臺灣博碩士論文加值系統

泌尿道上皮癌好發於60歲以上男性，早期發現一般是以手術治療為主，晚期發生則以Cisplatin為主的化學治療有著極高的感受性，然而病人整體存活僅有8到14個月。即使二線用藥paclitaxel，雖然也有50%以上的治療效果，但是病人的整體存活仍是相當低的。本實驗室之前的研究顯示，松杉靈芝免疫調節蛋白FIP-gts能抑制腫瘤生長及端粒酶活性。合併處理FIP-gts和bafilomycin A1可透過抑制細胞自噬造成泌尿道上皮癌細胞株NTUB1和鉑金抗藥性泌尿道上皮癌細胞株N/P的細胞凋亡。本實驗目的為探討FIP-gts結合溶酶小體抑制劑chloroquine，對於NTUB1及N/P細胞株之毒殺作用與機轉。研究結果顯示，使用CCK-8和西方點墨法分析，FIP-gts結合chloroquine會透過自噬小體大量堆積而有效抑制NTUB1與N/P細胞存活。使用流式細胞儀分析，FIP-gts與chloroquine共同處理會造成NTUB1與N/P細胞Sub G1累積，Annexin V/PI染色可得較多雙色螢光訊號與粒線體膜電位低下等現象。以西方墨點法與CCK8發現FIP-gts與chloroquine並不會活化caspase，caspase抑制劑z-VAD-fmk也不會回復FIP-gts結合chloroquine造成的細胞毒殺作用，顯示FIP-gts與chloroquine可能是透過caspase-independent細胞死亡來毒殺N/P細胞。以VSV-G pseudo-typed lentivirus抑制LC3(shLC3)表現建立細胞自噬缺陷細胞，發現此shLC3自噬缺陷細胞株可有效抑制FIP-gts與chloroquine導致之細胞死亡與Sub G1累積。使用流式細胞儀與H2DCFDA分析，發現FIP-gts與chloroquine並不產生ROS。但是使用ROS抑制劑N-acetyl-cysteine(NAC)可回復FIP-gts和chloroquine的細胞毒性和自噬小體堆積，卻無法挽救粒線體膜電位低下。進一步使用程序性壞死(Necroptosis)抑制劑Necrostatin-1證實FIP-gts結合chloroquine並不會造成程序性壞死，所以我們初步認為FIP-gts結合chloroquine是藉由大量的細胞自噬小體堆積而造成的caspase-independent細胞死亡。

Urothelial carcinoma (UC) is the most common cancer in Taiwan men. The average age at the time of diagnosis is over 60. Surgery of transurethral resection using cytoscope is considered to be an option only when the disease is in its early stages. Cisplatin-based chemotherapy is highly effective for metastatic urothelial carcinoma. However, the progression free survival is short and the median overall survival ranges from only 8 to 14 months in metastatic UC patients. Effective salvage therapy in patients in whom cisplatin based regimens fail was lacking until the introduction of new active cytotoxic agents, such as paclitaxel, in recent years. Although the treatment have approximately 50% response, the median overall survival is poor. In our previous study, FIP-gts, a fungal immunomodulatory protein, was cloned from Ganoderma tsugae and purified. FIP-gts possesses antitumor activity against solid tumors and inhibits telomerase activity. In additional, FIP-gts and Bafilomycin A1 combined treatment was found to lead to enhancement of apoptosis along with inhibition of autophagy in parental, NTUB1 and cisplatin-resistant, N/P cells. The purpose of this study was to examine the effects of FIP-gts and chloroquine co-treatment on inhibiting urothelial cancer cell viability. Using CCK-8 assay and Western blot, chloroquine increased FIP-gts-induced cytotoxicity and autophagosome accumulation in parental and cisplatin-resistant urothelial cancer cell lines. On flow cytometry, chloroquine enhanced FIP-gts-mediated sub-G1 accumulation, annexin V positive signal and mitochondrial membrane potential loss. Combination treatment with FIP-gts and chloroquine did not elicit the cleavage of caspase-3/7. Furthermore, z-VAD-fmk, a pan-caspase inhibitor, did not reverse the FIP-gts and chloroquine-suppressed cell viability. This suggest that FIP-gts and chloroquine induced caspase-independent cell death. The capabilities of FIP-gts and chloroquine to induce cytotoxicity and sub-G1 phase accumulation were abolished in autophagy-defective cells (LC3 shRNA).
Using H2DCFDA staining and flow cytometry, FIP-gts and chloroquine did not induce ROS production. N-acetyl cysteine, a ROS scavenger, inhibited the cytotoxicity and LC3-II accumulation in FIP-gts and chloroquine-treated N/P cells. Necrostatin-1, a necroptosis inhibitor, did not reverse the FIP-gts and chloroquine-suppressed cell viability.
We conclude that Chloroquine enhanced FIP-gts-induced autophagy dependent caspase-independent cell death via abundant autophagosome accumulation.

第一章 緒論(Introduction) 1
第一節 泌尿道上皮癌的分類與治療方針 1
第二節 依賴型劊蛋白(caspase-dependent)與非依賴型
劊蛋白(caspase-independent)之細胞死亡 4
第三節 細胞自體吞噬(autophagy) 5
第四節 靈芝免疫調節蛋白之分類與抗癌研究 9
第五節 氯奎寧(chloroquine)之分子機制與臨床應用 11 第六節 酮康唑(ketoconazole) 之分子機制與臨床應用 12
第二章 縮寫表(Abbreviation) 15
第三章 研究動機(Motivation) 17
第四章 實驗材料與方法(Material and methods) 18
第一節 藥品與試劑 18
第二節 實驗儀器設備 21
第三節 抗體資料 22
第四節 實驗方法 24
一、細胞培養(Cell culture) 24
二、細胞存活試驗 26
三、西方點墨法(Western blotting) 28
四、質體抽取與轉染作用 31
五、RNA干擾(RNA interference；RNAi) 33
六、活性氧物種(reactive oxygen species；ROS)之測定 35
七、粒線體膜電位(mitochondrial membrane potential；
MMP)之測定 36
八、單染色Propidium iodide測定Sub-G1與細胞週期 37
九、雙染色Annexin V/Propidium iodide測定細胞凋亡 38
第五章 結果(Results) 39
一、靈芝免疫調節蛋白FIP-gts和chloroquine抑制泌尿道上皮癌
細胞存活 39
二、FIP-gts和chloroquine共同作用促使泌尿道上皮癌細
胞產生細胞自體吞噬現象並造成自噬小體大量堆積 39
三、FIP-gts和chloroquine共同作用促進Sub-G1以及
Annexin V (+)/PI(+)訊號產生 40
四、FIP-gts和chloroquine共同作用促使粒線體膜電位下降 42
五、FIP-gts和chloroquine共同作用對細胞週期調控蛋白的影響 43
六、FIP-gts和chloroquine共同作用導致capase-independent細
胞死亡 43
七、使用caspases抑制劑並不會抑制FIP-gts和chloroquine導致
的細胞死亡 45
八、FIP-gts和chloroquine共同作用並不會誘發ROS產生 45
九、使用ROS抑制劑(NAC)並不會抑制FIP-gts和chloroquine
促使粒線體膜電位下降之情形 46
十、使用ROS抑制劑(NAC)會降低FIP-gts和chloroquine的細
胞毒殺作用 46
十一、使用Necroptosis抑制劑並不會抑制FIP-gts和chloroquine
導致的細胞死亡 47
十二、過度表現Survivin並不會抑制FIP-gts和chloroquine導致
的細胞死亡 48
十三、以VSV-G pseudotyped lentivirus-shRNA系統降低LC3表
現量大幅減緩FIP-gts和chloroquine的細胞毒殺能力 49
十四、以VSV-G pseudotyped lentivirus-shRNA系統降低LC3表
現量明顯減緩FIP-gts和chloroquine促進的Sub-G1訊號產生 49
十五、FIP-gts對裸鼠腫瘤形成的影響 50
十六、FIP-gts增加泌尿道上皮癌對ketoconazole的感受性 51
十七、FIP-gts與ketoconazole共同作用對於細胞週期之影響 51
十八、FIP-gts和ketoconazole共同作用無法活化Caspase3/7導致
細胞凋亡 52
十九、FIP-gts誘發細胞自噬加強ketoconazole造成的細胞死亡 52
二十、ROS抑制劑及shLC3部分降低FIP-gts和chloroquine的細
胞敏感性 53
第六章 討論(Discussion) 54
第七章 圖表(Tables and Figures) 62
第八章 參考文獻(References) 97

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