臺灣博碩士論文加值系統

缺氧誘導性因子-1α(HIF-1α) 為一種對於氧分壓敏感的轉錄因子，在正常氧分壓下HIF-1α很快就被prolyl hydroxylase氫氧化進入26S蛋白酶體降解，當細胞處理缺氧環境時HIF-1α會累積並與HIF-1β形成複合物進行轉錄活化的作用；HIF-1α所調控的基因包括影響能量供給、細胞生長、血管新生等，被認為在腫瘤發育過程扮演促進者的角色。 米諾黴素(Minocycline)為一種廣效性四環黴素類的抗生素，藉由與原核生物之30S核醣體結合干擾蛋白質合成進而抑制細菌的生長；近年來許多研究指出米諾黴素除了抗菌外，對於真核細胞的發炎反應、細胞凋亡及血管新生皆有所影響，且能抑制腫瘤的生長與微血管生成；本研究希望能夠透過進一步實驗，觀察處於缺氧微環境的腫瘤細胞處理米諾黴素時，對於缺氧適應反應調控者、血管新生促進者－HIF-1α是否會造成影響及可能牽涉的相關訊息機轉。本研究採用非小細胞肺癌細胞株A549和H1299做為材料，以西方墨點法分析，發現在正常氧分壓下單獨處理米諾黴素，並不影響HIF-1α表現；但若使用氯化亞鈷(II)或0.5%氧分壓的缺氧環境下使HIF-1α大量表現時，前處理米諾黴素則可抑制HIF-1α累積的現象，以MTT試驗觀察米諾黴素並未對細胞產生毒性；米諾黴素不會影響HIF-1αmRNA的表現，而利用蛋白轉譯抑制劑cycloheximide觀察HIF-1α降解情況，顯示米諾黴素能加速HIF-1α蛋白的降解速率，的確米諾黴素造成缺氧誘導因子-1α累積表現減少的現象會隨著26S蛋白酶體抑制劑MG132的處理而消失；此外米諾黴素的處理不改變細胞中pVHL蛋白(E3 ubiquitin ligase)表現量但增加pVHL與HIF-1α間的鍵結程度；訊息傳遞路徑方面，米諾黴素能夠抑制由缺氧所活化之AKT、ERK磷酸化現象；HIF-1α下游基因：VEGF、MMP-2的表現也會受到米諾黴素的影響，並能藉由抑制HIF-1α的表現達到抑制內皮細胞管狀構造的形成。總結以上結果，米諾黴素直接或間接影響增加AKT、ERK訊息傳遞，促進pVHL蛋白與HIF-1α間的鍵結加速HIF-1α降解速率，並降低其下游基因表現及抑制血管新生體外模式－HUVECs管狀形成。

HIF-1α is an oxygen-regulated protein and is recognized as a positive factor to tumorigenesis. During nomoxia, HIF-1α is rapidly hydroxylated by prolyl hydroxylase then goes into 26S proteasome degradation pathway. During hypoxia, HIF-1α accumulates and dimerizes with HIF-1β to transactivate hypoxia-responsive gene expression. There are more than 100 HIF-1 downstream genes identified with varying functions, including glucose metabolism, angiogenesis, cell proliferation/apoptosis. Minocycline is a second-generation tetracycline, and remains widely used clinically. Several studies have demonstrated that it also affects many cellular functions relevant to human vascular diseases including inflammation, matrix remodeling and angiogenesis. Moreover, minocycline also can repress tumor growth and angiogenesis. In this study, we found that minocycline will not affect on HIF-1α expression during nomoxia ,but significantly inhibit HIF-1α accumulation during hypoxia and CoCl2 treatment without affecting cell viability. By using the translation inhibitor cycloheximide, we found that minocycline reduced HIF-1α protein expression via accelerating HIF-1α degradation rate, but had no significant effect on HIF-1α mRNA expression. Minocycline caused a decrease in the level of HIF-1α could be rescued by MG132. The pVHL(E3 ubiquitin ligase) protein levels were unchanged by minocycline, but pVHL binding to HIF-1α was increased. Minocycline also inhibited hypoxia-induced activation of Akt and ERK1/2. In addition, minocycline inhibited the mRNA level of hypoxia-responsive gene such as VEGF and MMP-2. Furthermore, Minocycline inhibited hypoxia-induced tubule formation in HUVECs. In conclusion, Minocycline can enhance pVHL binding to HIF-1α via Akt、ERK signaling pathway and thereby accelerate degradation of HIF-1α.This result leads to decrease VEGF and MMP-2 expression and inhibite hypoxia-induced endothelial cell tubule formation.

口試委員會審定書..……………………………………………….…………………….i
謝誌….……………………………………………….………………………………….ii
縮寫表(Abbreviations)..……………………………………………….………………iii
圖表目錄(Figures and Tables)…………………………...……………………………iv
中文摘要……………………………………………………………………………..... vi
英文摘要………………………………………………………………..…………...... vii
第一章 緒論(Introduction)
1.1腫瘤發育與缺氧…………………………………………………..……...………….2
1.2缺氧狀態的調節者－缺氧誘導因子-1(Hypoxia inducible factor-1；HIF-1)……….2
1.3缺氧誘導因子-1α(HIF-1α)的表現與調控……………………..……...……………4
1.4米諾黴素 (Minocycline)之背景、藥物動力學、毒理介紹..……...………………9
1.5米諾黴素今日之新應用…………………………………………..……...………10
1.6研究動機……………...……………………………………………...……………..13
第二章 實驗材料與方法 (Materials and Methods)
2.1實驗材料………………………………………………..……...……………..…….15
2.1.1藥品……………………………………………….…..……...……………..…….15
2.1.2抗體、酵素與試劑……………………………….…..……...……………..…….15
2.2實驗方法
2.2.1細胞培養和缺氧系統培養箱(Cell culture and Hypoxia treatment ) ……..…….16
2.2.2細胞毒性測試(Cell viability test/MTT assay) …….…..……...……………..…..17
2.2.3細胞總蛋白質液收集(Cell lysate collection) …….…..……...….…………..…..17
2.2.4西方墨點法(Western blot analysis) …….…..……...…………….…………..…..17
2.2.5免疫沉澱法(Immunoprecipitation)….…..……......…………….…………..…..18
2.2.6萃取細胞RNA(RNA extraction) …….…..……......…………….…………..…..18
2.2.7 反轉錄鏈鎖聚合酶反應(Reverse transcription polymerase chain reaction).…..19
2.2.8酵素免疫法測試VEGF蛋白表現(VEGF ELISA assay) ……….…………..…..20
2.2.9管狀形成實驗(In vitro matrigel capillaries formation) ……….…………..……..20
2.2.10統計分析(Statistic analysis) ……….…………..……………………………….20
第三章 實驗結果(Results)
3.1 在正常氧分壓下單獨處理米諾黴素不影響缺氧誘導因子1α表現…………….22
3.2 米諾黴素能抑制由氯化亞鈷(II)處理或處於缺氧狀態造成的缺氧誘導性因1α累積之現象……….…………..…………………………………………………...22
3.3 米諾黴素抑制缺氧誘導因子-1α累積之現象並非是由於細胞死亡所造成…....23
3.4 米諾黴素的處理會影響缺氧誘導因子1α的穩定性，加速其降解速率…...........24
3.5 米諾黴素抑制缺氧誘導因子1α累積之現象是藉由26S蛋白酶體路徑..............26
3.6 米諾黴素能增加pVHL蛋白與缺氧誘導因子-1α之間的鍵結但不影響細胞中pVHL蛋白的表現量…………..………………………………………………….27
3.7 米諾黴素能抑制由缺氧狀態刺激所引起的AKT與ERK磷酸………….……...28
3.8 米諾黴素能抑制由缺氧狀態所誘發的血管內皮生長因子表現量增加的現象..29
3.9 米諾黴素能抑制缺氧狀態誘導的人類臍靜脈內皮細胞管狀構造形成作用….31
第四章 討論(Discussion)
4.1 米諾黴素抑制由缺氧狀態所誘發的缺氧誘導因子-1α累積現象之可能機轉…35
4.2 米諾黴素抑制由缺氧狀態活化之Akt和ERK磷酸化…………………………38
4.3 米諾黴素對於缺氧狀態下血管新生相關機制的影響…….…………………….39
4.4 米諾黴素與缺氧狀態對A549與H1299細胞MMP-2 mRNA表現的影響…….40
第五章 結論(Conclusion) ……………………………………...……………………43
參考文獻(References) …………………………………………………………………45
圖表集(Figures and Tables) ….………………………………………………………..61

Agulnik, M., and Siu, L.L. (2004). An update on the systemic therapy of malignant salivary gland cancers: role of chemotherapy and molecular targeted agents. Curr Med Chem Anticancer Agents 4, 543-551.

Alfonso-Jaume, M.A., Bergman, M.R., Mahimkar, R., Cheng, S., Jin, Z.Q., Karliner, J.S., and Lovett, D.H. (2006). Cardiac ischemia-reperfusion injury induces matrix metalloproteinase-2 expression through the AP-1 components FosB and JunB. Am J Physiol Heart Circ Physiol 291, H1838-1846.

Alvarez-Tejado, M., Alfranca, A., Aragones, J., Vara, A., Landazuri, M.O., and del Peso, L. (2002). Lack of evidence for the involvement of the phosphoinositide 3-kinase/Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. J Biol Chem 277, 13508-13517.

Amato Giaccia, B.G.S.a.R.S.J. (2003). HIF-1 as a target for drug development. NATURE REVIEWS 2, 1-9.
An, W.G., Kanekal, M., Simon, M.C., Maltepe, E., Blagosklonny, M.V., and Neckers, L.M. (1998). Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature 392, 405-408.

Arnesen, T., Kong, X., Evjenth, R., Gromyko, D., Varhaug, J.E., Lin, Z., Sang, N., Caro, J., and Lillehaug, J.R. (2005). Interaction between HIF-1 alpha (ODD) and hARD1 does not induce acetylation and destabilization of HIF-1 alpha. FEBS Lett 579, 6428-6432.

Ashcroft, M., Taya, Y., and Vousden, K.H. (2000). Stress signals utilize multiple pathways to stabilize p53. Mol Cell Biol 20, 3224-3233.

Bae, M.K., Jeong, J.W., Kim, S.H., Kim, S.Y., Kang, H.J., Kim, D.M., Bae, S.K., Yun, I., Trentin, G.A., Rozakis-Adcock, M., et al. (2005). Tid-1 interacts with the von Hippel-Lindau protein and modulates angiogenesis by destabilization of HIF-1alpha. Cancer Res 65, 2520-2525.

Bae, S.H., Jeong, J.W., Park, J.A., Kim, S.H., Bae, M.K., Choi, S.J., and Kim, K.W. (2004). Sumoylation increases HIF-1alpha stability and its transcriptional activity. Biochem Biophys Res Commun 324, 394-400.
Bardos, J.I., and Ashcroft, M. (2005). Negative and positive regulation of HIF-1: a complex network. Biochim Biophys Acta 1755, 107-120.

Bardos, J.I., Chau, N.M., and Ashcroft, M. (2004). Growth factor-mediated induction of HDM2 positively regulates hypoxia-inducible factor 1alpha expression. Mol Cell Biol 24, 2905-2914.

Bennett, L.L., Jr., Ward, V.L., and Brockman, R.W. (1965). Inhibition of protein synthesis in vitro by cycloheximide and related glutarimide antibiotics. Biochim Biophys Acta 103, 478-485.

Bergman, M.R., Cheng, S., Honbo, N., Piacentini, L., Karliner, J.S., and Lovett, D.H. (2003). A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers. Biochem J 369, 485-496.

Berra, E., Benizri, E., Ginouves, A., Volmat, V., Roux, D., and Pouyssegur, J. (2003). HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia. EMBO J 22, 4082-4090.

Berra, E., Richard, D.E., Gothie, E., and Pouyssegur, J. (2001). HIF-1-dependent transcriptional activity is required for oxygen-mediated HIF-1alpha degradation. FEBS Lett 491, 85-90.

Bilton, R., Mazure, N., Trottier, E., Hattab, M., Dery, M.A., Richard, D.E., Pouyssegur, J., and Brahimi-Horn, M.C. (2005). Arrest-defective-1 protein, an acetyltransferase, does not alter stability of hypoxia-inducible factor (HIF)-1alpha and is not induced by hypoxia or HIF. J Biol Chem 280, 31132-31140.

Blagosklonny, M.V., An, W.G., Romanova, L.Y., Trepel, J., Fojo, T., and Neckers, L. (1998). p53 inhibits hypoxia-inducible factor-stimulated transcription. J Biol Chem 273, 11995-11998.

Brahimi-Horn, C., Mazure, N., and Pouyssegur, J. (2005). Signalling via the hypoxia-inducible factor-1alpha requires multiple posttranslational modifications. Cell Signal 17, 1-9.

Bruick, R.K., and McKnight, S.L. (2001). A conserved family of prolyl-4-hydroxylases that modify HIF. Science 294, 1337-1340.

Brusselmans, K., Compernolle, V., Tjwa, M., Wiesener, M.S., Maxwell, P.H., Collen, D., and Carmeliet, P. (2003). Heterozygous deficiency of hypoxia-inducible factor-2alpha protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia. J Clin Invest 111, 1519-1527.

Bussolino, F., Mantovani, A., and Persico, G. (1997). Molecular mechanisms of blood vessel formation. Trends Biochem Sci 22, 251-256.

Cai, Z., Manalo, D.J., Wei, G., Rodriguez, E.R., Fox-Talbot, K., Lu, H., Zweier, J.L., and Semenza, G.L. (2003). Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury. Circulation 108, 79-85.

Castanares, M., Vera, Y., Erkkila, K., Kyttanen, S., Lue, Y., Dunkel, L., Wang, C., Swerdloff, R.S., and Hikim, A.P. (2005). Minocycline up-regulates BCL-2 levels in mitochondria and attenuates male germ cell apoptosis. Biochem Biophys Res Commun 337, 663-669.

Chang, C.C., Lin, M.T., Lin, B.R., Jeng, Y.M., Chen, S.T., Chu, C.Y., Chen, R.J., Chang, K.J., Yang, P.C., and Kuo, M.L. (2006). Effect of connective tissue growth factor on hypoxia-inducible factor 1alpha degradation and tumor angiogenesis. J Natl Cancer Inst 98, 984-995.

Cheng, J., Kang, X., Zhang, S., and Yeh, E.T. (2007). SUMO-specific protease 1 is essential for stabilization of HIF1alpha during hypoxia. Cell 131, 584-595.

Choi, H.J., Eun, J.S., Kim, B.G., Kim, S.Y., Jeon, H., and Soh, Y. (2006). Vitexin, an HIF-1alpha inhibitor, has anti-metastatic potential in PC12 cells. Mol Cells 22, 291-299.
Choi, H.J., Song, B.J., Gong, Y.D., Gwak, W.J., and Soh, Y. (2008). Rapid degradation of hypoxia-inducible factor-1alpha by KRH102053, a new activator of prolyl hydroxylase 2. Br J Pharmacol 154, 114-125.

Cohen-Jonathan, E., Evans, S.M., Koch, C.J., Muschel, R.J., McKenna, W.G., Wu, J., and Bernhard, E.J. (2001). The farnesyltransferase inhibitor L744,832 reduces hypoxia in tumors expressing activated H-ras. Cancer Res 61, 2289-2293.

Conrad, P.W., Freeman, T.L., Beitner-Johnson, D., and Millhorn, D.E. (1999a). EPAS1 trans-activation during hypoxia requires p42/p44 MAPK. J Biol Chem 274, 33709-33713.

Conrad, P.W., Rust, R.T., Han, J., Millhorn, D.E., and Beitner-Johnson, D. (1999b). Selective activation of p38alpha and p38gamma by hypoxia. Role in regulation of cyclin D1 by hypoxia in PC12 cells. J Biol Chem 274, 23570-23576.

Cunha, B.A. (2006). New uses for older antibiotics: nitrofurantoin, amikacin, colistin, polymyxin B, doxycycline, and minocycline revisited. Med Clin North Am 90, 1089-1107.

Duivenvoorden, W.C., Hirte, H.W., and Singh, G. (1997). Use of tetracycline as an inhibitor of matrix metalloproteinase activity secreted by human bone-metastasizing cancer cells. Invasion Metastasis 17, 312-322.

Ema, M., Hirota, K., Mimura, J., Abe, H., Yodoi, J., Sogawa, K., Poellinger, L., and Fujii-Kuriyama, Y. (1999). Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. EMBO J 18, 1905-1914.

Epstein, A.C., Gleadle, J.M., McNeill, L.A., Hewitson, K.S., O''Rourke, J., Mole, D.R., Mukherji, M., Metzen, E., Wilson, M.I., Dhanda, A., et al. (2001). C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107, 43-54.

Eun-Jin Yeoa, Y.-S.C., Jong-Wan Park (2004). New anticancer strategies targeting HIF-1. Biochemical Pharmacology 68, 1061-1069.

Fang, J., Xia, C., Cao, Z., Zheng, J.Z., Reed, E., and Jiang, B.H. (2005). Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways. FASEB J 19, 342-353.

Fang, J., Zhou, Q., Liu, L.Z., Xia, C., Hu, X., Shi, X., and Jiang, B.H. (2007). Apigenin inhibits tumor angiogenesis through decreasing HIF-1alpha and VEGF expression. Carcinogenesis 28, 858-864.

Folkman, J. (1996). Fighting cancer by attacking its blood supply. Sci Am 275, 150-154.
Furst, D.E. (1998). Update on clinical trials in the rheumatic diseases. Curr Opin Rheumatol 10, 123-128.

Gasparini, G., and Gion, M. (2000). Molecular-targeted anticancer therapy: challenges related to study design and choice of proper endpoints. Cancer J 6, 117-131.

Gnarra, J.R., Ward, J.M., Porter, F.D., Wagner, J.R., Devor, D.E., Grinberg, A., Emmert-Buck, M.R., Westphal, H., Klausner, R.D., and Linehan, W.M. (1997). Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice. Proc Natl Acad Sci U S A 94, 9102-9107.

Gong, Y.D., Seo, J.S., Chon, Y.S., Hwang, J.Y., Park, J.Y., and Yoo, S.E. (2003). Construction of 6-amino-2,2-dimethyl-3,4,6-trisubstituted-2H-1-benzopyran library by solid phase synthesis. J Comb Chem 5, 577-589.

Grevstad, H.J., and Boe, O.E. (1995). Effect of doxycycline on surgically induced osteoclast recruitment in the rat. Eur J Oral Sci 103, 156-159.

Grossman, S.R. (2001). p300/CBP/p53 interaction and regulation of the p53 response. Eur J Biochem 268, 2773-2778.

Grossman, S.R., Perez, M., Kung, A.L., Joseph, M., Mansur, C., Xiao, Z.X., Kumar, S., Howley, P.M., and Livingston, D.M. (1998). p300/MDM2 complexes participate in MDM2-mediated p53 degradation. Mol Cell 2, 405-415.

Gu, Y.Z., Moran, S.M., Hogenesch, J.B., Wartman, L., and Bradfield, C.A. (1998). Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha. Gene Expr 7, 205-213.

Hanemaaijer, R., Sorsa, T., Konttinen, Y.T., Ding, Y., Sutinen, M., Visser, H., van Hinsbergh, V.W., Helaakoski, T., Kainulainen, T., Ronka, H., et al. (1997). Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline. J Biol Chem 272, 31504-31509.

Hanemaaijer, R., Visser, H., Koolwijk, P., Sorsa, T., Salo, T., Golub, L.M., and van Hinsbergh, V.W. (1998). Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Adv Dent Res 12, 114-118.
Heo, K., Cho, Y.J., Cho, K.J., Kim, H.W., Kim, H.J., Shin, H.Y., Lee, B.I., and Kim, G.W. (2006). Minocycline inhibits caspase-dependent and -independent cell death pathways and is neuroprotective against hippocampal damage after treatment with kainic acid in mice. Neurosci Lett 398, 195-200.

Hewitson, K.S., McNeill, L.A., Riordan, M.V., Tian, Y.M., Bullock, A.N., Welford, R.W., Elkins, J.M., Oldham, N.J., Bhattacharya, S., Gleadle, J.M., et al. (2002). Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem 277, 26351-26355.

Hoffman, E.C., Reyes, H., Chu, F.F., Sander, F., Conley, L.H., Brooks, B.A., and Hankinson, O. (1991). Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252, 954-958.

Hu, C.J., Sataur, A., Wang, L., Chen, H., and Simon, M.C. (2007). The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha. Mol Biol Cell 18, 4528-4542.

Hu, C.J., Wang, L.Y., Chodosh, L.A., Keith, B., and Simon, M.C. (2003). Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol 23, 9361-9374.

Ivan, M., and Kaelin, W.G., Jr. (2001). The von Hippel-Lindau tumor suppressor protein. Curr Opin Genet Dev 11, 27-34.

Iyer, N.V., Kotch, L.E., Agani, F., Leung, S.W., Laughner, E., Wenger, R.H., Gassmann, M., Gearhart, J.D., Lawler, A.M., Yu, A.Y., et al. (1998). Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 12, 149-162.

Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., et al. (2001). Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468-472.

Jeong, J.W., Bae, M.K., Ahn, M.Y., Kim, S.H., Sohn, T.K., Bae, M.H., Yoo, M.A., Song, E.J., Lee, K.J., and Kim, K.W. (2002). Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation. Cell 111, 709-720.
Jiang, B.H., Agani, F., Passaniti, A., and Semenza, G.L. (1997). V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression. Cancer Res 57, 5328-5335.

Karni, R., Dor, Y., Keshet, E., Meyuhas, O., and Levitzki, A. (2002). Activated pp60c-Src leads to elevated hypoxia-inducible factor (HIF)-1alpha expression under normoxia. J Biol Chem 277, 42919-42925.

Ke, Q., and Costa, M. (2006). Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 70, 1469-1480.

Keilhoff, G., Langnaese, K., Wolf, G., and Fansa, H. (2007). Inhibiting effect of minocycline on the regeneration of peripheral nerves. Dev Neurobiol 67, 1382-1395.

Kleinman, H.K., McGarvey, M.L., Liotta, L.A., Robey, P.G., Tryggvason, K., and Martin, G.R. (1982). Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21, 6188-6193.

Kline, D.D., Peng, Y.J., Manalo, D.J., Semenza, G.L., and Prabhakar, N.R. (2002). Defective carotid body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1 alpha. Proc Natl Acad Sci U S A 99, 821-826.

Koumenis, C., Alarcon, R., Hammond, E., Sutphin, P., Hoffman, W., Murphy, M., Derr, J., Taya, Y., Lowe, S.W., Kastan, M., et al. (2001). Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation. Mol Cell Biol 21, 1297-1310.

Kozma, S.C., and Thomas, G. (2002). Regulation of cell size in growth, development and human disease: PI3K, PKB and S6K. Bioessays 24, 65-71.

Kraus, R.L., Pasieczny, R., Lariosa-Willingham, K., Turner, M.S., Jiang, A., and Trauger, J.W. (2005). Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity. J Neurochem 94, 819-827.

Kwon, S.J., Song, J.J., and Lee, Y.J. (2005). Signal pathway of hypoxia-inducible factor-1alpha phosphorylation and its interaction with von Hippel-Lindau tumor suppressor protein during ischemia in MiaPaCa-2 pancreatic cancer cells. Clin Cancer Res 11, 7607-7613.

Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J., and Whitelaw, M.L. (2002). Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 295, 858-861.

Lee, K., Roth, R.A., and LaPres, J.J. (2007). Hypoxia, drug therapy and toxicity. Pharmacol Ther 113, 229-246.

Li, C.H. (2005). Studies on the Mechanisms of Chloramphenicol Induced Senescence and Carcinogenesis. 國立台灣大學毒理學研究所博士論文.

Li, C.H., Tzeng, S.L., Cheng, Y.W., and Kang, J.J. (2005). Chloramphenicol-induced mitochondrial stress increases p21 expression and prevents cell apoptosis through a p21-dependent pathway. J Biol Chem 280, 26193-26199.

Lim, J.H., Park, J.W., and Chun, Y.S. (2006a). Human arrest defective 1 acetylates and activates beta-catenin, promoting lung cancer cell proliferation. Cancer Res 66, 10677-10682.

Lim, J.H., Park, J.W., Kim, M.S., Park, S.K., Johnson, R.S., and Chun, Y.S. (2006b). Bafilomycin induces the p21-mediated growth inhibition of cancer cells under hypoxic conditions by expressing hypoxia-inducible factor-1alpha. Mol Pharmacol 70, 1856-1865.

Lim, J.H., Park, J.W., Kim, S.J., Kim, M.S., Park, S.K., Johnson, R.S., and Chun, Y.S. (2007). ATP6V0C competes with von Hippel-Lindau protein in hypoxia-inducible factor 1alpha (HIF-1alpha) binding and mediates HIF-1alpha expression by bafilomycin A1. Mol Pharmacol 71, 942-948.

Lin, S., Zhang, Y., Dodel, R., Farlow, M.R., Paul, S.M., and Du, Y. (2001). Minocycline blocks nitric oxide-induced neurotoxicity by inhibition p38 MAP kinase in rat cerebellar granule neurons. Neurosci Lett 315, 61-64.

Liu, Y.V., Baek, J.H., Zhang, H., Diez, R., Cole, R.N., and Semenza, G.L. (2007). RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha. Mol Cell 25, 207-217.

Lorne M. Golub, N.S.R., and Thomas F. McNamara (1991). Tetracyclines Inhibit Connective Tissue Breakdown: New Therapeutic Implications for an Old Family of Drugs. Critical Reviews in Oral Biology and Medicine 2, 297-322.

Makino, Y., Kanopka, A., Wilson, W.J., Tanaka, H., and Poellinger, L. (2002). Inhibitory PAS domain protein (IPAS) is a hypoxia-inducible splicing variant of the hypoxia-inducible factor-3alpha locus. J Biol Chem 277, 32405-32408.

Maltepe, E., Keith, B., Arsham, A.M., Brorson, J.R., and Simon, M.C. (2000). The role of ARNT2 in tumor angiogenesis and the neural response to hypoxia. Biochem Biophys Res Commun 273, 231-238.

Maltepe, E., Schmidt, J.V., Baunoch, D., Bradfield, C.A., and Simon, M.C. (1997). Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 386, 403-407.

Mason, G.G., Witte, A.M., Whitelaw, M.L., Antonsson, C., McGuire, J., Wilhelmsson, A., Poellinger, L., and Gustafsson, J.A. (1994). Purification of the DNA binding form of dioxin receptor. Role of the Arnt cofactor in regulation of dioxin receptor function. J Biol Chem 269, 4438-4449.

Matsuki, S., Iuchi, Y., Ikeda, Y., Sasagawa, I., Tomita, Y., and Fujii, J. (2003). Suppression of cytochrome c release and apoptosis in testes with heat stress by minocycline. Biochem Biophys Res Commun 312, 843-849.

Maxwell, P.H., Wiesener, M.S., Chang, G.W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R., and Ratcliffe, P.J. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271-275.

Mazure, N.M., Chen, E.Y., Yeh, P., Laderoute, K.R., and Giaccia, A.J. (1996). Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression. Cancer Res 56, 3436-3440.

McNeill, L.A., Hewitson, K.S., Claridge, T.D., Seibel, J.F., Horsfall, L.E., and Schofield, C.J. (2002). Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the beta-carbon of asparagine-803. Biochem J 367, 571-575.

Metz, L.M., Zhang, Y., Yeung, M., Patry, D.G., Bell, R.B., Stoian, C.A., Yong, V.W., Patten, S.B., Duquette, P., Antel, J.P., et al. (2004). Minocycline reduces gadolinium-enhancing magnetic resonance imaging lesions in multiple sclerosis. Ann Neurol 55, 756.

Minet, E., Michel, G., Mottet, D., Raes, M., and Michiels, C. (2001). Transduction pathways involved in Hypoxia-Inducible Factor-1 phosphorylation and activation. Free Radic Biol Med 31, 847-855.

Moeller, B.J., Dreher, M.R., Rabbani, Z.N., Schroeder, T., Cao, Y., Li, C.Y., and Dewhirst, M.W. (2005). Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity. Cancer Cell 8, 99-110.

Morimoto, N., Shimazawa, M., Yamashima, T., Nagai, H., and Hara, H. (2005). Minocycline inhibits oxidative stress and decreases in vitro and in vivo ischemic neuronal damage. Brain Res 1044, 8-15.

NELSON, M.L. (1998). Chemical and biological dynamics of tetracyclines. Adv Dent Res 12, 5-11.

Neshat, M.S., Mellinghoff, I.K., Tran, C., Stiles, B., Thomas, G., Petersen, R., Frost, P., Gibbons, J.J., Wu, H., and Sawyers, C.L. (2001). Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A 98, 10314-10319.

O''Dell, J.R., Blakely, K.W., Mallek, J.A., Eckhoff, P.J., Leff, R.D., Wees, S.J., Sems, K.M., Fernandez, A.M., Palmer, W.R., Klassen, L.W., et al. (2001). Treatment of early seropositive rheumatoid arthritis: a two-year, double-blind comparison of minocycline and hydroxychloroquine. Arthritis Rheum 44, 2235-2241.
Ohbayashi, H. (2002). Matrix metalloproteinases in lung diseases. Curr Protein Pept Sci 3, 409-421.

Parangi, S., O''Reilly, M., Christofori, G., Holmgren, L., Grosfeld, J., Folkman, J., and Hanahan, D. (1996). Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc Natl Acad Sci U S A 93, 2002-2007.

Paul, S.A., Simons, J.W., and Mabjeesh, N.J. (2004). HIF at the crossroads between ischemia and carcinogenesis. J Cell Physiol 200, 20-30.

Peng, P.L., Hsieh, Y.S., Wang, C.J., Hsu, J.L., and Chou, F.P. (2006). Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Toxicol Appl Pharmacol 214, 8-15.

Philip, S., and Kundu, G.C. (2003). Osteopontin induces nuclear factor kappa B-mediated promatrix metalloproteinase-2 activation through I kappa B alpha /IKK signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways. J Biol Chem 278, 14487-14497.

Pi, R., Li, W., Lee, N.T., Chan, H.H., Pu, Y., Chan, L.N., Sucher, N.J., Chang, D.C., Li, M., and Han, Y. (2004). Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways. J Neurochem 91, 1219-1230.

Podar, K., Tai, Y.T., Davies, F.E., Lentzsch, S., Sattler, M., Hideshima, T., Lin, B.K., Gupta, D., Shima, Y., Chauhan, D., et al. (2001). Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration. Blood 98, 428-435.

Powers, M.V., and Workman, P. (2006). Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors. Endocr Relat Cancer 13 Suppl 1, S125-135.

Powis, G., and Kirkpatrick, L. (2004). Hypoxia inducible factor-1alpha as a cancer drug target. Mol Cancer Ther 3, 647-654.

Rankin, E.B., and Giaccia, A.J. (2008). The role of hypoxia-inducible factors in tumorigenesis. Cell Death Differ 15, 678-685.

Redin, G.S. (1966). Antibacterial activity in mice of minocycline, a new tetracycline. Antimicrob Agents Chemother (Bethesda) 6, 371-376.

Rifkin, B.R., Vernillo, A.T., and Golub, L.M. (1993). Blocking periodontal disease progression by inhibiting tissue-destructive enzymes: a potential therapeutic role for tetracyclines and their chemically-modified analogs. J Periodontol 64, 819-827.

Rudek, M.A., Figg, W.D., Dyer, V., Dahut, W., Turner, M.L., Steinberg, S.M., Liewehr, D.J., Kohler, D.R., Pluda, J.M., and Reed, E. (2001a). Phase I clinical trial of oral COL-3, a matrix metalloproteinase inhibitor, in patients with refractory metastatic cancer. J Clin Oncol 19, 584-592.

Rudek, M.A., Horne, M., Figg, W.D., Dahut, W., Dyer, V., Pluda, J.M., and Reed, E. (2001b). Reversible sideroblastic anemia associated with the tetracycline analogue COL-3. Am J Hematol 67, 51-53.

Ryan, H.E., Lo, J., and Johnson, R.S. (1998). HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J 17, 3005-3015.

Saikali, Z., and Singh, G. (2003). Doxycycline and other tetracyclines in the treatment of bone metastasis. Anticancer Drugs 14, 773-778.

Scarabelli, T.M., Stephanou, A., Pasini, E., Gitti, G., Townsend, P., Lawrence, K., Chen-Scarabelli, C., Saravolatz, L., Latchman, D., Knight, R., et al. (2004). Minocycline inhibits caspase activation and reactivation, increases the ratio of XIAP to smac/DIABLO, and reduces the mitochondrial leakage of cytochrome C and smac/DIABLO. J Am Coll Cardiol 43, 865-874.

Semenza, G.L., and Wang, G.L. (1992). A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 12, 5447-5454.

Tamargo, R.J., Bok, R.A., and Brem, H. (1991). Angiogenesis inhibition by minocycline. Cancer Res 51, 672-675.

Tanguay, R.L., Andreasen, E., Heideman, W., and Peterson, R.E. (2000). Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions. Biochim Biophys Acta 1494, 117-128.

Temes, E., Martin-Puig, S., Acosta-Iborra, B., Castellanos, M.C., Feijoo-Cuaresma, M., Olmos, G., Aragones, J., and Landazuri, M.O. (2005). Activation of HIF-prolyl hydroxylases by R59949, an inhibitor of the diacylglycerol kinase. J Biol Chem 280, 24238-24244.
Terranova, V.P., Liotta, L.A., Russo, R.G., and Martin, G.R. (1982). Role of laminin in the attachment and metastasis of murine tumor cells. Cancer Res 42, 2265-2269.

Tikka, T., Fiebich, B.L., Goldsteins, G., Keinanen, R., and Koistinaho, J. (2001). Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 21, 2580-2588.

Wang, J., Wei, Q., Wang, C.Y., Hill, W.D., Hess, D.C., and Dong, Z. (2004). Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 279, 19948-19954.

Wang, X., Zhu, S., Drozda, M., Zhang, W., Stavrovskaya, I.G., Cattaneo, E., Ferrante, R.J., Kristal, B.S., and Friedlander, R.M. (2003). Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington''s disease. Proc Natl Acad Sci U S A 100, 10483-10487.

Welsh, S.J., Bellamy, W.T., Briehl, M.M., and Powis, G. (2002). The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis. Cancer Res 62, 5089-5095.

Welsh, S.J., Williams, R.R., Birmingham, A., Newman, D.J., Kirkpatrick, D.L., and Powis, G. (2003). The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit hypoxia-induced factor 1alpha and vascular endothelial growth factor formation. Mol Cancer Ther 2, 235-243.

Wewer, U.M., Liotta, L.A., Jaye, M., Ricca, G.A., Drohan, W.N., Claysmith, A.P., Rao, C.N., Wirth, P., Coligan, J.E., Albrechtsen, R., et al. (1986). Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin. Proc Natl Acad Sci U S A 83, 7137-7141.

Whitelaw, M.L., Gustafsson, J.A., and Poellinger, L. (1994). Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation. Mol Cell Biol 14, 8343-8355.

Whiteway, M., and Szostak, J.W. (1985). The ARD1 gene of yeast functions in the switch between the mitotic cell cycle and alternative developmental pathways. Cell 43, 483-492.

Wiesener, M.S., Jurgensen, J.S., Rosenberger, C., Scholze, C.K., Horstrup, J.H., Warnecke, C., Mandriota, S., Bechmann, I., Frei, U.A., Pugh, C.W., et al. (2003). Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs. FASEB J 17, 271-273.

Williams, S., Barnes, J., Wakisaka, A., Ogasa, H., and Liang, C.T. (1999). Treatment of osteoporosis with MMP inhibitors. Ann N Y Acad Sci 878, 191-200.
Williams, S., Wakisaka, A., Zeng, Q.Q., Barnes, J., Martin, G., Wechter, W.J., and Liang, C.T. (1996). Minocycline prevents the decrease in bone mineral density and trabecular bone in ovariectomized aged rats. Bone 19, 637-644.

Williams, S., Wakisaka, A., Zeng, Q.Q., Barnes, J., Seyedin, S., Martin, G., Wechter, W.J., and Liang, C.T. (1998). Effect of minocycline on osteoporosis. Adv Dent Res 12, 71-75.

Yamakawa, M., Liu, L.X., Date, T., Belanger, A.J., Vincent, K.A., Akita, G.Y., Kuriyama, T., Cheng, S.H., Gregory, R.J., and Jiang, C. (2003). Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors. Circ Res 93, 664-673.

Yao, J.S., Chen, Y., Zhai, W., Xu, K., Young, W.L., and Yang, G.Y. (2004). Minocycline exerts multiple inhibitory effects on vascular endothelial growth factor-induced smooth muscle cell migration: the role of ERK1/2, PI3K, and matrix metalloproteinases. Circ Res 95, 364-371.

Ye V. Liu, G.L.S. (2007). RACK1 vs. HSP90 Competition for HIF-1α Degradation vs. Stabilization. Cell Cycle 6, 656-659.

Yrjänheikki, J., S A, J., Keinanen, R., Pellikka, M., Hokfelt, T., and Koistinaho, J. (1998). Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci 95, 15769-15774.

Yrjänheikki, J., Tikka, T., Keinanen, R., Goldsteins, G., Chan, P.H., and Koistinaho, J. (1999). A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci 96, 13496-13500.
Yu, F., White, S.B., Zhao, Q., and Lee, F.S. (2001). Dynamic, site-specific interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor protein. Cancer Res 61, 4136-4142.

Yuan, Y., Hilliard, G., Ferguson, T., and Millhorn, D.E. (2003). Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 278, 15911-15916.

Zhang, Q., Tang, X., Lu, Q., Zhang, Z., Rao, J., and Le, A.D. (2006). Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia- and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Mol Cancer Ther 5, 1227-1238.