臺灣博碩士論文加值系統

中文摘要
放射治療是癌症治療中一個主要治療方式，而肺癌的預後不好主要是因為其發現時常已局部侵犯或轉移，而且對放射或化學治療不敏感。因此如何發展出新的方法來抑制肺癌的快速生長及增加對治療敏感度對改進肺癌治療結果是重要的。此外，由於放射治療的主要限制是正常組織的耐受性，而肺炎為胸部放射治療最主要的併發症，因此如何降低放射治療引起的併發症或增加肺部對放射治療的耐受性也是非常重要的。
放射治療可產生反應性氧分子(reactive oxygen species: ROS) ，而且放射治療所產生生物傷害有三分之二是由反應性氧分子造成。而反應性氧分子所造成的氧化壓力在生物體內的發炎反應、去氧核糖核酸的傷害及許多基因的表現皆佔有重要角色。因此細胞內的氧化還原狀態，和細胞增生或死亡及放射治療的反應之間存有密切的相關性。
Peroxiredoxin I (Prx I) 為一大類細胞內的抗氧化劑中的一員，其可降低細胞內反應性氧分子且在很多類腫瘤中皆會過度表現，但Prx I在腫瘤細胞內所扮演的角色並不清楚。在第一章的研究中，我們希望探討Prx I 在肺癌細胞的生長及放射線治療的敏感度所扮演的角色。首先經由免疫組織染色及蛋白質測定證實肺癌腫瘤及細胞株皆有Prx I過度表現，之後利用基因轉植製造出Prx I 表現很低的 A549 (p53+) Prx I antisense (AS) 及H1299 Prx I AS 轉植細胞株，再去觀察它們和A549及H1299 wild type 的差異性。結果在細胞實驗中我们發現抑制Prx I 的表現會造成肺癌細胞的生長速度變慢，尤其是在A549肺癌細胞。此種抑制A549肺癌細胞生長的現象，在裸鼠實驗中得到再次確定。進而探討其機轉，可能是經由p53造成細胞周期進展停滯。因此在沒有p53的H1299腫瘤細胞株，抑制Prx I造成的生長抑制現象並不明顯。此外在裸鼠實驗中發現抑制Prx I也可降低肺癌細胞的侵犯性，其機轉可能是經由降低VEGF 的分泌，但同樣可發現抑制Prx I降低肺癌細胞侵犯性的程度在A549及H1299是不同的，意味著p53可能在其中仍扮演某種角色。抑制Prx I無論在細胞實驗或裸鼠實驗皆發現可增加A549及H1299肺癌細胞放射線治療的敏感度，其機轉包含降低肺癌細胞清除反應性氧分子的能力及降低細胞對損害的去氧核糖核酸的修補能力。經由本次研究結果發現Prx I的表現的確對肺癌細胞的生長、侵犯性及對治療的耐受性扮演重要的角色，而p53則可能是肺癌的預後因子。改變Prx I的表現可能是未來治療肺癌一個新的方向。
有很多文獻指出放射治療對組織的併發症主要是經由放射治療所引起發炎反應造成。一般認為，早期處理其發炎反應有助於降低放射治療併發症的產生。放射治療對去氧核糖核酸的傷害會引發NF-κB的活化，而NF-κB即是調整發炎反應程度的重要決定因子，其可刺激很多發炎基因的表現。咖啡酸苯乙酯（caffeic acid phenethyl ester，以下簡稱 CAPE）是蜂膠中的一種具生物活性的成分，也是NF-κB的抑制因子。第二章的研究主旨即是在探討當接受胸部放射治療時，給予CAPE是否可有效抑制NF-κB的活化並減少肺部發炎反應。在細胞實驗中，我們發現也是抗氧化劑的CAPE可對人類肺癌A549細胞造成明顯的細胞毒性與放射致敏感性，但對正常肺部纖維細胞並不會有此現象。其機轉可能是經由CAPE降低肺癌A549細胞內的NF-κB及GSH所造成。在活體動物實驗中，我們對BALB/c雄性老小鼠有或沒有接受CAPE處理後，接受10 Gy以及20 Gy的全胸腔照射。照射之後，進行NF-κB活化與急性發炎細胞激素的表現分析。結果發現CAPE可降低放射線照射後的發炎激素表現，包含IL-1α，IL-1β, IL-6, TNF-α以及TGF-β。除此之外，在組織學與免疫學的實驗結果顯示，CAPE可減少放射線引起的間質肺炎與TGF-β的表現。經由本研究顯示CAPE可在不造成正常肺部毒性下，減少胸部放射線照射後的連鎖發炎反應。此研究結果，提供了未來有關於合併CAPE與胸部放射線照射，在肺癌治療臨床試驗的理論基礎。

Radiotherapy is an important modality of cancer treatment. The poor prognosis of lung cancer is commonly associated with the advanced stage of the disease and the resistance to chemo- and radiotherapy. Therefore, development of new approaches to overcome rapid growth and radiation resistance of lung cancer cells is critical for improving the outcome of cancer therapy. Furthermore, the cell-killing induced by radiation is not tumor or cell-type specific. The major limitation for radiotherapy is the tolerance of normal tissue. Radiation pneumonitis is a major obstacle of thoracic irradiation. It is therefore important to determine how the incidence of radiation-induced complication might be decreased and how the dose that normal lung can tolerate might be increased.
About two thirds of the biologic damage by x-rays is caused by indirect action of free radicals. Ionizing radiation is capable of generating ROS such as H2O2 and OH. Oxidative stress induced by free radical plays an important role in inflammation, DNA damage and genes expression. The level of ROS generated could determine whether the activated cell will proliferate or undergo apoptosis. There is a close relationship between the cellular redox state, cell fate and the effects of irradiation.
Peroxiredoxins (Prxs) belong to a special antioxidant family which contain essential catalytic cysteine residues and use thioredoxin as electron donor to reduce peroxides. Prx I is a member of the 2-Cys subfamily and is ubiquitously present in cytosol. Prx I can inhibit the activation of NF-κB and reduce the intracellular H2O2 induced by platelet-derived growth factor (PDGF) or tumor necrosis factor-α. Furthermore, Prx I is overexpressed in several tumor types. However, its functions in cancer are largely unknown. In the results described in Chapter I, we examined the role of Prx I in lung cancer cell growth in vitro and in vivo, and its influence on these tumor cells’ sensitivity to irradiation therapy. We demonstrated that the overexpression of Prx I in human lung cancer tissues and cell lines. Decrease of Prx I protein led to similar levels of changes in many biological functions. Both cell lines had less capacity to scavenge ROS, suffered similar higher levels of DNA damage as indicated by 8-oxoguanine, lost much ability to perform NHEJ to repair irradiation-induced damage, and subsequently showed less ability to survive from irradiation as measured by clonogenic assay. However, the p53+ A549 cells and p53- H1299 did show significant differences after Prx I antisense knock down of the protein production. H1299 Prx I antisense transfectant showed less slow down of cell growth in culture, fewer cells arrested in G1 phase, and less sensitive than its A549 counterpart to irradiation treatment, which may be contributed by its lack of p53. Despite these discrepancies between the two cell lines, reduction in Prx I protein did affect lung cancer xenograft metastasic potential and its sensitivity to irradiation. Results of these studies indicated that overexpression of Prx I may be important for lung cancer cell growth, metastasis, repairing of DNA damage, and even resistance to ROS and irradiation. Its interplays with p53 and other tumor survival genes may be good prognosticators for lung patient survival in irradiation therapy. Furthermore, manipulation of Prx I may provide new a direction for therapeutic approaches of lung cancer.
Radiation induces an acute pro-inflammatory response in many normal cells and tissues. Various investigators have demonstrated that radiation-induced proinflammatory cytokines contributed significant complications associated with radiotherapy. The persistent accumulation and activation of immune cells are a hallmark of chronic inflammation and late effects. Early manipulation of inflammatory responses could be useful in modifying subsequent late effect. Moreover, DNA damage induced by irradiation has been reported to activate the NF-κB in cells. NF-κB is a key regulator of immune and inflammatory response and emerged as a decisive factor in the cell response to apoptotic challenge. The putative target genes of NF-κB are mainly involved in immune and inflammatory response. Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of honeybee propolis and a potent and specific inhibitor of NF-κB activation. In the results described in Chapter II, we investigated the efficacy of CAPE in inhibiting NF-κB activation and proinflammatory production induced by irradiation. The data of in vitro study revealed that CAPE, a known antioxidant, induced significant cytotoxicity, apoptosis, and radiosensitization in a human lung adenocarcinoma A549 cell line, in contrast to that noted in normal lung fibroblasts. The mechanism underpinning this differential cytotoxicity and radiosensitization for lung cancer induced by CAPE appears to be related to intracellular NF-κB and GSH, respectively. The results from in vivo study showed that CAPE treatment decreased the expressions of inflammatory cytokines including IL-1α and β, IL-6, TNF-α and TGF-β, after irradiation. Moreover, histological and immunochemical data revealed that CAPE decreased radiation-induced interstitial pneumonitis and TGF-β expression. Taken together, our results suggest that CAPE decreases the cascade of inflammatory responses induced by thoracic irradiation without causing toxicity in normal lung tissue. This provides a rationale for combining CAPE and thoracic radiotherapy for lung cancer treatment in further clinical studies.

Table of Contents
Table of contents--------------------------------------------------------------1
Abbreviations------------------------------------------------------------------5
Abstract------------------------------------------------------------------------ 7
中文摘要-----------------------------------------------------------------------11
Introduction-----------------------------------------------------------------------14
1.1 Radiation and cell death -----------------------------------------------------------------------14
1.2 Reduction oxidation state -----------------------------------------------------------------------16
1.3 Peroxiredoxin I and cancer -----------------------------------------------------------------------18
1.4 Cell cycle-----------------------------------------------------------------------20
1.5 DNA repair -----------------------------------------------------------------------22
1.6 Distant metastasis and angiogenesis-----------------------------------------------------------------------23
1.7 Caffeic acid phenethyl ester (CAPE)-----------------------------------------------------------------------25
1.8 NF-κB -----------------------------------------------------------------------26
1.9 Radiation induced responses of normal tissues and radiation pneumonitis-----------------------------------------------------------------------27
Chapter I: Inhibition of lung tumor growth and augmentation of radiosensitivity by decreasing peroxiredoxin I expression
1. Rationale, experimental models and approaches-----------------------------------------------------------------------30
2. Material and Methods-----------------------------------------------------------------------32
3. Results
3.1 Overexpression of Prx I in human lung cancer tissues and cell lines-----------------------------------------------------------------------39
3.2 Expression of Prx I proteins in Prx I antisense transfected cells and xenograft tumors-----------------------------------------------------------------------39
3.3 Effect of Prx I antisense on cell growth and tumor xenografts growth-----------------------------------------------------------------------40
3.4 Effect of Prx I antisense on cell cycle and apoptosis-----------------------------------------------------------------------41
3.5 Effects of Prx I antisense on radiation sensitivity-----------------------------------------------------------------------42
3.6 Effect of Prx I antisense on intracellular ROS scavenging and on oxidative DNA damage-----------------------------------------------------------------------43
3.7 Effect of Prx I antisense on G2M progression after irradiation-----------------------------------------------------------------------43
3.8 Prx I antisense inhibits in vitro end rejoining-----------------------------------------------------------------------44
3.9 Effect of p53 expression-----------------------------------------------------------------------45
3.10Effect of Prx I expression on spontaneous metastatic properties -----------------------------------------------------------------------45
3.11Prx I antisense modulates bcl-2 and VEGF expression-----------------------------------------------------------------------46
4. Discussion
4.1 Prx I and cancer -----------------------------------------------------------------------47
4.2 The effect on cell growth and apoptosis of Prx I antisense-----------------------------------------------------------------------48
4.3 Prx I and p53-----------------------------------------------------------------------49
4.4 Prx I and metastasis-----------------------------------------------------------------------50
4.5 Prx I antisense and radiosensitivity
4.5.1 ROS and p53 -----------------------------------------------------------------------52
4.5.2 Cell cycle -----------------------------------------------------------------------54
4.5.3 DNA repair -----------------------------------------------------------------------56
5. Conclusion-----------------------------------------------------------------------57
Chapter II: Caffeic acid phenethyl ester decreases acute pneumonitis after irradiation in vitro and in vivo
1. Rationale, experimental models and approaches-----------------------------------------------------------------------59
2. Material and Methods-----------------------------------------------------------------------61
3. Results
3.1 CAPE induces differential growth inhibition and cytotoxicity between A549 and WI-38 cells-----------------------------------------------------------------------69
3.2 Effect of CAPE on the induction of apoptosis -----------------------------------------------------------------------69
3.3 Effect of CAPE on cell cycle progression -----------------------------------------------------------------------70
3.4 CAPE cause no cytotoxicity or radiosensitization in normal lung cells-----------------------------------------------------------------------70
3.5 Sensitization of A549 cells to radiation treatment-----------------------------------------------------------------------71
3.6 Effect of CAPE on intracellular H2O2 and GSH level-----------------------------------------------------------------------71
3.7 Different gene expression profile after CAPE treatment-----------------------------------------------------------------------72
3.8 Confirmation of the expression levels of cyclin B1 and Cox-2 protein-----------------------------------------------------------------------73
3.9 CAPE attenuates NF-κB expression differentially in normal lung and lung cancer cells-----------------------------------------------------------------------73
3.10Effect of CAPE on the radiation-induced expression of proinflammatory cytokines as revealed by RPA and real-time PCR----------------------------------------------74
3.11CAPE attenuates the induction of pulmonary inflammation by irradiation-----------------------------------------------------------------------75
4. Discussion
4.1 Effective dose of CAPE for lung cancer cell line-----------------------------------------------------------------------76
4.2 CAPE and redox state -----------------------------------------------------------------------76
4.3 CAPE and apoptotic machinery-----------------------------------------------------------------------77
4.4 CAPE and radiosensitization for lung cancer-----------------------------------------------------------------------79
4.5 CAPE and NF-κB
4.5.1 Differential cytotoxicity -----------------------------------------------------------------------80
4.5.2 Radiosensitization -----------------------------------------------------------------------81
4.5.3 Radiation-induced pulmonary inflammation -----------------------------------------------------------------------81
4.6 CAPE and radiation pneumonitis
4.6.1 Histology of radiation pneumonitis -----------------------------------------------------------------------83
4.6.2 Inflammatory cytokines and radiation pneumonitis-----------------------------------------------------------------------83
4.6.3 CAPE reduced radiation pneumonitis -----------------------------------------------------------------------84
5. Conclusion-----------------------------------------------------------------------85
Future Prespectives-----------------------------------------------------------------------86
References -----------------------------------------------------------------------88
Figures
Chapter I-----------------------------------------------------------------------107
Chapter II-----------------------------------------------------------------------130
Appendix: List of publications during the period of receiving my PhD training year -----------------------------------------------------------------------143

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