- 學位論文
探討一氧化氮釋放劑對於促進與抑制白血病細胞生長的影響
The effect of nitric oxide donors on promoting or impeding cell growth in leukemia cells
摘要
一氧化氮(nitric oxide，NO)在體內可以調節許多的生理功能，如: 神經訊息傳遞、血管擴張、血管平滑肌舒張及抑制血小板凝集等作用。而在腫瘤治療方面，其扮演著雙重的角色：具有促進腫瘤生長的活性，也具有抑制腫瘤生長的作用。一氧化氮因配位化學不安定，因此容易與細胞中的鐵結合而形成穩定複合物 ( dinitrosyl-dithiol iron complex，DNIC)，其可以使NO在生理中儲存與運輸較為安定，但同時也可能使細胞中的鐵釋出，影響鐵的平衡而造成細胞毒性的產生，其在腫瘤治療上NO影響細胞中的鐵，也可能會影響腫瘤細胞的生長。 目前除了生理功能誘導一氧化氮的釋放之外，也可藉由外加給予一氧化氮釋放劑(NO donor)，使其進入生理中再釋放出NO，進而影響生理功能及腫瘤細胞之生長。本研究之目的為投予不同的NO donors進入白血病細胞之後，對於白血病細胞造成的增生與抑制作用，是否與NO影響細胞中鐵離子的平衡有關。 本實驗中使用五種NO donors，分別為: SNAP、SIN-1、Nonoate、GSNO及Cys-NO，將其投予至三種白血病相關的細胞株 : K562、CCRF- CEM及NCIH929之中，觀察其對於腫瘤細胞所造成的影響。由細胞存活率試驗，評估NO donors對於細胞造成的增生及抑制作用；再藉由比色定量分析法直接檢測細胞中鐵的變化。 細胞存活率試驗中，當K562及CCRF-CEM細胞投予SNAP 、 GSNO及Nonoate的濃度相對較低 ( 50 - 800 ?嵱 )時有細胞增生作用，而NCIH929細胞則沒有細胞增生的現象，其細胞增生的作用為SNAP > GSNO > Nonoate；而當濃度高於1000 ?嵱則有細胞毒性的產生。NO donors對於細胞所造成的影響，主要透過產生NO來參與作用。促使細胞增生的作用可能為NO有抗氧化活性，可抑制自由基傷害；而血清的albumin也可以清除自由基避免SNAP對於細胞造成傷害； GSH ( glutathione ) 為重要的抗氧化物，抑制GSH使其無法清除生理環境中自由基等氧化物質的傷害，進而則會提升細胞毒性的產生。 而NO donors合併三價鐵(ferric ammonium citrate，FAC)，可使鐵進入細胞內而調節鐵的恆定，因此減少RSNO造成的細胞毒性；相對地，合併鐵的螯合劑(deferoxamine，DFO)，使其螯合細胞中的鐵，則會影響細胞鐵的平衡，而增加NO donors造成的細胞毒性。另外，使用比色定量分析法檢測細胞中的鐵，也發現投予NO donors會使細胞中測得的鐵減少，得知NO donors可能會影響細胞內的鐵。 總結以上結果，NO donors 對於細胞所造成的影響，可能低濃度NO具有抗氧化活性，可以避免自由基的傷害；高濃度的NO則會造成細胞毒性。另外 NO donors 也可能會影響細胞內的鐵的平衡。
並列摘要
Nitric oxide (NO) can regulate many physiological functions, including neurotransmission, vasodilation, smooth muscle relaxation, and inhibition of platelet aggregation. Nitric oxide has biphasic roles in oncology as a pro-neoplastic and/or an anti-neoplastic agent. NO possesses a rich coordination chemistry with iron (Fe) and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. DNICs can not only make NO more stable in the physical storage and transportation, but also cause iron release from cells, thus affecting the balance of iron in the cell may cause cytotoxicity. In the cancer therapy, NO affecting the balance of iron in the cells, may also affect the growth of tumor cells. In addition to the physiological functions induced nitric oxide release, NO can also be given by intriducing nitric oxide releasing agent (NO donor) into cells, which may affect the physiological function and tumor cell growth. The aim of this study was to observe the effect of NO donors on promoting or impeding leukemia cell lines, and relationship between NO balance and the cellular iron. Three leukemia-related cell lines, K562, CCRF-CEM, and NCI- H929 were used in the study. Cells were treated with five NO donors, S-Nitroso-N-acetylpenicillamine (SNAP), S-Nitrosoglutathione (GSNO), S-nitrosocysteine (Cys-NO), 3-morpholinosydnonimine hydrochloride (SIN-1) and Spermine NONOate. After cells were treated with each NO donors, the cell viability was evaluated by MTS assay. The amount of cellular iron was then measured by colorimetric ferrozine-based assay. Cell viability experiments showed that SNAP、GSNO and Nonoate induced cell proliferation in relatively low concentrations (50-800 ?嵱) in K562 and CCRF-CEM；however, NCIH929 didn’t have the phenomenon of cell proliferation. The cell proliferation enhancement effect was most obvious in SNAP, comparing with GSNO and Nonoate. Concentrations higher than 1000 ?嵱 of NO donors were cytotoxic. The influence of cell growth may be related with mainly the production of NO. Promote cell proliferation may be NO have the role of antioxidant activity and inhibit free radical damage. Albumin can remove free radicals and avoid SNAP damage to the cell. Glutathione, an antioxidants, when inhibit of GSH may makes it can’t remove the free radicals and enhance the cytotoxicity. The studies of NO donors combined with ferric ions (ferric ammonium citrate，FAC) showed that FAC can protect K562 cell from RSNO drugs induced cytotoxicity, which may be due to make the iron into the cell to adjust the iron's constant. In contrast, results in NO donors combined with iron chelator (Deferoxamine，DFO) found that DFO can chelate the intracellular iron and increase cytotoxicity. The amount of cellular iron was then measured by colorimetric ferrozine-based assay, also found that NO donors decrease the amount of cellular iron. That NO donors may affect the balance of iron within the cell. In conclusion, NO donors induced cell proliferation in relatively low concentrations may because NO have the role of antioxidant activity and inhibit free radical damage；navertheless, relatively higher concentrations were cytotoxic. In addition, NO donors may also affect the balance of iron within the cell.