三氧化二砷 (Arsenic trioxide, ATO) 是一種天然毒性化合物，可作為急性前骨髓性白血病的治療藥物，許多研究都證實三氧化二砷誘發多種腫瘤細胞的細胞凋亡。三氧化二砷可透過活化 ROS、p38 以及其他許多途徑誘導細胞凋亡的發生。我們先前的研究證實胃幽門螺旋桿菌 (Helicobacter pylori, Hp) 感染以及 SUMO-1 的大量表現均可活化 p38，並且可在細胞核中發現 SUMO-1 和 p-p38 共同形成核點。因此我們將於本研究中探討 p38 蛋白及相撲蛋白在三氧化二砷誘發 AGS 細胞凋亡的角色。我們發現三氧化二砷會活化 SUMO-1、SUMO-2 和 p38 的基因及蛋白表現。此外三氧化二砷可透過 ROS、p38/p53/BAX 以及 caspase 8/tBID 途徑誘發細胞凋亡。而外源性的相撲蛋白可加成性地增加三氧化二砷、胃幽門螺旋桿菌以及 diamide 所誘導的細胞凋亡，此外相撲蛋白參與細胞凋亡的機制與 p38 所調控的途徑有所關聯。

Arsenic trioxide (ATO), an anti-cancer drug for acute promyelocytic leukemia (APL), was able to induce apoptosis in several cancer cell lines including solid tumors, myeloma and lymphoma. Previous researches showed that ATO triggered apoptosis via p38 MAPK pathway, and p38 was activated by environmental stress such as ROS. Our previous studies have established that phosphorylation of p38 was elevated in AGS cells during Helicobacter pylori (Hp) infection and overexpression of SUMO-1. Moreover, SUMO-1 was co-localized with p-p38 in nucleus during overexpression of SUMO-1. In this study, we evaluated the mechanisms and the association of SUMOs and p38 of ATO-induced apoptosis. Present results exhibited that ATO induced apoptosis, and elevated p38 and SUMOs in transcript and protein levels. In addition, up-regulation of p-p38, p53, BAX, cleaved caspase 8 and t-BID were also observed during exposure of ATO. Moreover, inhibitors of p38 and caspase 8 and ROS scavenger, N-acetyl-cysteine (NAC), protected AGS from ATO-induced apoptosis. These results suggested that ATO triggered apoptosis via ROS-, p38- and caspase 8-mediated pathway. Furthermore, our data established that SUMOs enhanced p38-mediated apoptosis.

Verification from thesis oral defense committee …………………i
Abstract in Chinese ……………………………………………ii
Abstract in English ………………………………………iii
Contents ………………………………………………iv
List of figures ……………………………………………………ix
Abbreviations ………………………………………………x
I. Introduction ………………………………………………1
1. Apoptosis …………………………………………………1
1.1 Extrinsic and intrinsic pathways ……………………………1
2. Arsenic trioxide ……………………………………5
2.1 Tumorigenesis of arsrnic trioxide ………………………5
2.2 Clinical application and apoptotic mechanisms of arsenic trioxide …6
3. p38 mitogen-activated protein kinase (p38 MAPK) ………10
3.1 Regulation of p38 ……………………………………11
3.2 p38-mediated apoptosis ……………………………………12
4. Small ubiquitin-related modifiers (SUMOs) ……………14
4.1 SUMOylation ………………………………14
4.2 Localization of SUMOs ………………………………17
4.3 Functions of SUMOs in cellular processes …………………20
4.3.1 Regulation of nuclear bodies formation ……………21
4.3.2 Regulation of proteins activity and stabilization …………………22
4.3.3 SUMOs-related apoptosis ……………………………………23
5. p53 regulation and apoptosis ………………………………25
5.1 Stabilization and activation of p53 …………………25
5.2 p53-mediated apoptosis ………………………………27
II. Objectives ……………………………………………………29
III. Materials and methods ………………………………………30
1. Cell culture ……………………………………………30
1.1 Materials …………………………………………………30
1.2 Methods …………………………………………………31
2. Transfection ……………………………………………33
2.1 Materials …………………………………………33
2.2 Methods ……………………………………………………33
3. MTT cell viability assay ………………………………35
3.1 Materials ………………………………………………35
3.2 Machine ………………………………………………36
3.3 Methods ……………………………………………………36
3.3.1 Preparation of ATO solution ………………………………36
3.3.2 ATO-induced cell death …………………………………36
3.3.3 Inhibition of cell death by inhibitors of caspase 8 and MAPK ………37
3.3.4 Contribution of SUMO-1, SUMO-2 and p38 in ATO-, diamide- or Hp 43504-induced apoptosis …………………………38
3.3.5 Contribution of triptolide in ATO-, diamide- or Hp 43504-induced apoptosis ……………………………………………39
4. Reverse Transcription-PCR (RT-PCR) …………………41
4.1 Materials …………………………………………41
4.2 Methods ………………………………………………………42
4.2.1 Treatment of ATO …………………………………………42
4.2.2 RNAs extraction ……………………………………………43
4.2.3 cDNA synthesis (Reverse transcription, RT) ……………44
4.2.4 Polymerase Chain Reaction (PCR) ………………………45
5. Apoptosis assay ………………………………………47
5.1 Materials ……………………………………………46
5.2 Machine ……………………………………………47
5.3 Software …………………………………………………47
5.4 Methods …………………………………………48
5.4.1 Cell cycle analysis ……………………………………48
6. Western blotting …………………………………………50
6.1 Materials ……………………………………………50
6.2 Primary antibodies ………………………………………51
6.3 Secondary antibodies …………………………………52
6.4 Methods ………………………………………53
6.5 SDS-PAGE …………………………………………53
6.5.1 Materials ……………………………………53
6.5.2 Machine ………………………………………54
7. Statistical analysis …………………………………55
IV. Results ……………………………………………56
1. ATO induced cell death in a dose- and time-dependent manner ……56
2. ATO enhanced the gene expression of SUMO-1, SUMO-2 and p38 …59
3. p38 and caspase 8 pathways were activated by ATO ………65
4. ATO induced apoptosis in AGS cells …………………………………68
5. ATO induced apoptosis via ROS-, p38- and caspase 8-mediated pathway ……………………………………………………71
6. ATO-induced apoptosis was elevated in p38, SUMO-1 or SUMO-2 overexpressing AGS cells ……………………………76
7. Triptolide enhanced p38-mediated apoptosis ……………………84
V. Discussion …………………………………………………87
VI. Future works ……………………………………………………92
VII. References …………………………………………………93
VIII. Supplements …………………………………………………111

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