2016-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655998摘要：轉移性黑色素細胞癌以高侵犯性著稱，且易對治療產生抗性。大多數的化學療法 是以誘導細胞凋亡的機制造成細胞毒性，但黑色素細胞癌不易產生細胞凋亡現象。 因此，在黑色素細胞癌需要尋求另外的細胞死亡路徑以做為新的治療策略。自栽 的特點為將細胞質内的蛋白質及胞器局限在自弒體中，再由溶腺體做整體分解。 自弒在細胞遇到環境壓力如生長因子缺乏或氧化壓力時會被調控增加。雖然一開 始自弒的增加是細胞為了保護自己避免細胞死亡，但長期過度的自弒會造成細胞 不可逆的傷害最後導致自弒細胞死亡。我們之前的研究發現ARMS(ankyrin repeat-rich membrane spanning)是一個經膜蛋白平台，在黑色素細胞癌有過量表現。 減少ARMS的表現會使自弒體形成增加，並增加過氧化氫造成的非凋亡性細胞 死亡。因黑色素細胞癌的基礎自弒現象比正常黑色素細胞高，因此我們假設減少 ARMS表現合併可促進自弒的小分子藥物治療，會造成黑色素細胞癌的自弒性 細胞死亡，這點可能有潛力做為黑色素細胞癌的新治療策略。本計晝第一年將著 重於探討自弒在ARMS表現減少的黑色素細胞癌細胞遭受氧化壓力時，決定細 胞生死的角色。我們會研究自弒的流量、長半衰期蛋白質的分解，以及在抑制自 弒相關蛋白時對細胞存活的影響，以證實自弒的功能為細胞保護或造成細胞死亡。 在計晝的第二年我們將闡明因減少ARMS表現而增加自弒這條新穎訊息傳遞 路徑的分子機制，會著重於mTORC1的訊息傳導依賴性及type III PI3 kinase 的角色貢獻。為了找尋新穎治療藥物，我們和Baylor醫學院合作由藥物篩選發 現一種T型約通道阻斷劑flunarizine可有效活化自試。我們將進一步釐清 flunarizine在減少ARMS表現的細胞誘發自試現象的分子機制。最後一年之研 究將根據前兩年之研究結果，以黑色素細胞癌動物模式為研究方法，評估減少 ARMS表現合併可促進自弒的小分子藥物的新治療模式。我們將以過去文獻已 證實可在黑色素細胞癌經由活化AMPK引起自試細胞死亡的metformin做為 正控制組，研究flunarizine對腫瘤的治療效果。藉由本計晝的研究完成，我們 將提供一種藉由合併兩種路徑誘發自弒細胞死亡以治療黑色素細胞癌的新方 向。<br> Abstract: Metastatic melanoma is notoriously aggressive and treatment-resistant. Most chemotherapeutic drugs exert their cytotoxic effect by inducing apoptosis, which, however, is often deficient in melanoma cells. Therefore, it is appropriate to develop new therapeutic strategies targeting alternative cell death pathways. Autophagy, characterized by the sequestration of cytoplasmic proteins and organelles within autophagosomes for bulk degradation by lysosomes, is upregulated when cells encounter environmental stresses, including growth factor withdrawal as well as oxidative stress. Although initially acting as a protector for cell death, prolonged and extensive autophagy will lead to irreversible cellular demise and result in autophagic cell death. We have identified that depletion of ARMS (ankyrin repeat-rich membrane spanning), a transmembrane scaffolding protein overexpressed in melanoma, significantly enhanced autophagosome formation and increased hydrogen peroxide (H2〇2)-induced cell death via a non-apoptotic mechanism. We hypothesize that, with the cell context of an autophagy-primed nature in melanoma, combination treatment with ARMS silencing and small molecule autophagy enhancer can induce autophagic cell death, which could be a promising strategy in melanoma therapy. During the first year of the study, we will focus on elucidating the role of autophagy in life or death in ARMS-depleted melanoma cells under oxidative stress; the flux of autophagy, the extent of long-lived protein degradation, and the cell fate after autophagy inhibition will be evaluated. In the second year, we will characterize the signaling mechanisms in this novel autophagy-enhancing pathway resulting from ARSM knockdown. The dependence of mTORC1 signaling and the contribution type III PI3 kinase will be addressed. From a drug library screen collaborated with Baylor College of Medicine, we have identified a T-type calcium blocker named flunarizine that could efficiently activate autophagy. We will further characterize the molecular mechanisms related to autophagy in flunarizine while treating ARMS-depleted melanoma cells. During the final year of this project, novel treatment strategies for melanoma through the combination of ARMS depletion and autophagy-enhancing small molecule treatment, including flunarizine and metformin, will be evaluated in melanoma animal models. Metformin will serve as a positive control in which the effect of autophagic cell death in melanoma through AMP-activated protein kinase (AMPK) activation has been demonstrated. Upon the completion of this proposal, we will provide a new direction for combination treatment of melanoma by enhancing autophagy-mediated cell death through two different routes.ARMS自弒黑色素細胞癌mTOR氧化壓力ARMSautophagymelanomamTORoxidative stress