑制劑;除了抑制自噬作用(以3-MA、GW9662、WGP)外,過度提升自噬作用(以Rapamycin)亦可破壞H460細胞對ROSI之抗藥性,在抗癌藥物之選擇上須特別注意;另外,由於WGP是一副作用低的飲品,又有抑制自噬作用破壞癌細胞抗藥性的功能,未來可考慮做為肺癌化療時的佐劑,用以降低高副作用化療藥物之劑量。
Non-small cell lung cancer (NSCLC) often develops multiple drug resistance during chemotherapy, which has become the biggest obstacle for clinical treatment of lung cancer. Rosiglitazone (ROSI) is a PPAR-γ agonist and has been used in diabetes and cancer treatment. Cisplatin is the first line chemotherapeutic drug for cancer. Our laboratory has recently found that NSCLC cells (H460) have developed higher drug resistance to both ROSI and Cisplatin, via different working mechanisms. Autophagy is a self-eating process to degrade defect or excessive proteins or organelles to protect cell from extreme environmental changes. It has been indicated to involve in the drug resistance of many cancer cells. The role of autophagy in regulating of multidrug resistance of H460 cells was still unclear. The primary goal of the study was to know if autophagy contributes to the drug resistances of H460 cells to ROSI and cisplatin, and its application in lung cancer therapy. The specific aims of the study included: to clarify if ROSI and cisplatin act through autophagy to promote the growth (or survival) and drug resistance of H460 cells; to confirm if ROSI act through PPAR-γ to initiate the PKCδ/JNK-1/BcL-2/Beclin-1 signaling pathway to stimulate autophagy; to know if water extract of Graptopetalum paraguayense leaves (WGP) inhibits autophagy to break drug resistance of H460 cells to ROSI and cisplatin; and to clarify if rapamycin (autophagy activator) aggravates ROSI-induced autophagy to trigger the apoptosis of H460 cells. The techniques applied in the study included, the MTT assay, Western blotting, Annexin V/PI staining, DAPI staining, DiOC6 staining and EMSA. The results showed that ROSI activated the PPAR-γ/JNK-1/BcL-2/Beclin-1 signaling pathway to stimulate autophagy, which in turn promoted the growth and drug resistance of H460 cells. Cisplatin also stimulated autophagy to inhibit apoptosis of H460 cells and lead to drug resistance. Autophagy inhibitor (3-MA), PPAR-γ inhibitor (GW9662) and WGP, all inhibited autophagy to break drug resistance of H460 cells and revealed a potential to become anticancer drugs. Rapamycin-aggravated autophagy in ROSI-treated H460 cells can indeed trigger apoptosis and break drug resistance to ROSI. In overall, the major contributions of this study were to demonstrate for first time that autophagy played a critical role in the regulation of multiple drug resistance of H460 cells; GW9662 and WGP can block ROSI-activated PPAR-γ/JNK-1/BcL-2/Beclin-1 signaling pathway to server as new autophagy inhibitors; autophagy inhibition (by 3-MA, GW9662 or WGP) and excessive induction of autophagy (by rapamycin) can both break drug resistance of H460 cells to ROSI, a critical concern for anticancer drug selection; furthermore, since WGP is safe to drink and can block the anticancer drug-induced autophagy to break drug resistance of H460 cells, it may be considered to be used as an adjuvant during chemotherapy to lower down dose of the anticancer drug applied and adverse side effects.
文章公開時間: 10000-01-01
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