Mechanism of Proteasome Inhibitor MG132-Induced Apoptosis in Non-Small Cell Lung Cancer Cell Line (H1299)

摘 要 在真核細胞中，ubiquitin-proteasome pathway 為一主要的蛋白質分解系統。此路徑不但負責一些短生命期及長生命期蛋白質的分解，對於一些腫瘤抑制因子、轉錄因子及細胞週期的調控蛋白亦具同樣作用。改變這些蛋白質的分解，對於腫瘤的生長及細胞凋亡有著深遠的影響。 在本篇研究中，主要是探討26S proteasome 抑制劑MG132 對於p53缺乏的 human non-small cell lung cancer (NSCLC)-H1299的細胞凋亡作用。細胞在處理MG132後，會造成細胞產生reactive oxygen species (ROS)、G2/M arrest 及細胞凋亡，同時亦使細胞內之細胞週期調控蛋白質p21Waf/Cip1 及p27Kip1 表現增加，而對於抗細胞凋亡的bcl-2家族蛋白質，則會使其表現量減少。而以抗氧化劑如Glutathione、N-acetylcysteine 或 catalase抑制 ROS 的生成，都能阻止MG132引起的G2/M arrest ，但對於細胞凋亡則不具影響作用，此結果顯示，ROS 的產生是造成MG132引起G2/M arrest的原因。然而，以siRNA transfection的方式減低細胞內p21 Waf/Cip1 的表現卻不足以阻止MG132引起的 G2/M arrest，顯示還有其他路徑參與此調控作用。 在西方墨點法的結果亦顯示，MG132 能夠活化stress kinase JNK 及引起c-Jun磷酸化，而以JNK-AP-1路徑專一性抑制劑 Sp600125處理細胞，則可有效抑制MG132 引起的c-Jun 磷酸化，但卻只能輕微減少p21 Waf/Cip1的表現。另外，以 Sp600125處理細胞、或以p21 Waf/Cip1siRNA 及 p21 Waf/Cip1 antisense oligonucleotide transfection的方式，都會增加MG132引起的細胞凋亡現象，這樣的結果說明了，不管是JNK 活化或p21的表現增加，都並沒有參與MG132引起的細胞凋亡作用，相反地，他們可能都在此作用中扮演著保護的角色。 除此以外，MG132亦會導致cytochrome c的釋出、caspase-3活化，繼而使poly-ADP ribose polymerase(PARP)分解。而以caspase之廣效性抑制劑z-VAD-FMK 處理，則能回復MG132引起的細胞凋亡作用。所有這些結果顯示，在H1299細胞中，因proteasome抑制所引起的細胞凋亡作用，極可能是透過粒線體的訊息傳遞路徑來達成，同時，caspases在整個過程中亦可能扮演著重要的角色。Abstract The ubiquitin-proteasome pathway is the major proteolytic system in all eukaryotic cells. This pathway is responsible not only for the degradation of short and long-lived proteins but also tumor suppressors, transcription factors and cell cycle proteins. Altered degradation of these proteins should have profound effects on tumor growth and apoptosis. In present study, the effect of MG132, an inhibitor of 26S proteasome, on p53-deficient human non-small cell lung cancer (NSCLC), H1299 was investigated. Treatment of the cells with MG132 induced reactive oxygen species (ROS) production, G2/M arrest and apoptosis in conjunction with up-regulated levels of cell cycle regulation proteins, p21 Waf/Cip1 and p27Kip1, and down-regulation of anti-apoptotic bcl-2 families. Inhibitions of the ROS production by antioxidants such as glutathione, N-acetylcysteine or catalase prevented the G2/M arrest but have no effect on apoptosis induced by MG132. This data suggested that ROS production was involved in MG132-induced G2/M arrest. However, diminished the expression of p21 Waf/Cip1 by siRNA seems insufficient to prevent MG132-mediated G2/M arrest, suggested that other pathways might involved. Western blot analysis also showed that MG132 activated the stress kinase JNK and triggered phosphorylation of c-Jun. Sp600125, a specific inhibitor of the JNK-AP-1 pathway, inhibited the MG132 induction of c-Jun phosphorylation but only slightly attenuated the p21 expression. The apoptosis induced by MG132 was augmented by Sp600125 treatment, p21 Waf/Cip1 siRNA and p21 Waf/Cip1antisense oligonucleotide transfection. These data suggested that neither JNK activation nor p21 up-regulation was involved in MG132 induced apoptosis. In contrast, both of them might play a protective role in this effect. Furthermore, MG132 also caused the released of cytochrome c, activation of caspase-3 with the consequent degradation of poly-ADP ribose polymerase (PARP). In addition, the broad-spectrum caspase inhibitor, z-VAD-FMK markedly reversed apoptosis induced by MG132. All these data suggested that the apoptotic effect of proteasome inhibition in H1299 cells might be through the mitochondria pathway and caspases might play an important role in this signaling pathway.目錄 頁碼 附圖目錄…………………………………………………………….. 5 附表目錄…………………………………………………………….. 6 縮寫表……………………………………………………………….. 7 中文摘要…………………………………………………………….. 8 英文摘要…………………………………………………………….. 10 口試委員問答集…………………………………………………….. 12 第一章 緒論 1-1 細胞內蛋白質分解的方式……………………………………… 14 1-1.1 Lysosomal apparatus 及ubiquitin proteasome pathway. 14 1-1.2 26S proteasome 的結構……………………………….. 15 1-2 Ubiquitin-proteasome system 的功能…………………………. 16 1-2.1 細胞週期的調控 (Cell Cycle Regulation)……………. 16 1-2.2 轉錄作用的調控 (Transcriptional Regulation)……….. 18 1-2.3 細胞凋亡 (Apoptosis)…………………………………. 19 1-3 26S proteasome 的抑制劑………………………………. 21 1-4 26S proteasome 抑制劑的生物效應…………………………… 23 1-4.1 抑制蛋白質分解 (Inhibition of Protein Degradation)… 23 1-4.2 引發熱休克反應 (Induction of Heat Shock Response).. 23 1-4.3 抗發炎活性 (Anti-inflammatory Activity)…………… 24 1-4.4 細胞毒性 (Cytotoxicity)……………………………… 24 1-4.5 抗腫瘤作用 (Anti-tumor Effects)…………………….. 25 1-5 研究動機……………………………………………………….. 26 第二章 實驗材料與方法 2-1 實驗材料……………………………………………………….. 35 2.1.1 實驗藥品………………………………………………. 35 2.1.2 抗體、載體與質體……………………………………. 36 2-2 實驗方法……………………………………………………….. 37 2-2.1 Cell Line and Culture Condition………………………. 37 2-2.2 Assessment of Apoptosis and Cell Cycle Analysis……. 37 DNA Content Analysis………………………………... 37 Annexin V-FITC and PI Double Stain………………… 38 2-2.3 Measurement of Mitochondrial Transmembrane Potential (ΔΨm)……………………………………….. 38 2-2.4 Determination of Production of H2O2 and Hydroxyl Radical………………………………………………… 39 2-2.5 Measurement of Caspase Activation…………………... 39 2-2.6 DNA Fragmentation Assay on Agarose Gel…………… 40 2-2.7 Western Blot Analysis…………………………………. 41 2-2.8 Cytochrome c Release and PARP Cleavage…………… 43 2-2.9 Reverse transcription-polymerase Chain Reaction (RT-PCR)……………………………………………… 44 2-2.10 Transfection and Luciferase Activity Assay…………… 47 2-2.11 Transfection of p21 Sense and Antisense Oligonucleotide……………………………………….. 49 2-2.11 Transfection of p21 siRNA……………………………. 50 第三章 結果 3-1 MG132引發人類肺癌細胞產生細胞凋亡的現象…………… 51 3-2 MG132 對細胞週期的影響…………………………………… 53 3-3 自由基在MG132引起的細胞凋亡中所扮演的角色………… 54 3-3.1 MG132引發細胞內自由基增加……………………... 54 3-3.2 抗氧化劑對MG132引起之細胞凋亡及細胞週期改 變之影響……………………………………………… 55 3-4 MG132對於細胞週期調控蛋白的影響……………………… 56 3-4.1 MG132對p21 Waf/Cip1表現的調控作用………………. 57 3-4.2 p21 Waf/Cip1抗細胞凋亡的作用………………………… 59 3-4.3 p21 Waf/Cip1對於細胞週期的調控作用………………… 61 3-5 JNK/AP1 訊息傳遞路徑在MG132引起的細胞凋亡中所 扮演的角色……………………………………………. 62 3-5.1 MG132對JNK/AP1 訊息傳遞路徑的影響………… 62 3-5.2 MAPK inhibitior對於MG132引發p21 Waf/Cip1表現 的影響………………………………………………… 63 3-5.3 JNK /stress activation kinase抗細胞凋亡的作用……. 63 3-6 MG132對細胞粒線體的影響………………………………… 64 3-6.1 MG132不會造成細胞粒線體膜電位的下降………... 64 3-6.2 MG132導致cytochorme c從粒線體釋出，並活化 Caspase 3及造成PARP cleavage……………………. 65 3-6.3 MG132 對於Bcl-2家族蛋白的影響……………….. 66 3-7 Pancaspase inhibitor 抑制MG132引起的細胞凋亡………… 67 3-8 SP600125 對於MG132引發細胞凋亡的加成作用………… 68 第四章 討論 4-1 Proteasome抑制劑的細胞毒性作用…………………………. 99 4-2 MG132引發細胞內自由基產生……………………………... 101 4-3 MG132對於p21 Waf/Cip1的調控作用………………………..... 105 4-4 p21 Waf/Cip1在調控細胞週期及細胞凋亡中扮演的角色……… 107 4-5 JNK/AP1 訊息傳遞路徑在MG132引起的細胞凋亡中 的作用………………………………………………………… 110 4-6 MG132引起粒線體依賴性細胞凋亡路徑 (Mitochondria-dependent Apoptosis Pathway)……………… 113 第五章 結論…………………………………………………….. 117 參考文獻……………………………………………………………119 附圖目錄 Figure 1-1 Ubiquitin-proteasome pathway………………………… 28 Figure 1-2 26S Proteosome 的組成………………………………. 29 Figure 1-3 引發細胞凋亡的不同路徑……………………………. 30 Figure 1-4 五類主要之proteasome 抑制劑……………………… 31 Figure 1-5 Proteasome inhibitors引發細胞凋亡的可能機轉……. 32 Figure 3-1.1 Phosphatidylserine extrernalization were determined by Annexin-V-FITC staining……………………………… 69 Figure 3-1.2 Induction of apoptosis by MG132 on H1299 cells……. 70 Figure 3-2 MG132 induced G2/M arrest in H1299 cells ………….. 73 Figure 3-3.1 MG132 induced ROS production in a time and dose- dependent manner and effect of antioxidant on MG132- induced ROS production………………………………..75 Figure 3-3.2 Effects of antioxidant on MG132 induced apoptosis and G2/M arrest…………………………………………….. 77 Figure 3-4 Effects of MG132 on p21, p27expression in H1299 Cell................................................................................. 79 Figure 3-4.1 MG132 enhanced p21 mRNA and protein expression by increased p21 mRNA and protein stability in H1299 cells…………………………………………………… 80 Figure 3-4.2 MG132-mediated apoptosis enhanced by abolished p21 expression in H1299 cells…………………………. 83 Figure 3-4.3 Inhibition of MG132-mediated G2/M arrest by Abolished p21 expression in H1299 cells……………… 85 Figure 3-5.1 MG132 induced activation of the JNK/AP-1 pathway in H1299cells………………………………………….. 86 Figure 3-5.2 Effects of MAP kinase inhibition on MG132-induced p21 expression in H1299 cells…………………………. 87 Figure 3-5.3 MG132-induced apoptosis enhanced by specific JNK inhibitor Sp600125……………………………………. 89 Figure 3-6.1 Effects of MG132 and SP600125 on mitochondria membrane potential in H1299 cells…………………….. 90 Figure 3-6.2 MG132 caused cytochrome c release, caspase 3 Activation and PARP cleavage…………………………. 92 Figure 3-6.3 Western blotting analysis of Bcl-xL, Bcl-2 in total cell lysate and mitochondria fraction of H1299 cells……... 94 Figure 3-7 Effect of a pan caspase inhibitor on MG132 induced apoptosis……………………………………………….. 96 Figure 3-8 Effects of specific JNK inhibitor SP600125 on MG132 mediated changed of Bcl-2 family, cytochrome c release, and PARP cleavage……………………………………… 97 Figure 5 Possible mechanism of MG132 induced apoptosis…… 118 附表目錄 Table 1-1 Ubiquitin-proteasome pathway的生理功能及相關 之蛋白質………………………………………………….. 33 Table 1-2 各類proteasome inhibitors的特性……………………….3