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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/34639

Title: AMPK 涉及調控 Ceramide 誘導膀胱癌細胞死亡
AMPK is Essential Kinase for Ceramide-Induced Cell Death in Bladder Cancer
Authors: Yi-An Chen
陳奕安
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 腺苷磷酸活化激酶;神經醯胺;膀胱癌
AMPK;Ceramide;Bladder cancer
Date: 2012
Issue Date: 2013-10-07T02:44:42Z
Abstract: 脂質代謝已經被認為不只維持細胞膜功能並還有訊息傳遞來控制細胞活性。Ceramide 是一種疏水性神經鞘脂,是由神經鞘氨醇與長或短脂肪酸組成。Ceramide 具有生物活性可以調控細胞死亡以及細胞週期停滯。細胞內 ceramide 其碳鍊長介於 16 到 26 ,取決於不同 ceramide synthase 異構酶。許多短鍊 ceramide 類似物如 C2-ceramide、C6-ceramide、C8-cermiade 因為對於膜穿透性好,也常被當作研究 ceramide 特性的材料。 實驗結果發現,利用 C2-ceramide 成功誘發老鼠膀胱癌 MBT2 細胞株、人類HEK293T 細胞株死亡。為了鑑別 ceramide 在 MBT2 細胞株模式中是以 apoptosis 或 necrosis 機制造成細胞死亡,以 Rhodamine 123 染劑和流式細胞儀檢測其粒線體膜電位變化,結果發現 ceramide 會造成粒線體膜電位喪失。為了釐清粒線體膜電位喪失是否為 Bcl family 或 ROS 所造成粒線體功能損失。以 RT-PCR 檢測其 RNA 表現,發現 ceramide 會提昇 pro-apoptotic Bcl family Bad ,減少 anti-apoptotic Bcl family Bcl-xL、Bik mRNA 表現。並且在 H2DCFDA 染劑和流式細胞儀實驗結果發現 ROS 的在粒線體膜電位喪失後才產生。這些證據支持 ceramide 調控 Bcl family 使粒線體膜電位喪失。儘管如此,實驗結果發現,經 caspase inhibitor 前處理後再處裡 ceramide 的細胞發現 caspase inhibitor 並不能減少 ceramide 誘發細胞死亡。結果顯示 ceramide 在 MBT2 細胞株模式中致死機制是 caspase-independent 機制。根據最近的研究指出 ceramide 處理細胞之後,會引發細胞飢餓。主要是因為 ceramide 使細胞膜表現營養受器減少,並減少 autophagy 數量。造成細胞產生嚴重代謝壓力。因此,我們推測 ceramide 造成細胞原因可能跟能量代謝有關,並以 AMPK 作為觀測對象。AMPK 是一種 serine-theronine kinase,在細胞代謝功能為能量感測,並維持細胞內能量恆定性。實驗結果發現,AMPK 抑制劑 compound C 可以成功抑制 ceramide 誘發細胞死亡。根據實驗結果推測,直接活化 AMPK 應該也能使細胞死亡。實驗以 AMPK 促進劑 AICAR 處理加藥 ceramide 細胞。但結果發現,未加入 ceramide 而直接以 AMPK 促進劑 AICAR 活化 AMPK 並無法殺死細胞。為了探討為何抑制 AMPK 可以抑制 ceramide 誘發細胞死亡,以 Acridine Orange 檢測 autophagy,發現處理 compound C 會產生大量 autophagy。由實驗結果推測,autophagy 可能是compound C 抑制 ceramide 誘發細胞死亡的原因。實驗結果發現, autophagy 抑制劑 Hydroxychloroquine 可以抑制 compound C的藥效使 ceramide 誘發細胞死亡。這個發現支持這項推測。 總結來說,ceramide 在 MBT2 細胞株模式也可以誘導細胞死亡。透過調控 Bcl family 導致粒線體功能損傷釋放粒線體內物質,造成 caspase-independent apoptosis。並且在此模式中,AMPK 是 ceramide 造成細胞死亡中不可或缺的必要條件,但非充分條件。且經由實驗結果推測 autophagy 可能是抑制 ceramide 誘發細胞死亡的潛在因子。
Lipid metabolism has been recognized for participation in membrane functions and signaling that control cellular activities. Ceramides are one of sphingoil lipid, hydrophobic molecule; composited by sphingosine with long or short chain fatty acid, and it has bioative to promote cell death and growth arrest. Cellular ceramides, long N-acyl chains ranging from 16 to 26 carbons in length depending on the different ceramide synthase isoform. Moreover, the short chain ceramide analogs: N-acetyl-sphingosine (C2-ceramide); N-hexanoylsphingosine (C6-ceramide) and N-octanoylsphingosine (C8-ceramide) are also used due to more membrane permeable. Our results show that C2-ceramide induces cell death in MBT2 cell line, mouse bladder cancer cells, and HET293T cell line, transformed human cells. To identify whether ceramide-induced cell death is apoptosis or necrosis in MBT2 cells, the membrane potential was measured by flowcytometry with Rhodamine 123 staining. This result shows that ceramide induce mitochondrial dysfunction. To recognize the role of mitochondrial dysfunction, RNA expressions level was measured by RT-PCR. The results show that the Bad, pro-apoptotic Bcl family, mRNA level increase and anti-apoptotic Bcl family such as Bcl-xL and Bik mRNA level decrease after treatment of ceramide. Moreover, ROS was detected after mitochondrial dysfunction measured by flowcytometry with H2DCFDA staining. These results prove that ceramide-induced mitochondrial dysfunction regulated by Bcl family; however, our result shows that the caspase inhibitor can't inhibit ceramide-induced cell death. According the results, we suggest that the mechanism of ceramide-induced cell death is caspase-independent manner in MBT2 cell lines. Although the roles of ceramide-related Bcl family regulation are still not fully understand, recent studies show that ceramide can induce cellular starvation because ceramide decrease cellular nutrient receptor and autophagy causing severe metabolic stress. According these studies, we suggest that ceramide-induced cell death may be related by energy metabolism which correlates AMPK activity. AMPK is serine-theronine kinase, thought as energy sensor, and keep homeostasis. Our data shows that compound C, AMPK inhibitor, can inhibit ceramide-induced cell death. Although we suggest activate AMPK without ceramide may also induced cell death, the data shows that AICAR, AMPK activator, activate AMPK without ceramide by can't induce cell death. To investigate the mechanism that compound C inhibit ceramide-induced cell death, we detected autophagy in cells by Acridine Orange staining. The result shows that compound C induce autophagy predominately which may explain the effect of compound C which inhibit ceramide-induced cell death. Moreover, Hydroxychloroquine, autophagy inhibitor, can mask the effect of compound C causing ceramide-induced cell death which result supports our suggestion. In conclusion, our study shows that ceramide can also induce cell death in MBT2 cell line by regulating Bcl family causing mitochondrial dysfunction in caspase-independent apoptosis manner. Our findings show that AMPK activity is essential but not enough for induce ceramide-induced cell death. While autophagy rescues ceramide-induced cell death since AMPK activity is inhibited by compound C.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0019936010
http://ntour.ntou.edu.tw/handle/987654321/34639
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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