|Abstract: ||急性發炎反應 (acute inflammation) 是當宿主受到如致病微生物感染、化學刺激或創傷造成組織損害，會聚集免疫細胞或其他發炎細胞，在發炎部位對抗感染。研究指出，慢性發炎反應 (chronic inflammation) 造成發炎細胞產生ROS、一氧化氮 (nitric oxide, NO) 及前發炎性細胞激素 (pro-inflammatory cytokine) 提供癌症多發環境 (cancer-prone microenvironment)，藉促進癌細胞增生和存活、血管增生、遷移造成癌症的發展。此外慢性發炎反應，降低癌細胞對荷爾蒙及化學治療藥物反應。許多研究顯示，長期發炎反應與動脈硬化、慢性腸炎、氣喘等過敏疾病、或類風濕等自體免疫疾病致病有關。在神經退化疾病中，發炎反應是造成神經退化疾病發生和神經細胞死亡重要因素。 微神經膠細胞 (microglia) 發炎反應在阿茲海默症 (Alzheimer’s disease)、帕金森氏 (Parkinson’s disease) 和多發性硬化症 (Multiple sclerosis) 等的神經退化性疾病扮演重要角色。研究指出cyclin-dependent kinase (CDK) 之化學抑制劑，可經抑制細胞週期防止細胞進展和降低發炎反應所誘導之細胞死亡。但CDK抑制劑在抑制微神經膠細胞活性或神經細胞死亡之機制尚未清楚。 以 CDK抑制劑olomoucine探討在經脂多醣 (lipopolysaccharide, LPS) 所誘導BV2微神經膠細胞發炎反應影響。在LPS誘導BV2微神經膠細胞中，olomoucine抑制細胞增生和NO產生。以luciferase報導基因分析iNOS和NF-kappa B轉錄活性，結果顯示olomoucine抑制經LPS所誘導iNOS活性及降低NF-kappa B和E2F轉錄活性。在LPS誘導之BV2微神經膠細胞中，NO為LPS誘導細胞死亡的主要因子。以olomoucine處理可降低經LPS和NO所誘導之細胞死亡。Olomoucine降低BV2微神經膠細胞中pro-apoptotic Bcl-2家族之BNIP3蛋白質。Knockdown BNIP3表現降低NO所誘導之死亡和粒線體損害。 本研究證明CDK抑制劑olomoucine，經降低LPS所誘導之前發炎性反應降低細胞中NO降低細胞死亡。研究結果顯示CDK抑制olomoucine可負調控細胞BNIP3表現降低現體損害，具有保護細胞能力。 膀胱癌是一種泌尿道系統常見的癌症，在世界癌症排名占第八位。膀胱癌是一個高復發性的癌症，約將近50％到60％患者會復發。因此開發新的療法改善膀胱癌的治癒率是非常重要的。目前在膀胱癌中，Bacille Calmette-Guerin (BCG) 免疫療法是一種常見的療法，但約20％患者對BCG治療無效，然而迄今這療法對膀胱癌之作用機制仍不明瞭。目前已知BCG灌注後會使巨噬細胞聚集到膀胱，誘發前發炎性分子產生發炎反應，達到毒殺癌細胞效果。 因此本研究擬以LPS刺激老鼠巨噬細胞 Raw264.7，模擬BCG灌注後吸引巨噬細胞所產生的前發炎性細胞激素和化學激素 (chemokine)；收集LPS處理12小時後的Raw264.7老鼠巨噬細胞細胞培養液conditioned medium (CM-LPS)，探討對膀胱癌細胞死亡之機制。結果顯示CM-LPS會造成MBT-2細胞株大量的死亡。 推測在前發炎性細胞激素中，腫瘤壞死因子-alpha (tumor necrosis factor-alpha, TNF-alpha) 為可能造成細胞死亡之因子。因此以50 ng/ml的TNF-alpha處理MBT-2細胞12小時，顯示可降低約10細胞存活，caspase-8和caspase-all抑制劑會降低TNF-alpha所降低之細胞存活。以西方墨點法分析AKT磷酸化情況，發現TNF-alpha增加MBT-2細胞內AKT活性。顯示AKT活化後，抑制TNF-alpha誘導的細胞死亡。神經醯胺 (ceramide) 為發炎反應中產生之非蛋白質分子，外加短鍊神經鞘脂代謝物c2-神經醯胺，顯示其可以誘導MBT-2細胞死亡，增加MBT-2細胞ROS產生及造成粒線體膜電位降低。以lentiviral病毒粒子感染MBT-2細胞knockdown Bad後會降低c2-神經醯胺所誘導產生ROS和粒線體膜電位降低。共同處理TNF-和c2-神經醯胺顯著增加細胞之染色質濃縮和DNA片段化提升MBT-2細胞死亡，且AKT活性降低。在AKT活性降低同時，Bad磷酸化降低。此外在MBT-2細胞中，c2-神經醯胺可透過AKT降低活性影響細胞Bad活性，進一步調控細胞粒線體調控之內生性細胞凋亡，促進TNF-alpha所誘導細胞凋亡。 以小分子非胜肽類之BH3 mimetics ABT-737，模擬以BH3區域取代Bad和Bcl-2、Bcl-xL、Bcl-w之anti-apoptotic蛋白質結合特性，探討其對TNF-所誘導之細胞死亡之影響。Hochest3334.2結果顯示，TNF-alpha和ABT-737共同處理可增加誘導MBT-2細胞死亡；以caspase-8、caspase-9及caspase-all抑制劑會降低TNF-alpha和ABT-737之細胞死亡。TNF-alpha和ABT-737可顯著增加caspase-8和caspase-9活性。TNF-和ABT-737共同處理顯示增加MBT-2細胞內Bim蛋白質表現。TNF-alpha和ABT-737會造成MBT-2細胞粒線體膜電位降低，增加細胞質中cytochrome c和粒線體中Bim蛋白質。證實在MBT-2細胞中以ABT-737直接取代Bad和細胞內anti-apoptotic蛋白質結合，可以誘導MBT-2細胞內之粒線體所調控之內生性細胞死亡，促進TNF-alpha誘導細胞死亡。在我們結果顯示，未來可以利用之臨床試驗階段之ABT-737衍生物，應用於膀胱癌治療上，改善對於BCG治療無效之患者對BCG的效果。 由本研究結果顯示，以CDK抑制劑降低微神經膠細胞發炎反應造成NF-kappa B活性及NO產生，降低發炎反應誘導微神經膠細胞死亡；相反在膀胱癌，以發炎反應產生之前發炎性細胞素TNF-alpha與神經醯胺或ABT-737共同處理，可毒殺膀胱癌細胞，顯示發炎反應於不同細胞具有不同面像，調控細胞死亡和存活。|
Acute inflammatory resulting from infection of microbial pathogen, chemical irritation or wounding may recruit inflammatory cells to elicit pro-inflammatory response to against infect. Moreover, chronic inflammation have reported to produce reactive oxygen species (ROS), nitric oxide (NO) and pro-inflammatory cytokines to provide cancer-prone microenvironment, promoting cancer cell proliferation and survival, angiogenesis, migration and invasion leading to cancer progression. The pro-inflammatory response also involve in etiology of arteriosclerosis, bowel disease, asthma, autoimmune disease and neurodegenerative disease. The microglia-inflammatory response is play important role in neurodegenerative disease, including Alzheimer’s disease, Parkinson’s disease and multiple sclerosis disease. Chemical inhibitors of cyclin-dependent kinase (CDK) may prevent progression through the cell cycle and decrease inflammation-induced cell death. The mechanism of the protective effect of CDK inhibitors remains unexplored in microglia. Olomoucine was used to treat BV2 microglial cells, and the differential gene expression was examined by microarray. Western blotting analyzed the levels of inducible nitric oxide synthase (iNOS), cytochrome c and BNIP3. The promoter activity of iNOS and the transcriptional activity of E2F and NF-kappa B were measured by luciferase assay. Cell cycle and mitochondrial activity were examined by flow cytometry. Knock-down of BNIP3 was performed by lentivirus shRNA. We demonstrated that olomoucine inhibits cell proliferation, decreases NO production, reduces iNOS promoter activity, and alleviates NF-B and E2F transcriptional activation in lipopolysaccharide (LPS)-stimulated BV2 cells. The cell death elicited by LPS results from NO, and olomoucine can reduces LPS- and NO-induced cell death. Furthermore, olomoucine decreases the expression of BNIP3, a pro-apoptotic Bcl-2, and that knock-down of the gene promotes cell survival after NO treatment. CDK inhibitor reduces LPS-induced pro-inflammatory responses leading to the decrease of NO, which elicits cell death. Moreover, the protective effect of the inhibitor is due to the down-regulation of BNIP3, which involves mitochondrial disruption. Bladder cancer, a malignant urinary system tumor, is in the eighth position of the international cancer charts. Nowadays, Bacillus-Calmette-Guerin (BCG) immune therapy is the most common way for the treatment of bladder cancer. However, there are around 20 % patients unable to get any benefit from this, and the main mechanism of the effect of BCG is still unclear. Since BCG treatment have reported that macrophages might be recruited into the bladder to induce cancer cell death by the pro-inflammatory response, we further study the mechanism of the response involving cell death. We used LPS to treat macrophage Raw264.7 for the generation of conditioned medium (CM-LPS). Our results demonstrated that CM-LPS might cause cell death via a caspase-dependent manner in MBT-2 bladder cancer cells. As compared with CM-LPS, TNF-alpha involves the cell death. Using the 50 ng/ml of TNF-alpha was induced about 10 the cell death in MBT-2 cell, which decreasing by caspase-8 and pan caspase inhibitors. Moreover, TNF-alpha-induced AKT activation was found in MBT-2 cell by western blot, implying that the activating AKT may have anti-apoptotic activity. Since ceramide is generated after inflammation, we propose that ceramide may increase TNF-alpha-induced cell death in bladder cancer. C2-ceramide was induced the cell death via increasing the ROS production and loss of the mitochondrial membrane potential. Knockdown of the Bad was decreased the cell death, ROS and loss of the mitochondrial membrane potential. TNF-alpha combining with ceramide was exhibited chromatin condensation and DNA fragmentation by Hochest3334.2 staining assay. Moreover, using western blot to analyze AKT expression in MBT-2 bladder cell were exhibited that the AKT activation was decreased at the same time. We suggest that ceramide were promoted TNF-alpha-induced cell death via decreasing AKT activity and increasing Bad de-phosphorylation that involves cell death via mitochondrial disruption. These results are demonstrated that ceramide-mediated AKT inactivation may improve TNF-alpha-induced cell death in MBT-2 bladder cancer cells. Instead of Bad, ABT-737 can interact with the anti-apoptotic family of Bcl-2, Bcl-x/l and Bcl-w protein by BH3 domain. To study the role of the non-peptide BH3 mimetic ABT-737 in TNF--induced cell death. TNF- combining with ABT-737 was exhibited the cell death, the activity of caspase-8 and caspase-9 were increased in MBT-2 bladder cancer cell. Moreover, TNF-alpha and ABT-737 co-treatment were induced the Bim protein expression. And co-treatment were decreased the mitochondrial membrane potential, cytochrome c release and translocate the Bim to mitochondria. Our findings suggest that ABT-737 can promote the TNF-alpha-induced cell death by controlling the mitochondrial-mediation intrinsic cell death pathway. The future can improve the bladder cancer therapy, using the pre-clinical tests phase ABT-737 derivative combine with TNF-alpha to promote the BCG resistant bladder cancer cell death. In our results, we demonstrate that CDK inhibitor repressed the inflammatory by reducing NF-kappa B activity and NO production in BV2 microglia cell to decrease LPS-induce microglia cell death. In addition, TNF-/ceramide or ABT-737 was able to induce MBT-2 bladder cancer cell death. In this study, our findings display the multiple role of inflammatory response in different cell types in the protection or promotion of cell death.