- 學位論文
靈芝免疫調節蛋白與氯奎寧經由細胞自體吞噬和非依賴型劊蛋白路徑誘發鉑金抗藥性泌尿道上皮癌細胞死亡之機制
Combination of FIP-gts and Chloroquine Induces Cell Death via Autophagy and Caspase-independent Pathway in Cisplatin-resistant Urothelial Cancer Cells
摘要
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。
並列摘要
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.
參考文獻
延伸閱讀
- 王文君（2011）。探討新型組織蛋白去乙醯酶抑制劑PBHA降低內毒素脂多醣體誘發人類單核球細胞基質蛋白酵素活化之作用機轉〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-1707201111120300
- 朱建華、葉舒平、吳慧中、邱啟銘（2015）。天生靈芝萃取液可透過NF-kB路徑抑制細菌脂多醣誘發小鼠RAW264.7免疫細胞株的一氧化氮生成。MC-Transaction on Biotechnology，7(1)，1-11。https://www.airitilibrary.com/Article/Detail?DocID=22255184-201503-201504230002-201504230002-1-11
- Kuan, C. C. (2006). 類固醇皂苷元diosgenin促進卵白蛋白免疫小鼠表現抗原專一性IgG2a抗體和伽馬干擾素 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2006.02826
- 陳美晨（2015）。Molecular Mechanism of Combining Cisplatin and Aspirin on Human Urothelial Cell Carcinoma and Cisplatin-Resistant Sublines〔碩士論文，高雄醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0011-2708201511043200
- Yang, D., Ji, H. F., Zhang, X. M., Yue, H., Lin, L., Ma, Y. Y., Huang, X. N., Fu, J., & Wang, W. Z. (2014). Protective Effect of Cytosolic Phospholipase A2 Inhibition against Inflammation and Degeneration by Promoting Regulatory T Cells in Rats with Experimental Autoimmune Encephalomyelitis. Mediators of Inflammation, (2014-9), 67-73. https://doi.org/10.1155/2014/890139