2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648829摘要：惡性黑色素細胞癌是最具侵襲性的皮膚癌，轉移性黑色素細胞癌大多數對細胞凋亡有拮抗性，對化學療法之效果也因而不彰。我們之前的研究顯示，ARMS在黑色素細胞癌有過量表現，ARMS 可藉由MEK/ERK 路徑，抑制紫外線B 光照射誘發之細胞凋亡。減少ARMS 在黑色素細胞癌細胞的表現，則會促進紫外線誘發之細胞凋亡。我們尚未發表的研究結果顯示，減少ARMS 的表現可顯著增加過氧化氫引起的自噬性(autophagic)細胞死亡，自噬性細胞死亡為第二型細胞計劃死亡，由溶小體(lysosome)執行細胞內容物及胞器的分解。本研究之目的在於藉由活體外及活體內實驗，釐清減少ARMS 的表現，是否可藉由細胞凋亡或自噬性細胞死亡的機轉，增加黑色素細胞癌對化學治療之敏感性。我們在此研究設計中提出三個方向。首先，我們會使用對照組和以RNA 干擾法建立ARMS 表現減少的小鼠黑色素細胞癌B16F10 穩定細胞株。以各種轉移性黑色素細胞癌常用的化學治療藥物處理，並輔以藥物劑量及處理時間之調整，以群落形成測試法(Colony-forming assay)及存活測試法，比較實驗組及對照組細胞對特定化療藥物的敏感性差異。為了於活體內實驗探討減少ARMS 的表現是否可增加黑色素細胞癌對化學療法之敏感性，我們利用B57BL/6 小鼠，以尾部靜脈注射實驗組或對照組黑色素細胞癌細胞的肺轉移動物模式，給予其接受化學治療，在療程完成後，比較兩者間肺腫瘤數量之差異及病理變化。接著，為了了解ARMS 在調控化療藥物誘發之細胞死亡的分子機轉，我們會運用數種探討細胞凋亡及自噬的方法，並分析其訊息傳遞路徑。最後，為了發展以降低ARMS 表現為導向的黑色素細胞癌治療策略，我們將藉由經腹腔或腫瘤內注射siRNA/polyethylenimine(PEI)複合物，建立活體內siRNA 全身性投予系統。在表現ARMS 的B16BL6 黑色素細胞癌自發性轉移小鼠模式中，投予化療藥物、ARMS-siRNA/PEI 複合物、或化療藥物加上ARMS-siRNA/PEI 複合物，觀察小鼠皮下及肺臟腫瘤的變化。此研究可提供惡性黑色素細胞癌一種針對降低ARMS 表現為基礎的新穎治療。<br> Abstract: Melanoma is the most aggressive form of skin cancer. Metastatic melanomas arelargely resistant to apoptosis and consequently to most chemotherapy. Our previouswork has shown that ARMS is overexpressed in malignant melanoma and can conferapoptotic resistance to UVB-induced apoptotic cell death through activatingMEK/ERK pathway. Depletion of ARMS in melanoma cells can promote apoptoticdeath in response to UVB. Our unpublished data also showed that depletion of ARMSsignificantly increased H2O2-induced autophagic cell death; a type II programmed celldeath involving lysosomal degradation of cellular contents and organelles. The goal ofthis study is to investigate whether downregulation of ARMS expression may enhancechemosensitivity of melanoma, either through apoptosis or autophagic cell death, byin vitro and in vivo studies. We propose three directions for the experimental study.First, we will exploit control and RNA interference-mediated ARMS-knockdownB16F10 melanoma stable cell lines and compare for their susceptibility to variouschemotherapeutic agents frequently used for metastatic melanoma, with time and doseadjustment. Colony forming assays and survival assays will be used. To evaluate theeffect of ARMS knockdown on enhancing chemotherapy-mediated cell death in vivo,experimental melanoma lung metastases with or without ARMS expression inC57BL/6 mice will be used. These mice will receive the indicated chemotherapy andtheir tumors in the lungs will be compared and send for histopathologic examination.Second, to decipher the molecular mechanism that ARMS involves in the regulationof chemotherapy-mediated cell death, various apoptotic or autophagic assays will beemployed. The signal transduction pathways related to cell death will be analyzed.Finally, to develop a therapeutic ARMS-silencing strategy for malignant melanoma,we will establish an in vivo systemic siRNA delivery system through intraperitonealor intratumoral injection of siRNA/polyethylenimine (PEI) complexes. In a melanomaspontaneous metastases mouse model using B16BL6 melanoma cells with ARMSexpression, the systemic chemotherapy alone, or ARMS-siRNA/PEI complexes alone,or the combination of both will be administrated. The subcutaneous melanoma andthe metastatic pulmonary tumors will be examined. This study will offer an avenuefor the development of a novel ARMS knockdown-based therapeutical application formalignant melanomaARMS細胞凋亡自噬化學治療黑色素細胞癌轉移活體內 siRNA 投予ARMSapoptosisautophagychemotherapymelanomametastasisin vivo siRNA delivery