STUDIES OF THE ANTICANCER POTENTIAL OF ANDROGRAPHOLIDE IN HUMAN CANCER CELLS ZHOU JING (M. Med. China Academy of Chinese Medical Sciences; P. R. China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF EPIDEMIOLOGY AND PUBLIC HEALTH NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to express my deepest respect and acknowledgements to my supervisor, Prof. Shen Han-Ming, for his professional and enthusiastic guidance, as well as the encouragement, patience and instructive discussions throughout my study. I also would like to gratefully acknowledge Prof. Ong Choon-Nam for his consistent support and constructive suggestions on my study. Their guidance not only introduced me into this exciting biological area, but also taught me the right way of doing scientific research. What I have learned from them will benefit my future career and life. It was also a great pleasure for me to study in such a warm and harmonious family of Department of Epidemiology and Public Health. I was surrounded by a group of friendly people who helped me carrying out my study smoothly. I would like to thank Prof. David Koh for his general guidance and support during my study in this department. A special thank goes to our laboratory staff: Mr. Ong Her Yam, Mr. Ong Yeong Bing, Ms. Zhao Min and Ms. Su Jin for their technical support and kind help in the process of laboratory work. I also would like to extend my appreciation to my bench mates Dr. Zhang Siyuan, Dr. Won Yen Kim, Dr. Huang Qing, Dr. Shi Ranxin, Ms. Ong Chye Sun, Mr. Wu Youtong, Ms. Tan Huiling, Ms Shi Jie, Ms Zhuang Qiushi, Mr. Zhao Wei and Ms. Ng Shukie for their useful comments and suggestions on my study. I would also like to thank all other staff and graduate students in our department, for their generous help and encouragement. Especially, I would like to express my deepest appreciation to my husband, my parents and dear sister, for their love, understanding and continuing support. ii TABLE OF CONTENTS Title Page Acknowledgements ii Table of Contents iii Summary ix List of Tables and Figures xi Abbreviations xiii List of Publications xvii CHAPTER INTRODUCTION 1.1 Andrographolide (Andro) 1.1.1 Andrographis paniculata and Andro 1.1.2 Chemical structure and metabolism of Andro 1.1.3 Pharmacological properties of Andro 1.1.3.1 Inhibitory effects on inflammation 1.1.3.2 Immuno-regulatory effects 1.1.3.3 Anti-HIV effects 1.1.3.4 Hepatoprotective effects 1.1.3.5 Cardiovascular protective effect 1.1.3.6 Anti-diabetes effects 1.1.3.7 Anticancer potential of Andro 10 1.1.3.7.1 Inhibition of cancer cell proliferation and induction of cell cycle arrest 1.1.3.7.2 Induction of apoptotic cell death 10 11 iii 1.1.3.7.3 Anti-angiogenesis, anti-adhesion and other effects 1.1.4 Molecular mechanisms involved in the effects of Andro 12 12 1.1.4.1 Effect on NF-κB signaling pathway 12 1.1.4.2 Effects on Mitogen-activated protein kinase (MAPK) pathway 14 1.1.4.3 Effects on AKT/PKB survival pathways 15 1.2 Apoptosis and Apoptosis regulation 17 1.2.1 General introduction about apoptosis 17 1.2.2 Caspases 21 1.2.3 Bcl-2 protein family and mitochondria 22 1.2.3.1 Bcl-2 protein family 22 1.2.3.2 The central role of mitochondria in apoptosis process 24 1.2.3.3 Mechanisms of Bcl-2 proteins regulating apoptosis at mitochondrial level 1.2.4 p53 26 29 1.2.4.1 Regulation of p53 expression and function in apoptosis 29 1.2.4.2 Role of p53 in apoptosis 31 1.2.5 Reactive Oxygen Species (ROS) 33 1.2.5.1 ROS and ROS-activated molecules 33 1.2.5.2 Involvement of ROS in apoptosis 35 1.2.6 STATs signaling pathway 38 1.2.6.1 STAT family members and their functional domains 38 1.2.6.2 Activation of STATs signaling 39 1.2.6.3 Negative regulation of STATs signaling 40 1.2.6.4 STAT3 as an oncogene in cancer therapy 42 1.2.7 Dys-regulated apoptosis in cancer 44 iv 1.2.8 TRAIL and TRAIL-induced apoptosis 45 1.2.8.1 Introduction 45 1.2.8.2 Regulation of TRAIL-induced cell death 46 1.3 Objectives of the study 49 CHAPTER THE CRITICAL ROLE OF PRO-APOPTOTIC BCL-2 FAMILY MEMBERS IN ANDRO-INDUCED APOPTOSIS IN HUMAN CANCER CELLS 2.1 Introduction 52 2.2 Materials and Methods 54 2.2.1 Chemicals and reagents 54 2.2.2 Cell culture and treatments 54 2.2.3 Detection of Apoptosis 55 2.2.4 Caspase 3/7 activity assay 55 2.2.5 Cell subfractionation 56 2.2.6 Immunoprecipitation and western blot 56 2.2.7 Transient transfection and siRNA-mediated protein knock-down 57 2.2.8 Immunofluorescence and confocal microscopy 57 2.2.9 Statistical analysis 58 2.3 Results 58 2.3.1 Andro induces apoptosis in human cancer cells 58 2.3.2 Caspase cascade in Andro-induced apoptosis 59 2.3.3 Andro induces Bid cleavage following caspase-8 activation 64 2.3.4 Andro induces Bax conformational change 64 2.3.5 Andro induces Bax translocation and cytochrome c release 65 2.3.6 Knockdown of Bid expression protects cell from Andro-induced cell death 65 v 2.3.7 Bcl-2 and CrmA over-expression block Andro-induced apoptosis 2.4 Discussion 70 72 CHAPTER ANDRO SENSITIZES CANCER CELLS TO TRAIL-INDUCED APOPTOSIS VIA P53-MEDIATED DEATH RECEPTOR UP-REGULATION 3.1 Introduction 78 3.2 Materials and Methods 79 3.2.1 Chemicals, reagents and antibodies 79 3.2.2 Cell culture and treatments 80 3.2.3 Detection of apoptosis 80 3.2.4 RNA interference 80 3.2.5 Immunoblot analysis 81 3.2.6 Measurement of intracellular ROS 81 3.2.7 Measurement of cell surface expression of death receptors 81 3.2.8 RNA extraction and reverse transcription-PCR 81 3.3 Results 82 3.3.1 Andro sensitizes TRAIL-induced apoptosis 82 3.3.2 Andro promotes TRAIL-induced caspase activation 85 3.3.3 Andro promotes FLIP-L cleavage and XIAP down-regulation as a result of enhanced caspase activation 88 3.3.4 Andro sensitizes TRAIL-induced apoptosis via DR4 up-regulation 91 3.3.5 DR4 up-regulation is critical for the sensitization effect of Andro 91 3.3.6 p53 is required for the DR4 up-regulation and enhanced apoptosis induced by Andro 94 3.3.7 Andro elevates p53 expression through JNK-mediated p53 Thr81 vi phosphorylation 99 3.4 Discussion 104 CHAPTER INHIBITION OF CONSTITUTIVE STAT3 ACTIVITY BY ANDRO ENHANCES CHEMO-SENSITIVITY OF CANCER CELLS TO DOXORUBICIN 4.1 Introduction 110 4.2 Materials and Methods 112 4.2.1 Cell culture and reagents 112 4.2.2 Luciferase assay 112 4.2.3 Preparation of nuclear and cytosolic extracts 113 4.2.4 Immunofluorescence and confocal microscopy 113 4.2.5 DNA transfection and immunoprecipitation 114 4.2.6 Colony formation assay 114 4.3 Results 4.3.1 Andro suppresses constitutive STAT3 activation in human cancer cells 114 114 4.3.2 Andro inhibits IL-6-inducible STAT3 phosphorylation and nuclear translocation in human cancer cells 4.3.3 Andro inhibits STAT phosphorylation through JAK1/2 suppression 117 120 4.3.4 Constitutively active STAT3 confers resistance to doxorubicin-induced cytotoxicity in cancer cells 4.3.5 Andro enhances doxorubicin-induced apoptosis in human cancer cells 123 127 4.3.6 Andro sensitizes doxorubicin-induced apoptosis via suppression of STAT3 131 4.4 Discussion 134 vii CHAPTER GENERAL DISCUSSION AND CONCLUSIONS 5.1 Pro-apoptotic Bcl-2 members play a critical role in Andro-induced apoptosis 141 5.2 Andro sensitizes TRAIL-induced apoptosis in human cancer cells 144 5.3 Andro inhibits STAT3 activation and sensitizes doxorubicin-induced cell death in human cancer cells 149 5.4 The interlinks among molecular mechanisms involved in Andro anticancer property 152 5.5 Conclusions 153 CHAPTER REFERENCES References 156 viii SUMMARY Andrographolide (Andro) is one of the major active components in Andrographis paniculata, a traditional herbal medicine which has been widely used in treatment of various disorders including respiratory infection, bacterial dysentery, diarrhea, and fever. Andro, a bicyclic diterpenoid lactone, has been shown to possess potent antiinflammatory effect, which is executed by inhibiting NF-κB pathway and regulating expression of various cytokines. Recently, Andro was proposed to be able to induce apoptosis in cancer cells, suggesting the anticancer potential of this compound. However, the molecular mechanisms are largely unknown. Therefore, in order to systematically investigate the anticancer properties of Andro, the following investigations have been conducted in this study: (i) identification of the molecular mechanisms involved in Andro-induced apoptosis; (ii) evaluation of the anti-tumor potential of Andro by investigating its sensitization ability on TRAIL-induced apoptosis; (iii) investigation of the combined effect of Andro with an established cancer theratpeutic agent doxorubicin in cancer cells. First, I found that Andro could induce caspase-dependent apoptosis in various human cancer cells. To elucidate the mechanisms of Andro-induced apoptosis, a series of experiments were conducted to demonstrate the critical role of pro-apoptotic Bcl-2 proteins (Bid and Bax) in relaying the cell death signaling initiated by Andro from caspase-8 to mitochondria, leading to the release of cytochrome c and activation of caspase cascade, and eventually resulting in apoptotic cell death in cancer cells. Next I investigated the anticancer potential by evaluating the synergistic effect of Andro on TRAIL-induced apoptosis. Andro significantly sensitized various cancer cells in response to TRAIL-mediated apoptosis. Such sensitization was achieved ix through the up-regulation of DR4. Furthermore, p53 protein which was stabilized and activated by ROS-mediated JNK phosphorylation, was found to be responsible for the DR4 up-regulation and Andro-sensitized apoptosis in cancer cells. In addition to the sensitization effect of Andro and TRAIL, I also investigated the synergistic effect of Andro on the anticancer activities of doxorubicin, a potent chemotherapeutic agent that has been widely used in cancer therapy. Our data demonstrated that the sensitization effect was due to Andro-mediated inhibition on STAT3 activity. As a result, Andro caused the suppression of STAT3-mediated transcription of anti-apoptotic genes (Bcl-2 and Mcl-1), which contributed to the enhanced sensitivity of cancer cells in response to doxorubicin. In conclusion, the present study provides a new insight of the anticancer property of Andro. Andro could be used alone as an anticancer agent through inducing apoptosis in various cancer cells. More importantly, this study provides convincing evidence showing that Andro is capable of sensitizing cancer cells to TRAIL and doxorubicin-induced apoptosis. Such findings support the potential application of Andro as a potent apoptosis inducer and chemo-sensitizer in cancer therapy. x diabetes. Curr Mol Med 7, 674-686. Kapil, A., Koul, I.B., Banerjee, S.K., and Gupta, B.D. (1993). Antihepatotoxic effects of major diterpenoid constituents of Andrographis paniculata. Biochem Pharmacol 46, 182-185. Kasahara, Y., Iwai, K., Yachie, A., Ohta, K., Konno, A., Seki, H., Miyawaki, T., and Taniguchi, N. (1997). Involvement of reactive oxygen intermediates in spontaneous and CD95 (Fas/APO-1)-mediated apoptosis of neutrophils. Blood 89, 1748-1753. Kaufmann, S.H., and Earnshaw, W.C. (2000). Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256, 42-49. Kaufmann, S.H., and Hengartner, M.O. (2001). Programmed cell death: alive and well in the new millennium. Trends Cell Biol 11, 526-534. Kaufmann, S.H., and Steensma, D.P. (2005). On the TRAIL of a new therapy for leukemia. Leukemia 19, 2195-2202. Kerr, J.F., Wyllie, A.H., and Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239257. Kim, H., Kim, E.H., Eom, Y.W., Kim, W.H., Kwon, T.K., Lee, S.J., and Choi, K.S. (2006). Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant hepatoma cells to TRAIL-induced apoptosis through reactive oxygen species-mediated up-regulation of DR5. Cancer Res 66, 1740-1750. Kim, H.Y., Park, E.J., Joe, E.H., and Jou, I. (2003). Curcumin suppresses Janus kinase-STAT inflammatory signaling through activation of Src homology domaincontaining tyrosine phosphatase in brain microglia. J Immunol 171, 6072-6079. Kim, T.G., Hwi, K.K., and Hung, C.S. (2005). Morphological and biochemical changes of andrographolide-induced cell death in human prostatic adenocarcinoma PC-3 cells. In Vivo 19, 551-557. Kim, Y.S., and Milner, J.A. (2005). Targets for indole-3-carbinol in cancer prevention. J Nutr Biochem 16, 65-73. Kokontis, J.M., Wagner, A.J., O'Leary, M., Liao, S., and Hay, N. (2001). A transcriptional activation function of p53 is dispensable for and inhibitory of its apoptotic function. Oncogene 20, 659-668. Korsmeyer, S.J. (1992). Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80, 879-886. Korsmeyer, S.J., Wei, M.C., Saito, M., Weiler, S., Oh, K.J., and Schlesinger, P.H. (2000). Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 7, 1166-1173. 169 Kroemer, G., and Reed, J.C. (2000). Mitochondrial control of cell death. Nat Med 6, 513-519. Kumar, R.A., Sridevi, K., Kumar, N.V., Nanduri, S., and Rajagopal, S. (2004). Anticancer and immunostimulatory compounds from Andrographis paniculata. J Ethnopharmacol 92, 291-295. Kumar, S. (2007). Caspase function in programmed cell death. Cell Death Differ 14, 32-43. Kumar, S., Rabson, A.B., and Gelinas, C. (1992). The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein. Mol Cell Biol 12, 3094-3106. Kuo, P.L., Hsu, Y.L., Lin, T.C., Lin, L.T., and Lin, C.C. (2004). Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by prodelphinidin B-2 3,3'-di-O-gallate from Myrica rubra via Fas-mediated pathway. J Pharm Pharmacol 56, 1399-1406. Kurbanov, B.M., Fecker, L.F., Geilen, C.C., Sterry, W., and Eberle, J. (2007). Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-kappaB but is related to downregulation of initiator caspases and DR4. Oncogene 26, 3364-3377. Kuwana, T., Bouchier-Hayes, L., Chipuk, J.E., Bonzon, C., Sullivan, B.A., Green, D.R., and Newmeyer, D.D. (2005). BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 17, 525-535. Lang, G.A., Iwakuma, T., Suh, Y.A., Liu, G., Rao, V.A., Parant, J.M., Valentin-Vega, Y.A., Terzian, T., Caldwell, L.C., Strong, L.C., et al. (2004). Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119, 861-872. LeBlanc, H.N., and Ashkenazi, A. (2003). Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10, 66-75. Leist, M., Single, B., Naumann, H., Fava, E., Simon, B., Kuhnle, S., and Nicotera, P. (1999). Inhibition of mitochondrial ATP generation by nitric oxide switches apoptosis to necrosis. Exp Cell Res 249, 396-403. Levy, D.E., and Darnell, J.E., Jr. (2002). Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3, 651-662. Lewis, T.S., Shapiro, P.S., and Ahn, N.G. (1998). Signal transduction through MAP kinase cascades. Adv Cancer Res 74, 49-139. Li, H., Zhu, H., Xu, C.J., and Yuan, J. (1998). Cleavage of BID by caspase mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491-501. Li, H.H., Cai, X., Shouse, G.P., Piluso, L.G., and Liu, X. (2007a). A specific PP2A regulatory subunit, B56gamma, mediates DNA damage-induced dephosphorylation of 170 p53 at Thr55. Embo J 26, 402-411. Li, J., Cheung, H.Y., Zhang, Z., Chan, G.K., and Fong, W.F. (2007b). Andrographolide induces cell cycle arrest at G2/M phase and cell death in HepG2 cells via alteration of reactive oxygen species. Eur J Pharmacol 568, 31-44. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S.I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., et al. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943-1947. Lim, C.P., and Cao, X. (1999). Serine phosphorylation and negative regulation of Stat3 by JNK. J Biol Chem 274, 31055-31061. Lin, D., and Takemoto, D.J. (2005). Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions. J Biol Chem 280, 13682-13693. Lin, W.W., and Karin, M. (2007). A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest 117, 1175-1183. Liu, H., Colavitti, R., Rovira, II, and Finkel, T. (2005). Redox-dependent transcriptional regulation. Circ Res 97, 967-974. Liu, X., Yue, P., Khuri, F.R., and Sun, S.Y. (2004). p53 upregulates death receptor expression through an intronic p53 binding site. Cancer Res 64, 5078-5083. Lu, G.D., Shen, H.M., Chung, M.C., and Ong, C.N. (2007). Critical role of oxidative stress and sustained JNK activation in aloe-emodin-mediated apoptotic cell death in human hepatoma cells. Carcinogenesis 28, 1937-1945. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998). Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481-490. MacFarlane, M., Inoue, S., Kohlhaas, S.L., Majid, A., Harper, N., Kennedy, D.B., Dyer, M.J., and Cohen, G.M. (2005a). Chronic lymphocytic leukemic cells exhibit apoptotic signaling via TRAIL-R1. Cell Death Differ 12, 773-782. MacFarlane, M., Kohlhaas, S.L., Sutcliffe, M.J., Dyer, M.J., and Cohen, G.M. (2005b). TRAIL receptor-selective mutants signal to apoptosis via TRAIL-R1 in primary lymphoid malignancies. Cancer Res 65, 11265-11270. Marchenko, N.D., Zaika, A., and Moll, U.M. (2000). Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem 275, 16202-16212. Matsuda, T., Kuroyanagi, M., Sugiyama, S., Umehara, K., Ueno, A., and Nishi, K. (1994). Cell differentiation-inducing diterpenes from Andrographis paniculata Nees. Chem Pharm Bull (Tokyo) 42, 1216-1225. 171 Matsumoto, A., Seki, Y., Kubo, M., Ohtsuka, S., Suzuki, A., Hayashi, I., Tsuji, K., Nakahata, T., Okabe, M., Yamada, S., et al. (1999). Suppression of STAT5 functions in liver, mammary glands, and T cells in cytokine-inducible SH2-containing protein transgenic mice. Mol Cell Biol 19, 6396-6407. McCubrey, J.A., Lahair, M.M., and Franklin, R.A. (2006). Reactive oxygen speciesinduced activation of the MAP kinase signaling pathways. Antioxid Redox Signal 8, 1775-1789. Melino, G., Bernassola, F., Knight, R.A., Corasaniti, M.T., Nistico, G., and FinazziAgro, A. (1997). S-nitrosylation regulates apoptosis. Nature 388, 432-433. Merchant, M.S., Yang, X., Melchionda, F., Romero, M., Klein, R., Thiele, C.J., Tsokos, M., Kontny, H.U., and Mackall, C.L. (2004). Interferon gamma enhances the effectiveness of tumor necrosis factor-related apoptosis-inducing ligand receptor agonists in a xenograft model of Ewing's sarcoma. Cancer Res 64, 8349-8356. Meyer, M., Schreck, R., and Baeuerle, P.A. (1993). H2O2 and antioxidants have opposite effects on activation of NF-kappa B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. Embo J 12, 2005-2015. Michael, D., and Oren, M. (2003). The p53-Mdm2 module and the ubiquitin system. Semin Cancer Biol 13, 49-58. Micheau, O., Lens, S., Gaide, O., Alevizopoulos, K., and Tschopp, J. (2001). NFkappaB signals induce the expression of c-FLIP. Mol Cell Biol 21, 5299-5305. Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P., and Moll, U.M. (2003). p53 has a direct apoptogenic role at the mitochondria. Mol Cell 11, 577590. Miller, D.K. (1997). The role of the Caspase family of cysteine proteases in apoptosis. Semin Immunol 9, 35-49. Minana, J.B., Gomez-Cambronero, L., Lloret, A., Pallardo, F.V., Del Olmo, J., Escudero, A., Rodrigo, J.M., Pelliin, A., Vina, J.R., Vina, J., et al. (2002). Mitochondrial oxidative stress and CD95 ligand: a dual mechanism for hepatocyte apoptosis in chronic alcoholism. Hepatology 35, 1205-1214. Miyashita, T., and Reed, J.C. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293-299. Moll, U.M., Wolff, S., Speidel, D., and Deppert, W. (2005). Transcription-independent pro-apoptotic functions of p53. Curr Opin Cell Biol 17, 631-636. Momand, J., Zambetti, G.P., Olson, D.C., George, D., and Levine, A.J. (1992). The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53mediated transactivation. Cell 69, 1237-1245. Murphy, K.M., Streips, U.N., and Lock, R.B. (2000). Bcl-2 inhibits a Fas-induced 172 conformational change in the Bax N terminus and Bax mitochondrial translocation. J Biol Chem 275, 17225-17228. Naka, T., Narazaki, M., Hirata, M., Matsumoto, T., Minamoto, S., Aono, A., Nishimoto, N., Kajita, T., Taga, T., Yoshizaki, K., et al. (1997). Structure and function of a new STAT-induced STAT inhibitor. Nature 387, 924-929. Nakano, K., and Vousden, K.H. (2001). PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 7, 683-694. Nam, S.Y., Jung, G.A., Hur, G.C., Chung, H.Y., Kim, W.H., Seol, D.W., and Lee, B.L. (2003). Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAILinduced apoptosis in human gastric cancers. Cancer Sci 94, 1066-1073. Nanduri, S., Nyavanandi, V.K., Thunuguntla, S.S., Kasu, S., Pallerla, M.K., Ram, P.S., Rajagopal, S., Kumar, R.A., Ramanujam, R., Babu, J.M., et al. (2004). Synthesis and structure-activity relationships of andrographolide analogues as novel cytotoxic agents. Bioorg Med Chem Lett 14, 4711-4717. Natoni, A., Macfarlane, M., Inoue, S., Walewska, R., Majid, A., Knee, D., Stover, D.R., Dyer, M.J., and Cohen, G.M. (2007). TRAIL signals to apoptosis in chronic lymphocytic leukaemia cells primarily through TRAIL-R1 whereas cross-linked agonistic TRAIL-R2 antibodies facilitate signalling via TRAIL-R2. Br J Haematol. Nesterov, A., Lu, X., Johnson, M., Miller, G.J., Ivashchenko, Y., and Kraft, A.S. (2001). Elevated AKT activity protects the prostate cancer cell line LNCaP from TRAIL-induced apoptosis. J Biol Chem 276, 10767-10774. Nguyen, M., Millar, D.G., Yong, V.W., Korsmeyer, S.J., and Shore, G.C. (1993). Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence. J Biol Chem 268, 25265-25268. Nicholson, S.E., De Souza, D., Fabri, L.J., Corbin, J., Willson, T.A., Zhang, J.G., Silva, A., Asimakis, M., Farley, A., Nash, A.D., et al. (2000). Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp130. Proc Natl Acad Sci U S A 97, 6493-6498. Nieboer, P., de Vries, E.G., Mulder, N.H., and van der Graaf, W.T. (2005). Relevance of high-dose chemotherapy in solid tumours. Cancer Treat Rev 31, 210-225. Niu, G., Bowman, T., Huang, M., Shivers, S., Reintgen, D., Daud, A., Chang, A., Kraker, A., Jove, R., and Yu, H. (2002). Roles of activated Src and Stat3 signaling in melanoma tumor cell growth. Oncogene 21, 7001-7010. Niu, G., Wright, K.L., Ma, Y., Wright, G.M., Huang, M., Irby, R., Briggs, J., Karras, J., Cress, W.D., Pardoll, D., et al. (2005). Role of Stat3 in regulating p53 expression and function. Mol Cell Biol 25, 7432-7440. Nobel, C.S., Burgess, D.H., Zhivotovsky, B., Burkitt, M.J., Orrenius, S., and Slater, A.F. (1997). Mechanism of dithiocarbamate inhibition of apoptosis: thiol oxidation by 173 dithiocarbamate disulfides directly inhibits processing of the caspase-3 proenzyme. Chem Res Toxicol 10, 636-643. Novotny-Diermayr, V., Zhang, T., Gu, L., and Cao, X. (2002). Protein kinase C delta associates with the interleukin-6 receptor subunit glycoprotein (gp) 130 via Stat3 and enhances Stat3-gp130 interaction. J Biol Chem 277, 49134-49142. Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino, T., Taniguchi, T., and Tanaka, N. (2000). Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, 1053-1058. Okuno, H., Akahori, A., Sato, H., Xanthoudakis, S., Curran, T., and Iba, H. (1993). Escape from redox regulation enhances the transforming activity of Fos. Oncogene 8, 695-701. Olive, K.P., Tuveson, D.A., Ruhe, Z.C., Yin, B., Willis, N.A., Bronson, R.T., Crowley, D., and Jacks, T. (2004). Mutant p53 gain of function in two mouse models of LiFraumeni syndrome. Cell 119, 847-860. Oltvai, Z.N., Milliman, C.L., and Korsmeyer, S.J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609-619. Oya, M., Ohtsubo, M., Takayanagi, A., Tachibana, M., Shimizu, N., and Murai, M. (2001). Constitutive activation of nuclear factor-kappaB prevents TRAIL-induced apoptosis in renal cancer cells. Oncogene 20, 3888-3896. Ozben, T. (2007). Oxidative stress and apoptosis: impact on cancer therapy. J Pharm Sci 96, 2181-2196. Ozoren, N., Fisher, M.J., Kim, K., Liu, C.X., Genin, A., Shifman, Y., Dicker, D.T., Spinner, N.B., Lisitsyn, N.A., and El-Deiry, W.S. (2000). Homozygous deletion of the death receptor DR4 gene in a nasopharyngeal cancer cell line is associated with TRAIL resistance. Int J Oncol 16, 917-925. Palacios, C., Yerbes, R., and Lopez-Rivas, A. (2006). Flavopiridol induces cellular FLICE-inhibitory protein degradation by the proteasome and promotes TRAILinduced early signaling and apoptosis in breast tumor cells. Cancer Res 66, 8858-8869. Panossian, A., Hovhannisyan, A., Mamikonyan, G., Abrahamian, H., Hambardzumyan, E., Gabrielian, E., Goukasova, G., Wikman, G., and Wagner, H. (2000). Pharmacokinetic and oral bioavailability of andrographolide from Andrographis paniculata fixed combination Kan Jang in rats and human. Phytomedicine 7, 351-364. Pathak, A.K., Bhutani, M., Nair, A.S., Ahn, K.S., Chakraborty, A., Kadara, H., Guha, S., Sethi, G., and Aggarwal, B.B. (2007). Ursolic acid inhibits STAT3 activation pathway leading to suppression of proliferation and chemosensitization of human multiple myeloma cells. Mol Cancer Res 5, 943-955. Pearl, S.H., and Kanat, I.O. (1988). Diabetes and healing: a review of the literature. J 174 Foot Surg 27, 268-270. Pearson, G., Robinson, F., Beers Gibson, T., Xu, B.E., Karandikar, M., Berman, K., and Cobb, M.H. (2001). Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22, 153-183. Pedranzini, L., Dechow, T., Berishaj, M., Comenzo, R., Zhou, P., Azare, J., Bornmann, W., and Bromberg, J. (2006). Pyridone 6, a pan-Janus-activated kinase inhibitor, induces growth inhibition of multiple myeloma cells. Cancer Res 66, 9714-9721. Pelicano, H., Carney, D., and Huang, P. (2004). ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 7, 97-110. Peng, G.Y., Zhou, F., Ding, R.L., Li, H.D., and Yao, K. (2002). [Modulation of lianbizi injection (andrographolide) on some immune functions]. Zhongguo Zhong Yao Za Zhi 27, 147-150. Pinton, P., Ferrari, D., Magalhaes, P., Schulze-Osthoff, K., Di Virgilio, F., Pozzan, T., and Rizzuto, R. (2000). Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells. J Cell Biol 148, 857-862. Poolsup, N., Suthisisang, C., Prathanturarug, S., Asawamekin, A., and Chanchareon, U. (2004). Andrographis paniculata in the symptomatic treatment of uncomplicated upper respiratory tract infection: systematic review of randomized controlled trials. J Clin Pharm Ther 29, 37-45. Puri, A., Saxena, R., Saxena, R.P., Saxena, K.C., Srivastava, V., and Tandon, J.S. (1993). Immunostimulant agents from Andrographis paniculata. J Nat Prod 56, 995999. Qin, L.H., Kong, L., Shi, G.J., Wang, Z.T., and Ge, B.X. (2006). Andrographolide inhibits the production of TNF-alpha and interleukin-12 in lipopolysaccharidestimulated macrophages: role of mitogen-activated protein kinases. Biol Pharm Bull 29, 220-224. Rahman-Roblick, R., Roblick, U.J., Hellman, U., Conrotto, P., Liu, T., Becker, S., Hirschberg, D., Jornvall, H., Auer, G., and Wiman, K.G. (2007). p53 targets identified by protein expression profiling. Proc Natl Acad Sci U S A 104, 5401-5406. Rajagopal, S., Kumar, R.A., Deevi, D.S., Satyanarayana, C., and Rajagopalan, R. (2003). Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata. J Exp Ther Oncol 3, 147-158. Rajasingh, J., Raikwar, H.P., Muthian, G., Johnson, C., and Bright, J.J. (2006). Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia. Biochem Biophys Res Commun 340, 359-368. Ravi, R., Bedi, G.C., Engstrom, L.W., Zeng, Q., Mookerjee, B., Gelinas, C., Fuchs, 175 E.J., and Bedi, A. (2001). Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB. Nat Cell Biol 3, 409-416. Ravizza, R., Gariboldi, M.B., Molteni, R., and Monti, E. (2008). Linalool, a plantderived monoterpene alcohol, reverses doxorubicin resistance in human breast adenocarcinoma cells. Oncol Rep 20, 625-630. Rebbaa, A., Chou, P.M., and Mirkin, B.L. (2001). Factors secreted by human neuroblastoma mediated doxorubicin resistance by activating STAT3 and inhibiting apoptosis. Mol Med 7, 393-400. Reddy, V.L., Reddy, S.M., Ravikanth, V., Krishnaiah, P., Goud, T.V., Rao, T.P., Ram, T.S., Gonnade, R.G., Bhadbhade, M., and Venkateswarlu, Y. (2005). A new bisandrographolide ether from Andrographis paniculata nees and evaluation of anti-HIV activity. Nat Prod Res 19, 223-230. Renzing, J., Hansen, S., and Lane, D.P. (1996). Oxidative stress is involved in the UV activation of p53. J Cell Sci 109 ( Pt 5), 1105-1112. Ricci, M.S., Kim, S.H., Ogi, K., Plastaras, J.P., Ling, J., Wang, W., Jin, Z., Liu, Y.Y., Dicker, D.T., Chiao, P.J., et al. (2007). Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAILinduced death. Cancer Cell 12, 66-80. Rupniewska, Z., and Bojarska-Junak, A. (2004). [Apoptosis: mitochondrial membrane permeabilization and the role played by Bcl-2 family proteins]. Postepy Hig Med Dosw (Online) 58, 538-547. Sabuncu, T., Vural, H., Harma, M., and Harma, M. (2001). Oxidative stress in polycystic ovary syndrome and its contribution to the risk of cardiovascular disease. Clin Biochem 34, 407-413. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998). Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. Embo J 17, 2596-2606. Samali, A., Nordgren, H., Zhivotovsky, B., Peterson, E., and Orrenius, S. (1999). A comparative study of apoptosis and necrosis in HepG2 cells: oxidant-induced caspase inactivation leads to necrosis. Biochem Biophys Res Commun 255, 6-11. Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I., and Yoshimura, A. (2000). CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2. J Biol Chem 275, 29338-29347. Sasser, A.K., Sullivan, N.J., Studebaker, A.W., Hendey, L.F., Axel, A.E., and Hall, B.M. (2007). Interleukin-6 is a potent growth factor for ER-alpha-positive human breast cancer. Faseb J 21, 3763-3770. Sato, T., Hanada, M., Bodrug, S., Irie, S., Iwama, N., Boise, L.H., Thompson, C.B., Golemis, E., Fong, L., Wang, H.G., et al. (1994). Interactions among members of the 176 Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci U S A 91, 9238-9242. Satyanarayana, C., Deevi, D.S., Rajagopalan, R., Srinivas, N., and Rajagopal, S. (2004). DRF 3188 a novel semi-synthetic analog of andrographolide: cellular response to MCF breast cancer cells. BMC Cancer 4, 26. Sawa, T., and Ohshima, H. (2006). Nitrative DNA damage in inflammation and its possible role in carcinogenesis. Nitric Oxide 14, 91-100. Sax, J.K., Fei, P., Murphy, M.E., Bernhard, E., Korsmeyer, S.J., and El-Deiry, W.S. (2002). BID regulation by p53 contributes to chemosensitivity. Nat Cell Biol 4, 842849. Schimmer, A.D., Dalili, S., Batey, R.A., and Riedl, S.J. (2006). Targeting XIAP for the treatment of malignancy. Cell Death Differ 13, 179-188. Schindler, C., and Darnell, J.E., Jr. (1995). Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem 64, 621-651. Schoemaker, M.H., Ros, J.E., Homan, M., Trautwein, C., Liston, P., Poelstra, K., van Goor, H., Jansen, P.L., and Moshage, H. (2002). Cytokine regulation of pro- and antiapoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein (cIAP2) prevents apoptosis. J Hepatol 36, 742-750. Scorrano, L., Oakes, S.A., Opferman, J.T., Cheng, E.H., Sorcinelli, M.D., Pozzan, T., and Korsmeyer, S.J. (2003). BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300, 135-139. Shankar, S., and Srivastava, R.K. (2004). Enhancement of therapeutic potential of TRAIL by cancer chemotherapy and irradiation: mechanisms and clinical implications. Drug Resist Updat 7, 139-156. Sheeja, K., Guruvayoorappan, C., and Kuttan, G. (2007). Antiangiogenic activity of Andrographis paniculata extract and andrographolide. Int Immunopharmacol 7, 211221. Sheeja, K., and Kuttan, G. (2006). Protective effect of Andrographis paniculata and andrographolide on cyclophosphamide-induced urothelial toxicity. Integr Cancer Ther 5, 244-251. Sheeja, K., and Kuttan, G. (2007a). Activation of cytotoxic T lymphocyte responses and attenuation of tumor growth in vivo by Andrographis paniculata extract and andrographolide. Immunopharmacol Immunotoxicol 29, 81-93. Sheeja, K., and Kuttan, G. (2007b). Modulation of natural killer cell activity, antibody-dependent cellular cytotoxicity, and antibody-dependent complementmediated cytotoxicity by andrographolide in normal and Ehrlich ascites carcinomabearing mice. Integr Cancer Ther 6, 66-73. 177 Sheikh, M.S., Burns, T.F., Huang, Y., Wu, G.S., Amundson, S., Brooks, K.S., Fornace, A.J., Jr., and el-Deiry, W.S. (1998). p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha. Cancer Res 58, 1593-1598. Shen, H.M., and Liu, Z.G. (2006). JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radic Biol Med 40, 928-939. Shen, Y.C., Chen, C.F., and Chiou, W.F. (2002). Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its antiinflammatory effect. Br J Pharmacol 135, 399-406. Shi, R.X., Ong, C.N., and Shen, H.M. (2005). Protein kinase C inhibition and x-linked inhibitor of apoptosis protein degradation contribute to the sensitization effect of luteolin on tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cancer cells. Cancer Res 65, 7815-7823. Shi, Y. (2001). A structural view of mitochondria-mediated apoptosis. Nat Struct Biol 8, 394-401. Shi, Y. (2002). Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9, 459-470. Shieh, S.Y., Ikeda, M., Taya, Y., and Prives, C. (1997). DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325-334. Shin, D.S., Kim, H.N., Shin, K.D., Yoon, Y.J., Kim, S.J., Han, D.C., and Kwon, B.M. (2009). Cryptotanshinone inhibits constitutive signal transducer and activator of transcription function through blocking the dimerization in DU145 prostate cancer cells. Cancer Res 69, 193-202. Shoshan-Barmatz, V., Israelson, A., Brdiczka, D., and Sheu, S.S. (2006). The voltagedependent anion channel (VDAC): function in intracellular signalling, cell life and cell death. Curr Pharm Des 12, 2249-2270. Shuai, K., Schindler, C., Prezioso, V.R., and Darnell, J.E., Jr. (1992). Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Science 258, 1808-1812. Singh, T.R., Shankar, S., and Srivastava, R.K. (2005). HDAC inhibitors enhance the apoptosis-inducing potential of TRAIL in breast carcinoma. Oncogene 24, 4609-4623. Singha, P.K., Roy, S., and Dey, S. (2003). Antimicrobial activity of Andrographis paniculata. Fitoterapia 74, 692-694. Singha, P.K., Roy, S., and Dey, S. (2007). Protective activity of andrographolide and arabinogalactan proteins from Andrographis paniculata Nees. against ethanol-induced toxicity in mice. J Ethnopharmacol 111, 13-21. 178 Sinibaldi, D., Wharton, W., Turkson, J., Bowman, T., Pledger, W.J., and Jove, R. (2000). Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling. Oncogene 19, 5419-5427. Sohal, R.S., Mockett, R.J., and Orr, W.C. (2002). Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic Biol Med 33, 575-586. Song, H., Hollstein, M., and Xu, Y. (2007). p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM. Nat Cell Biol 9, 573-580. Speidel, D., Helmbold, H., and Deppert, W. (2006). Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress. Oncogene 25, 940-953. Sprick, M.R., Rieser, E., Stahl, H., Grosse-Wilde, A., Weigand, M.A., and Walczak, H. (2002). Caspase-10 is recruited to and activated at the native TRAIL and CD95 deathinducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8. EMBO J 21, 4520-4530. Strasser, A. (2005). The role of BH3-only proteins in the immune system. Nat Rev Immunol 5, 189-200. Strasser, A., O'Connor, L., and Dixit, V.M. (2000). Apoptosis signaling. Annu Rev Biochem 69, 217-245. Suh, W.S., Kim, Y.S., Schimmer, A.D., Kitada, S., Minden, M., Andreeff, M., Suh, N., Sporn, M., and Reed, J.C. (2003). Synthetic triterpenoids activate a pathway for apoptosis in AML cells involving downregulation of FLIP and sensitization to TRAIL. Leukemia 17, 2122-2129. Suliman, A., Lam, A., Datta, R., and Srivastava, R.K. (2001). Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene 20, 2122-2133. Sussman, R.T., Ricci, M.S., Hart, L.S., Sun, S.Y., and El-Deiry, W.S. (2007). Chemotherapy-resistant side-population of colon cancer cells has a higher sensitivity to TRAIL than the non-SP, a higher expression of c-Myc and TRAIL-receptor DR4. Cancer Biol Ther 6, 1490-1495. Takeda, K., Stagg, J., Yagita, H., Okumura, K., and Smyth, M.J. (2007). Targeting death-inducing receptors in cancer therapy. Oncogene 26, 3745-3757. Takimoto, R., and El-Deiry, W.S. (2000). Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site. Oncogene 19, 1735-1743. Taylor, W.R., and Stark, G.R. (2001). Regulation of the G2/M transition by p53. Oncogene 20, 1803-1815. Testa, J.R., and Bellacosa, A. (2001). AKT plays a central role in tumorigenesis. Proc 179 Natl Acad Sci U S A 98, 10983-10985. Thamlikitkul, V., Dechatiwongse, T., Theerapong, S., Chantrakul, C., Boonroj, P., Punkrut, W., Ekpalakorn, W., Boontaeng, N., Taechaiya, S., Petcharoen, S., et al. (1991). Efficacy of Andrographis paniculata, Nees for pharyngotonsillitis in adults. J Med Assoc Thai 74, 437-442. Thisoda, P., Rangkadilok, N., Pholphana, N., Worasuttayangkurn, L., Ruchirawat, S., and Satayavivad, J. (2006). Inhibitory effect of Andrographis paniculata extract and its active diterpenoids on platelet aggregation. Eur J Pharmacol 553, 39-45. Thome, M., and Tschopp, J. (2001). Regulation of lymphocyte proliferation and death by FLIP. Nat Rev Immunol 1, 50-58. Toledano, M.B., and Leonard, W.J. (1991). Modulation of transcription factor NFkappa B binding activity by oxidation-reduction in vitro. Proc Natl Acad Sci U S A 88, 4328-4332. Tomasetti, M., Andera, L., Alleva, R., Borghi, B., Neuzil, J., and Procopio, A. (2006). Alpha-tocopheryl succinate induces DR4 and DR5 expression by a p53-dependent route: implication for sensitisation of resistant cancer cells to TRAIL apoptosis. FEBS Lett 580, 1925-1931. Trivedi, N.P., Rawal, U.M., and Patel, B.P. (2007). Hepatoprotective effect of andrographolide against hexachlorocyclohexane-induced oxidative injury. Integr Cancer Ther 6, 271-280. Truneh, A., Sharma, S., Silverman, C., Khandekar, S., Reddy, M.P., Deen, K.C., McLaughlin, M.M., Srinivasula, S.M., Livi, G.P., Marshall, L.A., et al. (2000). Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor. J Biol Chem 275, 23319-23325. Tsai, H.R., Yang, L.M., Tsai, W.J., and Chiou, W.F. (2004). Andrographolide acts through inhibition of ERK1/2 and Akt phosphorylation to suppress chemotactic migration. Eur J Pharmacol 498, 45-52. Turkson, J., Zhang, S., Mora, L.B., Burns, A., Sebti, S., and Jove, R. (2005). A novel platinum compound inhibits constitutive Stat3 signaling and induces cell cycle arrest and apoptosis of malignant cells. J Biol Chem 280, 32979-32988. Usatyuk, P.V., Vepa, S., Watkins, T., He, D., Parinandi, N.L., and Natarajan, V. (2003). Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells. Antioxid Redox Signal 5, 723-730. van Geelen, C.M., de Vries, E.G., Le, T.K., van Weeghel, R.P., and de Jong, S. (2003). Differential modulation of the TRAIL receptors and the CD95 receptor in colon carcinoma cell lines. Br J Cancer 89, 363-373. Vaquero, E.C., Edderkaoui, M., Pandol, S.J., Gukovsky, I., and Gukovskaya, A.S. 180 (2004). Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells. J Biol Chem 279, 34643-34654. Vaux, D.L., and Silke, J. (2005). IAPs, RINGs and ubiquitylation. Nat Rev Mol Cell Biol 6, 287-297. Ventura, J.J., Cogswell, P., Flavell, R.A., Baldwin, A.S., Jr., and Davis, R.J. (2004). JNK potentiates TNF-stimulated necrosis by increasing the production of cytotoxic reactive oxygen species. Genes Dev 18, 2905-2915. Vignais, P.V. (2002). The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell Mol Life Sci 59, 1428-1459. Vigneron, A., Roninson, I.B., Gamelin, E., and Coqueret, O. (2005). Src inhibits adriamycin-induced senescence and G2 checkpoint arrest by blocking the induction of p21waf1. Cancer Res 65, 8927-8935. Visen, P.K., Shukla, B., Patnaik, G.K., and Dhawan, B.N. (1993). Andrographolide protects rat hepatocytes against paracetamol-induced damage. J Ethnopharmacol 40, 131-136. Voelkel-Johnson, C. (2003). An antibody against DR4 (TRAIL-R1) in combination with doxorubicin selectively kills malignant but not normal prostate cells. Cancer Biol Ther 2, 283-290. Vogelstein, B., Lane, D., and Levine, A.J. (2000). Surfing the p53 network. Nature 408, 307-310. Wang, L., Wu, Q., Qiu, P., Mirza, A., McGuirk, M., Kirschmeier, P., Greene, J.R., Wang, Y., Pickett, C.B., and Liu, S. (2001). Analyses of p53 target genes in the human genome by bioinformatic and microarray approaches. J Biol Chem 276, 43604-43610. Wang, S., and El-Deiry, W.S. (2003). TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 22, 8628-8633. Wang, Y.J., Wang, J.T., Fan, Q.X., and Geng, J.G. (2007). Andrographolide inhibits NF-kappaBeta activation and attenuates neointimal hyperplasia in arterial restenosis. Cell Res 17, 933-941. Waterman, M.J., Stavridi, E.S., Waterman, J.L., and Halazonetis, T.D. (1998). ATMdependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nat Genet 19, 175-178. Wei, L.H., Kuo, M.L., Chen, C.A., Chou, C.H., Cheng, W.F., Chang, M.C., Su, J.L., and Hsieh, C.Y. (2001). The anti-apoptotic role of interleukin-6 in human cervical cancer is mediated by up-regulation of Mcl-1 through a PI 3-K/Akt pathway. Oncogene 20, 5799-5809. Wei, M.C., Lindsten, T., Mootha, V.K., Weiler, S., Gross, A., Ashiya, M., Thompson, C.B., and Korsmeyer, S.J. (2000). tBID, a membrane-targeted death ligand, 181 oligomerizes BAK to release cytochrome c. Genes Dev 14, 2060-2071. Willis, S.N., and Adams, J.M. (2005). Life in the balance: how BH3-only proteins induce apoptosis. Curr Opin Cell Biol 17, 617-625. Willis, S.N., Fletcher, J.I., Kaufmann, T., van Delft, M.F., Chen, L., Czabotar, P.E., Ierino, H., Lee, E.F., Fairlie, W.D., Bouillet, P., et al. (2007). Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak. Science 315, 856-859. Woo, A.Y., Waye, M.M., Tsui, S.K., Yeung, S.T., and Cheng, C.H. (2008). Andrographolide up-regulates cellular-reduced glutathione level and protects cardiomyocytes against hypoxia/reoxygenation injury. J Pharmacol Exp Ther 325, 226-235. Wu, G.S. (2004). The functional interactions between the p53 and MAPK signaling pathways. Cancer Biol Ther 3, 156-161. Wu, G.S., Burns, T.F., McDonald, E.R., 3rd, Jiang, W., Meng, R., Krantz, I.D., Kao, G., Gan, D.D., Zhou, J.Y., Muschel, R., et al. (1997). KILLER/DR5 is a DNA damageinducible p53-regulated death receptor gene. Nat Genet 17, 141-143. Wu, G.S., Kim, K., and el-Deiry, W.S. (2000). KILLER/DR5, a novel DNA-damage inducible death receptor gene, links the p53-tumor suppressor to caspase activation and apoptotic death. Adv Exp Med Biol 465, 143-151. Wu, W.S., Tsai, R.K., Chang, C.H., Wang, S., Wu, J.R., and Chang, Y.X. (2006). 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 4, 747-758. Wudarczyk, J., Debska, G., and Lenartowicz, E. (1996). Relation between the activities reducing disulfides and the protection against membrane permeability transition in rat liver mitochondria. Arch Biochem Biophys 327, 215-221. Xia, Y.F., Ye, B.Q., Li, Y.D., Wang, J.G., He, X.J., Lin, X., Yao, X., Ma, D., Slungaard, A., Hebbel, R.P., et al. (2004). Andrographolide attenuates inflammation by inhibition of NF-kappa B activation through covalent modification of reduced cysteine 62 of p50. J Immunol 173, 4207-4217. Xu, Y., Chen, A., Fry, S., Barrow, R.A., Marshall, R.L., and Mukkur, T.K. (2007). Modulation of immune response in mice immunised with an inactivated Salmonella vaccine and gavaged with Andrographis paniculata extract or andrographolide. Int Immunopharmacol 7, 515-523. Yamaguchi, H., Paranawithana, S.R., Lee, M.W., Huang, Z., Bhalla, K.N., and Wang, H.G. (2002). Epothilone B analogue (BMS-247550)-mediated cytotoxicity through induction of Bax conformational change in human breast cancer cells. Cancer Res 62, 466-471. Yamamoto, K., Ichijo, H., and Korsmeyer, S.J. (1999). BCL-2 is phosphorylated and 182 inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol 19, 8469-8478. Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P., and Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132. Yoshikawa, H., Matsubara, K., Qian, G.S., Jackson, P., Groopman, J.D., Manning, J.E., Harris, C.C., and Herman, J.G. (2001). SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 28, 29-35. Youle, R.J., and Strasser, A. (2008). The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9, 47-59. Yu, B.C., Chang, C.K., Su, C.F., and Cheng, J.T. (2007a). Mediation of betaendorphin in andrographolide-induced plasma glucose-lowering action in type I diabetes-like animals. Naunyn Schmiedebergs Arch Pharmacol. Yu, B.C., Hung, C.R., Chen, W.C., and Cheng, J.T. (2003). Antihyperglycemic effect of andrographolide in streptozotocin-induced diabetic rats. Planta Med 69, 1075-1079. Yu, C.L., Meyer, D.J., Campbell, G.S., Larner, A.C., Carter-Su, C., Schwartz, J., and Jove, R. (1995). Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science 269, 81-83. Yu, H., and Jove, R. (2004). The STATs of cancer--new molecular targets come of age. Nat Rev Cancer 4, 97-105. Yu, H., Kortylewski, M., and Pardoll, D. (2007b). Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nat Rev Immunol 7, 41-51. Yu, Q. (2006). Restoring p53-mediated apoptosis in cancer cells: new opportunities for cancer therapy. Drug Resist Updat 9, 19-25. Zamzami, N., and Kroemer, G. (2001). The mitochondrion in apoptosis: how Pandora's box opens. Nat Rev Mol Cell Biol 2, 67-71. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S.J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 87, 619-628. Zhang, L., and Fang, B. (2005). Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther 12, 228-237. Zhang, S., Ong, C.N., and Shen, H.M. (2004). Involvement of proapoptotic Bcl-2 family members in parthenolide-induced mitochondrial dysfunction and apoptosis. Cancer Lett 211, 175-188. 183 Zhang, S., Shen, H.M., and Ong, C.N. (2005). Down-regulation of c-FLIP contributes to the sensitization effect of 3,3'-diindolylmethane on TRAIL-induced apoptosis in cancer cells. Mol Cancer Ther 4, 1972-1981. Zhao, H.L., Tong, P.C., and Chan, J.C. (2006). Traditional Chinese medicine in the treatment of diabetes. Nestle Nutr Workshop Ser Clin Perform Programme 11, 15-25; discussion 25-19. Zhao, R., Gish, K., Murphy, M., Yin, Y., Notterman, D., Hoffman, W.H., Tom, E., Mack, D.H., and Levine, A.J. (2000). Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. Genes Dev 14, 981-993. Zhong, Z., Wen, Z., and Darnell, J.E., Jr. (1994). Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264, 95-98. Zhou, P., Qian, L., Kozopas, K.M., and Craig, R.W. (1997). Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions. Blood 89, 630-643. Zhu, W., Cowie, A., Wasfy, G.W., Penn, L.Z., Leber, B., and Andrews, D.W. (1996). Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types. Embo J 15, 4130-4141. 184 [...]... unknown As apoptosis-inducing ability of Andro is directly related to its potential of anticancer property, it is important to further investigate the mechanisms of Andro-induced apoptosis and the molecular targets of 11 this compound On the other hand, the proapoptotic effect of Andro in cancer cells seems contradictory to the anti-apoptotic effect found in normal epithelial and immune cells (Chapter 1.1.3.2)... morphological changes in Andro-treated human prostate cancer PC-3 cells: membrane blebbing, chromosome fragmentation and formation of apoptotic bodies The author further suggested that the activation of caspase-8 and caspase-3 was involved in Andro-induced apoptosis Another group (Cheung et al., 2005) explored the apoptosis-inducing effect of Andro in human leukemic HL-60 cells and indicated the Andro-induced chromosome... death Some studies have been carried out to demonstrate the potent anticancer activities of Andro and the possible molecular mechanisms In the following sections, these findings will be summarized accordingly 1.1.3.7.1 Inhibition of cancer cell proliferation and induction of cell cycle arrest The effect of Andro on cell proliferation and viability are less reported The anticancer property of Andro was... the effect of Andro on the ERK signaling pathway is stimulus- or cell text-dependent At present, the knowledge about the potential effects of Andro on MAPKs other family members, including JNK and p38, are rather limited The exact role of MAPK pathway in the bioactivities of Andro remains to be further investigated 1.1.4.3 Effects on AKT/PKB survival pathways The serine/threonine protein kinase Akt,... Fluorescein isothiothyanate FLIP FLICE inhibitory protein GAPDH glyceraldehyde-3-phosphate dehydrogenase GSH reduced glutathione H2O2 hydrogen peroxide HIV human immunodeficiency virus IAPs inhibitor of apoptosis proteins IL Interleukin Iκ B NF-κB inhibitory protein IKK IκB kinase INF-γ interferon-γ iNOS inducible nitric oxide synthase JAB Janus kinase binding protein JAKs Janus protein-tyrosine kinases... He et al., 2003) The identification of Andro metabolites is important in reflecting the in vivo metabolic fate and disposition of Andro in human Most absorbed Andro was found to be intensively metabolized in vivo, mainly as sulfonic acid adducts and sulfate compounds These formations may increase the polarity of the mother compound and in turn help its excretion in urine and feces In addition, glucuronide... accompanied by disappearance of mitochondrial cytochrome c and increased expression of Bax but decreased expression of Bcl-2 proteins, suggesting that Andro induced the intrinsic mitochondria-dependent pathway of apoptosis Both studies pointed out that, Andro could inhibit cell growth by inducing apoptosis in cancer cells However, the detailed molecular mechanisms of Androinduced apoptosis are still... down-regulation of CDK4 (Rajagopal et al., 2003) 1.1.3.7.2 Induction of apoptotic cell death Andro may have dual effects on apoptosis: in cancer cells Andro could stimulate apoptosis while in normal epithelial and immune cells Andro prevents apoptosis induction The latter effect has been discussed in Chapter 1.1.3.2 The induction of apoptotic death in cancer cells has been revealed by two recent studies In the. .. JAK1/JAK2 inhibition 122 Figure 4.5 The chemoresistance of tumor cells to doxorubicin-induced cytotoxicity is correlated to STAT3 activity 125 Figure 4.6 Constitutively active STAT3 plays a crucial role in chemoresistance of tumor cells to doxorubicin 126 Figure 4.7 Andro enhances doxorubicin-induced cytotoxicity in cancer cells 129 Figure 4.8 Andro sensitizes human cancer cells to doxorubicin-induced... the inflammatory symptoms of sinusitis It is worth noting that no adverse effects were reported in these studies Several mechanism studies implied that the anti-inflammation effect of Andro possibly resulted from its inhibition of nuclear factor-kappaB (NF-κB), suppression of the activation of immunocompetent cells and repression of production of key proinflammatory cytokines, such as TNF-α, IL-1, . STUDIES OF THE ANTICANCER POTENTIAL OF ANDROGRAPHOLIDE IN HUMAN CANCER CELLS ZHOU JING (M. Med. China Academy of Chinese Medical Sciences; P. R. China) A THESIS SUBMITTED. sensitizes TRAIL-induced apoptosis in human cancer cells 144 5.3 Andro inhibits STAT3 activation and sensitizes doxorubicin-induced cell death in human cancer cells 149 5.4 The interlinks among. suggesting the anticancer potential of this compound. However, the molecular mechanisms are largely unknown. Therefore, in order to systematically investigate the anticancer properties of Andro, the