Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92212
標題: 香茅醇經由TNF-α路徑促使RIP3活化導致ROS累積進而誘導人類非小細胞肺癌程序性細胞壞死
Citronellol Induces the Necroptosis of Human Lung Cancer Cell via TNF-α pathway Through RIP3 Activation and Reactive Oxygen Species Accumulation
作者: 洪善為
Shan-Wei Hung
關鍵字: 香茅醇;非小細胞肺癌;程序性細胞壞死;活性氧自由基;Citronellol;Necroptosis;Human Lung Cancer Cell;TNF-α;RIP3;ROS
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摘要: 
香茅醇是玫瑰天竺葵的主要成分,而玫瑰天竺葵精油可以誘發人類非小細胞癌細胞壞死,希望能夠利用香茅醇來更有效的抑制肺癌,並找出導致細胞死亡的相關機制。
首先利用香茅醇對於肺癌(A549、H1299、H23)、乳癌(BT-20)及前列腺癌細胞(PC3)細胞毒殺性測試,發現對H1299的毒殺性最是顯著。接著加藥後細胞經PI染劑單染處理,以流式細胞儀檢測顯示H1299有G1期細胞滯留的現象。進一步探討香茅醇誘導H1299細胞株的死亡方式,運用Annexin V及PI雙染發現AnnexinV-/PI+大幅增加,且未偵測到AnnexinV+/PI-的細胞,故香茅醇是藉由細胞壞死方式誘導H1299細胞死亡。由於ROS過表現會導致細胞壞死,利用H2DCF-DA螢光染劑測定加藥後ROS產量,分析結果發現香茅醇誘導H1299內ROS的濃度和時間增加有正相關。
在細胞程序性壞死發生時,ROS會快速累積造成DNA損傷及導致LMP及修補DNA的PARP快速活化最終細胞能量耗盡,溶酶體被破壞並釋放cathepsinB、D,這三種蛋白會隨著時間及濃度的增加而上升。過去研究顯示當TNF-α活化時,下游的RIP3會活化並與RIP1結合,若死亡途徑走向Necroptosis,則Caspase不會被活化。實驗發現加入香茅醇後ROS上升,TNF-α蛋白隨著濃度提高而表現量上升,且RIP3亦是如此。然而Caspase-3卻沒有被活化,驗證香茅醇是經由TNF-α路徑促使RIP3活化導致ROS累積,進而誘導H1299非小細胞肺癌Necroptosis細胞死亡。

Our previous studies showed that the essential oils of Pelargonium capitatum (EOPC) could induce the necroptosis of human non-small lung cancer cells. Citronellol, the major compound in EOPC, has anti-bacterial, anti-oxidant and anti- inflammatory effects. In this research, we would like to prove that citronellol may have noteworthy anticancer activity parallel to that of EOPC.
Citronellol was used to treat lung cancers (A549, H1299, H23), breast cancer (BT-20), prostate cancer (PC3) and breast normal cell (MCF10A) to measure the cytotoxicity of citronellol with MTT assay. Among the six cell lines, human non-small lung cancer H1299 was shown the most susceptibility to citronellol (24/48/72 hr IC50=48 μg/mL, 39 μg/mL, 26 μg/mL). PI staining experiment with flow cytometry were applied to citronellol-treated H1299 cells.We found that citronellol could inhibit the cell growth and arrest the cell cycle at G1 phase. Further, citronellol could induce the necroptosis of H1299 cells by PI/Annexin-V double staining experiment. Due to necroptosis was resulted from ROS accumulation, H2DCF-DA fluorescent staining was applied to detect ROS products in H1299 cells after treated cells with citronellol and found that ROS concentration was accumulated in cancer cellswith time-dependent manner.
The references indicated that when the necroptosis happened, TNF-α would be activated, down-stream RIP3 would complex with RIP1, and then ROS would accumulate quickly in cells. ROS accumulation caused DNA damage, followed by LMP and PARP over-activation andcellular energy depletion. Moreover, lysosome would be destroyed, and caused cathepsin B/D proteins to release into cytoplasm. If the necroptosis of signal pathway occurred, caspase would not be activated. Data of western blotting experiments showed that citronellol regulated the cell cycle of cancer cells by decreasing the protein levels of cyclin E, and promoted intracellular ROS accumulation. Citronellol could also increase the protein levels of TNF-α, RIP3, pro-PARP and cathepsin B/D in treated cells. However the protein levels of caspase-3 was not activated. We confirm that citronellol could induce the necroptosis of human lung cancer cell H1299 via TNF-α pathway through the RIP3 activation and ROS accumulation.
URI: http://hdl.handle.net/11455/92212
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