臺灣博碩士論文加值系統

研究背景 : 肺癌是國人罹患率及致死率極高的癌症，而非小細胞肺癌(non-small cell lung cancer, NSCLC)約佔所有肺癌80%，其中大細胞肺癌(large cell carcinoma)具高度侵略性、多重抗藥性及高度轉移能力。因此，從眾多的新穎抗癌藥物中篩選出能夠對抗大細胞肺癌是目前藥物設計首重標的。喹啉環化合物具有多樣化的生物活性，而目前喹啉環所衍生的抗癌藥物已廣泛地於臨床上應用。然而，此類化合物仍存在著細胞毒性不足與溶解度不佳而侷限其應用性，且抗癌機轉仍著重於癌細胞毒殺性單方面探討。有鑒於此，提升喹啉環衍生化合物抗癌的應用價值與改善此類化合物缺點將是研究者欲突破的目標。

研究目的 : 本研究著重於探討新穎的喹啉環化合衍生物2,9-bis(2-(pyrrolidin-1-yl)ethoxy)-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-11H-indeno[1,2-c]quinolin-11-one，簡稱為BPIQ，透過細胞實驗設計以評估BPIQ對肺癌細胞毒殺與轉移能力並探討其機轉。

研究結果 : 我們發現在BPIQ處理下，能夠有效率地抑制肺癌細胞增生，進一步地發現BPIQ能降低週期素cyclin A、cyclin B和CDK 1的蛋白表現及遲緩DNA複製的效率，最終導致細胞停滯於G2/M 週期，並且啟動細胞凋亡的產生。深入探討發現BPIQ能誘導促進凋亡蛋白(pro-apoptotic) Bad與Bim的累積，但降低促進生存(pro-survival) XIAP與survivin蛋白的含量，可能致使粒線體膜電位(mitochondria membrane potential, MMP)失去恆定，進而促進細胞色素c (cytochrome c)大量釋放到細胞質，活化凋亡酵素caspase-9 和 -3而啟動細胞凋亡。同時，本研究也發現抑制ERK活性會降低BPIQ造成的增生抑制作用及細胞凋亡現象，推測ERK在BPIQ抑制肺癌細胞增生及凋亡中扮演非常重要的角色。且進一步透過p-ATM、p-ATR、p-CHK 1、p-CHK 2和γH2AX的活化後發現BPIQ可能是經DNA損害導致細胞凋亡。再者，BPIQ能夠在非毒性劑量(non-cytotoxic dose)下，透過削弱內生性的活性氧化物(reactive oxygen species, ROS)進而降低轉錄因子SP-1蛋白表現，並抑制β-catenin蛋白核轉位作用(nuclear translocation)後，使降低下游COX-2蛋白表現量以及細胞轉移相關基質金屬蛋白酵素(matrix metalloproteinases, MMPs)的MMP-2和-9活性明顯下降，而抑制細胞轉移(cell migration)作用。同時，研究也發現以ERK及p38專一抑制劑分別前處理後，能增加BPIQ對H1299肺癌細胞20%抑制轉移的能力，此結果顯示BPIQ具有能夠拮抗ERK與p38在細胞內促進癌細胞轉移能力，預期未來搭配ERK及p38抑制劑將可能具有達到加成抑制癌細胞轉移的潛力。

結論 : 本研究以細胞實驗證實新穎合成喹啉衍生物BPIQ能透過不同劑量操作，產生雙重抗肺癌機轉特性，是一個能抑制肺癌生長與誘導細胞凋亡及抑制細胞轉移的專一性藥物。我們的研究證實BPIQ除可透過引發DNA損害與粒線體調節而啟動細胞凋亡作為途徑，更進一步地有效的抑制轉移相關蛋白表現量與轉錄因子核轉位(nuclear translocation)。另一方面，非毒性劑量的BPIQ能削弱H1299細胞內生性活性氧化物濃度，並顯著性地抑制肺癌細胞的轉移能力。未來我們將更深入進行動物實驗模式，以驗證BPIQ在活體內對抗肺腫瘤的生長與轉移能力。

2,9-bis(2-(pyrrolidin-1-yl)ethoxy)-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-11H-indeno[1,2-c]quinolin-11-one (BPIQ) is a derivative of 6-arylindeno[1,2-c]quinoline, which exhibits anti-cancer effects. In our study, BPIQ efficiently inhibited cell proliferation of human lung cancer cells. Furthermore, BPIQ reduced the levels of cell cycle-associated proteins, such as cyclin A, cyclin B and CDK1, and subsequently arrested DNA replication. As a result, the arrest of G2/M phase by BPIQ was observed. Moreover, the up-regulation of pro-apoptotic Bad, Bim and down-regulation of pro-survival XIAP and survivin were observed following BPIQ treatment. The intracellular imbalance of pro-apoptotic and pro-survival proteins may lead to the loss of mitochondrial membrane potential (ΔΨm), sequential release of cytochrome c and activation of caspase-9 and -3. Finally, BPIQ-induced apoptosis was characterized by phosphatidylserine (PS) externalization. The role of mitogen-activated protein kinase (MAPK) in BPIQ-induced apoptosis was also examined, and our results showed that the extracellular signal-regulated kinase (ERK), a member of MAPK family, was activated in BPIQ-induced H1299 cells. Additionally, blockade of ERK rescued BPIQ-induced proliferative inhibition and apoptotic cell death. Our study further confirmed that BPIQ-induced apoptosis was involved in activating the signaling pathway of DNA damage repair system, including γ-H2AX, ATM/ATR, CHK1 and CHK2.
On the other hand, the part II of our study found that non-cytotoxic dose (ten-fold below the concentration of IC50) of BPIQ inhibited migration of H1299 cells efficiently. Additionally, the reduced level of intracellular reactive oxygen species was correlated with cytosolic accumulation of β-catenin as well as the decreased level of migration-associated transcription factor SP-1. Likewise, the activities of matrix metalloproteinases (MMPs) MMP-2 and -9, as well as the expression of cyloxygenase-2 (COX-2) were decreased. The role of MAPK in non-cytotoxic dose of BPIQ-induced migration inhibition was also evaluated, and our results indicated that the ERK and p38 play antagonizing roles against BPIQ-induced inhibition on cell migration. As expected, blockade of ERK and p38 exhibited 20% enhancement on original BPIQ-induced inhibition on cell migration. Taken together, our results demonstrated a dual effect of BPIQ on anti-lung cancer, and it might be an advantage for lung cancer therapeutics in the future.

中文摘要........................i
ABSTRACT.......................iv
ABBREVIATION...................vii
PART I.........................1
1.INTRODUCTION.................2
2.MATERIALS AND METHODS........3
3.RESULTS......................6
4.DISCUSSION...................9
PART II........................13
1.INTRODUCTION.................14
2.MATERIALS AND METHODS........16
3.RESULTS......................20
4.DISCUSSION...................26
CONCLUSION.....................31
REFERENCES.....................32
FIGURES........................39

1.Ettinger, D.S., et al., Non-small cell lung cancer. J Natl Compr Canc Netw, 2010. 8(7): p. 740-801.
2.Pirker, R. and W. Minar, Chemotherapy of advanced non-small cell lung cancer. Front Radiat Ther Oncol, 2010. 42: p. 157-63.
3.Wagner, T.D. and G.Y. Yang, The role of chemotherapy and radiation in the treatment of locally advanced non-small cell lung cancer (NSCLC). Curr Drug Targets, 2010. 11(1): p. 67-73.
4.O''Rourke, N., et al., Concurrent chemoradiotherapy in non-small cell lung cancer. Cochrane Database Syst Rev, 2010(6): p. CD002140.
5.Tseng, C.H., et al., Synthesis and antiproliferative evaluation of certain indeno[1,2-c]quinoline derivatives. Part 2. J Med Chem, 2010. 53(16): p. 6164-79.
6.Roepe, P.D., Molecular and physiologic basis of quinoline drug resistance in Plasmodium falciparum malaria. Future Microbiol, 2009. 4(4): p. 441-55.
7.Kumar, S., S. Bawa, and H. Gupta, Biological activities of quinoline derivatives. Mini Rev Med Chem, 2009. 9(14): p. 1648-54.
8.Tseng, C.H., et al., Synthesis and antiproliferative evaluation of certain indeno[1,2-c]quinoline derivatives. Bioorg Med Chem, 2008. 16(6): p. 3153-62.
9.Simanek, E., Cancer Therapies Utilizing the Camptothecins: A Review of in Vivo Literature. Molecular pharmaceutics, 2010.
10.Venditto, V. and E. Simanek, Cancer Therapies Utilizing the Camptothecins: A Review of the in Vivo Literature. Molecular pharmaceutics, 2010. 7(2): p. 307-349.
11.Pommier, Y., Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer, 2006. 6(10): p. 789-802.
12.Tseng, C.H., et al., Synthesis and antiproliferative evaluation of 6-arylindeno [1, 2-c] quinoline derivatives. Bioorg Med Chem, 2009. 17(21): p. 7465-7476.
13.Chiu, C.C., et al., p38 MAPK and NF-kappaB pathways are involved in naphtho[1,2-b] furan-4,5-dione induced anti-proliferation and apoptosis of human hepatoma cells. Cancer Lett, 2010. 295(1): p. 92-9.
14.Wilkinson, R.W., et al., AZD1152, a selective inhibitor of Aurora B kinase, inhibits human tumor xenograft growth by inducing apoptosis. Clin Cancer Res, 2007. 13(12): p. 3682-8.
15.Sliwinska, M.A., et al., Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells. Mech Ageing Dev, 2009. 130(1-2): p. 24-32.
16.Chang, J.H. and H.Y. Kwon, Expression of 14-3-3delta, cdc2 and cyclin B proteins related to exotoxin A-induced apoptosis in HeLa S3 cells. Int Immunopharmacol, 2007. 7(9): p. 1185-91.
17.Wang, T., et al., Tissue inhibitor of metalloproteinase-1 protects MCF-7 breast cancer cells from paclitaxel-induced apoptosis by decreasing the stability of cyclin B1. Int J Cancer, 2010. 126(2): p. 362-70.
18.Narayan, S., et al., Expression of apoptosis regulators Bcl-2 and Bax in childhood acute lymphoblastic leukemia. Hematology, 2007. 12(1): p. 39-43.
19.Zecchin, K.G., et al., High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H2O2-induced apoptosis via calcineurin pathways. J Bioenerg Biomembr, 2007. 39(2): p. 186-94.
20.Saed, G.M., et al., Modulation of the BCL-2/BAX ratio by interferon-gamma and hypoxia in human peritoneal and adhesion fibroblasts. Fertil Steril, 2008. 90(5): p. 1925-30.
21.Lee, S.H., et al., Induction of apoptosis in human leukemia U937 cells by anthocyanins through down-regulation of Bcl-2 and activation of caspases. Int J Oncol, 2009. 34(4): p. 1077-83.
22.Zhao, Y.N., et al., Pro-apoptotic protein BIM in apoptosis of glucocorticoid-sensitive and -resistant acute lymphoblastic leukemia CEM cells. Med Oncol, 2010.
23.Gang, Z. and F. Lai-yun, Effect of matrine on apoptosis and Bad expression of colorectal cancer cells in vitro. Chongqing Medicine, 2009.
24.Carrasco, R.A., et al., Antisense Inhibition of Survivin Expression as a Cancer Therapeutic. Mol Cancer Ther, 2011. 10(2): p. 221.
25.Chanvorachote, P., et al., Curcumin sensitizes lung cancer cells to cisplatin-induced apoptosis through superoxide anion-mediated Bcl-2 degradation. Cancer Invest, 2009. 27(6): p. 624-35.
26.Fan, J., et al., Effect of Bcl-2 and Bax on survival of side population cells from hepatocellular carcinoma cells. World J Gastroenterol, 2007. 13(45): p. 6053-9.
27.Salakou, S., et al., Increased Bax/Bcl-2 ratio up-regulates caspase-3 and increases apoptosis in the thymus of patients with myasthenia gravis. In Vivo, 2007. 21(1): p. 123-32.
28.Coulthard, L.R., et al., p38(MAPK): stress responses from molecular mechanisms to therapeutics. Trends Mol Med, 2009. 15(8): p. 369-79.
29.Michaelidis, B., et al., Stress activated protein kinases, JNKs and p38 MAPK, are differentially activated in ganglia and heart of land snail Helix lucorum (L.) during seasonal hibernation and arousal. Comp Biochem Physiol A Mol Integr Physiol, 2009.
30.Watson, J.L., et al., Curcumin-induced apoptosis in ovarian carcinoma cells is p53-independent and involves p38 mitogen-activated protein kinase activation and downregulation of Bcl-2 and survivin expression and Akt signaling. Mol Carcinog, 2009.
31.Rosini, P., et al., NGF withdrawal induces apoptosis in CESS B cell line through p38 MAPK activation and Bcl-2 phosphorylation. Biochem Biophys Res Commun, 2000. 278(3): p. 753-9.
32.Xia, Z., et al., Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 1995. 270(5240): p. 1326-31.
33.Geest, C.R., et al., Tight control of MEK-ERK activation is essential in regulating proliferation, survival and cytokine production of CD34+ derived neutrophil progenitors. Blood, 2009.
34.Wang, X., J.L. Martindale, and N.J. Holbrook, Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem, 2000. 275(50): p. 39435-43.
35.Bacus, S.S., et al., Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene, 2001. 20(2): p. 147-55.
36.Chang, L. and M. Karin, Mammalian MAP kinase signalling cascades. Nature, 2001. 410(6824): p. 37-40.
37.Ikuta, K., et al., Defects in apoptotic signal transduction in cisplatin-resistant non-small cell lung cancer cells. Oncol Rep, 2005. 13(6): p. 1229-34.
38.Seo, M., et al., Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-kappaB in H1299 human lung cancer cells. Biochem Biophys Res Commun, 2009. 381(2): p. 153-8.
39.Shatz, M., et al., Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells. Exp Cell Res, 2010. 316(10): p. 1748-62.
40.Zhang, J.J., et al., Pyrrolidine dithiocarbamate inhibits nuclear factor-kappaB pathway activation, and regulates adhesion, migration, invasion and apoptosis of endometriotic stromal cells. Mol Hum Reprod, 2011. 17(3): p. 175-81.
41.Yue, X., et al., Interruption of beta-catenin suppresses the EGFR pathway by blocking multiple oncogenic targets in human glioma cells. Brain Res, 2010. 1366: p. 27-37.
42.Akamatsu, H., et al., Intestinal metastasis from non-small-cell lung cancer initially detected by (1)F-fluorodeoxyglucose positron emission tomography. Jpn J Radiol, 2010. 28(9): p. 684-7.
43.Kobayashi, M., et al., Nuclear translocation of beta-catenin in colorectal cancer. Br J Cancer, 2000. 82(10): p. 1689-93.
44.Wang, H. and Q. Zhou, [E-cadherin/beta-catenin and the invasion and metastasis of lung cancer]. Zhongguo Fei Ai Za Zhi, 2010. 13(3): p. 254-9.
45.Tang, X., et al., [A study on the relationship between E-cadherin, beta-catenin expression and metastasis and prognosis in non-small cell lung cancer.]. Zhongguo Fei Ai Za Zhi, 2002. 5(4): p. 263-267.
46.Lai, T.Y., et al., beta-catenin plays a key role in metastasis of human hepatocellular carcinoma. Oncol Rep, 2011.
47.Ushio-Fukai, M. and Y. Nakamura, Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Lett, 2008. 266(1): p. 37-52.
48.Clerkin, J.S., et al., Mechanisms of ROS modulated cell survival during carcinogenesis. Cancer Lett, 2008. 266(1): p. 30-6.
49.Wu, X.J. and X. Hua, Targeting ROS: selective killing of cancer cells by a cruciferous vegetable derived pro-oxidant compound. Cancer Biol Ther, 2007. 6(5): p. 646-7.
50.Giles, G.I., The redox regulation of thiol dependent signaling pathways in cancer. Curr Pharm Des, 2006. 12(34): p. 4427-43.
51.Chiu, W.T., et al., Contribution of reactive oxygen species to migration/invasion of human glioblastoma cells U87 via ERK-dependent COX-2/PGE(2) activation. Neurobiol Dis, 2010. 37(1): p. 118-29.
52.Wu, W.S., et al., Reactive oxygen species mediated sustained activation of protein kinase C alpha and extracellular signal-regulated kinase for migration of human hepatoma cell Hepg2. Mol Cancer Res, 2006. 4(10): p. 747-58.
53.Nam, C., K. Doi, and H. Nakayama, Etoposide induces G2/M arrest and apoptosis in neural progenitor cells via DNA damage and an ATM/p53-related pathway. Histol Histopathol, 2010. 25(4): p. 485-93.
54.Cappella, P., et al., Miniaturizing bromodeoxyuridine incorporation enables the usage of flow cytometry for cell cycle analysis of adherent tissue culture cells for high throughput screening. Cytometry A, 2010. 77(10): p. 953-61.
55.Ouyang, G., et al., Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways. Cell Biol Int, 2009. 33(12): p. 1237-44.
56.Zhang, R., et al., Benzyl isothiocyanate-induced DNA damage causes G2/M cell cycle arrest and apoptosis in human pancreatic cancer cells. J Nutr, 2006. 136(11): p. 2728-34.
57.Geback, T., et al., TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays. Biotechniques, 2009. 46(4): p. 265-74.
58.Jorgensen, E.D., et al., DNA damage response induced by exposure of human lung adenocarcinoma cells to smoke from tobacco- and nicotine-free cigarettes. Cell Cycle, 2010. 9(11).
59.Darzynkiewicz, Z., D.H. Halicka, and T. Tanaka, Cytometric assessment of DNA damage induced by DNA topoisomerase inhibitors. Methods Mol Biol, 2009. 582: p. 145-53.
60.Bass, D.A., et al., Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol, 1983. 130(4): p. 1910-7.
61.Li, A., et al., Collagen type I regulates beta-catenin tyrosine phosphorylation and nuclear translocation to promote migration and proliferation of gastric carcinoma cells. Oncol Rep, 2010. 23(5): p. 1247-55.
62.Bourguignon, L.Y., et al., Hyaluronan-CD44 interaction with neural Wiskott-Aldrich syndrome protein (N-WASP) promotes actin polymerization and ErbB2 activation leading to beta-catenin nuclear translocation, transcriptional up-regulation, and cell migration in ovarian tumor cells. J Biol Chem, 2007. 282(2): p. 1265-80.
63.Bae, I.H., et al., Bcl-w promotes gastric cancer cell invasion by inducing matrix metalloproteinase-2 expression via phosphoinositide 3-kinase, Akt, and Sp1. Cancer Res, 2006. 66(10): p. 4991-5.
64.Qin, H., Y. Sun, and E.N. Benveniste, The transcription factors Sp1, Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells. J Biol Chem, 1999. 274(41): p. 29130-7.
65.Benelli, R., et al., The chemopreventive retinoid 4HPR impairs prostate cancer cell migration and invasion by interfering with FAK/AKT/GSK3beta pathway and beta-catenin stability. Mol Cancer, 2010. 9: p. 142.
66.Hung, W.C. and H.C. Chang, Indole-3-carbinol inhibits Sp1-induced matrix metalloproteinase-2 expression to attenuate migration and invasion of breast cancer cells. J Agric Food Chem, 2009. 57(1): p. 76-82.
67.Xin, D., et al., The MIF homologue D-dopachrome tautomerase promotes COX-2 expression through beta-catenin-dependent and -independent mechanisms. Mol Cancer Res, 2010. 8(12): p. 1601-9.
68.Estrada, Y., J. Dong, and L. Ossowski, Positive crosstalk between ERK and p38 in melanoma stimulates migration and in vivo proliferation. Pigment Cell Melanoma Res, 2009. 22(1): p. 66-76.
69.Hwang, Y.P., et al., Suppression of EGF-induced tumor cell migration and matrix metalloproteinase-9 expression by capsaicin via the inhibition of EGFR-mediated FAK/Akt, PKC/Raf/ERK, p38 MAPK, and AP-1 signaling. Mol Nutr Food Res, 2011. 55(4): p. 594-605.
70.Kondziella, D., et al., Lamotrigine increases the number of BrdU-labeled cellsinthe rat hippocampus. Neuroreport, 2010.
71.Jazayeri, A., et al., ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat Cell Biol, 2006. 8(1): p. 37-45.
72.Lukas, J., C. Lukas, and J. Bartek, Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair (Amst), 2004. 3(8-9): p. 997-1007.
73.Bartek, J. and J. Lukas, Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr Opin Cell Biol, 2001. 13(6): p. 738-47.
74.Wang, J. and J. Yi, Cancer cell killing via ROS: to increase or decrease, that is the question. Cancer Biol Ther, 2008. 7(12): p. 1875-84.
75.Lim, S.D., et al., Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate, 2005. 62(2): p. 200-7.
76.Ferraro, D., et al., Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS). Oncogene, 2006. 25(26): p. 3689-98.
77.Pelicano, H., D. Carney, and P. Huang, ROS stress in cancer cells and therapeutic implications. Drug Resist Updat, 2004. 7(2): p. 97-110.
78.Bangaru, M.L., et al., Curcumin (diferuloylmethane) induces apoptosis and blocks migration of human medulloblastoma cells. Anticancer Res, 2010. 30(2): p. 499-504.
79.Hoogeboom, D. and B.M.T. Burgering, Should I stay or should I go:[beta]-catenin decides under stress. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, 2009. 1796(2): p. 63-74.
80.Guo, Q., et al., NGX6 inhibits cell invasion and adhesion through suppression of Wnt/beta-catenin signal pathway in colon cancer. Acta Biochim Biophys Sin (Shanghai), 2010. 42(7): p. 450-6.
81.Kobayashi, T., et al., Transient gene silencing of galectin-3 suppresses pancreatic cancer cell migration and invasion through degradation of beta-catenin. Int J Cancer, 2011.
82.Lai, T.Y., et al., 17beta-estradiol inhibits prostaglandin E2-induced COX-2 expressions and cell migration by suppressing Akt and ERK1/2 signaling pathways in human LoVo colon cancer cells. Mol Cell Biochem, 2010. 342(1-2): p. 63-70.
83.Itoh, T., et al., Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res, 1998. 58(5): p. 1048-51.
84.Fojas de Borja, P., et al., Cyclin A-CDK phosphorylates Sp1 and enhances Sp1-mediated transcription. EMBO J, 2001. 20(20): p. 5737-47.
85.Jacamo, R., et al., FAK phosphorylation at Ser-843 inhibits Tyr-397 phosphorylation, cell spreading and migration. J Cell Physiol, 2007. 210(2): p. 436-44.
86.Hwang, Y.P., et al., Suppression of EGF-induced tumor cell migration and matrix metalloproteinase-9 expression by capsaicin via the inhibition of EGFR-mediated FAK/Akt, PKC/Raf/ERK, p38 MAPK, and AP-1 signaling. Mol Nutr Food Res, 2011.