探討多胺誘發血癌細胞凋亡之作用機制

Study the Apoptotic Effect of Polyamine on Leukemia Cells and the Underline Mechanisms

吳婉鈴(Wan-Ling Wu)

指導教授 ： 張基隆

高雄醫學大學/醫學院/生物化學研究所/碩士(2013年)

https://doi.org/10.6832/KMU.2013.00098

摘要

多胺代謝( polyamine metabolism )在人體代謝中具重要角色，其參與了細胞生長、維持DNA染色質之結構、調控基因轉錄、轉譯等功能。在癌細胞生長過程中高度活化多胺代謝促進癌細胞生長，但其作用機制如何仍待釐清。
本研究使用三株人類血癌細胞株U937( monocyte )、Raji ( B cell )、Jurkat ( T cell )，以精胺( spermine )進行細胞毒殺實驗，實驗結果發現在精胺20 μM作用下，Jurkat細胞相較於U937及Raji細胞株具有較高的敏感性，死亡率達50.0 %，再進一步分析細胞週期，結果發現Jurkat細胞在精胺濃度20 μM作用下Sub-G1比例達40.0 %，Jurkat細胞胞內之活性氧化物( ROS )明顯增加，也明顯產生粒線體膜電位下降情形。以Western blot實驗發現其pro-caspase-3、pro-caspase-9表現明顯減少，且抗氧化分子Nrf-2之表現有明顯下降。但加入抗氧化劑( N-acetyl-L-cysteine；NAC )合併精胺作用於Jurkat細胞後，研究結果發現NAC明顯對Jurkat細胞產生保護的效果，並使細胞存活率回升至90.0 %以上。而經由超氧化物歧化酶( SOD )及穀胱甘肽含量( glutathione )檢測中發現U937及Raji細胞株之抗氧化能力明顯較Jurkat細胞佳，此外，精胺氧化酶( spermine oxidase；SMO )參與在精胺( spermine )代謝亞精胺( spermidine )的過程，並同時產生副產物過氧化氫( H2O2 )，我們測定三株細胞精胺氧化酶之表現量，結果發現Jurkat細胞精胺氧化酶之表現量明顯較U937及Raji細胞高70.0 %，此外加入精胺氧化酶抑制劑( MDL 72527 )，實驗結果發現Jurkat細胞細胞存活率回升至接近100.0 %，因此由以上實驗結果推測氧化壓力是造成Jurkat細胞產生細胞凋亡的重要原因。
綜合以上結果得知，Jurkat細胞對精胺具有較高敏感性，其抗氧化能力明顯較U937及Raji細胞差，主要乃細胞內精胺氧化酶活性高，增加過氧化氫( H2O2 )產生，造成氧化壓力並進一步攻擊細胞，進而caspase-3、caspase-9活性增強而造成凋亡現象。

關鍵字

多胺代謝 ； 血癌 ； 精胺

並列摘要

The metabolic pathway of polyamine plays important roles in cell growth, chromosome conformation maintenance, and gene transcription as well as translation. Previous studies indicated it would support cancer cells survival; however, the real mechanism was unclear. This study was designed to explore the effects of spermine on leukemia cell lines of U937( monocyte ), Raji ( B cell ), and Jurkat ( T cell ) cells.
Results showed Jurkat cell was the most sensitive to spermine treatment in these three types of cells. Treatment with 20 μM spermine, Jurkat cells were arrested at cell cycle sub-G1 phase by 40%, meanwhile, reactive oxygen species (ROS) was increased and mitochrodrial membrane protential was lost. In addition, western blotting showed cytochrome c was released, caspase-9 and caspase-3 expressions were increased as well as Nrf-2 expression was decreased. Furthermore, Co-addition of N-acetyl-L-cysteine(NAC) with spermine would restore Jurkat cell survival. At the same time, we found superoxide dismutase and glutathione levels were lower in Jurkat cell than in U937 cell or Raji cell. Those indicate low anti-oxidative capacity of leukemia cells results in higher sensitive to spermine treatment.
Spermine oxidase (SMO) oxidizes spermine to spermidine and hydrogen peroxide (H2O2). We also found Jurkat cell had higher SMO activity than the other two cells did. Addition of MDL 72527, a SMO inhibitor, would inhibit spermine toxicity on Jurkat cells. It suggests that production of oxidative stress (H2O2) is the cause of spermine toxicity of Jurkat cell.

並列關鍵字

polyamine ； leukemia ； spermine

參考文獻

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