臺灣博碩士論文加值系統

癌症是近年來國人的頭號健康殺手，尤其肺癌一直是癌症死亡率的第一名；肺癌分為小細胞肺癌與非小細胞肺癌，非小細胞肺癌其中的肺腺癌特別盛行於不吸菸的亞洲女性，因為其容易復發與轉移之特性而難以治癒，成為威脅國人健康的隱形殺手。以往研究多從天然植物藥草中萃取及開發具潛力之藥物用於癌症之化學治療，較少把研究焦點放在海洋生物上。Sinulariolide (SNL)係為從養殖型珊瑚Sinularia flexibilis 所萃取之化合物，文獻研究指出SNL已可有效抑制膀胱癌及皮膚癌細胞之增生。基於拓展其抗癌應用及改善抗癌藥物用量問題，本論文之目的與動機即針對目前越來越多的非小細胞肺腺癌來著手，以SNL為標的抗癌藥物試驗與開發對其治療之應用。吾人以高壓電場系統製備天然高分子/ SNL奈米聚合物，期能藉由此親水性之奈米聚合物改善SNL難溶於水、生物利用性低之缺點，提升SNL之臨床療效及降低使用劑量之實際問題。奈米微粒具有優異的表面積/體積比、擴散性佳等優點，形式上的應用也較無限制，因此被廣泛應用於各生醫領域。研究結果顯示能成功利用天然高分子包覆SNL成奈米微粒聚合物，其包覆率可約達90%，所製備的透明質酸包覆SNL奈米微粒聚合物粒徑約為33~ 77 nm；細胞活性結果測試得知SNL藥物的IC50濃度為75μg/mL。為了得知此奈米聚合物對肺腺癌細胞的毒殺效果與作用機轉，經由細胞培養結果顯示透明質酸所包覆SNL藥物濃度為25μg/mL之奈米微粒聚合物能於24小時內有效殺死A549 肺腺癌細胞，並達顯著上的差異。流式細胞儀結果：PI, JC-1及 Annexin-V結果顯示，透明質酸包覆SNL可降低所使用的SNL藥物劑量，且隨著SNL藥物濃度增加其細胞凋亡數目越多。西方墨點法之實驗結果發現透明質酸包覆SNL奈米微粒之細胞毒殺作用係與細胞凋亡之傳導路徑以及粒線體路徑相關。在以裸鼠動物作為in vivo研究實驗對象探討透明質酸包覆SNL奈米微粒是否可治療由A549 肺腺癌細胞所誘導的腫瘤，經由切除腫瘤組織之RT-PCR分析顯示透明質酸包覆SNL（濃度25 μg/mL及50 μg/mL）奈米微粒可降低腫瘤組織內細胞之survivin表現，同時相較於單獨使用SNL產生更明顯之細胞壞死的現象；初步結果顯示透明質酸包覆SNL奈米微粒不僅治療結果較佳，也可減少SNL藥物劑量。但在IHC實驗中並無明顯的毒殺作用產生。經由探討in vitro細胞實驗結果中，SNL於細胞週期前期顯示較明顯的A549細胞生長抑制，在毒殺的作用上則較為不明顯。且SNL藥物的IC50濃度高達75 μg/mL，顯示其對A549細胞毒性較低。SNL在針對A549細胞的直接毒殺及以A549細胞所引起的腫瘤的治療效果上並不明顯,但其在抑制A549細胞增生及腫瘤組織的生長有其一定的作用與效果。或許SNL藥物在肺癌上可作為控制腫瘤生長之輔助藥劑。當然，本研究還需更多後續研究以驗證此推論，最終目的是希望本論文的研究成果可朝肺癌治療臨床應用上來發展。

Lung cancer is one of the most clinically challenging malignant diseases worldwide. Lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Among NSCLC, adenocarcinoma is prevalent, especially in Asia non-smoking women. Sinulariolide (SNL), extracted from the farmed coral species Sinularia flexibilis, has been used for suppressing malignant cells. For developing anticancer therapeutic agents, we aimed to find an alternative for lung adenocarcinoma treatment by using SNL as the target drug. We investigated the SNL bioactivity on A549 lung cancer cells by conjugating SNL with hyaluronan nanoparticles to form HA/SNL nano-aggregates by using a high-voltage electrostatic field system. SNL was toxic on A549 cells with an IC50 of 75 µg/mL. The anticancer effects of HA/SNL nano-aggregates were assessed through cell viability assay, apoptosis assays, cell cycle analyses, and Western blotting. The size of HA/SNL nano-aggregates was approximately 33-77 nm in diameter with a thin continuous layer after aggregating numerous HA nanoparticles. Flow cytometric analysis revealed that the HA/SNL nano-aggregate-induced apoptosis was more effective at a lower SNL dose of 25 µg/mL than pure SNL. Western blotting indicated that caspases-3, -8, and -9 and Bcl-xL and Bax played crucial roles in the apoptotic signal transduction pathway. All the in vitro data suggested that HA/SNL nano-aggregates exerted stronger anticancer effects on A549 cells than did pure SNL via mitochondria-related pathways. In animal models, we examined the anti-cancer effects of HA/SNL aggregates on tumors induced by A549 cells in nude mice. The expression of survivin mRNA in HA/SNL nano-aggregate groups was significantly lower compared to that in pure SNL groups according to the data from RT-PCR. The results of H&E stain also revealed that the tumor tissues of nude mice treated with HA/SNL nano-aggregates had larger area percentage of tissue necrosis. The initial findings indicated that HA/SNL nano-aggregates can lead to better treatment efficacies with lower SNL doses. However, there were no obvious cytotoxicities from IHC experiments. According to the analyses that SNL can induce cell cycle arrest in G2/M phase of A549 cells in vitro and its IC50 was as high as 75 μg/mL, we could infer that SNL was not highly cytotoxic on A549 cells. Therefore, SNL can evidently inhibit both the proliferation of A549 cells and the growth of A549 tumors, but there was no direct cytotoxic effects observed in vivo. SNL may be used as an adjuvant anti-cancer therapeutic agent for lung adenocarcinoma. Further studies are need to access whether SNL and HA/SNL nano-aggregate are beneficial in treating lung cancer clinically.

中文摘要 …………………………………………………………………………… ii
Abstract……………………………………………………………………………… iv
List of Contents…………………………………………………………………… vi
List of Figures …………………………………………………………………… vii
List of Tables ……………………………………………………………………… viii
Introduction………………………………………………………… 1
1.1 Current challenge on lung cancer treatment 1
1.2 Sinulariolide and nanoparticles ………… 3
1.3 Specific aim of this proposal ………………………………… 5
Methods and Materials…………………………………………………………… 7
2.1 Production of hyaluronan/sinulariolide aggregates ……… 7
2.2 SNL incorporation efficiency and in vitro release study…………… 7
2.3 Cell culture and HA/SNL nano-aggregates treatment…… 8
2.4 Flow cytometric analysis of apoptosis……………… 9
2.5 Cell cycle analysis ……………………………………………………… 10
2.6 Western blotting analysis……………………………………………… 10
2.7 Wound healing assay…………………………………………………… 11
2.8 Animal studies………………………………………………………… 11
2.9 Real-time quantitative reverse transcription polymerase chain reaction …………… 12
2.10 Statistical analysis ………………………………………… 12
Results ……………………………………………………………………………… 13
3.1 Characteristics of HA/SNL nano-aggregates … 13
3.2 Hyaluronan nanoparticle cytotoxicity assay … 14
3.3 IC50 of SNL and in vitro cell viability ………… 14
3.4 Live/dead assay of A549 cells ………………………………… 14
3.5 Antimigratory effect of SNL and HA/SNL nano-aggregates on A549 cells ………………………………………………………… 15
3.6 Apoptosis assay through flow cytometry ………… 15
3.7 Cell cycle analysis ……………………………………………………… 16
3.8 Western blot analysis …………………………………………………… 16
3.9 Tumor cell necrosis and apoptosis in animal model ……… 18
Conclusions ………………………………………………………………………… 23
4.1 Issue needs to be addressed ……………………………………… 24
4.2 Future work …………………………………………………………… 27
References ………………………………………………………………………… 29

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