| Abstract: | 研究背景:口腔癌為台灣男性癌症發生率及死亡率的第四位。90%的口腔癌為鱗狀細胞癌，其特性為容易復發及局部淋巴結轉移轉移之特性。近年來研究發現癌症幹細胞(cancer stem cells, CSCs)」，是造成口腔癌患者對治療產生抗藥性、腫瘤容易復發及癌症轉移的重要關鍵。為此，標靶癌幹細胞特性，在口腔癌輔助治療方面，或許是一個很好的方向。本轉譯計畫利用奈米藥物結合光動力療法，期待透過調降癌幹細胞特性，應用於口腔癌治療。 研究目標: 第一年目標: 5-胺基酮戊酸 (ALA)奈米化載體合成製備。包含: (1)介孔洞二氧化矽奈米微粒（Mesoporous silica nanoparticles，MSNPs）製備; (2) 異硫氰基螢光素（Fluorescein isothiocyanate，FITC）標的MSNPs製備; (3)製備介孔洞二氧化矽5-胺基酮戊酸奈米微粒(MSNPs-ALA)。(4)MSNPs-ALA對口腔正常 細胞毒性評估;(5)利用共軛焦顯微鏡（評估MSNPs–ALA對口腔細胞釋放效率。 第二年目標: MSNPs–ALA光動力治療對於活體外及活體內癌症幹細胞特性影響。(1)利用MTT assay評估MSNPs-ALA光動力治療對於口腔癌幹細胞增生是否具備抑制力; (2)MSNPs-ALA光動力治療對於口腔癌幹細胞標記ALDH1, CD133, CD44, Oct4, Nanog表現量之影響;(3)利用幹細胞球體試驗評估MSNPs-ALA光動力治療對於口腔癌幹細胞自我更新能力之影響;(4) MSNPs-ALA光動力治療對於 口腔癌幹細胞in vitro腫瘤生成特性(如爬行、侵襲)之影響;(5)MSNPs–ALA光動 力治療對於口腔癌幹細胞細胞凋亡能力之影響;(6) 探討MSNPs-ALA光動力治療 搭配傳統化療藥物(如cisplatin or Fluorouracil)協同作用於口腔癌幹細胞，評估MSNPs-ALA光動力治療是否能提昇口腔癌幹細胞之化療敏感性及降低抗藥性標記;(7) 建立口腔癌幹細胞腫瘤生成及癌轉移老鼠模式;(8)MSNPs-ALA光動力治 療搭配傳統化學療法評估裸鼠腫瘤大小、轉移能力、及存活時間;(9)利用免疫組 織染色法評估MSNPs-ALA光動力治療於老鼠之腫瘤後，其癌幹細胞及抗藥性標記改變量。第三年目標: MSNPs–ALA光動力治療於口腔癌幹細胞之分子機轉探討。包含 : (1) 研究MSNPs-ALA光動力治療於癌幹細胞調控之微型 RNA(microRNAs); (2)利用訊息傳遞路徑的抑制劑及或利用及系統生物資訊學方式來了解在MSNPs-ALA光動力治療所造成癌幹細胞特性下降及提昇化療敏感性之主因及調控機轉; (3)利用基因降解(knockdown)以及過度表現(overexpression)方式探討MSNPs-ALA光動力治療調控路徑專一性; (4)以Luciferase assay測試 MSNPs–ALA光動力治療治療協合傳統化療藥物調控下游之基因活性，並以EMSA及 Immunoprecipitation 方式探討其直接調控標的基因。 重要性: 本研究強調基礎研究與臨床應用接軌的重要性，結合奈米藥物與光動力療法標靶癌幹細胞，期待有助於口腔癌的治療。
Background Oral cancer ranks the fourth leading malignancy and cancer death in male population in Taiwan. Metastasis to lymph nodes and distant sites is the major cause of mortality in patients with oral cancer. Most patients relapse within months after current therapeutic treatments. A subpopulation of cells called cancer stem cells (CSCs) possessing stemness properties was shown to enrich after therapy, resulting in the relapse and metastasis of tumors. Therefore, targeting CSCs appears to be a promising therapeutic modality for oral cancer. Based on the previous studies, the purpose of the translational study is to investigate that nanoparticle-mediated photodynamic therapy (PDT) enhances chemosensitization of oral cancer through eliminating CSCs property. Experimental method and specific aims In first year, we will prepare 5-aminolevulinic acid-incorporated mesoporous silica nanoparticles, involving (1) Synthesis of mesoporous silica nanoparticles (MSNPs); (2) Synthesis of fluorescent mesoporous silica nanoparticles （FITC-MSNPs）; (3) Synthesis of 5-aminolevulinic acid-incorporated mesoporous silica nanoparticles (MSNPs–ALA); (4) Evaluation of on MSNPs–ALA toxicity of normal oral cells by MTT assay; (5) Assessment of release efficiency of MSNPs-ALA in OC-CSCs by confocal microscope. In the second year study, we will evaluate the role of MSNPs–ALA in the modulation of the CSCs properties and markers of oral cancer-derived cancer stem cells (OC-CSCs) in vitro and in vivo, involving (1) Evaluation of MSNPs-ALA on proliferation rate of OC-CSCs; (2) Examination of MSNPs-ALA on targeting CSCs markers including ALDH1 activity, CD133, CD44, Oct4, and Nanog expression in OC-CSCs; (3) Assessment of MSNPs-ALA on self-renewal property of OC-CSCs; (4) Determination of MSNPs-ALA on in vitro tumorigenic properties in OC-CSCs; (5) Evaluation of MSNPs-ALA on apoptotic ability of OC-CSCs; (6) Examination of MSNPs-ALA on chemosensitization of OC-CSCs; (7) Establish of the OC-CSCs-derived tumorigenic and metastatic animal models; (8) Validation of anti-tumor and survival rate in nude mice with xenografts of OC-CSCs treated with MSNPs-ALA alone or combined with chemotherapy in developed severe tumors; (9) Evaluation of MSNPs-ALA in vivo anti-CSCs ability with ex vivo biopsies and immunohistochemistry of CSCs and drud-resistant markers in xenografts-derived tumor sections by MSNPs-ALA treatment combined with chemotherapy. In the third year study, we will clarify the functional involvement and molecular mechanisms of MSNPs-ALA-mediated CSCs properties, involving (1) Exploration of the specifically expressed microRNAs levels in MSNPs-ALA-treated OC-CSCs by microRNAs microarray and real-time RT-PCR analysis (2) Analysis of the detailed molecular mechanisms by which MSNPs-ALA mediating the cancer stemness and chemosensitization by chemical inhibitor and systemic bioinformatics. (3) Determination of MSNPs-ALA-regulated specific downstream pathways by lentivial-mediated overexpression or knockdown methodology. (4) Further examination of MSNPs-ALA-regulated genes and proteins interaction directly by luciferase activity, EMSA, and Immunoprecipitation. Significance In three years of the integration of basic and clinical translational research study, we expect to prove that MSNPs-ALA may be a potential chemo-adjuvant therapy against oral cancer and will be developed as a target therapy drug for personal therapy. |
