臺灣博碩士論文加值系統

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.

中文摘要 I
英文摘要 (Abstract) III
目錄 V
圖目錄…………………………………………………………………………… … VI
第一部分:探討olomoucine之CDK抑制劑經BNIP3降低BV2微神經膠細胞中LPS和NO誘導之細胞死亡 1
壹、緒論 2
一、神經退化性疾病 (neurodegenerative disease) 與發炎反應 2
二、細胞週期的調控 2
三、細胞週期抑制劑 (cyclin-dependent kinase inhibitor,CDK inhibitor) 與神經保護作用 (neuroprotection) 4
貳、研究動機 5
叁、材料與方法 5
一、實驗材料 5
二、實驗方法與步驟 7
肆、結果 16
一、Olomoucine降低BV2微神經膠細胞增生和E2F轉錄活性 16
二、Olomoucine降低LPS誘導之NO產生和細胞死亡 16
三、Olomoucine降低LPS誘導之iNOS表現和NF-B活性 16
四、Olomoucine降低NO誘導之細胞死亡 17
五、Olomoucine降低NO誘導之粒線體損害 17
六、Olomouicne降低BV2微神經膠細胞BNIP3表現 18
七、Knockdown BV2中BNIP3表現降低LPS和NO誘導之細胞死亡 18
伍、討論 19
陸、參考文獻…………………………………………………………………..20
柒、附圖………………………………………………………………………..25第二部分:探討神經醯胺降低之AKT和Bad活性促進TNF-alpha誘導膀胱癌細胞死亡的路徑………………………………………………………………………………………………………………..35
壹、緒論 36
一、膀胱癌 36
二、腫瘤壞死因子 (TNF) 38
三、神經醯胺 (Ceramide) 39
四、細胞凋亡 (Apoptosis) 41
五、Bcl-2 家族 42
六、AKT 44
貳、研究動機 46
叁、材料與方法 48
一、實驗材料 48
二、實驗方法與步驟 51
肆、結果 62
一、CM-LPS誘發MBT-2細胞死亡因子探討 62
二、TNF-alpha和c2-神經醯胺共同處理下細胞死亡路徑探討 64
三、BH3 mimetics ABT737對TNF-alpha所誘導之細胞死亡探討 65
伍、討論 67
一、TNF-在MBT-2細胞中扮演之角色 67
二、MBT-2細胞中TNF-alpha誘導之AKT活性是造成細胞無法顯著死亡 主因 68
三、神經醯胺所涉及之AKT活性和TNF-alpha誘導之細胞死亡關係 68
四、神經醯胺涉及AKT活性和MBT-2細胞內Bcl-2家族Bad蛋白質活性關係 69
五、以BH3 mimetics ABT-737取代Bad與 Bcl-2家族蛋白質結合後於TNF-誘導細胞死亡影響 69
六、在MBT-2細胞以smac mimetic活化細胞內caspase活性與TNF-所誘導細胞死亡關係 70
陸、參考文獻 72
柒、附圖 82
捌、附錄 93
玖、相關著作 94

Benada, J., Burdova, K., Lidak, T., von Morgen, P., and Macurek, L. (2015). Polo-like kinase 1 inhibits DNA damage response during mitosis. Cell cycle 14, 219-231.

Block, M.L., and Hong, J.S. (2005). Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Progress in neurobiology 76, 77-98.

Block, M.L., Zecca, L., and Hong, J.S. (2007). Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nature reviews Neuroscience 8, 57-69.

Bouchoux, C., and Uhlmann, F. (2011). A quantitative model for ordered Cdk substrate dephosphorylation during mitotic exit. Cell 147, 803-814.

Boutis, A., Papazisis, K., Pistevou-Gompaki, K., Lambropoulos, A., Sofroniadis, I., Papageorgiou, A., Destouni, E., and Kortsaris, A. (2006). Cyclin-dependent kinase (CDK) inhibitor olomoucine enhances gamma-irradiation-induced apoptosis and cell cycle arrest in Raji cells. Anticancer research 26, 3493-3498.

Brown, G.C., and Bal-Price, A. (2003). Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Molecular neurobiology 27, 325-355.

Byrnes, K.R., and Faden, A.I. (2007). Role of cell cycle proteins in CNS injury. Neurochemical research 32, 1799-1807.

Chavez-Valdez, R., Martin, L.J., Flock, D.L., and Northington, F.J. (2012). Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia-ischemia. Neuroscience 219, 192-203.

Gang, H., Dhingra, R., Wang, Y., Mughal, W., Gordon, J.W., and Kirshenbaum, L.A. (2011). Epigenetic regulation of E2F-1-dependent Bnip3 transcription and cell death by nuclear factor-kappaB and histone deacetylase-1. Pediatric cardiology 32, 263-266.

Garrofe-Ochoa, X., Melero-Fernandez de Mera, R.M., Fernandez-Gomez, F.J., Ribas, J., Jordan, J., and Boix, J. (2008). BAX and BAK proteins are required for cyclin-dependent kinase inhibitory drugs to cause apoptosis. Molecular cancer therapeutics 7, 3800-3806.

Holcakova, J., Muller, P., Tomasec, P., Hrstka, R., Nekulova, M., Krystof, V., Strnad, M., Wilkinson, G.W., and Vojtesek, B. (2014). Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy. PloS one 9, e89228.

Irizarry, R.A., Hobbs, B., Collin, F., Beazer-Barclay, Y.D., Antonellis, K.J., Scherf, U., and Speed, T.P. (2003). Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249-264.

Jhou, R.S., Sun, K.H., Sun, G.H., Wang, H.H., Chang, C.I., Huang, H.C., Lu, S.Y., and Tang, S.J. (2009). Inhibition of cyclin-dependent kinases by olomoucine and roscovitine reduces lipopolysaccharide-induced inflammatory responses via down-regulation of nuclear factor kappaB. Cell proliferation 42, 141-149.

Kolupaeva, V., and Janssens, V. (2013). PP1 and PP2A phosphatases--cooperating partners in modulating retinoblastoma protein activation. The FEBS journal 280, 627-643.

Kumar, S.K., LaPlant, B., Chng, W.J., Zonder, J., Callander, N., Fonseca, R., Fruth, B., Roy, V., Erlichman, C., Stewart, A.K., et al. (2015). Dinaciclib, a novel CDK inhibitor, demonstrates encouraging single-agent activity in patients with relapsed multiple myeloma. Blood 125, 443-448.

Lapenna, S., and Giordano, A. (2009). Cell cycle kinases as therapeutic targets for cancer. Nature reviews Drug discovery 8, 547-566.

Lawrie, C.M., Sulistijo, E.S., and MacKenzie, K.R. (2010). Intermonomer hydrogen bonds enhance GxxxG-driven dimerization of the BNIP3 transmembrane domain: roles for sequence context in helix-helix association in membranes. Journal of molecular biology 396, 924-936.

Lim, H.J., Lee, H.S., and Ryu, J.H. (2008). Suppression of inducible nitric oxide synthase and cyclooxygenase-2 expression by tussilagone from Farfarae flos in BV-2 microglial cells. Archives of pharmacal research 31, 645-652.

Liu, B., Gao, H.M., Wang, J.Y., Jeohn, G.H., Cooper, C.L., and Hong, J.S. (2002). Role of nitric oxide in inflammation-mediated neurodegeneration. Annals of the New York Academy of Sciences 962, 318-331.

Liu, B., and Hong, J.S. (2003). Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. The Journal of pharmacology and experimental therapeutics 304, 1-7.

Liu, J., Wang, J., and Zhou, Y. (2012). Upregulation of BNIP3 and translocation to mitochondria in nutrition deprivation induced apoptosis in nucleus pulposus cells. Joint, bone, spine : revue du rhumatisme 79, 186-191.

Lucas, S.M., Rothwell, N.J., and Gibson, R.M. (2006). The role of inflammation in CNS injury and disease. British journal of pharmacology 147 Suppl 1, S232-240.
Mazure, N.M., and Pouyssegur, J. (2009). Atypical BH3-domains of BNIP3 and BNIP3L lead to autophagy in hypoxia. Autophagy 5, 868-869.

Monaco, E.A., 3rd, Beaman-Hall, C.M., Mathur, A., and Vallano, M.L. (2004). Roscovitine, olomoucine, purvalanol: inducers of apoptosis in maturing cerebellar granule neurons. Biochemical pharmacology 67, 1947-1964.

Morris, L., Allen, K.E., and La Thangue, N.B. (2000). Regulation of E2F transcription by cyclin E-Cdk2 kinase mediated through p300/CBP co-activators. Nature cell biology 2, 232-239.

Nishitani, H., and Lygerou, Z. (2002). Control of DNA replication licensing in a cell cycle. Genes to cells : devoted to molecular & cellular mechanisms 7, 523-534.

Obaya, A.J., and Sedivy, J.M. (2002). Regulation of cyclin-Cdk activity in mammalian cells. Cellular and molecular life sciences : CMLS 59, 126-142.

Perry, V.H. (2004). The influence of systemic inflammation on inflammation in the brain: implications for chronic neurodegenerative disease. Brain, behavior, and immunity 18, 407-413.

Quinsay, M.N., Lee, Y., Rikka, S., Sayen, M.R., Molkentin, J.D., Gottlieb, R.A., and Gustafsson, A.B. (2010). Bnip3 mediates permeabilization of mitochondria and release of cytochrome c via a novel mechanism. Journal of molecular and cellular cardiology 48, 1146-1156.

Raynaud, F.I., Whittaker, S.R., Fischer, P.M., McClue, S., Walton, M.I., Barrie, S.E., Garrett, M.D., Rogers, P., Clarke, S.J., Kelland, L.R., et al. (2005). In vitro and in vivo pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine and CYC202. Clinical cancer research : an official journal of the American Association for Cancer Research 11, 4875-4887.

Sassone, J., Colciago, C., Marchi, P., Ascardi, C., Alberti, L., Di Pardo, A., Zippel, R., Sipione, S., Silani, V., and Ciammola, A. (2010). Mutant Huntingtin induces activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3). Cell death & disease 1, e7.

Schmidt-Kastner, R., Aguirre-Chen, C., Kietzmann, T., Saul, I., Busto, R., and Ginsberg, M.D. (2004). Nuclear localization of the hypoxia-regulated pro-apoptotic protein BNIP3 after global brain ischemia in the rat hippocampus. Brain research 1001, 133-142.

Shaw, J., Yurkova, N., Zhang, T., Gang, H., Aguilar, F., Weidman, D., Scramstad, C., Weisman, H., and Kirshenbaum, L.A. (2008). Antagonism of E2F-1 regulated Bnip3 transcription by NF-kappaB is essential for basal cell survival. Proceedings of the National Academy of Sciences of the United States of America 105, 20734-20739.

Tamrakar, S., Rubin, E., and Ludlow, J.W. (2000). Role of pRB dephosphorylation in cell cycle regulation. Frontiers in bioscience : a journal and virtual library 5, D121-137.

Tian, D.S., Wang, W., Xu, Y.L., Yu, Z.Y., Xie, M.J., Wang, P., and Zhang, G.B. (2006). [Effects of cyclin dependent protein kinase inhibitor olomoucine on the microenvironment of axonal regeneration after spinal cord injury: an experiment with rats]. Zhonghua yi xue za zhi 86, 901-905.

Tian, D.S., Xie, M.J., Yu, Z.Y., Zhang, Q., Wang, Y.H., Chen, B., Chen, C., and Wang, W. (2007). Cell cycle inhibition attenuates microglia induced inflammatory response and alleviates neuronal cell death after spinal cord injury in rats. Brain research 1135, 177-185.

Vande Velde, C., Cizeau, J., Dubik, D., Alimonti, J., Brown, T., Israels, S., Hakem, R., and Greenberg, A.H. (2000). BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Molecular and cellular biology 20, 5454-5468.

Vereshaga, Y.A., Volynsky, P.E., Pustovalova, J.E., Nolde, D.E., Arseniev, A.S., and Efremov, R.G. (2007). Specificity of helix packing in transmembrane dimer of the cell death factor BNIP3: a molecular modeling study. Proteins 69, 309-325.

Versijpt, J., Van Laere, K., Dierckx, R.A., Dumont, F., De Deyn, P.P., Slegers, G., and Korf, J. (2003). Scintigraphic visualization of inflammation in neurodegenerative disorders. Nuclear medicine communications 24, 209-221.

Wang, X., Chen, S., Ma, G., Ye, M., and Lu, G. (2005). Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration. Mechanisms of ageing and development 126, 1241-1254.

Yurkova, N., Shaw, J., Blackie, K., Weidman, D., Jayas, R., Flynn, B., and Kirshenbaum, L.A. (2008). The cell cycle factor E2F-1 activates Bnip3 and the intrinsic death pathway in ventricular myocytes. Circulation research 102, 472-479.

Zhang, S., Zhang, Z., Sandhu, G., Ma, X., Yang, X., Geiger, J.D., and Kong, J. (2007). Evidence of oxidative stress-induced BNIP3 expression in amyloid beta neurotoxicity. Brain research 1138, 221-230.

Aggarwal, B.B. (2003). Signalling pathways of the TNF superfamily: a double-edged sword. Nature reviews Immunology 3, 745-756.

Aggarwal, B.B., Gupta, S.C., and Kim, J.H. (2012). Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey. Blood 119, 651-665.

Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W., and Yuan, J. (1996). Human ICE/CED-3 protease nomenclature. Cell 87, 171.

Asnaghi, L., Calastretti, A., Bevilacqua, A., D'Agnano, I., Gatti, G., Canti, G., Delia, D., Capaccioli, S., and Nicolin, A. (2004). Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt. Oncogene 23, 5781-5791.

Beutler, B., and Cerami, A. (1986). Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature 320, 584-588.

Beverly, L.J., Howell, L.A., Hernandez-Corbacho, M., Casson, L., Chipuk, J.E., and Siskind, L.J. (2013). BAK activation is necessary and sufficient to drive ceramide synthase-dependent ceramide accumulation following inhibition of BCL2-like proteins. The Biochemical journal 452, 111-119.

Bhardwaj, A., and Aggarwal, B.B. (2003). Receptor-mediated choreography of life and death. Journal of clinical immunology 23, 317-332.

Birbes, H., Luberto, C., Hsu, Y.T., El Bawab, S., Hannun, Y.A., and Obeid, L.M. (2005). A mitochondrial pool of sphingomyelin is involved in TNFalpha-induced Bax translocation to mitochondria. The Biochemical journal 386, 445-451.

Bishr, M., Lattouf, J.B., Latour, M., and Saad, F. (2014). Tumour stage on re-staging transurethral resection predicts recurrence and progression-free survival of patients with high-risk non-muscle invasive bladder cancer. Canadian Urological Association journal = Journal de l'Association des urologues du Canada 8, E306-310.

Bose, S., Chandran, S., Mirocha, J.M., and Bose, N. (2006). The Akt pathway in human breast cancer: a tissue-array-based analysis. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 19, 238-245.

Brognard, J., Clark, A.S., Ni, Y., and Dennis, P.A. (2001). Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer research 61, 3986-3997.

Cai, Q., Sun, H., Peng, Y., Lu, J., Nikolovska-Coleska, Z., McEachern, D., Liu, L., Qiu, S., Yang, C.Y., Miller, R., et al. (2011). A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment. Journal of medicinal chemistry 54, 2714-2726.

Carswell, E.A., Old, L.J., Kassel, R.L., Green, S., Fiore, N., and Williamson, B. (1975). An endotoxin-induced serum factor that causes necrosis of tumors. Proceedings of the National Academy of Sciences of the United States of America 72, 3666-3670.

Che, X., Yang, D., Zong, H., Wang, J., Li, X., Chen, F., Chen, X., and Song, X. (2012). Nuclear cIAP1 overexpression is a tumor stage- and grade-independent predictor of poor prognosis in human bladder cancer patients. Urologic oncology 30, 450-456.

Chen, X., Wang, T., Yang, D., Wang, J., Li, X., He, Z., Chen, F., Che, X., and Song, X. (2013). Expression of the IAP protein family acts cooperatively to predict prognosis in human bladder cancer patients. Oncology letters 5, 1278-1284.

Cho, T.M., Kim, W.J., and Moon, S.K. (2014). AKT signaling is involved in fucoidan-induced inhibition of growth and migration of human bladder cancer cells. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 64, 344-352.

Clark, A.S., West, K., Streicher, S., and Dennis, P.A. (2002). Constitutive and inducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells. Molecular cancer therapeutics 1, 707-717.

Colell, A., Coll, O., Mari, M., Fernandez-Checa, J.C., and Garcia-Ruiz, C. (2002). Divergent role of ceramide generated by exogenous sphingomyelinases on NF-kappa B activation and apoptosis in human colon HT-29 cells. FEBS letters 526, 15-20.

Cosulich, S.C., Worrall, V., Hedge, P.J., Green, S., and Clarke, P.R. (1997). Regulation of apoptosis by BH3 domains in a cell-free system. Current biology : CB 7, 913-920.

Cristofanon, S., and Fulda, S. (2012). ABT-737 promotes tBid mitochondrial accumulation to enhance TRAIL-induced apoptosis in glioblastoma cells. Cell death & disease 3, e432.

Datta, S.R., Brunet, A., and Greenberg, M.E. (1999). Cellular survival: a play in three Akts. Genes & development 13, 2905-2927.

Devin, A., Cook, A., Lin, Y., Rodriguez, Y., Kelliher, M., and Liu, Z. (2000). The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity 12, 419-429.

Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000). Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33-42.

Duenas, M., Martinez-Fernandez, M., Garcia-Escudero, R., Villacampa, F., Marques, M., Saiz-Ladera, C., Duarte, J., Martinez, V., Gomez, M.J., Martin, M.L., et al. (2013). PIK3CA gene alterations in bladder cancer are frequent and associate with reduced recurrence in non-muscle invasive tumors. Molecular carcinogenesis, 1-11.

Feldmann, M., and Maini, R.N. (2003). Lasker Clinical Medical Research Award. TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases. Nature medicine 9, 1245-1250.

Fry, M.J. (2001). Phosphoinositide 3-kinase signalling in breast cancer: how big a role might it play? Breast cancer research : BCR 3, 304-312.

Fulda, S., and Vucic, D. (2012). Targeting IAP proteins for therapeutic intervention in cancer. Nature reviews Drug discovery 11, 109-124.

Galadari, S., Rahman, A., Pallichankandy, S., Galadari, A., and Thayyullathil, F. (2013). Role of ceramide in diabetes mellitus: evidence and mechanisms. Lipids in health and disease 12, 98.

Gazzaniga, P., Gradilone, A., Giuliani, L., Gandini, O., Silvestri, I., Nofroni, I., Saccani, G., Frati, L., and Agliano, A.M. (2003). Expression and prognostic significance of LIVIN, SURVIVIN and other apoptosis-related genes in the progression of superficial bladder cancer. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 14, 85-90.

Geley, S., Hartmann, B.L., and Kofler, R. (1997). Ceramides induce a form of apoptosis in human acute lymphoblastic leukemia cells that is inhibited by Bcl-2, but not by CrmA. FEBS letters 400, 15-18.

Gomes-Giacoia, E., Miyake, M., Goodison, S., Sriharan, A., Zhang, G., You, L., Egan, J.O., Rhode, P.R., Parker, A.S., Chai, K.X., et al. (2014). Intravesical ALT-803 and BCG treatment reduces tumor burden in a carcinogen induced bladder cancer rat model; a role for cytokine production and NK cell expansion. PloS one 9, e96705.

Gu, L., Huang, B., Shen, W., Gao, L., Ding, Z., Wu, H., and Guo, J. (2013). Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi. Journal of neuroinflammation 10, 109.

Harris, L.D., De La Cerda, J., Tuziak, T., Rosen, D., Xiao, L., Shen, Y., Sabichi, A.L., Czerniak, B., and Grossman, H.B. (2008). Analysis of the expression of biomarkers in urinary bladder cancer using a tissue microarray. Molecular carcinogenesis 47, 678-685.

Herbein, G., and O'Brien, W.A. (2000). Tumor necrosis factor (TNF)-alpha and TNF receptors in viral pathogenesis. Proceedings of the Society for Experimental Biology and Medicine Society for Experimental Biology and Medicine 223, 241-257.

Hsu, H., Huang, J., Shu, H.B., Baichwal, V., and Goeddel, D.V. (1996a). TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4, 387-396.

Hsu, H., Shu, H.B., Pan, M.G., and Goeddel, D.V. (1996b). TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299-308.

Jinesh, G.G., Chunduru, S., and Kamat, A.M. (2012). Smac mimetic enables the anticancer action of BCG-stimulated neutrophils through TNF-alpha but not through TRAIL and FasL. Journal of leukocyte biology 92, 233-244.

Jinesh, G.G., and Kamat, A.M. (2012). Redirecting neutrophils against bladder cancer cells by BCG and Smac mimetic combination. Oncoimmunology 1, 1161-1162.

Kashkar, H., Wiegmann, K., Yazdanpanah, B., Haubert, D., and Kronke, M. (2005). Acid sphingomyelinase is indispensable for UV light-induced Bax conformational change at the mitochondrial membrane. The Journal of biological chemistry 280, 20804-20813.

Kennedy, S.G., Wagner, A.J., Conzen, S.D., Jordan, J., Bellacosa, A., Tsichlis, P.N., and Hay, N. (1997). The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes & development 11, 701-713.

Kerr, J.F., Wyllie, A.H., and Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British journal of cancer 26, 239-257.

Konopleva, M., Contractor, R., Tsao, T., Samudio, I., Ruvolo, P.P., Kitada, S., Deng, X., Zhai, D., Shi, Y.X., Sneed, T., et al. (2006). Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer cell 10, 375-388.

Krasilnikov, M., Adler, V., Fuchs, S.Y., Dong, Z., Haimovitz-Friedman, A., Herlyn, M., and Ronai, Z. (1999). Contribution of phosphatidylinositol 3-kinase to radiation resistance in human melanoma cells. Molecular carcinogenesis 24, 64-69.

Kurinna, S.M., Tsao, C.C., Nica, A.F., Jiffar, T., and Ruvolo, P.P. (2004). Ceramide promotes apoptosis in lung cancer-derived A549 cells by a mechanism involving c-Jun NH2-terminal kinase. Cancer research 64, 7852-7856.

Lin, C.F., Chen, C.L., Chiang, C.W., Jan, M.S., Huang, W.C., and Lin, Y.S. (2007). GSK-3beta acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 in ceramide-induced mitochondrial apoptosis. Journal of cell science 120, 2935-2943.

Lin, X., Bohle, A.S., Dohrmann, P., Leuschner, I., Schulz, A., Kremer, B., and Fandrich, F. (2001). Overexpression of phosphatidylinositol 3-kinase in human lung cancer. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie 386, 293-301.

Luo, Y., Yamada, H., Chen, X., Ryan, A.A., Evanoff, D.P., Triccas, J.A., and O'Donnell, M.A. (2004). Recombinant Mycobacterium bovis bacillus Calmette-Guerin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity. Clinical and experimental immunology 137, 24-34.

Martinez, T.N., Chen, X., Bandyopadhyay, S., Merrill, A.H., and Tansey, M.G. (2012). Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons. Molecular neurodegeneration 7, 45.

Micheau, O., and Tschopp, J. (2003). Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181-190.

Mimeault, M. (2002). New advances on structural and biological functions of ceramide in apoptotic/necrotic cell death and cancer. FEBS letters 530, 9-16.

Monick, M.M., Mallampalli, R.K., Carter, A.B., Flaherty, D.M., McCoy, D., Robeff, P.K., Peterson, M.W., and Hunninghake, G.W. (2001). Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages. The Journal of Immunology 167, 5977-5985.

Morales, A., Eidinger, D., and Bruce, A.W. (1976). Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. The Journal of urology 116, 180-183.

Muslin, A.J., Tanner, J.W., Allen, P.M., and Shaw, A.S. (1996). Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889-897.

Oka, N., Tanimoto, S., Taue, R., Nakatsuji, H., Kishimoto, T., Izaki, H., Fukumori, T., Takahashi, M., Nishitani, M., and Kanayama, H.O. (2006). Role of phosphatidylinositol-3 kinase/Akt pathway in bladder cancer cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. Cancer science 97, 1093-1098.

Olmos, G., and Llado, J. (2014). Tumor necrosis factor alpha: a link between neuroinflammation and excitotoxicity. Mediators of inflammation 2014, 861231.

Polesel, J., Bosetti, C., di Maso, M., Montella, M., Libra, M., Garbeglio, A., Zucchetto, A., Turati, F., Talamini, R., La Vecchia, C., et al. (2014). Duration and intensity of tobacco smoking and the risk of papillary and non-papillary transitional cell carcinoma of the bladder. Cancer causes & control : CCC.

Pop, C., and Salvesen, G.S. (2009). Human caspases: activation, specificity, and regulation. The Journal of biological chemistry 284, 21777-21781.

Priulla, M., Calastretti, A., Bruno, P., Azzariti, A., Paradiso, A., Canti, G., and Nicolin, A. (2007). Preferential chemosensitization of PTEN-mutated prostate cells by silencing the Akt kinase. The Prostate 67, 782-789.

Rosenberg, J.E., and Hahn, W.C. (2009). Bladder cancer: modeling and translation. Genes & development 23, 655-659.

Sander, B., Damm, O., Gustafsson, B., Andersson, U., and Hakansson, L. (1996). Localization of IL-1, IL-2, IL-4, IL-8 and TNF in superficial bladder tumors treated with intravesical bacillus Calmette-Guerin. The Journal of urology 156, 536-541.

Shin, J.S., Park, J.H., Kim, J.D., Lee, J.M., and Kim, S.J. (1995). Induction of tumour necrosis factor-alpha (TNF-alpha) mRNA in bladders and spleens of mice after intravesical administration of bacillus Calmette-Guerin. Clinical and experimental immunology 100, 26-31.

Shu, H.B., Takeuchi, M., and Goeddel, D.V. (1996). The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proceedings of the National Academy of Sciences of the United States of America 93, 13973-13978.

Siegel, R., Ma, J., Zou, Z., and Jemal, A. (2014). Cancer statistics, 2014. CA: a cancer journal for clinicians 64, 9-29.

Siskind, L.J., Kolesnick, R.N., and Colombini, M. (2002). Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins. The Journal of biological chemistry 277, 26796-26803.

Slipicevic, A., Holm, R., Nguyen, M.T., Bohler, P.J., Davidson, B., and Florenes, V.A. (2005). Expression of activated Akt and PTEN in malignant melanomas: relationship with clinical outcome. American journal of clinical pathology 124, 528-536.

Srinivasula, S.M., Datta, P., Fan, X.J., Fernandes-Alnemri, T., Huang, Z., and Alnemri, E.S. (2000). Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway. The Journal of biological chemistry 275, 36152-36157.

Sugiki, H., Hozumi, Y., Maeshima, H., Katagata, Y., Mitsuhashi, Y., and Kondo, S. (2000). C2-ceramide induces apoptosis in a human squamous cell carcinoma cell line. The British journal of dermatology 143, 1154-1163.

Sun, H., Nikolovska-Coleska, Z., Yang, C.Y., Qian, D., Lu, J., Qiu, S., Bai, L., Peng, Y., Cai, Q., and Wang, S. (2008). Design of small-molecule peptidic and nonpeptidic Smac mimetics. Accounts of chemical research 41, 1264-1277.

Tang, J.M., He, Q.Y., Guo, R.X., and Chang, X.J. (2006). Phosphorylated Akt overexpression and loss of PTEN expression in non-small cell lung cancer confers poor prognosis. Lung cancer 51, 181-191.

Taniguchi, K., Koga, S., Nishikido, M., Yamashita, S., Sakuragi, T., Kanetake, H., and Saito, Y. (1999). Systemic immune response after intravesical instillation of bacille Calmette-Guerin (BCG) for superficial bladder cancer. Clinical and experimental immunology 115, 131-135.

Toker, A., and Newton, A.C. (2000). Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. The Journal of biological chemistry 275, 8271-8274.

Tracey, K.J., and Cerami, A. (1994). Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annual review of medicine 45, 491-503.

VanderWeele, D.J., Zhou, R., and Rudin, C.M. (2004). Akt up-regulation increases resistance to microtubule-directed chemotherapeutic agents through mammalian target of rapamycin. Molecular cancer therapeutics 3, 1605-1613.

Varfolomeev, E.E., and Ashkenazi, A. (2004). Tumor necrosis factor: an apoptosis JuNKie? Cell 116, 491-497.

Verhagen, A.M., Ekert, P.G., Pakusch, M., Silke, J., Connolly, L.M., Reid, G.E., Moritz, R.L., Simpson, R.J., and Vaux, D.L. (2000). Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43-53.

Wajant, H. (2003). Death receptors. Essays in biochemistry 39, 53-71.

Wang, H., Yang, Y.B., Shen, H.M., Gu, J., Li, T., and Li, X.M. (2012). ABT-737 induces Bim expression via JNK signaling pathway and its effect on the radiation sensitivity of HeLa cells. PloS one 7, e52483.

Woodcock, J. (2006). Sphingosine and ceramide signalling in apoptosis. IUBMB life 58, 462-466.

Wu, X., Obata, T., Khan, Q., Highshaw, R.A., De Vere White, R., and Sweeney, C. (2004). The phosphatidylinositol-3 kinase pathway regulates bladder cancer cell invasion. BJU international 93, 143-150.

Yamada, H., Kuroda, E., Matsumoto, S., Matsumoto, T., Yamada, T., and Yamashita, U. (2002). Prostaglandin E2 down-regulates viable Bacille Calmette-Guerin-induced macrophage cytotoxicity against murine bladder cancer cell MBT-2 in vitro. Clinical and experimental immunology 128, 52-58.

Yamada, H., Matsumoto, S., Matsumoto, T., Yamada, T., and Yamashita, U. (2000). Enhancing effect of an inhibitor of nitric oxide synthesis on bacillus Calmette-Guerin-induced macrophage cytotoxicity against murine bladder cancer cell line MBT-2 in vitro. Japanese journal of cancer research : Gann 91, 534-542.

Yan, J., Xiang, J., Lin, Y., Ma, J., Zhang, J., Zhang, H., Sun, J., Danial, N.N., Liu, J., and Lin, A. (2013). Inactivation of BAD by IKK inhibits TNFalpha-induced apoptosis independently of NF-kappaB activation. Cell 152, 304-315.

Zhang, T., Li, Y., Zou, P., Yu, J.Y., McEachern, D., Wang, S., and Sun, D. (2013). Physiologically based pharmacokinetic and pharmacodynamic modeling of an antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in a mouse xenograft model of human breast cancer. Biopharmaceutics & drug disposition 34, 348-359.

Zhang, X., Dong, L., Chapman, E., and Benedict, W.F. (2008). Conditioned medium from Ad-IFN-alpha-infected bladder cancer and normal urothelial cells is cytotoxic to cancer cells but not normal cells: further evidence for a strong bystander effect. Cancer gene therapy 15, 817-822.